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Sample records for frogs xenopus laevis

  1. Cutaneous acariasis in the African clawed frog (Xenopus laevis).

    Science.gov (United States)

    Ford, Timothy R; Dillehay, Dirck L; Mook, Deborah M

    2004-12-01

    Increased mortality was observed in a single colony of 50 Xenopus laevis. The frogs were used as oocyte donors in developmental biology studies. Necropsy findings included dermal erythema and petechiation consistent with red leg syndrome; dermal ulcerations and white, filamentous growths on the skin were consistent with Saprolegnia sp. Microscopic evaluation of the skin and fungus revealed an astigmatid mite similar to those of the genus Rhizoglyphus. The mite was also found in the water and the biological filter of the tanks housing the frogs. This mite is considered not to be a parasite of X. laevis; instead, it feeds off moss, fungi, and detritus. Subsequent evaluation of the sphagnum moss used for shipping the frogs from the supplier revealed the same mite in the moss. Our hypothesis is that the mite was introduced into the tank with the shipment of new frogs in sphagnum moss. The mites lived within the biological filter, and were only found after the growth of Saprolegnia sp. attracted the mites to the frogs. Laboratory animal care and veterinary personnel should consider non-pathogenic species of mites in the differential diagnosis of acariasis in Xenopus frogs.

  2. Eugenol for anesthesia of African clawed frogs (Xenopus laevis).

    Science.gov (United States)

    Guénette, Sarah A; Hélie, Pierre; Beaudry, Francis; Vachon, Pascal

    2007-05-01

    To determine the level of anesthesia attained in Xenopus laevis frogs with eugenol at different doses and by different routes of administration. Prospective experimental trial. Sixty X. laevis nonbreeding female frogs weighing between 90 and 140 g. Three different routes of administration were tested - subcutaneous injections into the dorsal lymph sacs, topical administration using a gauze patch, and immersion in a bath containing eugenol. Following the determination of the best route of administration, the acetic acid test, the withdrawal reflex, righting reflex, heart rate, and respiratory frequency were used to evaluate central nervous system depression following eugenol bath administration. In an additional group, the response to a surgical incision of the abdominal wall was evaluated. The pharmacokinetics of eugenol were determined following bath immersion administration, and pharmacokinetic parameters were calculated following blood concentration determination by tandem liquid chromatography/mass spectrometry analyses. It was not possible to induce anethesia with subcutaneous and patch administration, independent of the eugenol dose administered. The immersion bath was the only efficacious route for anesthesia inducing surgical anesthesia for at least 30 minutes with postoperative analgesia. Histopathology of selected tissues (heart, lung, liver, kidneys, eyes) showed no evidence of lesions 24 hours following bath immersion. The elimination half-life (T(1/2)) was 4 hours. When administered as a single-bath immersion (dose 350 mg L(-1)) for 15 minutes, eugenol may serve as an effective anesthetic in X. laevis frogs for short surgical procedures.

  3. Genome evolution in the allotetraploid frog Xenopus laevis

    Science.gov (United States)

    Session, Adam M.; Uno, Yoshinobu; Kwon, Taejoon; Chapman, Jarrod A.; Toyoda, Atsushi; Takahashi, Shuji; Fukui, Akimasa; Hikosaka, Akira; Suzuki, Atsushi; Kondo, Mariko; van Heeringen, Simon J.; Quigley, Ian; Heinz, Sven; Ogino, Hajime; Ochi, Haruki; Hellsten, Uffe; Lyons, Jessica B; Simakov, Oleg; Putnam, Nicholas; Stites, Jonathan; Kuroki, Yoko; Tanaka, Toshiaki; Michiue, Tatsuo; Watanabe, Minoru; Bogdanovic, Ozren; Lister, Ryan; Georgiou, Georgios; Paranjpe, Sarita S.; van Kruijsbergen, Ila; Shu, Shengquiang; Carlson, Joseph; Kinoshita, Tsutomu; Ohta, Yuko; Mawaribuchi, Shuuji; Jenkins, Jerry; Grimwood, Jane; Schmutz, Jeremy; Mitros, Therese; Mozaffari, Sahar; Suzuki, Yutaka; Haramoto, Yoshikazu; Yamamoto, Takamasa S.; Takagi, Chiyo; Heald, Rebecca; Miller, Kelly; Haudenschild, Christian; Kitzman, Jacob; Nakayama, Takuya; Izutsu, Yumi; Robert, Jacques; Fortriede, Joshua; Burns, Kevin; Lotay, Vaneet; Karimi, Kamran; Yasuoka, Yuuri; Dichmann, Darwin S.; Flajnik, Martin F.; Houston, Douglas W; Shendure, Jay; DuPasquier, Louis; Vize, Peter D.; Zorn, Aaron M.; Ito, Michihiko; Marcotte, Ed; Wallingford, John B.; Ito, Yuzuru; Asashima, Makoto; Ueno, Naoto; Matsuda, Yoichi; Veenstra, Gert Jan C.; Fujiyama, Asao

    2017-01-01

    To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We demonstrate the allotetraploid origin of X. laevis by partitioning its genome into two homeologous subgenomes, marked by distinct families of “fossil” transposable elements. Based on the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged ~34 million years ago (Mya) and combined to form an allotetraploid ~17–18 Mya. 56% of all genes are retained in two homeologous copies. Protein function, gene expression, and the amount of flanking conserved sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression. PMID:27762356

  4. Genome evolution in the allotetraploid frog Xenopus laevis.

    Science.gov (United States)

    Session, Adam M; Uno, Yoshinobu; Kwon, Taejoon; Chapman, Jarrod A; Toyoda, Atsushi; Takahashi, Shuji; Fukui, Akimasa; Hikosaka, Akira; Suzuki, Atsushi; Kondo, Mariko; van Heeringen, Simon J; Quigley, Ian; Heinz, Sven; Ogino, Hajime; Ochi, Haruki; Hellsten, Uffe; Lyons, Jessica B; Simakov, Oleg; Putnam, Nicholas; Stites, Jonathan; Kuroki, Yoko; Tanaka, Toshiaki; Michiue, Tatsuo; Watanabe, Minoru; Bogdanovic, Ozren; Lister, Ryan; Georgiou, Georgios; Paranjpe, Sarita S; van Kruijsbergen, Ila; Shu, Shengquiang; Carlson, Joseph; Kinoshita, Tsutomu; Ohta, Yuko; Mawaribuchi, Shuuji; Jenkins, Jerry; Grimwood, Jane; Schmutz, Jeremy; Mitros, Therese; Mozaffari, Sahar V; Suzuki, Yutaka; Haramoto, Yoshikazu; Yamamoto, Takamasa S; Takagi, Chiyo; Heald, Rebecca; Miller, Kelly; Haudenschild, Christian; Kitzman, Jacob; Nakayama, Takuya; Izutsu, Yumi; Robert, Jacques; Fortriede, Joshua; Burns, Kevin; Lotay, Vaneet; Karimi, Kamran; Yasuoka, Yuuri; Dichmann, Darwin S; Flajnik, Martin F; Houston, Douglas W; Shendure, Jay; DuPasquier, Louis; Vize, Peter D; Zorn, Aaron M; Ito, Michihiko; Marcotte, Edward M; Wallingford, John B; Ito, Yuzuru; Asashima, Makoto; Ueno, Naoto; Matsuda, Yoichi; Veenstra, Gert Jan C; Fujiyama, Asao; Harland, Richard M; Taira, Masanori; Rokhsar, Daniel S

    2016-10-20

    To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

  5. PHENOBARBITAL AFFECTS THYROID HISTOLOGY AND LARVAL DEVELOPMENT IN THE AFRICAN CLAWED FROG XENOPUS LAEVIS

    Science.gov (United States)

    The abstract highlights our recent study to explore endocrine disrupting effects of phenobarbital in the African clawed frog, Xenopus laevis. In mammals, this chemical is known to induce the biotransforming enzyme UDP-glucuronosyltransferase (UDPGT) resulting in increased thyroid...

  6. Developmental Toxicity of Drinking Water Disinfection By-Products to Embryos of the African Clawed Frog (Xenopus laevis)

    Science.gov (United States)

    2005-06-10

    developmental toxicity tests with embryos of the South African clawed frog Xenopus laevis used to evaluate four individual DWDB; bromodichloromethane...SUBJECT TERMS Developmental toxicity; FETAX; water disinfection by-products; frogs ; Xenopus laevis; embryo malformations; embryo mortality...Disinfection By-Products to Embryos of the African Clawed Frog (Xenopus laevis) L. M. Brennan,1 M. W. Toussaint,1 D. M. Kumsher,1 W. E. Dennis,’ A. B

  7. Parasites of the African clawed frog, Xenopus laevis, in southern California, U.S.A

    Science.gov (United States)

    Kuperman, Boris I.; Matey, Victoria E.; Fisher, Richard N.; Ervin, Edward L.; Warburton, Manna L.; Bakhireva, Ludmila; Lehman, Cynthia A.

    2004-01-01

    A total of 230 feral African clawed frogs, Xenopus laevis, from 3 localities in southern California were examined for parasites. The following species were found: 3 species of Protozoa, Nyctotherussp., Balantidium xenopodis, Protoopalina xenopodus; 2 species of Monogenea, Protopolystoma xenopodis, Gyrdicotylus gallieni; 1 species of Digenea, Clinostomum sp. (as metacercariae); 1 species of Cestoda, Cephalochlamys namaquensis; 2 species of Nematoda, Contracaecum sp. (as larvae), Eustrongylides sp. (as larvae); and 1 species of Acanthocephala, Acanthocephalus sp. (as cystacanth). Of these, the protozoans P. xenopodus and B. xenopodis, both monogeneans, and the cestode have an African origin. Contracaecum sp., Eustrongylides sp., and Acanthocephalus sp. have not been previously reported from X. laevis.

  8. Accelerated Gene Evolution and Subfunctionalization in thePseudotetraploid Frog Xenopus Laevis

    Energy Technology Data Exchange (ETDEWEB)

    Hellsten, Uffe; Khokha, Mustafa K.; Grammar, Timothy C.; Harland,Richard M.; Richardson, Paul; Rokhsar, Daniel S.

    2007-03-01

    Ancient whole genome duplications have been implicated in the vertebrate and teleost radiations, and in the emergence of diverse angiosperm lineages, but the evolutionary response to such a perturbation is still poorly understood. The African clawed frog Xenopus laevis experienced a relatively recent tetraploidization {approx} 40 million years ago. Analysis of the considerable amount of EST sequence available for this species together with the genome sequence of the related diploid Xenopus tropicalis provides a unique opportunity to study the genomic response to whole genome duplication.

  9. Inverse Effects on Growth and Development Rates by Means of Endocrine Disruptors in African Clawed Frog Tadpoles ("Xenopus Laevis")

    Science.gov (United States)

    Hackney, Zachary Carl

    2007-01-01

    Previous work on fish, frogs, and salamanders, showed the ability for estrogen (EE2) and anthropogenic endocrine disruptors to skew sex ratios and cause hermaphrodism. This study addressed the effects of estrogens on growth and development rates of African clawed frog tadpoles ("Xenopus laevis") during their gender determination stages. The…

  10. Isoflurane anesthesia in the African clawed frog (Xenopus laevis).

    Science.gov (United States)

    Smith, J M; Stump, K C

    2000-11-01

    Isoflurane is one of the safest and most accepted anesthetic agents for reptiles, birds, and mammals. It has also been used in terrestrial amphibians. The use of inhalation agents in an entirely aquatic frog presents a new dilemma for delivery in contrast to terrestrial species. The African Clawed Frog respires by using both transcutaneous gas exchange and air breathing. These frogs remain submerged for long periods of time, thus making standard inhalation techniques impractical. We tested five methods for delivering isoflurane: 1) bubbling isoflurane and oxygen in the water, 2) intracoelomic injection, 3) subcutaneous injection, 4) intramuscular injection, and 5) topical application. For the topical application, we developed a simple technique by using an absorptive pad with a vapor-barrier backing, saturating the pad with the liquid isoflurane, and placing the pad on the back of the frog while it was confined in a plastic bowl. Although two of the three injectable routes induced anesthesia, only the topical route produced rapid induction with consistent, safe recovery. Bubbling isoflurane with oxygen into water was unsuccessful. Topical application of isoflurane was most successful and appears to be a safe and practical method that can be used as an alternative to tricaine methylsulphonate, hypothermia, or other methods for anesthetizing African Clawed Frogs.

  11. Electroencephalographic and physiologic changes after tricaine methanesulfonate immersion of African clawed frogs (Xenopus laevis).

    Science.gov (United States)

    Lalonde-Robert, Vanessa; Desgent, Sébastien; Duss, Sandra; Vachon, Pascal

    2012-01-01

    The objective of this study was to determine electroencephalographic and complementary physiologic changes in Xenopus leavis frogs after bath immersion in MS222. We also evaluated the addition of sodium pentobarbital injected intracoelomi- cally 2 h after MS222 immersion to achieve euthanasia. Frogs (n = 9) weighing 105.5 ± 8.4 g (mean ± 1 SD) were immersed in MS222 at either 1 or 3 g/L until anesthesia was achieved; a conductive stainless steel screw then was implanted in the skull on top of the outer pial surface of the brain. Frogs were immersed again in MS222 at the same concentration as previously, and electroencephalograms, heart rate, oxygen saturation, and respiratory movements were recorded. Amplitude and mean frequency of the electroencephalographic signal were evaluated at 15-min intervals until a flat-line signal was achieved. At 2 h after induction, frogs were injected intracoelomically with sodium pentobarbital (0.5 mL; 240 mg/mL) to accelerate euthanasia. Immersion of frogs in 1 or 3 g/L of MS222 depressed cerebral activity within 30 min without a significant effect on cardiac function. Intracoelomic injection of sodium pentobarbital at 2 h after MS222 administration rapidly (3.2 ± 1.7 min) induced cardiac arrest. In conclusion, immersion in MS222 can be used for the collection of organs from X. laevis frogs, but the addition of pentobarbital is required to achieve euthanasia.

  12. Mortality and morbidity in African clawed frogs (Xenopus laevis) associated with construction noise and vibrations.

    Science.gov (United States)

    Felt, Stephen A; Cowan, Andrea M; Luong, Richard; Green, Sherril L

    2012-03-01

    In Spring 2008, 175 adult female Xenopus laevis were exposed to construction-related vibrations that caused overt water rippling in the frog tanks. The 3 affected tanks were custom-built static, 300-gal 'pond-style' tanks that sat on the floor of the housing room. The water in the tank developed visible ripples as a result of the vibrations transmitted through the floor during jack-hammering in an adjacent room that was approximately 10 ftaway. All frogs in the tanks displayed buoyancy problems, excessive air gulping, and skin sloughing; ultimately 7 frogs died. In addition, these 7 animals were bloated, and 5 of these 7 had regurgitated and everted their stomach and distal esophagus into the oral cavity, resulting in airway obstruction and death. Gross pathologic findings included regurgitation and eversion of the stomach of the distal portion of the esophagus into the oral cavity, obstruction of the airway, and lung overinflation. No significant histologic lesions were observed. Construction vibrations transmitted through the water appeared to have disrupted the mechanoreceptive function of the lateral line system, resulting in overstimulation of the noxious feeding response, regurgitation, and eversion of the stomach and distal esophagus into the oral cavity and subsequent suffocation due to airway obstruction. After immediate cessation of the jack-hammering and relocation of the remaining frogs, no additional morbidities or mortalities occurred.

  13. Extracellular Ca2+ Is Required for Fertilization in the African Clawed Frog, Xenopus laevis.

    Science.gov (United States)

    Wozniak, Katherine L; Mayfield, Brianna L; Duray, Alexis M; Tembo, Maiwase; Beleny, David O; Napolitano, Marc A; Sauer, Monica L; Wisner, Bennett W; Carlson, Anne E

    2017-01-01

    The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy. Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+. Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu.

  14. The pharmacokinetics of enrofloxacin in adult African clawed frogs (Xenopus laevis).

    Science.gov (United States)

    Howard, Antwain M; Papich, Mark G; Felt, Stephen A; Long, Charles T; McKeon, Gabriel P; Bond, Emmitt S; Torreilles, Stéphanie L; Luong, Richard H; Green, Sherril L

    2010-11-01

    Pharmacokinetics of enrofloxacin, a fluoroquinolone antibiotic, was determined in adult female Xenopus laevis after single-dose administration (10 mg/kg) by intramuscular or subcutaneous injection. Frogs were evaluated at various time points until 8 h after injection. Plasma was analyzed for antibiotic concentration levels by HPLC. We computed pharmacokinetic parameters by using noncompartmental analysis of the pooled concentrations (naive pooled samples). After intramuscular administration of enrofloxacin, the half-life was 5.32 h, concentration maximum was 10.85 μg/mL, distribution volume was 841.96 mL/kg, and area under the time-concentration curve was 57.59 μg×h/mL; after subcutaneous administration these parameters were 4.08 h, 9.76 μg/mL, 915.85 mL/kg, and 47.42 μg×h/mL, respectively. According to plasma pharmacokinetics, Xenopus seem to metabolize enrofloxacin in a manner similar to mammals: low levels of the enrofloxacin metabolite, ciprofloxacin, were detected in the frogs' habitat water and plasma. At necropsy, there were no gross or histologic signs of toxicity after single-dose administration; toxicity was not evaluated for repeated dosing. The plasma concentrations reached levels considered effective against common aquatic pathogens and suggest that a single, once-daily dose would be a reasonable regimen to consider when treating sick frogs. The treatment of sick frogs should be based on specific microbiologic identification of the pathogen and on antibiotic susceptibility testing.

  15. Impacts of Climate Change on the Global Invasion Potential of the African Clawed Frog Xenopus laevis.

    Science.gov (United States)

    Ihlow, Flora; Courant, Julien; Secondi, Jean; Herrel, Anthony; Rebelo, Rui; Measey, G John; Lillo, Francesco; De Villiers, F André; Vogt, Solveig; De Busschere, Charlotte; Backeljau, Thierry; Rödder, Dennis

    2016-01-01

    By altering or eliminating delicate ecological relationships, non-indigenous species are considered a major threat to biodiversity, as well as a driver of environmental change. Global climate change affects ecosystems and ecological communities, leading to changes in the phenology, geographic ranges, or population abundance of several species. Thus, predicting the impacts of global climate change on the current and future distribution of invasive species is an important subject in macroecological studies. The African clawed frog (Xenopus laevis), native to South Africa, possesses a strong invasion potential and populations have become established in numerous countries across four continents. The global invasion potential of X. laevis was assessed using correlative species distribution models (SDMs). SDMs were computed based on a comprehensive set of occurrence records covering South Africa, North America, South America and Europe and a set of nine environmental predictors. Models were built using both a maximum entropy model and an ensemble approach integrating eight algorithms. The future occurrence probabilities for X. laevis were subsequently computed using bioclimatic variables for 2070 following four different IPCC scenarios. Despite minor differences between the statistical approaches, both SDMs predict the future potential distribution of X. laevis, on a global scale, to decrease across all climate change scenarios. On a continental scale, both SDMs predict decreasing potential distributions in the species' native range in South Africa, as well as in the invaded areas in North and South America, and in Australia where the species has not been introduced. In contrast, both SDMs predict the potential range size to expand in Europe. Our results suggest that all probability classes will be equally affected by climate change. New regional conditions may promote new invasions or the spread of established invasive populations, especially in France and Great Britain.

  16. Impacts of Climate Change on the Global Invasion Potential of the African Clawed Frog Xenopus laevis.

    Directory of Open Access Journals (Sweden)

    Flora Ihlow

    Full Text Available By altering or eliminating delicate ecological relationships, non-indigenous species are considered a major threat to biodiversity, as well as a driver of environmental change. Global climate change affects ecosystems and ecological communities, leading to changes in the phenology, geographic ranges, or population abundance of several species. Thus, predicting the impacts of global climate change on the current and future distribution of invasive species is an important subject in macroecological studies. The African clawed frog (Xenopus laevis, native to South Africa, possesses a strong invasion potential and populations have become established in numerous countries across four continents. The global invasion potential of X. laevis was assessed using correlative species distribution models (SDMs. SDMs were computed based on a comprehensive set of occurrence records covering South Africa, North America, South America and Europe and a set of nine environmental predictors. Models were built using both a maximum entropy model and an ensemble approach integrating eight algorithms. The future occurrence probabilities for X. laevis were subsequently computed using bioclimatic variables for 2070 following four different IPCC scenarios. Despite minor differences between the statistical approaches, both SDMs predict the future potential distribution of X. laevis, on a global scale, to decrease across all climate change scenarios. On a continental scale, both SDMs predict decreasing potential distributions in the species' native range in South Africa, as well as in the invaded areas in North and South America, and in Australia where the species has not been introduced. In contrast, both SDMs predict the potential range size to expand in Europe. Our results suggest that all probability classes will be equally affected by climate change. New regional conditions may promote new invasions or the spread of established invasive populations, especially in France

  17. Amphibian (Xenopus laevis) tadpoles and adult frogs mount distinct interferon responses to the Frog Virus 3 ranavirus.

    Science.gov (United States)

    Wendel, Emily S; Yaparla, Amulya; Koubourli, Daphne V; Grayfer, Leon

    2017-03-01

    Infections of amphibians by Frog Virus 3 (FV3) and other ranavirus genus members are significantly contributing to the amphibian declines, yet much remains unknown regarding amphibian antiviral immunity. Notably, amphibians represent an important step in the evolution of antiviral interferon (IFN) cytokines as they are amongst the first vertebrates to possess both type I and type III IFNs. Accordingly, we examined the roles of type I and III IFNs in the skin of FV3-challenged amphibian Xenopus laevis) tadpoles and adult frogs. Interestingly, FV3-infected tadpoles mounted type III IFN responses, whereas adult frogs relied on type I IFN immunity. Subcutaneous administration of type I or type III IFNs offered short-term protection of tadpoles against FV3 and these type I and type III IFNs induced the expression of distinct antiviral genes in the tadpole skin. Moreover, subcutaneous injection of tadpoles with type III IFN significantly extended their survival and reduced FV3 dissemination. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Extracellular Ca2+ Is Required for Fertilization in the African Clawed Frog, Xenopus laevis

    Science.gov (United States)

    Duray, Alexis M.; Tembo, Maiwase; Beleny, David O.; Napolitano, Marc A.; Sauer, Monica L.; Wisner, Bennett W.

    2017-01-01

    Background The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy. Methodology/principal finding Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+. Conclusions/Significance Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu. PMID:28114360

  19. Nondestructive Imaging of Internal Structures of Frog (Xenopus laevis) Embryos by Shadow-Projection X-Ray Microtomography

    Science.gov (United States)

    Aoki, Sadao; Yoneda, Ikuo; Nagai, Takeharu; Ueno, Naoto; Murakami, Kazuo

    1994-04-01

    Nondestructive high-resolution imaging of frog ( Xenopus laevis) embryos has been developed by X-ray microtomography. Shadow-projection X-ray microtomography with a brilliant fine focus laboratory X-ray source could image fine structures of Xenopus embryos which were embedded in paraffin wax. The imaging system enabled us to not only distinguish endoderm from ectoderm at the gastrula stage, but also to obtain a cross-section view of the tail bud embryo showing muscle, notochord and neural tube without staining. Furthermore, the distribution of myosin was also imaged in combination with whole-mount immunohistochemistry.

  20. Temperature-independent energy expenditure in early development of the African clawed frog Xenopus laevis

    Science.gov (United States)

    Nagano, Yatsuhisa; Ode, Koji L.

    2014-08-01

    The thermal dissipation of activated eggs and embryos undergoing development from cleavage to the tailbud stage of the African clawed frog Xenopus laevis was measured as a function of incubation time at temperatures ranging from T = 288.2 K to 295.2 K, using a high-precision isothermal calorimeter. A23187-mediated activation of mature eggs induced stable periodic thermal oscillations lasting for 8-34 h. The frequency agreed well with the cell cycle frequency of initial cleavages at the identical temperature. In the developing embryo, energy metabolism switches from embryonic to adult features during gastrulation. The thermal dissipation after gastrulation fit well with a single modified Avrami equation, which has been used for modeling crystal-growth. Both the oscillation frequency of the activated egg and the growth rate of the embryo strongly depend on temperature with the same apparent activation energy of approximately 87 kJ mole-1. This result suggests that early development proceeds as a single biological time, attributable to a single metabolic rate. A temperature-independent growth curve was derived by scaling the thermogram to the biological time, indicating that the amount of energy expenditure during each developmental stage is constant over the optimal temperature range.

  1. Unequal contribution of native South African phylogeographic lineages to the invasion of the African clawed frog, Xenopus laevis, in Europe

    Directory of Open Access Journals (Sweden)

    Charlotte De Busschere

    2016-02-01

    Full Text Available Due to both deliberate and accidental introductions, invasive African Clawed Frog (Xenopus laevis populations have become established worldwide. In this study, we investigate the geographic origins of invasive X. laevis populations in France and Portugal using the phylogeographic structure of X. laevis in its native South African range. In total, 80 individuals from the whole area known to be invaded in France and Portugal were analysed for two mitochondrial and three nuclear genes, allowing a comparison with 185 specimens from the native range. Our results show that native phylogeographic lineages have contributed differently to invasive European X. laevis populations. In Portugal, genetic and historical data suggest a single colonization event involving a small number of individuals from the south-western Cape region in South Africa. In contrast, French invasive X. laevis encompass two distinct native phylogeographic lineages, i.e., one from the south-western Cape region and one from the northern regions of South Africa. The French X. laevis population is the first example of a X. laevis invasion involving multiple lineages. Moreover, the lack of population structure based on nuclear DNA suggests a potential role for admixture within the invasive French population.

  2. Unequal contribution of native South African phylogeographic lineages to the invasion of the African clawed frog, Xenopus laevis, in Europe.

    Science.gov (United States)

    De Busschere, Charlotte; Courant, Julien; Herrel, Anthony; Rebelo, Rui; Rödder, Dennis; Measey, G John; Backeljau, Thierry

    2016-01-01

    Due to both deliberate and accidental introductions, invasive African Clawed Frog (Xenopus laevis) populations have become established worldwide. In this study, we investigate the geographic origins of invasive X. laevis populations in France and Portugal using the phylogeographic structure of X. laevis in its native South African range. In total, 80 individuals from the whole area known to be invaded in France and Portugal were analysed for two mitochondrial and three nuclear genes, allowing a comparison with 185 specimens from the native range. Our results show that native phylogeographic lineages have contributed differently to invasive European X. laevis populations. In Portugal, genetic and historical data suggest a single colonization event involving a small number of individuals from the south-western Cape region in South Africa. In contrast, French invasive X. laevis encompass two distinct native phylogeographic lineages, i.e., one from the south-western Cape region and one from the northern regions of South Africa. The French X. laevis population is the first example of a X. laevis invasion involving multiple lineages. Moreover, the lack of population structure based on nuclear DNA suggests a potential role for admixture within the invasive French population.

  3. Effect of water hardness on oocyte quality and embryo development in the African clawed frog (Xenopus laevis).

    Science.gov (United States)

    Godfrey, Earl W; Sanders, George E

    2004-04-01

    Husbandry and health of the African clawed frog, Xenopus laevis, greatly influences the quality of oocytes produced. One factor affecting oocyte quality is the water conditions in which females are maintained. Dechlorination and adequate salt concentration are known to affect oocytes, but water hardness has not been considered an important factor in Xenopus husbandry by the research community. We found that, when females were kept in soft water or water with marine salts alone, even when it was cooled to 17 to 18 degrees C, the quality of oocytes decreased; only 20 to 25% of resulting embryos developed to tailbud stages. Survival and normal development of embryos increased significantly within one month of addition to the laboratory housing water of salts that mimic conditions in African Rift Valley lakes. These salts greatly increased water hardness; development of embryos to tailbud stages remained high (50 to 70% on average) for more than a year after their addition to the water housing females. Water from South African ponds where X. laevis are collected, and from wells used by the major suppliers of X. laevis, also was moderately to very hard. Our results suggest that X. laevis is naturally adapted to hard water, and indicate that increasing general hardness during laboratory housing is more important for oocyte quality and embryo development than is increasing carbonate hardness (alkalinity) in the water used to house females.

  4. Changes in contractile properties by androgen hormones in sexually dimorphic muscles of male frogs (Xenopus laevis).

    Science.gov (United States)

    Regnier, M; Herrera, A A

    1993-02-01

    1. Male frogs (Xenopus laevis) were castrated then given either empty or testosterone-filled implants to produce animals with low or high levels of circulating testosterone. Eight weeks later the contractile properties of an androgen-sensitive forelimb flexor, the flexor carpi radialis muscle (FCR), were measured in vitro. Another forelimb flexor muscle, the coracoradialis, and a hindlimb muscle, the iliofibularis, were analysed similarly. 2. Plasma testosterone levels were 0.9 +/- 0.3 ng/ml (+/- S.E.M.) in castrated frogs with blank implants (C) and 61.3 +/- 4.7 ng/ml in castrates with testosterone implants (CT). Unoperated males, sampled at various times of the year, ranged between 10.8 and 51.0 ng/ml. 3. With direct electrical stimulation of the FCR, contraction time of the isometric twitch was not affected by testosterone levels. Relaxation times were affected, however. Half- and 90% relaxation times were 27 and 42% longer, respectively, for CT compared to C muscles. 4. Testosterone also had no effect on the contraction time of twitches elicited by stimulation of the FCR nerve. Half- and 90% relaxation times were 51 and 76% longer, respectively, for CT compared to C muscles. 5. Tetanus tension, elicited by direct stimulation of the FCR at 50 Hz, was 86% greater in CT compared to C muscles. The average cross-sectional area of FCR muscle fibres was 84% greater in CT muscles. These results implied that testosterone treatment had no effect on specific muscle tension. 6. Stimulation of the FCR nerve at 50 Hz resulted in 53% less tension than the same stimulus applied directly to CT muscles. In C muscles the difference was only 14%. This suggested that testosterone treatment reduced synaptic efficacy. 7. In CT muscles, direct or nerve stimulation of fibres in the shoulder region of the FCR elicited twitches that contracted and relaxed more slowly than fibres in the elbow region. In C muscles there was no difference in contraction or relaxation time between fibres in

  5. Immune defenses against Batrachochytrium dendrobatidis, a fungus linked to global amphibian declines, in the South African clawed frog, Xenopus laevis.

    Science.gov (United States)

    Ramsey, Jeremy P; Reinert, Laura K; Harper, Laura K; Woodhams, Douglas C; Rollins-Smith, Louise A

    2010-09-01

    Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections.

  6. Analgesic effects of meloxicam, morphine sulfate, flunixin meglumine, and xylazine hydrochloride in African-clawed frogs (Xenopus laevis).

    Science.gov (United States)

    Coble, Dondrae J; Taylor, Douglas K; Mook, Deborah M

    2011-05-01

    We evaluated analgesic use and analgesiometry in aquatic African-clawed frogs (Xenopus laevis). We used the acetic acid test (AAT) to assess the analgesic potential of systemic xylazine hydrochloride, meloxicam, flunixin meglumine, and morphine sulfate after injection into the dorsal lymph sac. Flunixin meglumine provided better analgesia than did the other drugs, most evident at 5 and 9 h after administration. Because the AAT was associated with the development of dermal lesions, we discontinued use of this assay and chose the Hargreaves test as an alternative method of measuring nociception in Xenopus. This assay is commonly performed in rodents, but its efficacy in an aquatic species such as Xenopus was unknown prior to this study. We found that the Hargreaves test was an effective measure of nociception in Xenopus, and we used it to evaluate the effectiveness of the nonopiod agents xylazine hydrochloride, meloxicam, and flunixin meglumine both in the absence of surgery and after surgical oocyte harvest. Similar to findings from the AAT, flunixin meglumine provided better analgesia in the Hargreaves test than did the other agents when analyzed in the absence of surgical intervention. Results were equivocal after oocyte harvest. Although surgical oocyte harvest is a common procedure in Xenopus, and currently there are no published recommendations for analgesia after this invasive surgery. Future studies are needed to clarify the efficacy of nonsteroidal antiinflammatory drugs for that purpose.

  7. Analgesic Effects of Meloxicam, Morphine Sulfate, Flunixin Meglumine, and Xylazine Hydrochloride in African-Clawed Frogs (Xenopus laevis)

    Science.gov (United States)

    Coble, Dondrae J; Taylor, Douglas K; Mook, Deborah M

    2011-01-01

    We evaluated analgesic use and analgesiometry in aquatic African-clawed frogs (Xenopus laevis). We used the acetic acid test (AAT) to assess the analgesic potential of systemic xylazine hydrochloride, meloxicam, flunixin meglumine, and morphine sulfate after injection into the dorsal lymph sac. Flunixin meglumine provided better analgesia than did the other drugs, most evident at 5 and 9 h after administration. Because the AAT was associated with the development of dermal lesions, we discontinued use of this assay and chose the Hargreaves test as an alternative method of measuring nociception in Xenopus. This assay is commonly performed in rodents, but its efficacy in an aquatic species such as Xenopus was unknown prior to this study. We found that the Hargreaves test was an effective measure of nociception in Xenopus, and we used it to evaluate the effectiveness of the nonopiod agents xylazine hydrochloride, meloxicam, and flunixin meglumine both in the absence of surgery and after surgical oocyte harvest. Similar to findings from the AAT, flunixin meglumine provided better analgesia in the Hargreaves test than did the other agents when analyzed in the absence of surgical intervention. Results were equivocal after oocyte harvest. Although surgical oocyte harvest is a common procedure in Xenopus, and currently there are no published recommendations for analgesia after this invasive surgery. Future studies are needed to clarify the efficacy of nonsteroidal antiinflammatory drugs for that purpose. PMID:21640031

  8. Structure-related effects of pyrethroid insecticides on the lateral-line sense organ and on peripheral nerves of the clawed frog, Xenopus laevis

    NARCIS (Netherlands)

    Vijverberg, H.P.M.; Ruigt, GeS. F.; Bercken, J. van den

    1982-01-01

    The effects of seven different pyrethroid insecticides on the lateral-line sense organ and on peripheral nerves of the clawed frog, Xenopus laevis, were investigated by means of electrophysiological methods. The results show that two classes of pyrethroid can be clearly distinguished. (i) Pyrethroid

  9. An aryl hydrocarbon receptor repressor from Xenopus laevis: function, expression, and role in dioxin responsiveness during frog development.

    Science.gov (United States)

    Zimmermann, Anna L; King, Elizabeth A; Dengler, Emelyne; Scogin, Shana R; Powell, Wade H

    2008-07-01

    Xenopus laevis and other frogs are extremely insensitive to the toxicity of xenobiotic ligands of the aryl hydrocarbon receptor (AHR), including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Premetamorphic life stages are especially insensitive, and they are reported to be refractory to induction of Cytochrome P4501As, which are readily induced in older animals. The AHR repressor (AHRR) is a member of the AHR gene family. AHRR expression is induced by TCDD; it then represses AHR in an apparent negative feedback loop. In this study, we sought to test the hypothesis that constitutive AHRR expression underlies the lack of TCDD responsiveness in frog early life stages. We determined the sequence of an AHRR complimentary DNA encoding an 85.3-kDa protein sharing 52-55% identity with the bHLH/PAS domains of other AHRRs. In transient transfection assays, X. laevis AHRR inhibited TCDD-induced reporter gene expression mediated by either X. laevis AHR paralog, AHR1alpha or AHR1beta. AHRR messenger RNA was expressed at low levels in embryos (Nieuwkoop-Faber stage 33-38; approximately 52 h.p.f.) and was induced approximately twofold following TCDD exposure (42 ng/g wet weight). In contrast, AHRR exhibited higher constitutive expression and was induced more than threefold in tadpoles at stage 52-55 (prometamorphic; approximately 4 weeks postfertilization) and in isolated viscera of stage 62 tadpoles (in the metamorphic climax; approximately 7 weeks postfertilization). Although the magnitude of induction was smaller, the temporal pattern of AHRR expression and inducibility resembled that of CYP1A6. Thus, attenuated transcriptional activation of AHR target genes and low TCDD toxicity in X. laevis embryos cannot be explained by constitutive, high-level expression of AHRR.

  10. Effects of 17α-ethynylestradiol on sexual differentiation and development of the African clawed frog (Xenopus laevis).

    Science.gov (United States)

    Tompsett, Amber R; Wiseman, Steve; Higley, Eric; Pryce, Sara; Chang, Hong; Giesy, John P; Hecker, Markus

    2012-11-01

    Several studies have shown that exposure of amphibians, including the African clawed frog (Xenopus laevis), to potent estrogens at critical times during development results in feminization and/or demasculinization. However, genotyping of X. laevis has only recently become possible, so studies performed in the past were rarely able to make explicit linkages between genetic and phenotypic sex. Therefore, to further characterize this relationship, X. laevis tadpoles were exposed during development to 0.09, 0.84, or 8.81 μg/L 17α-ethynylestradiol (EE2), which is the estrogen analog commonly used in oral contraceptives. Exposure to all concentrations of EE2 tested resulted in significant delays in time to metamorphosis. Genotyping showed that genetic sex ratios were similar among treatments. However, morphological evaluation revealed that a significant number of individuals with a male genotype displayed mixed sex and abnormal phenotypes. Additionally, both genetic males and females exposed to EE2 exhibited greater presence of vitellogenin protein relative to the respective controls. Since estrogens function downstream of the initial molecular signals of sexual differentiation, it is likely that genetic male animals received mixed endogenous male and exogenous female signals that caused disordered sexual development. The production of vitellogenin was probably temporally separated and independent from primary effects on sexual differentiation, and might have contributed to delays in metamorphosis observed in individuals exposed to EE2. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Acute and Chronic Outcomes of Gas-Bubble Disease in a Colony of African Clawed Frogs (Xenopus laevis).

    Science.gov (United States)

    Tsai, Julia Y; Felt, Stephen A; Bouley, Donna M; Green, Sherril L

    2017-02-01

    Gas-bubble disease occurs in aquatic species that are exposed to water that is supersaturated with gases. In February 2007, municipal water supersaturated with gas was inadvertently pumped into the vivarium's aquatic housing systems and affected approximately 450 adult female Xenopus laevis. The inflow of supersaturated water was stopped immediately, the holding tanks aggressively aerated, and all experimental manipulations and feeding ceased. Within the first 6 h after the event, morbidity approached 90%, and mortality reached 3.5%. Acutely affected frogs showed clinical signs of gas-bubble disease: buoyancy problems, micro- and macroscopic bubbles in the foot webbing, hyperemia in foot webbing and leg skin, and loss of the mucous slime coat. All of the frogs that died or were euthanized had areas of mesenteric infarction, which resulted in intestinal epithelial necrosis and degeneration of the muscular tunic. Over the subsequent 2 wk, as gas saturation levels returned to normal, the clinical symptoms resolved completely in the remaining frogs. However, 3 mo later, 85% of them failed to lay eggs or produce oocytes, and the remaining 15% produced oocytes of low number and poor quality, yielding cytosolic extracts with poor to no enzymatic activity. Histology of the egg mass from a single 2- to 3-y-old frog at 3 mo after disease resolution revealed irregularly shaped oocytes, few large mature oocytes, and numerous small, degenerating oocytes. At 6 mo after the incident, the remaining frogs continued to fail to produce eggs of sufficient quantity or quality after hormonal priming. The researchers consequently opted to cull the remainder of the colony and repopulate with new frogs.

  12. Effects of low dose endosulfan exposure on brain neurotransmitter levels in the African clawed frog Xenopus laevis.

    Science.gov (United States)

    Preud'homme, Valérie; Milla, Sylvain; Gillardin, Virginie; De Pauw, Edwin; Denoël, Mathieu; Kestemont, Patrick

    2015-02-01

    Understanding the impact of pesticides in amphibians is of growing concern to assess the causes of their decline. Among pesticides, endosulfan belongs to one of the potential sources of danger because of its wide use and known effects, particularly neurotoxic, on a variety of organisms. However, the effect of endosulfan was not yet evaluated on amphibians at levels encompassing simultaneously brain neurotransmitters and behavioural endpoints. In this context, tadpoles of the African clawed frog Xenopus laevis were submitted to four treatments during 27 d: one control, one ethanol control, and two low environmental concentrations of endosulfan (0.1 and 1 μg L(-1)). Endosulfan induced a significant increase of brain serotonin level at both concentrations and a significant increase of brain dopamine and GABA levels at the lower exposure but acetylcholinesterase activity was not modified by the treatment. The gene coding for the GABA transporter 1 was up-regulated in endosulfan contaminated tadpoles while the expression of other genes coding for the neurotransmitter receptors or for the enzymes involved in their metabolic pathways was not significantly modified by endosulfan exposure. Endosulfan also affected foraging, and locomotion in links with the results of the physiological assays, but no effects were seen on growth. These results show that low environmental concentrations of endosulfan can induce adverse responses in X. laevis tadpoles. At a broader perspective, this suggests that more research using and linking multiple markers should be used to understand the complex mode of action of pollutants.

  13. Genetic Basis of Spectral Tuning in the Violet-Sensitive Visual Pigment of African Clawed Frog, Xenopus laevis

    Science.gov (United States)

    Takahashi, Yusuke; Yokoyama, Shozo

    2005-01-01

    Ultraviolet (UV) and violet vision in vertebrates is mediated by UV and violet visual pigments that absorb light maximally (λmax) at ∼360 and 390–440 nm, respectively. So far, a total of 11 amino acid sites only in transmembrane (TM) helices I–III are known to be involved in the functional differentiation of these short wavelength-sensitive type 1 (SWS1) pigments. Here, we have constructed chimeric pigments between the violet pigment of African clawed frog (Xenopus laevis) and its ancestral UV pigment. The results show that not only are the absorption spectra of these pigments modulated strongly by amino acids in TM I–VII, but also, for unknown reasons, the overall effect of amino acid changes in TM IV–VII on the λmax-shift is abolished. The spectral tuning of the contemporary frog pigment is explained by amino acid replacements F86M, V91I, T93P, V109A, E113D, L116V, and S118T, in which V91I and V109A are previously unknown, increasing the total number of critical amino acid sites that are involved in the spectral tuning of SWS1 pigments in vertebrates to 13. PMID:16079229

  14. Comparative study of diclofenac-induced embryotoxicity and teratogenesis in Xenopus laevis and Lithobates catesbeianus, using the frog embryo teratogenesis assay: Xenopus (FETAX).

    Science.gov (United States)

    Cardoso-Vera, Jesús Daniel; Islas-Flores, Hariz; SanJuan-Reyes, Nely; Montero-Castro, Elena Irabella; Galar-Martínez, Marcela; García-Medina, Sandra; Elizalde-Velázquez, Armando; Dublán-García, Octavio; Gómez-Oliván, Leobardo Manuel

    2017-01-01

    Water is an increasingly deteriorated, limited natural resource due to population increase and industrialization. Also, the widespread use of pharmaceuticals in modern society leads to their presence in domestic, hospital and industrial effluents. Due to their analgesic properties, some of the most commonly used pharmaceuticals are nonsteroidal anti-inflammatory drugs (NSAIDs). High concentrations of one these products, diclofenac (DCF), have been detected in effluents and water bodies of different countries, including Mexico. Diverse studies show that trace amounts (ngL(-1) to μgL(-1)) of this compound induce toxicity on aquatic organisms such as algae, microcrustaceans and fish. However, studies on its potential toxicity during development in species of commercial interest such as the American bullfrog Lithobates catesbeianus are scarce. The present study aimed to evaluate DCF-induced teratogenesis and embryotoxicity in Xenopus laevis and L. catesbeianus, a species marketed as a nutritional meat source in Mexico, using the frog embryo teratogenesis assay: Xenopus (FETAX). Oocytes in mid-blastula transition were exposed for 96h to 1, 4, 8, 16, 32 and 62.5mgDCFL(-1). The criteria evaluated were mortality, malformation and growth inhibition. The teratogenic index was 4.2 in L. catesbeianus, three-fold higher than the reference limit (1.5), and 3.9 in X. laevis. Diclofenac induced diverse malformations in both species, the most frequent of these being axial malformations in the tail and notochord, edema and stunted growth. Results indicate that DCF is a potentially teratogenic compound and is toxic during development in X. laevis and L. catesbeianus, a species which, due to its sensitivity, can be used to evaluate the toxicity of pharmaceutical products, using FETAX.

  15. Treatment of skin-invading capillarid nematode in a colony of South African clawed frogs (Xenopus laevis)

    Energy Technology Data Exchange (ETDEWEB)

    Cosgrove, G.E.; Jared, D.W.

    1977-08-01

    Pathological effects of a capillarid nematode on the skin of Xenopus are described. Eight frogs infested with the parasite were treated with thiabendazole in the aquarium water. Histological examinations of the skin of these frogs showed that the parasites had been eliminated. No toxicity was observed as a result of the drug therapy. (HLW)

  16. Facial Transplants in Xenopus laevis Embryos

    OpenAIRE

    Jacox, Laura A.; Dickinson, Amanda J.; Sive, Hazel

    2014-01-01

    Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo ...

  17. Specific in vivo and nonspecific in vitro alloreactivities of adult frogs (Xenopus laevis) that were thymectomized during early larval life.

    Science.gov (United States)

    Nagata, S; Cohen, N

    1983-07-01

    Thymectomy of very young Xenopus larvae abrogated in vitro proliferative responses to phytohemagglutinin (PHA) and allogeneic leukocytes. Thymectomized (Txd) frogs, however, still rejected first-set skin allografts chronically and second-set grafts (but not third-party grafts) more rapidly. The specific second-set rejection reaction by Txd frogs was transferable in vivo to secondary Txd hosts (with major histocompatibility complex identical to the cell donor) by the subcutaneous injection of a mixture of splenic and peripheral blood leukocytes. Spleen cells used in this adoptive transfer experiment (i.e., immune cells) were responsive to allogeneic cells from the original donor strain as well as from unrelated donors in mixed leukocyte culture in vitro but were still unreactive to PHA. The results are consistent with an extrathymic pathway of alloreactive cell differentiation in this species, although the physiological significance of such a pathway is not clear.

  18. Histological microstructure of the claws of the African clawed frog, Xenopus laevis (Anura: Pipidae): implications for the evolution of claws in tetrapods.

    Science.gov (United States)

    Maddin, Hillary C; Musat-Marcu, Sorin; Reisz, Robert R

    2007-05-15

    Claws are consistent components of amniote anatomy and may thus be implicated in the success of the amniote invasion of land. However, the evolutionary origin of these structures in tetrapods is unclear. Claws are present in certain extant non-amniotes, such as Xenopus laevis, the African clawed frog. The histology of the soft tissue component of the claws of X. laevis is described and compared with the amniote condition in order to gain new information on the question of homology of claws in these two groups based on patterns of keratinization. The X. laevis claw sheath is composed of a localized thickening of the corneous region of the epidermis that envelops the terminal phalanx. Noted differences between the non-cornified layers of the epidermis of the claw and non-claw region are the overall grainier appearance of the cells and an increased abundance of desmosomes in the intermediate spinosus cells. The biochemical identity of the sheath keratin(s) is inferred to be different from that of non-claw region epidermis, based on histological differences and differences in stain affinity between the two regions. The microstructure of the frog claw differs from that of amniotes in several respects, including the lack of a specified zone of growth near the base of the claw. Amphibians and amniotes, therefore, have very different patterns of claw sheath growth. Observations do not support homology of claws on a structural level in these two groups; however, further experimental work may confirm a conserved pattern of cornification in these structures in tetrapods. (c) 2006 Wiley-Liss, Inc.

  19. Aeromonas hydrophila infection in Xenopus laevis.

    Science.gov (United States)

    Hubbard, G B

    1981-06-01

    Aeromonas hydrophila caused severe disease in a group of Xenopus laevis within 3 weeks of receipt. The primary clinical signs were marked pallor of the skin, petechiation, lethargy, anorexia, and edema. The duration of the outbreak was approximately 45 days during which time 21 frogs became sick and 18 died, despite tetracycline therapy. Pale skin, subcutaneous edema and hemorrhages, ascites, and pale livers were seen at necropsy. Aeromonas hydrophila was isolated from skin, and the same organism was isolated in pure culture from skeletal muscle. Tetracycline treatment via stomach tube was effective if given early in the course of the disease. The outbreak was controlled by removing sick frogs, feeding twice per week, changing the water several hours after feeding, and maintaining the frogs where the ambient temperature was 22 degrees C or lower. The pallor of the skin and general malaise were produced experimentally by crowding normal frogs, changing the water infrequently, and increasing ambient temperatures. Mild disease was reproduced experimentally by subcutaneous injection of Aeromonas hydrophila into apparently healthy frogs.

  20. Pharmacologic parameters of MS222 and physiologic changes in frogs (Xenopus laevis) after immersion at anesthetic doses.

    Science.gov (United States)

    Lalonde-Robert, Vanessa; Beaudry, Francis; Vachon, Pascal

    2012-07-01

    We evaluated the anesthetic efficacy of MS222 (dose, 1 or 2 g/L; pH 7) administered as an immersion bath (duration, 20 min) for nonbreeding female Xenopus leavis frogs (n = 33; average body weight, 103 ± 16 g). The acid acetic test, the withdrawal reflex, righting behavior, heart rate, respiratory frequency, and blood oxygen saturation were used to evaluate the level of anesthesia. Acetic acid and withdrawal reflex responses were present at 30 and 60 min following immersion for the 1- and 2-g/L doses, respectively. MS222 had no effect on heart rate or oxygen saturation, but caused pronounced respiratory depression, as expected. Microscopic observations of selected tissues (heart, lung, liver, kidneys, and skin) showed no evidence of lesions at 24 h after immersion. In addition, we calculated the pharmacokinetics of MS222 in plasma and analyzed the drug by HPLC-tandem mass spectrometry. The calculated half-life of MS222 is 3.2 h. We conclude that MS222 administered at 1 or 2 g/mL via immersion bath for 20 min is an effective anesthetic that can be used for surgical procedures of less than 30 or 60 min, respectively, in Xenopus leavis.

  1. Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content.

    Science.gov (United States)

    Sun, Liangliang; Dubiak, Kyle M; Peuchen, Elizabeth H; Zhang, Zhenbin; Zhu, Guijie; Huber, Paul W; Dovichi, Norman J

    2016-07-05

    Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content. This progression forms a ladder of single-cell targets for the development of successively higher sensitivity instruments. In this manuscript, we performed bottom-up proteomics on single blastomeres isolated by microdissection from 2-, 4-, 8-, 16-, 32-, and 50-cell Xenopus laevis (African clawed frog) embryos. Over 1 400 protein groups were identified in single-run reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry from single balstomeres isolated from a 16-cell embryo. When the mass of yolk-free proteins in single blastomeres decreased from ∼0.8 μg (16-cell embryo) to ∼0.2 μg (50-cell embryo), the number of protein group identifications declined from 1 466 to 644. Around 800 protein groups were quantified across four blastomeres isolated from a 16-cell embryo. By comparing the protein expression among different blastomeres, we observed that the blastomere-to-blastomere heterogeneity in 8-, 16-, 32-, and 50-cell embryos increases with development stage, presumably due to cellular differentiation. These results suggest that comprehensive quantitative proteomics on single blastomeres isolated from these early stage embryos can provide valuable insights into cellular differentiation and organ development.

  2. Urotensin II receptor (UTR) exists in hyaline chondrocytes: a study of peripheral distribution of UTR in the African clawed frog, Xenopus laevis.

    Science.gov (United States)

    Konno, Norifumi; Fujii, Yuya; Imae, Haruka; Kaiya, Hiroyuki; Mukuda, Takao; Miyazato, Mikiya; Matsuda, Kouhei; Uchiyama, Minoru

    2013-05-01

    Urotensin II (UII) and UII-related peptide (URP) exhibit diverse physiological actions including vasoconstriction, locomotor activity, osmoregulation, and immune response through UII receptor (UTR), which is expressed in the central nervous system and peripheral tissues of fish and mammals. In amphibians, only UII has been identified. As the first step toward elucidating the actions of UII and URP in amphibians, we cloned and characterized URP and UTR from the African clawed frog Xenopus laevis. Functional analysis showed that treatment of UII or URP with Chinese hamster ovary cells transfected with the cloned receptor increased the intracellular calcium concentration in a concentration-dependent manner, whereas the administration of the UTR antagonist urantide inhibited UII- or URP-induced Ca(2+) mobilization. An immunohistochemical study showed that UTR was expressed in the splenocytes and leukocytes isolated from peripheral blood, suggesting that UII and URP are involved in the regulation of the immune system. UTR was also localized in the apical membrane of the distal tubule of the kidney and in the transitional epithelial cells of the urinary bladder. This result supports the view that the UII/URP-UTR system plays an important role in osmoregulation of amphibians. Interestingly, immunopositive labeling for UTR was first detected in the chondrocytes of various hyaline cartilages (the lung septa, interphalangeal joint and sternum). The expression of UTR was also observed in the costal cartilage, tracheal cartilages, and xiphoid process of the rat. These novel findings probably suggest that UII and URP mediate the formation of the cartilaginous matrix. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Polystyrene nanoparticles affect Xenopus laevis development

    Energy Technology Data Exchange (ETDEWEB)

    Tussellino, Margherita; Ronca, Raffaele [University of Naples Federico II, Department of Biology (Italy); Formiggini, Fabio [Italian Institute of Technology, Center for Advanced Biomaterials for Health Care IIT@CRIB (Italy); Marco, Nadia De [University of Naples Federico II, Department of Biology (Italy); Fusco, Sabato; Netti, Paolo Antonio [Italian Institute of Technology, Center for Advanced Biomaterials for Health Care IIT@CRIB (Italy); Carotenuto, Rosa, E-mail: rosa.carotenuto@unina.it [University of Naples Federico II, Department of Biology (Italy)

    2015-02-15

    Exposing living organisms to nanoparticulates is potentially hazardous, in particular when it takes place during embryogenesis. In this investigation, we have studied the effects of 50-nm-uncoated polystyrene nanoparticles (PSNPs) as a model to investigate the suitability of their possible future employments. We have used the standardized Frog Embryo Teratogenesis Assay-Xenopus test during the early stages of larval development of Xenopus laevis, and we have employed either contact exposure or microinjections. We found that the embryos mortality rate is dose dependent and that the survived embryos showed high percentage of malformations. They display disorders in pigmentation distribution, malformations of the head, gut and tail, edema in the anterior ventral region, and a shorter body length compared with sibling untreated embryos. Moreover, these embryos grow more slowly than the untreated embryos. Expressions of the mesoderm markers, bra (T-box Brachyury gene), myod1 (myogenic differentiation1), and of neural crest marker sox9 (sex SRY (determining region Y-box 9) transcription factor sox9), are modified. Confocal microscopy showed that the nanoparticles are localized in the cytoplasm, in the nucleus, and in the periphery of the digestive gut cells. Our data suggest that PSNPs are toxic and show a potential teratogenic effect for Xenopus larvae. We hypothesize that these effects may be due either to the amount of NPs that penetrate into the cells and/or to the “corona” effect caused by the interaction of PSNPs with cytoplasm components. The three endpoints of our study, i.e., mortality, malformations, and growth inhibition, suggest that the tests we used may be a powerful and flexible bioassay in evaluating pollutants in aquatic embryos.

  4. Polystyrene nanoparticles affect Xenopus laevis development

    Science.gov (United States)

    Tussellino, Margherita; Ronca, Raffaele; Formiggini, Fabio; Marco, Nadia De; Fusco, Sabato; Netti, Paolo Antonio; Carotenuto, Rosa

    2015-02-01

    Exposing living organisms to nanoparticulates is potentially hazardous, in particular when it takes place during embryogenesis. In this investigation, we have studied the effects of 50-nm-uncoated polystyrene nanoparticles (PSNPs) as a model to investigate the suitability of their possible future employments. We have used the standardized Frog Embryo Teratogenesis Assay- Xenopus test during the early stages of larval development of Xenopus laevis, and we have employed either contact exposure or microinjections. We found that the embryos mortality rate is dose dependent and that the survived embryos showed high percentage of malformations. They display disorders in pigmentation distribution, malformations of the head, gut and tail, edema in the anterior ventral region, and a shorter body length compared with sibling untreated embryos. Moreover, these embryos grow more slowly than the untreated embryos. Expressions of the mesoderm markers, bra (T-box Brachyury gene), myod1 (myogenic differentiation1), and of neural crest marker sox9 (sex SRY (determining region Y-box 9) transcription factor sox9), are modified. Confocal microscopy showed that the nanoparticles are localized in the cytoplasm, in the nucleus, and in the periphery of the digestive gut cells. Our data suggest that PSNPs are toxic and show a potential teratogenic effect for Xenopus larvae. We hypothesize that these effects may be due either to the amount of NPs that penetrate into the cells and/or to the "corona" effect caused by the interaction of PSNPs with cytoplasm components. The three endpoints of our study, i.e., mortality, malformations, and growth inhibition, suggest that the tests we used may be a powerful and flexible bioassay in evaluating pollutants in aquatic embryos.

  5. Cloning of an origin of DNA replication of Xenopus laevis

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    Watanabe, S.; Taylor, J.H.

    1980-09-01

    DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Eschrichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the (/sup 3/H)-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of th segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone. To determine the number of replications of pSW14, a novel method was employed. The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated (/sup 3/H)thymidine had replicated twice during the 4 hr in the unfertilized eggs of X. laevis. We conclude the pSW14 has a functional origin in the Xenopus DNA segment.

  6. [Do the variations in water carbon dioxide pressure and PH have an effect on the nature of end products of protein catabolism, ammonia and urea, in the clawed frog Xenopus laevis?].

    Science.gov (United States)

    Dejours, P; Armand, J; Beekenkamp, H

    1991-01-01

    The effects of PCO2 and pH changes in the ambient water on the nitrogen catabolism and the proportions of the excreted nitrogenous end products, ammonia and urea, were studied in the clawed frog, Xenopus laevis, at 24 degrees C. In animals living in artificial fresh water, the exposure to a hypocapnic alkalosis (PCO2 = 0.7 Torr instead of 10 Torr) did not entail any change in the nitrogen catabolism. In animals who lived in a water loaded with NaCl and had therefore a higher oxygen consumption, an intense nitrogen catabolism and a marked ureotelism, the hypocapnic alkalosis seems to have increased the intensity of the nitrogen catabolism. In neither group were there signs of ammonia toxicity.

  7. Connexins in the early development of the African clawed frog Xenopus laevis (Amphibia: The role of the connexin43 carboxyl terminal tail in the establishment of the dorso-ventral axis

    Directory of Open Access Journals (Sweden)

    Jaime Cofre

    2007-03-01

    Full Text Available Connexins are a family of related proteins identified in vertebrate forming gap junctions, which mediate cell-to-cell communication in early embryos, with an important role in establishing embryonic asymmetry and ‘communication compartments’. By in situ hybridization, immunocytochemistry, reverse transcriptase PCR (RT-PCR and western blotting we show that a Cx43-like molecule is present in oocytes and embryos of the African clawed frog Xenopus laevis, with specific localization in the animal-vegetal axis. This specific distribution is suggestive for an important role for this protein in the establishment of the dorso-ventral axis. Antisense RNA and antibodies directed against rat carboxyl terminal tail of the Cx43 (CT-Cx43 and injected in 1-cell stage Xenopus embryos, induced pronounced alterations in nervous system development, with a severe ventralization phenotype. Coherently, the overexpression of CT-Cx43 produced a dorsalization of the embryos. In antisense treated embryos, the expression of the beta-catenin gene is eliminated from the Nieuwkoop center, the pattern expression of the Chordin, Xnot and Xbra is modified, with no effect in expression of the Goosecoid gene. In CT-Cx43 mRNA treated embryos the pattern of expression of the beta-catenin, Chordin, Goosecoid, Xnot and engrailed-2 genes is modified. The expression of beta-catenin is increased in the Nieuwkoop center, the expression pattern of Chordin and Goosecoid is expanded to the posterior neural plate and engrailed-2 presents ectopic expression in the ventral region. Taken together our data suggest a role for CT-Cx43 as a maternal determinant with a critical function in the formation of the dorso-ventral axis in Xenopus laevis. The Cx43 may be one of the earliest markers of the dorso-ventral axis in these embryos and could possibly be acting through regionalization of factors responsible for the establishment of this axis.

  8. Limb Regeneration in Xenopus laevis Froglet

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    Makoto Suzuki

    2006-01-01

    Full Text Available Limb regeneration in amphibians is a representative process of epimorphosis. This type of organ regeneration, in which a mass of undifferentiated cells referred to as the “blastema” proliferate to restore the lost part of the amputated organ, is distinct from morphallaxis as observed, for instance, in Hydra, in which rearrangement of pre-existing cells and tissues mainly contribute to regeneration. In contrast to complete limb regeneration in urodele amphibians, limb regeneration in Xenopus, an anuran amphibian, is restricted. In this review of some aspects regarding adult limb regeneration in Xenopus laevis, we suggest that limb regeneration in adult Xenopus, which is pattern/tissue deficient, also represents epimorphosis.

  9. L-cysteine, N-acetyl-L-cysteine, and glutathione protect Xenopus laevis embryos against acrylamide-induced malformations and mortality in the frog embryo teratogenesis assay.

    Science.gov (United States)

    Rayburn, James R; Friedman, Mendel

    2010-10-27

    Dietary acrylamide is largely derived from heat-induced reactions between the amino group of the free amino acid asparagine and carbonyl groups of glucose and fructose during heat processing (baking, frying) of plant-derived foods such as potato fries and cereals. After consumption, acrylamide is absorbed into the circulation and is then distributed to various organs, where it can react with DNA, neurons, hemoglobin, and essential enzymes. In the present study, we explored the potential of L-cysteine (CySH), N-acetyl-L-cysteine (NAC), reduced glutathione (GSH), and the amino acid glycine (Gly) to protect frog embryos against acrylamide-induced developmental toxicity in the frog embryo teratogenesis assay - Xenopus (FETAX). To test the antiteratogenic potential, based on concentration-response study ranging from 0.07 to 4.22 mM acrylamide in FETAX solution (pH 8.1), we selected concentrations of acrylamide that induced 100% malformations and mortality. At the end of 96 h, we counted survivors and malformed embryos and measured embryo length. The data show that CySH, NAC, and GSH protected the embryos against acrylamide induced malformations and mortality to different degrees. CySH and GSH protected the embryos against both malformations and mortality, whereas NAC protected only against mortality. Gly had no protective effect. Possible mechanisms of the protective effects and the dietary significance of the results of this and related studies for food safety and human health are discussed.

  10. Skin wound healing in different aged Xenopus laevis.

    Science.gov (United States)

    Bertolotti, Evelina; Malagoli, Davide; Franchini, Antonella

    2013-08-01

    Xenopus froglets can perfectly heal skin wounds without scarring. To explore whether this capacity is maintained as development proceeds, we examined the cellular responses during the repair of skin injury in 8- and 15-month-old Xenopus laevis. The morphology and sequence of healing phases (i.e., inflammation, new tissue formation, and remodeling) were independent of age, while the timing was delayed in older frogs. At the beginning of postinjury, wound re-epithelialization occurred in form of a thin epithelium followed by a multilayered epidermis containing cells with apoptotic patterns and keratinocytes stained by anti-inducible nitric oxide synthase (iNOS) antibody. The inflammatory response, early activated by recruitment of blood cells immunoreactive to anti-tumor necrosis factor (TNF)-α, iNOS, transforming growth factor (TGF)-β1, and matrix metalloproteinase (MMP)-9, persisted over time. The dermis repaired by a granulation tissue with extensive angiogenesis, inflammatory cells, fibroblasts, and anti-α-SMA positive myofibroblasts. As the healing progressed, wounded areas displayed vascular regression, decrease in cellularity, and rearrangement of provisional matrix. The epidermis restored to a prewound morphology while granulation tissue was replaced by a fibrous tissue in a scar-like pattern. The quantitative PCR analysis demonstrated an up-regulated expression of Xenopus suppressor of cytokine signaling 3 (XSOCS-3) and Xenopus transforming growth factor-β2 (XTGF-β2) soon after wounding and peak levels were detected when granulation tissue was well developed with a large number of inflammatory cells. The findings indicate that X. laevis skin wound healing occurred by a combination of regeneration (in epidermis) and repair (in dermis) and, in contrast to froglet scarless wound healing, the growth to a more mature adult stage is associated with a decrease in regenerative capacity with scar-like tissue formation.

  11. Application of Xenopus laevis in ecotoxicology (I)--Introduction and quality control of laboratory animal

    Institute of Scientific and Technical Information of China (English)

    QIN Zhanfen; XU Xiaobai

    2006-01-01

    The aim of the series of papers is to discuss the application of Xenopus laevis, as model animal in biology, in ecotoxicology. X. laevis as model animal is wildly used in biological study and has provided a lot of relating data because of many advantages, such as living in water and being easily maintained, laying eggs in the whole year, and externally fertilizing and developing. Embryos and larvae of X. laevis like other amphibians are directly exposed in the aquatic environment and sensitive to pollutants. In addition, sex differentiation and sex organ development of X. laevis are sensitive to sex hormones and endocrine disruptors with sex hormone activities, which enable X. laevis to be used in studies on sex hormone disruption and reproductive toxicity of endocrine disruptors. Metamorphic development of X. laevis is very sensitive to thyroid hormones and thyroid disruptors, which enables X. laevis to be used for evaluating thyroid disruptors. Also, X. laevis ecotoxicology can be linked with amphibian population declines and malformed frog occurrence, being one of the hotspots in ecology. Thus, more and more laboratories have introduced X. laevis to ecotoxicological study. The quality of laboratory animals correlates with scientificity and reliability of results from animal experiments. It is especially important for toxicology. Quality control of X. laevis involving several factors such as water and food is discussed in this paper.

  12. The Genome of the Western Clawed Frog Xenopus tropicalis

    Energy Technology Data Exchange (ETDEWEB)

    Hellsten, Uffe; Harland, Richard M.; Gilchrist, Michael J.; Hendrix, David; Jurka, Jerzy; Kapitonov, Vladimir; Ovcharenko, Ivan; Putnam, Nicholas H.; Shu, Shengqiang; Taher, Leila; Blitz, Ira L.; Blumberg, Bruce; Dichmann, Darwin S.; Dubchak, Inna; Amaya, Enrique; Detter, John C.; Fletcher, Russell; Gerhard, Daniela S.; Goodstein, David; Graves, Tina; Grigoriev, Igor V.; Grimwood, Jane; Kawashima, Takeshi; Lindquist, Erika; Lucas, Susan M.; Mead, Paul E.; Mitros, Therese; Ogino, Hajime; Ohta, Yuko; Poliakov, Alexander V.; Pollet, Nicolas; Robert, Jacques; Salamov, Asaf; Sater, Amy K.; Schmutz, Jeremy; Terry, Astrid; Vize, Peter D.; Warren, Wesley C.; Wells, Dan; Wills, Andrea; Wilson, Richard K.; Zimmerman, Lyle B.; Zorn, Aaron M.; Grainger, Robert; Grammer, Timothy; Khokha, Mustafa K.; Richardson, Paul M.; Rokhsar, Daniel S.

    2009-10-01

    The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes over 20,000 protein-coding genes, including orthologs of at least 1,700 human disease genes. Over a million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like other tetrapods, the genome contains gene deserts enriched for conserved non-coding elements. The genome exhibits remarkable shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

  13. The genome of the Western clawed frog Xenopus tropicalis.

    Science.gov (United States)

    Hellsten, Uffe; Harland, Richard M; Gilchrist, Michael J; Hendrix, David; Jurka, Jerzy; Kapitonov, Vladimir; Ovcharenko, Ivan; Putnam, Nicholas H; Shu, Shengqiang; Taher, Leila; Blitz, Ira L; Blumberg, Bruce; Dichmann, Darwin S; Dubchak, Inna; Amaya, Enrique; Detter, John C; Fletcher, Russell; Gerhard, Daniela S; Goodstein, David; Graves, Tina; Grigoriev, Igor V; Grimwood, Jane; Kawashima, Takeshi; Lindquist, Erika; Lucas, Susan M; Mead, Paul E; Mitros, Therese; Ogino, Hajime; Ohta, Yuko; Poliakov, Alexander V; Pollet, Nicolas; Robert, Jacques; Salamov, Asaf; Sater, Amy K; Schmutz, Jeremy; Terry, Astrid; Vize, Peter D; Warren, Wesley C; Wells, Dan; Wills, Andrea; Wilson, Richard K; Zimmerman, Lyle B; Zorn, Aaron M; Grainger, Robert; Grammer, Timothy; Khokha, Mustafa K; Richardson, Paul M; Rokhsar, Daniel S

    2010-04-30

    The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

  14. Calpains expression during Xenopus laevis development.

    Science.gov (United States)

    Moudilou, E N; Mouterfi, N; Exbrayat, J-M; Brun, C

    2010-10-01

    Calpains are cytoplasmic proteases activated by calcium, implicated in cell differentiation and apoptosis. The best characterized enzymes are calpains 1-3. The aim of this work was to localize calpains 1-3 during the development of Xenopus laevis in order to clarify the function of these three proteases. For the first time, we detected the localization of the three proteases at the protein level between one-cell stage and adult age. Their expression was weak at early stages, then increased at tadpole stage and decreased through metamorphosis and adult life. The calpain's expression was maximal during the period characterized by the appearance of organs and modelling process. These observations suggest that calpains play a crucial role during development.

  15. Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

    Science.gov (United States)

    Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

    2016-08-01

    Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level.

  16. Plasticity of lung development in the amphibian, Xenopus laevis

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    Christopher S. Rose

    2013-10-01

    Contrary to previous studies, we found that Xenopus laevis tadpoles raised in normoxic water without access to air can routinely complete metamorphosis with lungs that are either severely stunted and uninflated or absent altogether. This is the first demonstration that lung development in a tetrapod can be inhibited by environmental factors and that a tetrapod that relies significantly on lung respiration under unstressed conditions can be raised to forego this function without adverse effects. This study compared lung development in untreated, air-deprived (AD and air-restored (AR tadpoles and frogs using whole mounts, histology, BrdU labeling of cell division and antibody staining of smooth muscle actin. We also examined the relationship of swimming and breathing behaviors to lung recovery in AR animals. Inhibition and recovery of lung development occurred at the stage of lung inflation. Lung recovery in AR tadpoles occurred at a predictable and rapid rate and correlated with changes in swimming and breathing behavior. It thus presents a new experimental model for investigating the role of mechanical forces in lung development. Lung recovery in AR frogs was unpredictable and did not correlate with behavioral changes. Its low frequency of occurrence could be attributed to developmental, physical and behavioral changes, the effects of which increase with size and age. Plasticity of lung inflation at tadpole stages and loss of plasticity at postmetamorphic stages offer new insights into the role of developmental plasticity in amphibian lung loss and life history evolution.

  17. A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes.

    Science.gov (United States)

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.

  18. Aryl Hydrocarbon Receptors in the frog Xenopus laevis: Two AHR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

    OpenAIRE

    Lavine, Jeremy A.; Rowatt, Ashley J.; Klimova, Tatyana; Whitington, Aric J.; Dengler, Emelyne; Beck, Catherine; Powell, Wade H.

    2005-01-01

    2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent developmental toxicant in most vertebrates. However, frogs are relatively insensitive to TCDD toxicity, especially during early life stages. Toxicity of TCDD and related halogenated aromatic hydrocarbons is mediated by the aryl hydrocarbon receptor (AHR), and specific differences in properties of the AHR signaling pathway can underlie differences in TCDD toxicity in different species. This study investigated the role of AHR in frog TCDD i...

  19. Effective RNAi-mediated β2-microglobulin loss of function by transgenesis in Xenopus laevis.

    Science.gov (United States)

    Nedelkovska, Hristina; Edholm, Eva-Stina; Haynes, Nikesha; Robert, Jacques

    2013-03-15

    To impair MHC class I (class I) function in vivo in the amphibian Xenopus, we developed an effective reverse genetic loss of function approach by combining I-SceI meganuclease-mediated transgenesis with RNAi technology. We generated transgenic outbred X. laevis and isogenetic laevis/gilli cloned lines with stably silenced expression of β2-microglobulin (b2m) critical for class I function. Transgenic F1 frogs exhibited decreased surface class I expression on erythrocytes and lymphocytes, decreased frequency of peripheral CD8 T cells and impaired CD8 T cell-mediated skin allograft rejection. Additionally, b2m knockdown increased susceptibility to viral infection of F0 transgenic larvae. This loss of function strategy offers new avenues for studying ontogeny of immunity and other developmental processes in Xenopus.

  20. Effective RNAi-mediated β2-microglobulin loss of function by transgenesis in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Hristina Nedelkovska

    2013-01-01

    To impair MHC class I (class I function in vivo in the amphibian Xenopus, we developed an effective reverse genetic loss of function approach by combining I-SceI meganuclease-mediated transgenesis with RNAi technology. We generated transgenic outbred X. laevis and isogenetic laevis/gilli cloned lines with stably silenced expression of β2-microglobulin (b2m critical for class I function. Transgenic F1 frogs exhibited decreased surface class I expression on erythrocytes and lymphocytes, decreased frequency of peripheral CD8 T cells and impaired CD8 T cell-mediated skin allograft rejection. Additionally, b2m knockdown increased susceptibility to viral infection of F0 transgenic larvae. This loss of function strategy offers new avenues for studying ontogeny of immunity and other developmental processes in Xenopus.

  1. Serum clinical biochemical and hematologic reference ranges of laboratory-reared and wild-caught Xenopus laevis.

    Science.gov (United States)

    Wilson, Sabrina; Felt, Stephen; Torreilles, Stéphanie; Howard, Antwain; Behan, Colleen; Moorhead, Roberta; Green, Sherril

    2011-09-01

    The South African clawed frogs Xenopus laevis and X. tropicalis are fully aquatic amphibians and well-established animal models. Because genetically engineered laboratory Xenopus are now being produced, the establishment of normal reference ranges for serum biochemical and hematologic parameters is essential for phenotyping and as a diagnostic aide. We determined normal reference ranges for hematologic values from 3 populations of X. laevis: wild-caught frogs (n = 43) and frogs from 2 commercial sources (A, n = 166; B, n = 109). For serum biochemistry, we determined normal reference ranges for frogs from source A and wild-caught frogs divided by sex and season. Significant differences across populations were found in WBC and RBC counts, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin concentration, and mean corpuscular volume. Among serum biochemical analytes, significant differences were found for albumin:globulin ratio, anion gap, and concentrations of albumin, globulin, total protein, lipase, alanine transaminase, γ-glutamyl transpeptidase; creatine phosphokinase; indirect, direct, and total bilirubin; cholesterol, low-density lipoprotein lipase, carbon dioxide, glucose, lactacte dehydrogenase, calcium, chloride, and sodium. We hypothesize that these differences can be attributed to differences in water quality, habitat, ambient temperature, diet, sex, recent transport or shipment, and genetic background. However, testing that hypothesis is beyond the scope of the current study. In addition, clinical chemistry and hematologic reference range values Xenopus laevis are quite distinct from those for other species and are most consistent with the only values published for another fully aquatic amphibian, the Eastern hellbender (Cryptobranchus alleganiensis).

  2. Plasticity in the melanotrope neuroendocrine interface of Xenopus laevis

    NARCIS (Netherlands)

    Jenks, B.G.; Kidane, A.H.; Scheenen, W.J.; Roubos, E.W.

    2007-01-01

    Melanotrope cells of the amphibian pituitary pars intermedia produce alpha-melanophore-stimulating hormone (alpha-MSH), a peptide which causes skin darkening during adaptation to a dark background. The secretory activity of the melanotrope of the South African clawed toad Xenopus laevis is regulated

  3. Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries.

    Science.gov (United States)

    Ruberti, I; Fragapane, P; Pierandrei-Amaldi, P; Beccari, E; Amaldi, F; Bozzoni, I

    1982-12-11

    Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved.

  4. Effect of methoxychlor on various life stages of Xenopus laevis.

    Science.gov (United States)

    Fort, Douglas J; Guiney, Patrick D; Weeks, John A; Thomas, John H; Rogers, Robert L; Noll, Andra M; Spaulding, Clinton D

    2004-10-01

    The toxicological effects of the organochlorine pesticide methoxychlor were evaluated at various life stages of the South African clawed frog, Xenopus laevis, in an effort to determine stage-specific sensitivity. A battery of four separate assays, including a short-term (4-day) early embryo-larval assay (FETAX) (NF stages 8-46 [Nieuwkoop and Faber, 1994]), 30-day hind limb development assay (NF stages 8-54), 18-day metamorphic climax assay (NF stages 58-66), and 30-day adult reproduction assay were performed. Test concentrations for the FETAX, hind limb development, metamorphic climax, and reproductive assays ranged from 0.0001-1.0 mg/l, 0.0001-0.1 mg/l, 0.0001-0.1 mg/l, and 0.001-0.1 mg/l, respectively. Results from the short-term embryo-larval assay indicated that increased embryo-lethality, malformation, and growth inhibition were not induced at methoxychlor (maximum soluble concentration). The 30-day hind limb development studies indicated methoxychlor exposure >/=0.01 mg/l delayed hind limb digit differentiation. Follicular hyperplasia of the thyroid glands was noted in specimens exposed to 0.1 mg/l methoxychlor. Results from the 18-day metamorphic climax assay indicated that methoxychlor inhibited the rate of tail resorption in a concentration-dependent manner. Whole body tissue triiodothyronine (T(3)) profiles showed a reduced and delayed surge during climax compared to controls. For the reproductive assessment, adult female X. laevis were super-ovulated and both female and male were then exposed to varying concentrations of methoxychlor. A concentration-dependent reduction in ovary weight and the number of viable oocytes was observed. In exposed male specimens, a concentration-dependent reduction in testis weight and sperm count was found. Methoxychlor was found to accumulate in the ovary, and to a lesser extent in the testis. Based on breeding studies in which exposed females were bred with control males and exposed males bred with control females, the

  5. The toxicity and teratogenicity of gibberellic acid (GA3) based on the frog embryo teratogenesis assay-Xenopus (FETAX)

    OpenAIRE

    BOĞA, Ayper; BİNOKAY, Seçil; SERTDEMİR, Yaşar

    2009-01-01

    This study investigated the developmental toxicity of a plant growth regulator (a type of pesticide) using the frog embryo teratogenesis assay-Xenopus (FETAX). Xenopus laevis embryos were exposed to 11 different concentrations of gibberellic acid (GA3), from stage 8 to 11, for 96 h under static renewal test conditions. The median lethal concentration (LC50), malformation (EC50), non-observed adverse effect concentration (NOAEC), and lowest observed adverse effect concentration (LOAEC) were ca...

  6. Probing the Xenopus laevis inner ear transcriptome for biological function

    Directory of Open Access Journals (Sweden)

    Powers TuShun R

    2012-06-01

    Full Text Available Abstract Background The senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. Amphibians have been widely used for physiological studies of mechanotransduction by sensory hair cells. In contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. Among amphibians, the genus Xenopus is a well-characterized genetic and developmental model that offers unique opportunities for inner ear research because of the amphibian capacity for tissue and organ regeneration. For these reasons, we implemented a functional genomics approach as a means to undertake a large-scale analysis of the Xenopus laevis inner ear transcriptome through microarray analysis. Results Microarray analysis uncovered genes within the X. laevis inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of Xenopus in cross-species genetic studies of the inner ear. The use of gene categories (inner ear tissue; deafness; ion channels; ion transporters; transcription factors facilitated the assignment of functional significance to probe set identifiers. We enhanced the biological relevance of our microarray data by using a variety of curation approaches to increase the annotation of the Affymetrix GeneChip® Xenopus laevis Genome array. In addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization. Conclusions We identified an abundance of targets for genetic analysis of auditory and vestibular function. The orthologues to human genes with known inner ear function and the highly expressed transcripts that lack annotation are particularly interesting candidates for future analyses. We used informatics approaches to impart biologically relevant information to the Xenopus inner ear transcriptome

  7. Xenopus laevis a success story of biological research in space

    Science.gov (United States)

    Horn, Eberhard R.

    2006-01-01

    The clawed toad Xenopus laevis is a common experimental animal used in many disciplines of life sciences, such as integrative, developmental and molecular biology or experimental medicine. Since 30 years, Xenopus is used in biological research in space. Important milestones were the years 1975, when Xenopus embryos flew for the first time on the Russian space station Salut-4 and 1994, when Xenopus eggs were successfully fertilized for the first time in space during the Japanese Spacelab mission STS-47 and developed in microgravity to vital tadpoles. Most Xenopus studies were related to embryogenesis and development. Observations during and after altered gravity revealed changes such as the thickening of the blastocoel roof, the dorsalization of the tail, and modifications of vestibular reflexes, fictive and freely swimming. Many changes were reversible even during microgravity exposure. Studies about the vestibuloocular reflex or synapse formation revealed an age-related sensitivity to altered gravity. Xenopus offers useful tools for studies about microgravity effects on living systems. Its oocyte is a suitable model to study ion channel function in space; the dorsalization model can be used to analyse growth factor sensibilities. Hardware for life support of adults, tadpoles and embryos (cf. SUPPLY unit in combination with miniaquaria) as well as for controlled experiments in space are prerequisites for an extension of research with Xenopus. The application aspect is based on the fact that fundamental research per se brings benefit to man.

  8. Genetic Mapping in Xenopus Laevis: Eight Linkage Groups Established

    OpenAIRE

    Graf, J. D.

    1989-01-01

    Inheritance of alleles at 29 electrophoretically detected protein loci and one pigment locus (albinism) was analyzed in Xenopus laevis by backcrossing multiply heterozygous individuals generated by intersubspecies hybridization. Pairwise linkage tests revealed eight classical linkage groups. These groups have been provisionally numbered from 1 to 8 in an arbitrarily chosen order. Linkage group 1 includes ALB-2 (albumin), ADH-1 (alcohol dehydrogenase), NP (nucleoside phosphorylase), and a(p) (...

  9. Regulative development of Xenopus laevis in microgravity

    Science.gov (United States)

    Black, S.; Larkin, K.; Jacqmotte, N.; Wassersug, R.; Pronych, S.; Souza, K.

    To test whether gravity is required for normal amphibian development, Xenopus leavis females were induced to ovulate aboard the orbiting Space Shuttle. Eggs were fertilized in vitro, and although early embryonic stages showed some abnormalities, the embryos were able to regulate and produce nearly normal larvae. These results demonstrate for the first time that a vertebrate can ovulate in the virtual absence of gravity, and that the eggs can develop to a free-living stage.

  10. Sterility and gene expression in hybrid males of Xenopus laevis and X. muelleri.

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    John H Malone

    Full Text Available BACKGROUND: Reproductive isolation is a defining characteristic of populations that represent unique biological species, yet we know very little about the gene expression basis for reproductive isolation. The advent of powerful molecular biology tools provides the ability to identify genes involved in reproductive isolation and focuses attention on the molecular mechanisms that separate biological species. Herein we quantify the sterility pattern of hybrid males in African Clawed Frogs (Xenopus and apply microarray analysis of the expression pattern found in testes to identify genes that are misexpressed in hybrid males relative to their two parental species (Xenopus laevis and X. muelleri. METHODOLOGY/PRINCIPAL FINDINGS: Phenotypic characteristics of spermatogenesis in sterile male hybrids (X. laevis x X. muelleri were examined using a novel sperm assay that allowed quantification of live, dead, and undifferentiated sperm cells, the number of motile vs. immotile sperm, and sperm morphology. Hybrids exhibited a dramatically lower abundance of mature sperm relative to the parental species. Hybrid spermatozoa were larger in size and accompanied by numerous undifferentiated sperm cells. Microarray analysis of gene expression in testes was combined with a correction for sequence divergence derived from genomic hybridizations to identify candidate genes involved in the sterility phenotype. Analysis of the transcriptome revealed a striking asymmetric pattern of misexpression. There were only about 140 genes misexpressed in hybrids compared to X. laevis but nearly 4,000 genes misexpressed in hybrids compared to X. muelleri. CONCLUSIONS/SIGNIFICANCE: Our results provide an important correlation between phenotypic characteristics of sperm and gene expression in sterile hybrid males. The broad pattern of gene misexpression suggests intriguing mechanisms creating the dominance pattern of the X. laevis genome in hybrids. These findings significantly

  11. Effects of agricultural pesticides on the immune system of Xenopus laevis and Rana pipiens

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    Christin, M.S.; Menard, L.; Gendron, A.D.; Ruby, S.; Cyr, D.; Marcogliese, D.J.; Rollins-Smith, L.; Fournier, M

    2004-03-30

    Over the last 30 years, there have been mass declines in diverse geographic locations among amphibian populations. Multiple causes have been suggested to explain this decline. Among these, environmental pollution is gaining attention. Indeed, some chemicals of environmental concern are known to alter the immune system. Given that amphibians are frequently exposed to agricultural pesticides, it is possible that these pollutants alter their immune system and render them more susceptible to different pathogens. In this study, we exposed two frog species, Xenopus laevis and Rana pipiens, for a short period of time to a mixture of pesticides (atrazine, metribuzine, endosulfan, lindane, aldicarb and dieldrin) representative in terms of composition and concentrations to what it is found in the environment of the southwest region of the province of Quebec. The pesticides were known to be present in surface water of many tributaries of the St. Lawrence River (Quebec, Canada). Our results demonstrate that the mixture of pesticides could alter the cellularity and phagocytic activity of X. laevis and the lymphocyte proliferation of R. pipiens. Taken together, these results indicate that agricultural pesticides can alter some aspects of the immune response in frogs and could contribute to their global decline by rendering them more susceptible to certain infections.

  12. Gene expression analysis of the ovary of hybrid females of Xenopus laevis and X. muelleri

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    Malone John H

    2008-03-01

    Full Text Available Abstract Background Interspecific hybrids of frogs of the genus Xenopus result in sterile hybrid males and fertile hybrid females. Previous work has demonstrated a dramatic asymmetrical pattern of misexpression in hybrid males compared to the two parental species with relatively few genes misexpressed in comparisons of hybrids and the maternal species (X. laevis and dramatically more genes misexpressed in hybrids compared to the paternal species (X. muelleri. In this work, we examine the gene expression pattern in hybrid females of X. laevis × X. muelleri to determine if this asymmetrical pattern of expression also occurs in hybrid females. Results We find a similar pattern of asymmetry in expression compared to males in that there were more genes differentially expressed between hybrids and X. muelleri compared to hybrids and X. laevis. We also found a dramatic increase in the number of misexpressed genes with hybrid females having about 20 times more genes misexpressed in ovaries compared to testes of hybrid males and therefore the match between phenotype and expression pattern is not supported. Conclusion We discuss these intriguing findings in the context of reproductive isolation and suggest that divergence in female expression may be involved in sterility of hybrid males due to the inherent sensitivity of spermatogenesis as defined by the faster male evolution hypothesis for Haldane's rule.

  13. Functional joint regeneration is achieved using reintegration mechanism in Xenopus laevis.

    Science.gov (United States)

    Tsutsumi, Rio; Yamada, Shigehito; Agata, Kiyokazu

    2016-02-01

    A functional joint requires integration of multiple tissues: the apposing skeletal elements should form an interlocking structure, and muscles should insert into skeletal tissues via tendons across the joint. Whereas newts can regenerate functional joints after amputation, Xenopus laevis regenerates a cartilaginous rod without joints, a "spike." Previously we reported that the reintegration mechanism between the remaining and regenerated tissues has a significant effect on regenerating joint morphogenesis during elbow joint regeneration in newt. Based on this insight into the importance of reintegration, we amputated frogs' limbs at the elbow joint and found that frogs could regenerate a functional elbow joint between the remaining tissues and regenerated spike. During regeneration, the regenerating cartilage was partially connected to the remaining articular cartilage to reform the interlocking structure of the elbow joint at the proximal end of the spike. Furthermore, the muscles of the remaining part inserted into the regenerated spike cartilage via tendons. This study might open up an avenue for analyzing molecular and cellular mechanisms of joint regeneration using Xenopus.

  14. Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis.

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    Tatini Rakshit

    Full Text Available Rhodopsin forms nanoscale domains (i.e., nanodomains in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates.

  15. D-Amino acid oxidase and presence of D-proline in Xenopus laevis.

    Science.gov (United States)

    Soma, Hiroki; Furuya, Ryuji; Kaneko, Ryo; Tsukamoto, Ayaka; Shirasu, Kazumitsu; Tanigawa, Minoru; Nagata, Yoko

    2013-10-01

    We purified D-amino acid oxidase (EC 1.4.3.3, DAO) from Xenopus laevis tadpoles. The optimal temperature and pH for enzyme activity were 35-40 °C and 8.3-9.0, respectively, depending on the substrate amino acids available to the enzyme; the highest activity was observed with D-proline followed by D-phenylalanine. Activity was significantly inhibited by p-hydroxymercuribenzoate, but only moderately by p-chloromercuribenzoate or benzoate. Enzyme activity was increased until the final tadpole stage, but was reduced to one-third in the adult and was localized primarily in the kidney. The tadpoles contained high concentrations of D-proline close to the final developmental stage and nearly no D-amino acids were detected in the adult frog, indicating that D-amino acid oxidase functions in metamorphosis.

  16. Mapping neurogenesis onset in the optic tectum of Xenopus laevis

    Science.gov (United States)

    Herrgen, Leah; Akerman, Colin J.

    2016-01-01

    Neural progenitor cells have a central role in the development and evolution of the vertebrate brain. During early brain development, neural progenitors first expand their numbers through repeated proliferative divisions and then begin to exhibit neurogenic divisions. The transparent and experimentally accessible optic tectum of Xenopus laevis is an excellent model system for the study of the cell biology of neurogenesis, but the precise spatial and temporal relationship between proliferative and neurogenic progenitors has not been explored in this system. Here we construct a spatial map of proliferative and neurogenic divisions through lineage tracing of individual progenitors and their progeny. We find a clear spatial separation of proliferative and neurogenic progenitors along the anterior-posterior axis of the optic tectum, with proliferative progenitors located more posteriorly and neurogenic progenitors located more anteriorly. Since individual progenitors are repositioned toward more anterior locations as they mature, this spatial separation likely reflects an increased restriction in the proliferative potential of individual progenitors. We then examined whether the transition from proliferative to neurogenic behavior correlates with cellular properties that have previously been implicated in regulating neurogenesis onset. Our data reveal that the transition from proliferation to neurogenesis is associated with a small change in cleavage plane orientation and a more pronounced change in cell cycle kinetics in a manner reminiscent of observations from mammalian systems. Our findings highlight the potential to use the optic tectum of Xenopus laevis as an accessible system for the study of the cell biology of neurogenesis. PMID:27358457

  17. Benchmarking Transcriptome Quantification Methods for Duplicated Genes in Xenopus laevis.

    Science.gov (United States)

    Kwon, Taejoon

    2015-01-01

    Xenopus is an important model organism for the study of genome duplication in vertebrates. With the full genome sequence of diploid Xenopus tropicalis available, and that of allotetraploid X. laevis close to being finished, we will be able to expand our understanding of how duplicated genes have evolved. One of the key features in the study of the functional consequence of gene duplication is how their expression patterns vary across different conditions, and RNA-seq seems to have enough resolution to discriminate the expression of highly similar duplicated genes. However, most of the current RNA-seq analysis methods were not designed to study samples with duplicate genes such as in X. laevis. Here, various computational methods to quantify gene expression in RNA-seq data were evaluated, using 2 independent X. laevis egg RNA-seq datasets and 2 reference databases for duplicated genes. The fact that RNA-seq can measure expression levels of similar duplicated genes was confirmed, but long paired-end reads are more informative than short single-end reads to discriminate duplicated genes. Also, it was found that bowtie, one of the most popular mappers in RNA-seq analysis, reports significantly smaller numbers of unique hits according to a mapping quality score compared to other mappers tested (BWA, GSNAP, STAR). Calculated from unique hits based on a mapping quality score, both expression levels and the expression ratio of duplicated genes can be estimated consistently among biological replicates, demonstrating that this method can successfully discriminate the expression of each copy of a duplicated gene pair. This comprehensive evaluation will be a useful guideline for studying gene expression of organisms with genome duplication using RNA-seq in the future.

  18. Identification and developmental expression of Xenopus laevis SUMO proteases.

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    Yonggang Wang

    Full Text Available SUMO proteins are small ubiquitin-related modifiers. All SUMOs are synthesized as propeptides that are post-translationally cleaved prior to conjugation. After processing, SUMOs become covalently conjugated to cellular targets through a pathway that is similar to ubiquitination. Ubiquitin like protein proteases/Sentrin specific proteases (Ulp/SENPs mediate both processing and deconjugation of SUMOs. The action of Ulp/SENPs makes SUMOylation a highly dynamic post-translational modification. To investigate how Ulp/SENPs are regulated in a developmental context, we isolated and characterized all Ulp/SENPs in Xenopus laevis. Xenopus possess homologues of mammalian SENP3, 5, 6 and 7. All of these enzymes reacted with HA-tagged vinyl sulfone derivatives of SUMO-2 (HA-SU2-VS but not SUMO-1 (HA-SU1-VS, suggesting that they act primarily on SUMO-2 and -3. In contrast, Xenopus possess a single member of the SENP1/SENP2 subfamily of Ulp/SENPs, most closely related to mammalian SENP1. Xenopus SENP1 reacted with HA-SU1-VS and HA-SU2-VS, suggesting that it acts on all SUMO paralogues. We analyzed the mRNA and protein levels for each of the Ulp/SENPs through development; we found that they show distinct patterns of expression that may involve both transcriptional and post-transcriptional regulation. Finally, we have characterized the developmental function of the most abundant Ulp/SENP found within Xenopus eggs, SENP3. Depletion of SENP3 using morpholino antisense oligonucleotides (morpholinos caused accumulation of high molecular weight SUMO-2/3 conjugated species, defects in developing embryos and changes in the expression of some genes regulated by the transforming growth factor beta (TGF-beta pathway. These findings collectively indicate that SUMO proteases are both highly regulated and essential for normal development.

  19. Budgett's frog (Lepidobatrachus laevis): A new amphibian embryo for developmental biology.

    Science.gov (United States)

    Amin, Nirav M; Womble, Mandy; Ledon-Rettig, Cristina; Hull, Margaret; Dickinson, Amanda; Nascone-Yoder, Nanette

    2015-09-15

    The large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett's frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett's frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett's tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett's frog model provides inimitable advantages for developmental studies-and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution.

  20. Basolateral Cl- uptake mechanisms in Xenopus laevis lung epithelium.

    Science.gov (United States)

    Berger, Jens; Hardt, Martin; Clauss, Wolfgang G; Fronius, Martin

    2010-07-01

    A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl(-) uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na(+)/K(+)/2 Cl(-) cotransporter (NKCC) and HCO(3)(-)/Cl(-) anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl(-)-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl(-) gradient. Further, we were interested in the involvement of the HCO(3)(-)/Cl(-) anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (I(SC)). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO(3)(-) concentrations. Cl(-) secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO(3)(-). These experiments indicate that the AE at least partially contributes to Cl(-) secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl(-) uptake, which contrasts with the common model for Cl(-) secretion in pulmonary epithelia.

  1. De novo Transcriptome Assemblies of Rana (Lithobates catesbeiana and Xenopus laevis Tadpole Livers for Comparative Genomics without Reference Genomes.

    Directory of Open Access Journals (Sweden)

    Inanc Birol

    Full Text Available In this work we studied the liver transcriptomes of two frog species, the American bullfrog (Rana (Lithobates catesbeiana and the African clawed frog (Xenopus laevis. We used high throughput RNA sequencing (RNA-seq data to assemble and annotate these transcriptomes, and compared how their baseline expression profiles change when tadpoles of the two species are exposed to thyroid hormone. We generated more than 1.5 billion RNA-seq reads in total for the two species under two conditions as treatment/control pairs. We de novo assembled these reads using Trans-ABySS to reconstruct reference transcriptomes, obtaining over 350,000 and 130,000 putative transcripts for R. catesbeiana and X. laevis, respectively. Using available genomics resources for X. laevis, we annotated over 97% of our X. laevis transcriptome contigs, demonstrating the utility and efficacy of our methodology. Leveraging this validated analysis pipeline, we also annotated the assembled R. catesbeiana transcriptome. We used the expression profiles of the annotated genes of the two species to examine the similarities and differences between the tadpole liver transcriptomes. We also compared the gene ontology terms of expressed genes to measure how the animals react to a challenge by thyroid hormone. Our study reports three main conclusions. First, de novo assembly of RNA-seq data is a powerful method for annotating and establishing transcriptomes of non-model organisms. Second, the liver transcriptomes of the two frog species, R. catesbeiana and X. laevis, show many common features, and the distribution of their gene ontology profiles are statistically indistinguishable. Third, although they broadly respond the same way to the presence of thyroid hormone in their environment, their receptor/signal transduction pathways display marked differences.

  2. Population-specific incidence of testicular ovarian follicles in Xenopus laevis from South Africa: A potential issue in endocrine testing

    Energy Technology Data Exchange (ETDEWEB)

    Du Preez, Louis H., E-mail: Louis.DuPreez@nwu.ac.za [School of Environmental Sciences and Development, North-West University, Potchefstroom 2531 (South Africa); Kunene, Nisile [School of Environmental Sciences and Development, North-West University, Potchefstroom 2531 (South Africa); Hanner, Robert [Department of Integrative Biology, University of Guelph, Guelph, ON, N1G 2W1 (Canada); Giesy, John P. [Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan (Canada); Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (Hong Kong); National Food Safety and Toxicology Center, Zoology Department, and Centre for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Solomon, Keith R., E-mail: ksolomon@uoguelph.ca [Centre for Toxicology and Department of Environmental Biology, University of Guelph, Guelph, ON, N1G 2W1 (Canada); Hosmer, Alan [Syngenta Crop Protection, Greensboro, NC 27419-8300 (United States); Van Der Kraak, Glen J. [Department of Integrative Biology, University of Guelph, Guelph, ON, N1G 2W1 (Canada)

    2009-10-19

    The African clawed frog (Xenopus laevis) has been identified as an appropriate sentinel for testing endocrine activity of existing chemicals in North America and Europe. Some reports suggest that the herbicide, atrazine (CAS Number [1912-24-9]) causes ovarian follicles to form in the testes of this frog. X. laevis collected from North East (NE) sites in South Africa had testicular ovarian follicles, irrespective of exposure to atrazine, while frogs from Southwest Western (SW) Cape region sites had none. Phylogenetic analysis of mitochondrial and nuclear genes indicates that frogs from the SW Cape are evolutionarily divergent from those from NE South Africa and the rest of sub-Saharan Africa. These findings provide a possible explanation for why conflicting results have been reported concerning the impact of atrazine on amphibian sexual differentiation and highlight the importance of understanding taxonomic status of the experimental animal. Even in common laboratory animals, there is a need for their correct taxonomic characterization before their use in tests for endocrine disruption.

  3. Bisphenol A induces feminization in Xenopus laevis tadpoles.

    Science.gov (United States)

    Levy, Gregor; Lutz, Ilka; Krüger, Angela; Kloas, Werner

    2004-01-01

    To evaluate possible estrogenic effects of bisphenol A (BPA) in an amphibian model, Xenopus laevis tadpoles were exposed to BPA and 17beta-estradiol (E2) during larval development. After metamorphosis, the gonadal phenotype was determined by gross morphology, and testes were further examined histologically to validate the results. BPA treatment altered the normal sex ratio toward females depending on the BPA concentrations added. Chemical analysis showed a time-dependent decline of BPA during semistatic exposure, indicating that BPA is taken up and metabolized to some extent by tadpoles. In addition, tadpoles were exposed to BPA and E2 for 2 weeks during sensitive stages of sexual differentiation. Afterward, the expression of an estrogenic biomarker, estrogen receptor (ER) mRNA, was assessed by semiquantitative RT-PCR. Both BPA and E2 up-regulated ER mRNA significantly. In conclusion, these results show clear evidence that BPA induces feminization in X. laevis tadpoles, revealing an estrogenic potency of BPA that influences sexual development in amphibians.

  4. Islet-1 Immunoreactivity in the Developing Retina of Xenopus laevis

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    Guadalupe Álvarez-Hernán

    2013-01-01

    Full Text Available The LIM-homeodomain transcription factor Islet1 (Isl1 has been widely used as a marker of neuronal differentiation in the developing visual system of different classes of vertebrates, including mammals, birds, reptiles, and fish. In the present study, we analyzed the spatial and temporal distribution of Isl1-immunoreactive cells during Xenopus laevis retinal development and its relation to the formation of the retinal layers, and in combination with different markers of cell differentiation. The earliest Isl1 expression appeared at St29-30 in the cell nuclei of sparse differentiating neuroblasts located in the vitreal surface of the undifferentiated retina. At St35-36, abundant Isl1-positive cells accumulated at the vitreal surface of the neuroepithelium. As development proceeded and through the postmetamorphic juveniles, Isl1 expression was identified in subpopulations of ganglion cells and in subsets of amacrine, bipolar, and horizontal cells. These data together suggest a possible role for Isl1 in the early differentiation and maintenance of different retinal cell types, and Isl1 can serve as a specific molecular marker for the study of retinal cell specification in X. laevis.

  5. Significant modulation of the hepatic proteome induced by exposure to low temperature in Xenopus laevis

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    Kazumichi Nagasawa

    2013-08-01

    The African clawed frog, Xenopus laevis, is an ectothermic vertebrate that can survive at low environmental temperatures. To gain insight into the molecular events induced by low body temperature, liver proteins were evaluated at the standard laboratory rearing temperature (22°C, control and a low environmental temperature (5°C, cold exposure. Using nano-flow liquid chromatography coupled with tandem mass spectrometry, we identified 58 proteins that differed in abundance. A subsequent Gene Ontology analysis revealed that the tyrosine and phenylalanine catabolic processes were modulated by cold exposure, which resulted in decreases in hepatic tyrosine and phenylalanine, respectively. Similarly, levels of pyruvate kinase and enolase, which are involved in glycolysis and glycogen synthesis, were also decreased, whereas levels of glycogen phosphorylase, which participates in glycogenolysis, were increased. Therefore, we measured metabolites in the respective pathways and found that levels of hepatic glycogen and glucose were decreased. Although the liver was under oxidative stress because of iron accumulation caused by hepatic erythrocyte destruction, the hepatic NADPH/NADP ratio was not changed. Thus, glycogen is probably utilized mainly for NADPH supply rather than for energy or glucose production. In conclusion, X. laevis responds to low body temperature by modulating its hepatic proteome, which results in altered carbohydrate metabolism.

  6. Expression of spasmolysin (FIM-A.1): an integumentary mucin from Xenopus laevis.

    Science.gov (United States)

    Hauser, F; Gertzen, E M; Hoffmann, W

    1990-08-01

    In the past, a unique type of precursor for a secretory protein was discovered. It contains a central repetitive domain rich in threonine residues and terminal cysteine-rich domains. Due to striking homologies of these terminal domains with pancreatic spasmolytic polypeptide, originally the name "prepro-spasmolysin" was proposed. Here we show that the mature protein has a MW of about 130 kDa, consisting of about 70% carbohydrate and 30% protein. Similar O-linked glycoproteins have been found in mucins from human intestine. For this and numerous other reasons we decided to rename this glycoprotein "frog integumentary mucin A.1" (FIM-A.1). Furthermore, analysis of the protein with specific antibodies against the predicted C-terminal end indicates that FIM-A.1 is probably not processed at pairs of basic residues. In situ hybridization as well as immunofluorescence studies revealed that FIM-A.1 is expressed and stored exclusively in mature mucous glands of Xenopus laevis skin. Only cone cells at the proximal part of these glands do not synthesize FIM-A.1. In contrast, all other physiologically active peptides from X. laevis skin investigated so far are synthesized in granular glands. A hypothetical function of FIMs for defense against microbial infections is discussed.

  7. Protein pattern of Xenopus laevis embryos grown in simulated microgravity.

    Science.gov (United States)

    Tedeschi, Gabriella; Pagliato, Lara; Negroni, Manuela; Montorfano, Gigliola; Corsetto, Paola; Nonnis, Simona; Negri, Armando; Rizzo, Angela Maria

    2011-03-01

    Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.

  8. Expression of rat liver cell membrane transporters for thyroid hormone in Xenopus laevis oocytes

    NARCIS (Netherlands)

    R. Docter (Roel); E.C.H. Friesema (Edith); P.G.J. van Stralen (Paul); E.P. Krenning (Eric); M.E. Everts (Maria); T.J. Visser (Theo); G. Hennemann

    1997-01-01

    textabstractThe present study was conducted to explore the possible use of Xenopus laevis oocytes for the expression cloning of cell membrane transporters for iodothyronines. Injection of stage V-VI X. laevis oocytes with 23 ng Wistar rat liver polyadenylated RNA (mRNA)

  9. Xenopus laevis - A success story in biological research in Space

    Science.gov (United States)

    Horn, E.

    A feature of sensory, neuronal and motor systems is the existence of a critical period during their development. Environmental modifications, in particular stimulus depri-vation, during this period of life affects development in a long-term manner. For gravity sensory systems, space flights offer the only opportunity for deprivation conditions. Studies in the amphibian Xenopus laevis presented the most complete picture. The presentation demonstrates the importance of Xenopus laevis as an ex-perimental model animal in the past and even for future research in Space. Studies are presented which range from fertilization in Space and anatomical studies during early development under weightlessness up to post-flight studies on the anatomy of the peripheral sense organ, the spinal motor activity and behavior. Gravity depriva-tion induces anatomical as well as behavioral and neurophysiological modifications, which are normalized either during flight (thickening of the blastocoel roof) or after reentry in 1g-conditions (swimming and reflex behavior, spinal motor activity). The physiological changes can be explained by mechanisms of physiological adaptation. However, the studies also revealed stages which were insensitive to gravity depriva-tion; they point to the existence of a critical period. Observations on morphological mal-formations are described which are reversible after termination of microgravity and which are linked to a depression of vestibular reflex behavior. They might be caused by a competition between dorsalization and ventralization inducing growth factors. This observation offers the possibility for a genetic approach in finding ba-sics for microgravity effects on the development of Xenopus, and in a general frame, on the development of vertebrates including men. At the present stage of research, it remains open whether adaptive processes during exposure to altered gravity or the existence of a critical period in vestibular development are responsible for

  10. Epithelial cell division in the Xenopus laevis embryo during gastrulation.

    Science.gov (United States)

    Hatte, Guillaume; Tramier, Marc; Prigent, Claude; Tassan, Jean-Pierre

    2014-01-01

    How vertebrate epithelial cells divide in vivo and how the cellular environment influences cell division is currently poorly understood. A sine qua non condition to study cell division in situ is the ease of observation of cell division. This is fulfilled in the Xenopus embryo at the gastrula stage where polarized epithelial cells divide with a high frequency at the surface of the organism. Recently, using this model system, we have shown that epithelial cells divide by asymmetric furrowing and that the mode of cell division is regulated during development. Here, we further characterize epithelial cell division in situ. To this end, we used confocal microscopy to study epithelial cell division in the ectoderm of the Xenopus laevis gastrula. Cell division was followed either by indirect immunofluorescence in fixed embryos or by live imaging of embryos transiently expressing diverse fluorescent proteins. Here, we show that during cytokinesis, the plasma membranes of the two daughter cells are usually separated by a gap. For most divisions, daughter cells make contacts basally at a distance from the furrow tip which creates an inverted teardrop-like shaped volume tightly associated with the furrow. At the end of cytokinesis, the inverted teardrop is resorbed; thus it is a transient structure. Several proteins involved in cytokinesis are localized at the tip of the inverted teardrop suggesting that the formation of the gap could be an active process. We also show that intercalation of neighboring cells between daughter cells occasionally occurs during cytokinesis. Our results reveal an additional level of complexity in the relationship between dividing cells and also with their neighboring cells during cytokinesis in the Xenopus embryo epithelium.

  11. A transgenic Xenopus laevis reporter model to study lymphangiogenesis

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    Annelii Ny

    2013-07-01

    The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP Xenopus laevis reporter lines expressing green fluorescent protein (GFP in blood- and lymph vessels driven by the Flk1 (VEGFR-2 promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening.

  12. The embryonic development of Xenopus laevis under a low frequency electric field.

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    Boga, Ayper; Binokay, Secil; Emre, Mustafa; Sertdemir, Yasar

    2012-06-01

    The aim of this study was to determine the effects of a low frequency electric field on the early embryonic development of frogs. The embryos of African clawed toads, Xenopus laevis, were exposed to a 20-μA electric current during the cleavage stages. The developmental processes of embryos during and after electric field exposure were monitored for teratogenic effects. All the embryos continuously exposed to the electric field died without undergoing any developmental processes. However, when the embryos were exposed to the electric field for 20-min periods (four times/over 2 d), the embryos developed into both normal tadpoles (70 %) and malformed tadpoles with light edema, reduced pigmentation, or axial anomalies, such as crooked tails. After exposure, the control embryos were at development stage 35.5 (2 d 2 h), while the normal embryos of the assay group were at developmental stage 41(3 d 4 h). There was a 1 d 2 h difference between the two developmental stages, revealing the importance of that time period for embryogenesis. In conclusion, the effects of electric current on Xenopus embryos are dependent on the initial developmental stage and the duration of exposure.

  13. A comparison of electronic and traditional cigarette butt leachate on the development of Xenopus laevis embryos.

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    Parker, Tatiana Tatum; Rayburn, James

    2017-01-01

    Potential developmental toxicities of three different cigarette butt leachates were evaluated using the frog embryo teratogenesis assay-Xenopus (FETAX). Xenopus laevis embryos were exposed to regular cigarette butt (RCB), menthol (MCB) and electronic (ECB) in concentrations ranging from 0 to 4 butts/l for RCB and MCB and 0-10 butts/l for ECB. The embryos were from stage 8 to 11 and were exposed for a 96-h period in static renewal test conditions. Median lethal concentration (LC50), malformation (EC50), non-observed adverse effect concentration (NOAEC), and lowest observed adverse effect concentration (LOAEC) were calculated. Results from these studies suggest that each tested leachate is teratogenic for X. laevis embryos. The lowest LC50 was determined for ECB exposure at 17.9 cigarette butts/L. The LC50 value was the highest with RCB and MCB having LC50 s of approximately 1 cigarette butt/L. There were notable EC50 differences with RCB having the highest and ECB the lowest. The NOAEC and LOAEC levels for RCB and MCB were below 1 cigarette butt/L for both mortality and malformations; over 8 butts/L for ECB mortality and over 4 butts/L for malformations. From these results, we conclude that RCB leachate is the most toxic compound, while MCB leachate has the higher teratogenicity. ECB leachate has the lowest toxic and teratogenic effects on embryos but there were still noticeable effects. The results confirmed that the FETAX assay can be useful in an integrated biological hazard assessment for the preliminary screening for ecological risks of cigarette butts, and electronic cigarettes, in aquatic environment.

  14. Myosin heavy chain isoform composition and stretch activation kinetics in single fibres of Xenopus laevis iliofibularis muscle.

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    Andruchova, Olena; Stephenson, Gabriela M M; Andruchov, Oleg; Stephenson, D George; Galler, Stefan

    2006-07-01

    Skeletal muscle is composed of specialized fibre types that enable it to fulfil complex and variable functional needs. Muscle fibres of Xenopus laevis, a frog formerly classified as a toad, were the first to be typed based on a combination of physiological, morphological, histochemical and biochemical characteristics. Currently the most widely accepted criterion for muscle fibre typing is the myosin heavy chain (MHC) isoform composition because it is assumed that variations of this protein are the most important contributors to functional diversity. Yet this criterion has not been used for classification of Xenopus fibres due to the lack of an effective protocol for MHC isoform analysis. In the present study we aimed to resolve and visualize electrophoretically the MHC isoforms expressed in the iliofibularis muscle of Xenopus laevis, to define their functional identity and to classify the fibres based on their MHC isoform composition. Using a SDS-PAGE protocol that proved successful with mammalian muscle MHC isoforms, we were able to detect five MHC isoforms in Xenopus iliofibularis muscle. The kinetics of stretch-induced force transients (stretch activation) produced by a fibre was strongly correlated with its MHC isoform content indicating that the five MHC isoforms confer different kinetics characteristics. Hybrid fibre types containing two MHC isoforms exhibited stretch activation kinetics parameters that were intermediate between those of the corresponding pure fibre types. These results clearly show that the MHC isoforms expressed in Xenopus muscle are functionally different thereby validating the idea that MHC isoform composition is the most reliable criterion for vertebrate skeletal muscle fibre type classification. Thus, our results lay the foundation for the unequivocal classification of the muscle fibres in the Xenopus iliofibularis muscle and for gaining further insights into skeletal muscle fibre diversity.

  15. Transient effects of microgravity on early embryos of Xenopus laevis

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    de Mazière, A.; Gonzalez-Jurado, J.; Reijnen, M.; Narraway, J.; Ubbels, G. A.

    In order to study the role of gravity on the early development of the clawed toad Xenopus laevis, we performed an experiment on the Maser-6 sounding rocket launched from Kiruna (Sweden) on 4 Nov 1993. The aim was to find out whether a short period of microgravity (mug) during fertilization and the first few minutes of development does indeed result in abnormal axis formation as was suggested by a pilot experiment on the Maser 3 in 1989. On the Maser 6 we used two new technical additions in the Fokker CIS unit, viz. a 1-g control centrifuge and a video recording unit which both worked successfully. The 1-g control centrifuge was used to discriminate between the influences of flight perturbations and mug. After fertilization shortly before launch, one of the first indications of successful egg activation, the cortical contraction, was registered in mug and on earth. Analysis of the video tapes revealed that the cortical contraction in mug starts earlier than at 1 g on earth. After recovery of the eggs fertilized in mug and culture of the embryos on earth, the morphology of the blastocoel has some consistent differences from blastulae from eggs fertilized in the 1-g centrifuge of the rocket. However from the gastrula stage onward, the mug embryos apparently recover and resume normal development: the XBra gene is normally expressed, and histological examination shows normal axis formation.

  16. Identification of genes associated with regenerative success of Xenopus laevis hindlimbs

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    Barker Donna

    2008-06-01

    Full Text Available Abstract Background Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate their tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus (strain N1 in which the BMP inhibitor Noggin can be over-expressed at any time during development. Activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles. Results In the current study, we have taken advantage of the N1 transgenic line to directly compare morphology and gene expression in same stage regenerating vs. BMP signalling deficient non-regenerating hindlimb buds. The wound epithelium of N1 transgenic hindlimb buds, which forms over the cut surface of the limb bud after amputation, does not transition normally into the distal thickened apical epithelial cap. Instead, a basement membrane and dermis form, indicative of mature skin. Furthermore, the underlying mesenchyme remains rounded and does not expand to form a cone shaped blastema, a normal feature of successful regeneration. Using Affymetrix Gene Chip analysis, we have identified genes linked to regenerative success downstream of BMP signalling, including the BMP inhibitor Gremlin and the stress protein Hsp60 (no blastema in zebrafish. Gene Ontology analysis showed that genes involved in embryonic development and growth are significantly over-represented in regenerating early hindlimb buds and that successful regeneration in the Xenopus hindlimb correlates with the induction of

  17. Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

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    Morin, R D; Chang, E; Petrescu, A; Liao, N; Kirkpatrick, R; Griffith, M; Butterfield, Y; Stott, J; Barber, S; Babakaiff, R; Matsuo, C; Wong, D; Yang, G; Smailus, D; Brown-John, M; Mayo, M; Beland, J; Gibson, S; Olson, T; Tsai, M; Featherstone, R; Chand, S; Siddiqui, A; Jang, W; Lee, E; Klein, S; Prange, C; Myers, R M; Green, E D; Wagner, L; Gerhard, D; Marra, M; Jones, S M; Holt, R

    2005-10-31

    Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence between the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.

  18. Homoeologous chromosomes of Xenopus laevis are highly conserved after whole-genome duplication.

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    Uno, Y; Nishida, C; Takagi, C; Ueno, N; Matsuda, Y

    2013-11-01

    It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD.

  19. Distinct expression profiles of transcriptional coactivators for thyroid hormone receptors during Xenopus laevis metamorphosis

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    BINDU D PAUL; YUN-BO SHI

    2003-01-01

    The biological effects of thyroid hormone(T3)are mediated by the thyroid hormone receptor(TR).Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3.T3 regulates a series of orchestrated developmental changes,which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog.T3 is presumed to bind to TRs,which in turn recruit coactivators,leading to gene activation.The best-studied coactivators belong to the p 160 or SRC family.Members of this family include SRC 1/NCoA- 1,SRC2/TIF2/GRIP 1,and SRC3/pCIP/ACTR/AIB- 1/RAC-3/TRAM- 1.These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP.Here,we studied the expression patterns of these coactivators during various stages of development.Amongst the coactivators cloned in Xenopus laevis,SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis,and SRC2 and p300 are expressed throughout postembryonic development with little change in their expression levels.These results support the view that these coactivators participate in gene regulation by TR during metamorphosis.

  20. Tail regeneration in Xenopus laevis as a model for understanding tissue repair.

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    Tseng, A-S; Levin, M

    2008-09-01

    Augmentation of regenerative ability is a powerful strategy being pursued for the biomedical management of traumatic injury, cancer, and degeneration. While considerable attention has been focused on embryonic stem cells, it is clear that much remains to be learned about how somatic cells may be controlled in the adult organism. The tadpole of the frog Xenopus laevis is a powerful model system within which fundamental mechanisms of regeneration are being addressed. The tadpole tail contains spinal cord, muscle, vasculature, and other terminally differentiated cell types and can fully regenerate itself through tissue renewal--a process that is most relevant to mammalian healing. Recent insight into this process has uncovered fascinating molecular details of how a complex appendage senses injury and rapidly repairs the necessary morphology. Here, we review what is known about the chemical and bioelectric signals underlying this process and draw analogies to evolutionarily conserved pathways in other patterning systems. The understanding of this process is not only of fundamental interest for the evolutionary and cell biology of morphogenesis, but will also generate information that is crucial to the development of regenerative therapies for human tissues and organs.

  1. The polymorphic integumentary mucin B.1 from Xenopus laevis contains the short consensus repeat.

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    Probst, J C; Hauser, F; Joba, W; Hoffmann, W

    1992-03-25

    The frog integumentary mucin B.1 (FIM-B.1), discovered by molecular cloning, contains a cysteine-rich C-terminal domain which is homologous with von Willebrand factor. With the help of the polymerase chain reaction, we now characterize a contiguous region 5' to the von Willebrand factor domain containing the short consensus repeat typical of many proteins from the complement system. Multiple transcripts have been cloned, which originate from a single animal and differ by a variable number of tandem repeats (rep-33 sequences). These different transcripts probably originate solely from two genes and are generated presumably by alternative splicing of an huge array of functional cassettes. This model is supported by analysis of genomic FIM-B.1 sequences from Xenopus laevis. Here, rep-33 sequences are arranged in an interrupted array of individual units. Additionally, results of Southern analysis revealed genetic polymorphism between different animals which is predicted to be within the tandem repeats. A first investigation of the predicted mucins with the help of a specific antibody against a synthetic peptide determined the molecular mass of FIM-B.1 to greater than 200 kDa. Here again, genetic polymorphism between different animals is detected.

  2. A Tunable Silk Hydrogel Device for Studying Limb Regeneration in Adult Xenopus Laevis.

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    Anne Golding

    Full Text Available In certain amphibian models limb regeneration can be promoted or inhibited by the local wound bed environment. This research introduces a device that can be utilized as an experimental tool to characterize the conditions that promotes limb regeneration in the adult frog (Xenopus laevis model. In particular, this device was designed to manipulate the local wound environment via a hydrogel insert. Initial characterization of the hydrogel insert revealed that this interaction had a significant influence on mechanical forces to the animal, due to the contraction of the hydrogel. The material and mechanical properties of the hydrogel insert were a factor in the device design in relation to the comfort of the animal and the ability to effectively manipulate the amputation site. The tunable features of the hydrogel were important in determining the pro-regenerative effects in limb regeneration, which was measured by cartilage spike formation and quantified by micro-computed tomography. The hydrogel insert was a factor in the observed morphological outcomes following amputation. Future work will focus on characterizing and optimizing the device's observed capability to manipulate biological pathways that are essential for limb regeneration. However, the present work provides a framework for the role of a hydrogel in the device and a path forward for more systematic studies.

  3. Hepatic confinement of newly produced erythrocytes caused by low-temperature exposure in Xenopus laevis.

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    Maekawa, Shun; Iemura, Hitomi; Kuramochi, Yuko; Nogawa-Kosaka, Nami; Nishikawa, Hironori; Okui, Takehito; Aizawa, Youichi; Kato, Takashi

    2012-09-01

    Diminished erythrocyte count and erythropoiesis have been reported during hypothermia in some ectothermic animals. In this study, the African clawed frog, Xenopus laevis, was used to investigate the cause of hypothermia-induced anemia. We developed a new model of hypothermia at 5°C and monitored blood cell count and erythropoiesis on several days. Erythrocyte count declined by 30% on the first day following cold exposure (5°C) and mRNA expression of hemeoxygenase-1 was enhanced 10-fold; accumulation of iron as a result of heme degradation was observed in the liver. One day after low-temperature exposure, erythropoietin mRNA expression was elevated in the liver and lung compared with that at normal temperature (22°C) by qRT-PCR analysis. Examination of liver sections (i.e. the erythropoietic organ) showed an increase in o-dianisidine-positive erythrocytes in the hepatic sinusoid 5 days after the onset of low-temperature exposure compared with normal liver. Peripheral erythrocyte count remained low, indicating that newly produced erythrocytes did not migrate from the liver to the circulation during hypothermia. In conclusion, this study reveals hypothermic anemia as being associated with hepatic erythrocyte destruction; prolonged anemia during low-temperature exposure is concomitant with newly produced erythrocytes being confined to the liver and may lead to new insights into vertebrate hematopoiesis.

  4. Sequential Turnovers of Sex Chromosomes in African Clawed Frogs (Xenopus Suggest Some Genomic Regions Are Good at Sex Determination

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    Benjamin L. S. Furman

    2016-11-01

    Full Text Available Sexual differentiation is fundamentally important for reproduction, yet the genetic triggers of this developmental process can vary, even between closely related species. Recent studies have uncovered, for example, variation in the genetic triggers for sexual differentiation within and between species of African clawed frogs (genus Xenopus. Here, we extend these discoveries by demonstrating that yet another sex determination system exists in Xenopus, specifically in the species Xenopus borealis. This system evolved recently in an ancestor of X. borealis that had the same sex determination system as X. laevis, a system which itself is newly evolved. Strikingly, the genomic region carrying the sex determination factor in X. borealis is homologous to that of therian mammals, including humans. Our results offer insights into how the genetic underpinnings of conserved phenotypes evolve, and suggest an important role for cooption of genetic building blocks with conserved developmental roles.

  5. PEX11β induces peroxisomal gene expression and alters peroxisome number during early Xenopus laevis development

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    Damjanovski Sashko

    2011-04-01

    Full Text Available Abstract Background Peroxisomes are organelles whose roles in fatty acid metabolism and reactive oxygen species elimination have contributed much attention in understanding their origin and biogenesis. Many studies have shown that de novo peroxisome biogenesis is an important regulatory process, while yeast studies suggest that total peroxisome numbers are in part regulated by proteins such as Pex11, which can facilitate the division of existing peroxisomes. Although de novo biogenesis and divisions are likely important mechanisms, the regulation of peroxisome numbers during embryonic development is poorly understood. Peroxisome number and function are particularly crucial in oviparous animals such as frogs where large embryonic yolk and fatty acid stores must be quickly metabolized, and resulting reactive oxygen species eliminated. Here we elucidate the role of Pex11β in regulating peroxisomal gene expression and number in Xenopus laevis embryogenesis. Results Microinjecting haemagglutinin (HA tagged Pex11β in early embryos resulted in increased RNA levels for peroxisome related genes PMP70 and catalase at developmental stages 10 and 20, versus uninjected embryos. Catalase and PMP70 proteins were found in punctate structures at stage 20 in control embryos, whereas the injection of ectopic HA-Pex11β induced their earlier localization in punctate structures at stage 10. Furthermore, the peroxisomal marker GFP-SKL, which was found localized as peroxisome-like structures at stage 20, was similarly found at stage 10 when co-microinjected with HA-Pex11β. Conclusions Overexpressed Pex11β altered peroxisomal gene levels and induced the early formation of peroxisomes-like structures during development, both of which demonstrate that Pex11β may be a key regulator of peroxisome number in early Xenopus embryos.

  6. Negative effects of low dose atrazine exposure on the development of effective immunity to FV3 in Xenopus laevis.

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    Sifkarovski, Jason; Grayfer, Leon; De Jesús Andino, Francisco; Lawrence, B Paige; Robert, Jacques

    2014-11-01

    The recent dramatic increase of the prevalence and range of amphibian host species and populations infected by ranaviruses such as Frog Virus 3 (FV3) raises concerns about the efficacies of amphibian antiviral immunity. In this context, the potential negative effects of water contaminants such as the herbicide atrazine, at environmentally relevant levels, on host antiviral immunity remains unclear. Here we describe the use of the amphibian Xenopus laevis as an ecotoxicology platform to elucidate the consequences of exposure to ecologically relevant doses of atrazine on amphibian antiviral immunity. X. laevis were exposed at tadpole and adult stages as well as during metamorphosis to atrazine (range from 0.1 to 10.0 ppb) prior to infection with FV3. Quantitative analysis of gene expression revealed significant changes in the pro-inflammatory cytokine, TNF-α and the antiviral type I IFN gene in response to FV3 infection. This was most marked in tadpoles that were exposed to atrazine at doses as low 0.1 ppb. Furthermore, atrazine exposure significantly compromised tadpole survival following FV3 infections. In contrast, acute atrazine exposure of mature adult frogs did not induce detectable effects on anti-FV3 immunity, but adults that were exposed to atrazine during metamorphosis exhibited pronounced defects in FV3-induced TNF-α gene expression responses and slight diminution in type I IFN gene induction. Thus, even at low doses, atrazine exposure culminates in impaired development of amphibian antiviral defenses.

  7. Negative Effects of Low Dose Atrazine Exposure on the Development of Effective Immunity to FV3 in Xenopus laevis

    Science.gov (United States)

    Sifkarovski, Jason; Grayfer, Leon; De Jesús Andino, Francisco; Lawrence, B. Paige; Robert, Jacques

    2014-01-01

    The recent dramatic increase of the prevalence and range of amphibian host species and populations infected by ranaviruses such as Frog Virus 3 (FV3) raises concerns about the efficacies of amphibian antiviral immunity. In this context, the potential negative effects of water contaminants such as the herbicide atrazine, at environmentally relevant levels, on host antiviral immunity remains unclear. Here we describe the use of the amphibian Xenopus laevis as an ecotoxiciology platform to elucidate the consequences of exposure to ecologically relevant doses of atrazine on amphibian antiviral immunity. X. laevis were exposed at tadpole and adult stages as well as during metamorphosis to atrazine (range from 0.1 to 10.0 ppb) prior to infection with FV3. Quantitative analysis of gene expression revealed significant changes in the pro-inflammatory cytokine, TNF-α and the antiviral type I IFN gene in response to FV3 infection. This was most marked in tadpoles that were exposed to atrazine at doses as low 0.1 ppb. Furthermore, atrazine exposure significantly compromised tadpole survival following FV3 infections. In contrast, acute atrazine exposure of mature adult frogs did not induce detectable effects on anti-FV3 immunity, but adults that were exposed to atrazine during metamorphosis exhibited pronounced defects in FV3-induced TNF-α gene expression responses and slight diminution in type I IFN gene induction. Thus, even at low doses, atrazine exposure culminates in impaired development of amphibian antiviral defenses. PMID:24984115

  8. A New Nomenclature of Xenopus laevis Chromosomes Based on the Phylogenetic Relationship to Silurana/Xenopus tropicalis.

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    Matsuda, Yoichi; Uno, Yoshinobu; Kondo, Mariko; Gilchrist, Michael J; Zorn, Aaron M; Rokhsar, Daniel S; Schmid, Michael; Taira, Masanori

    2015-01-01

    Xenopus laevis (XLA) is an allotetraploid species which appears to have undergone whole-genome duplication after the interspecific hybridization of 2 diploid species closely related to Silurana/Xenopus tropicalis (XTR). Previous cDNA fluorescence in situ hybridization (FISH) experiments have identified 9 sets of homoeologous chromosomes in X. laevis, in which 8 sets correspond to chromosomes 1-8 of X. tropicalis (XTR1-XTR8), and the last set corresponds to a fusion of XTR9 and XTR10. In addition, recent X. laevis genome sequencing and BAC-FISH experiments support this physiological relationship and show no gross chromosome translocation in the X. laevis karyotype. Therefore, for the benefit of both comparative cytogenetics and genome research, we here propose a new chromosome nomenclature for X. laevis based on the phylogenetic relationship and chromosome length, i.e. XLA1L, XLA1S, XLA2L, XLA2S, and so on, in which the numbering of XLA chromosomes corresponds to that in X. tropicalis and the postfixes 'L' and 'S' stand for 'long' and 'short' chromosomes in the homoeologous pairs, which can be distinguished cytologically by their relative size. The last chromosome set is named XLA9L and XLA9S, in which XLA9 corresponds to both XTR9 and XTR10, and hence, to emphasize the phylogenetic relationship to X. tropicalis, XLA9_10L and XLA9_10S are also used as synonyms.

  9. Molecular Cloning of phd1 and Comparative Analysis of phd1, 2, and 3 Expression in Xenopus laevis

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    Dandan Han

    2012-01-01

    Full Text Available Intensive gene targeting studies in mice have revealed that prolyl hydroxylase domain proteins (PHDs play important roles in murine embryonic development; however, the expression patterns and function of these genes during embryogenesis of other vertebrates remain largely unknown. Here we report the molecular cloning of phd1 and systematic analysis of phd1, phd2, and phd3 expression in embryos as well as adult tissues of Xenopus laevis. All three phds are maternally provided during Xenopus early development. The spatial expression patterns of phds genes in Xenopus embryos appear to define a distinct synexpression group. Frog phd2 and phd3 showed complementary expression in adult tissues with phd2 transcription levels being high in the eye, brain, and intestine, but low in the liver, pancreas, and kidney. On the contrary, expression levels of phd3 are high in the liver, pancreas, and kidney, but low in the eye, brain, and intestine. All three phds are highly expressed in testes, ovary, gall bladder, and spleen. Among three phds, phd3 showed strongest expression in heart.

  10. Neural transduction in Xenopus laevis lateral line system.

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    Strelioff, D; Honrubia, V

    1978-03-01

    1. The process of neural excitation in hair cell systems was studied in an in vitro preparation of the Xenopus laevis (African clawed toad) lateral line organ. A specially designed stimulus chamber was used to apply accurately controlled pressure, water movement, or electrical stimuli, and to record the neural responses of the two afferent fibers innervating each organ or stitch. The objective of the study was to determine the characteristics of the neural responses to these stimuli, and thus gain insight into the transduction process. 2. A sustained deflection of the hair cell cilia due to a constant flow of water past the capula resulted in a maintained change in the mean firing rate (MFR) of the afferent fibers. The data also demonstrated that the neural response was proportional to the velocity of the water flow and indicated that both deflection and movement of the cilia were the effective physiological stimuli for this hair cell system. 3. The preparations responded to sinusoidal water movements (past the capula) over the entire frequency range of the stimulus chamber, 0.1-130 Hz, and were most sensitive between 10 and 40 Hz. The variation of the MFR and the percent modulation indicated that the average dynamic range of each organ was 23.5 dB. 4. The thresholds, if any, for sustained pressure changes and for sinusoidal pressure variations in the absence of water movements were very high. Due to the limitations of the stimulus chamber it was not possible to generate pressure stimuli of sufficient magnitude to elicit a neural response without also generating suprathreshold water-movement stimuli. Sustained pressures had no detectable effect on the neural response to water-movement stimuli. 5. The preparations were very sensitive to electrical potentials applied across the toad skin on which the hair cells were located. Potentials which made the ciliated surfaces of the hair cells positive with respect to their bases increased the MFR of the fibers, whereas

  11. Regulation of cyclin E stability in Xenopus laevis embryos

    Science.gov (United States)

    Brandt-(Webb), Yekaterina

    Cyclin-Cdk complexes positively regulate cell cycle progression. Cyclins are regulatory subunits that bind to and activate cyclin-dependent kinases or Cdks. Cyclin E associates with Cdk2 to mediate G1/S phase transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how this protein is downregulated during development in order to determine if these mechanisms are disrupted during tumorigenesis, leading to its overexpression. Using Xenopus laevis embryos as a model, we have shown previously that during the first 12 embryonic cell cycles Cyclin E levels remain constant yet Cdk2 activity oscillates twice per cell cycle. Cyclin E is abruptly destabilized by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT). Based on work our work and work by others, we have hypothesized that differential phosphorylation and a change in localization result in Cyclin E degradation by the 26S proteasome at the MBT. To test this, we generated a series of point mutations in conserved threonine/serine residues implicated in degradation of human Cyclin E. Using Western blot analysis, we show that similarly to human Cyclin E, mutation of these residues to unphosphorylatable alanine stabilizes Cyclin E past the MBT when they are expressed in vivo. Cyclin E localization was studied by immunofluorescence analysis of endogenous and exogenous protein in pre-MBT, MBT, and post-MBT embryos. In addition, we developed a novel method of conjugating recombinant His6-tagged Cyclin E to fluorescent (CdSe)ZnS nanoparticles (quantum dots) capped with dihydrolipoic acid. Confocal microscopy was used to visualize His6Cyclin E-quantum dot complexes inside embryo cells in real time. We found that re-localization at the MBT from the cytoplasm to the nucleus

  12. Translational control of Connexin 30 and 41 m RNAs in Xenopus laevis embryos

    NARCIS (Netherlands)

    Meijer, Hedda Arlinde

    2001-01-01

    During the early stages of Xenopus laevis development no transcription occurs. Many cell divisions take place in about 9 h, a process orchestrated by maternal mRNAs. When the embryo contains about thousand cells, zygotic transcription initiates during the so-called mid blastula transition or MBT. Be

  13. Expression and physiological regulation of BDNF receptors in the neuroendocrine melanotrope cell of Xenopus laevis

    NARCIS (Netherlands)

    Kidane, A.H.; Dooren, S.H. van; Roubos, E.W.; Jenks, B.G.

    2007-01-01

    Brain-derived neurotrophic factor (BDNF) and alpha-melanophore-stimulating hormone (alpha-MSH) are co-sequestered in secretory granules in melanotrope cells of the pituitary pars intermedia of the amphibian Xenopus laevis. alpha-MSH is responsible for pigment dispersion in dermal melanophores during

  14. Twitch and tetanic tension during culture of mature xenopus laevis single muscle fibres

    NARCIS (Netherlands)

    Jaspers, R.T.; Feenstra, H.M.; Lee-de Groot, M.B.E.; Huijing, P.A.; Laarse, W.J.

    2001-01-01

    Investigation of the mechanisms of muscle adaptation requires independent control of the regulating factors. The aim of the present study was to develop a serum-free medium to culture mature single muscle fibres of Xenopus laevis. As an example, we used the culture system to study adaptation of twit

  15. Comparative effects of DDT, allethrin, dieldrin and aldrin-transdiol on sense organs of Xenopus laevis

    NARCIS (Netherlands)

    Akkermans, L.M.A.; Bercken, J. van den; Versluijs-Helder, M.

    1975-01-01

    The effects of DDT, allethrin, dieldrin and aldrin-transdiol were studied in two different sense organs of Xenopus laevis; the lateral-line organ and the cutaneous touch receptors. DDT and allethrin produced pronounced repetitive firing in both preparations. Dieldrin and aldrin-transdiol, on the oth

  16. Comparative effects of DDT, allethrin, dieldrin and aldrin-transdiol on sense organs of Xenopus laevis

    NARCIS (Netherlands)

    Akkermans, L.M.A.; Bercken, J. van den; Versluijs-Helder, M.

    1975-01-01

    The effects of DDT, allethrin, dieldrin and aldrin-transdiol were studied in two different sense organs of Xenopus laevis; the lateral-line organ and the cutaneous touch receptors. DDT and allethrin produced pronounced repetitive firing in both preparations. Dieldrin and aldrin-transdiol, on the

  17. Atrazine and malathion shorten the maturation process of Xenopus laevis oocytes and have an adverse effect on early embryo development.

    Science.gov (United States)

    Ji, Qichao; Lee, Jessica; Lin, Yu-Huey; Jing, Guihua; Tsai, L Jillianne; Chen, Andrew; Hetrick, Lindsay; Jocoy, Dylan; Liu, Junjun

    2016-04-01

    The use of pesticides has a negative impact on the environment. Amphibians have long been regarded as indicator species to pollutants due to their permeable skin and sensitivity to the environment. Studies have shown that population declines of some amphibians are directly linked with exposure to agricultural contaminants. In the past, much of the studies have focused on the toxic effect of contaminants on larvae (tadpoles), juvenile and adult frogs. However, due to the nature of their life cycle, amphibian eggs and early embryos are especially susceptible to the contaminants, and any alteration during the early reproductive stages may have a profound effect on the health and population of amphibians. In this study, we analyzed the effect of atrazine and malathion, two commonly used pesticides, on Xenopus laevis oocyte maturation and early embryogenesis. We found that both atrazine and malathion shortened the frog oocyte maturation process and resulted in reduced Emi2 levels at cytostatic factor-mediated metaphase arrest, and a high level of Emi2 is critically important for oocyte maturation. Furthermore, frog embryos fertilized under the influence of atrazine and/or malathion displayed a higher rate of abnormal division that eventually led to embryo death during early embryogenesis.

  18. The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.

    Science.gov (United States)

    Miyamoto, Kei; Suzuki, Ken-Ichi T; Suzuki, Miyuki; Sakane, Yuto; Sakuma, Tetsushi; Herberg, Sarah; Simeone, Angela; Simpson, David; Jullien, Jerome; Yamamoto, Takashi; Gurdon, J B

    2015-01-01

    Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.

  19. The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.

    Directory of Open Access Journals (Sweden)

    Kei Miyamoto

    Full Text Available Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0 needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN mRNA to oocytes at the germinal vesicle (GV stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.

  20. Optimized transgenesis in Xenopus laevis/gilli isogenetic clones for immunological studies.

    Science.gov (United States)

    Nedelkovska, Hristina; Robert, Jacques

    2012-03-01

    Xenopus laevis provides a unique animal model, alternative to mouse, to study immunology. Even though, several methodologies have been developed for the generation of transgenic Xenopus, to date none have been adapted for the X. laevis/gilli (LG) isogenetic clones that are essential for immunological studies. Since LG clones are generated via gynogenesis, transgenic methods using transgene integration into the sperm nuclei are not suited. Therefore, we have tested three alternative methods for LG transgenesis: the phiC31 integrase, the Sleeping Beauty transposase, and the I-SceI meganuclease. All three techniques produced transgenic LG clones; however, the I-SceI meganuclease was most effective. It resulted in high transgenesis efficiency (35-50%), bright nonmosaic GFP expression as well as stable germline transmission with 100% of the progeny carrying the transgene. Production of transgenic LG clones will allow us to modulate immune gene expression and further strengthen X. laevis as a biomedical model.

  1. Bioconcentration kinetics of PCBs in various parts of the lifecycle of the tadpoles Xenopus laevis

    Institute of Scientific and Technical Information of China (English)

    ZHAO Rong-biao; SUN Da-yu; FU Shan; WANG Xiao-fei; ZHAO Ru-song

    2007-01-01

    Polychlorinated biphenyls(PCBs)in Xenopus laevis have been reported only for a few congeners.Additionally,there is very little information on the abilitv of Xenopus laevis to bioconcentrate PCBs.To address these issues,the tadpole Xenopus laevis was exposed to Aroclorl254 mixtures in water at room temperature for 110 d followed by an additional 110 d of nonspiked PCBs in the water for the control group.During the whole process,bioconcentration factors(BCFs)of PCBs ranged from 1180 to 15670.For most PCB congeners.the highest and lowest bioconcentrations of the kinetic curves were found to be remarkably simultaneous,respectively.All 141 PCB congeners under the same experimental conditions had no linear correlation on the lgBCF versus lgKow relationship.The relationship between lgBCFs and lgKow followed a parabolic pattern indicative of selective bioconcentration,suggesting that the kinetic curves of the PCB congeners observed in the lifecycle of the tadpoles may be concentrated due to the amphibian special species and internal metabolism.In contrast,IgBCFs for PCBs were inversely related to lgKow,suggesting that a metabolism of the higher Kow'PCB congeners occurred.These results support the author'S conclusion that the tadpole Xenopus laevis plays major roles in the bioconcentration of PCB congeners,and demonstrated that the exposure kinetic curves of PCB congeners are complex.Besides the amphibian metamorphous development,the lifecycle of the tadpole Xenopus laevis also may be of importance in determining the bioconcentration of PCB congeners.

  2. Extracellular quaternary ammonium blockade of transient receptor potential vanilloid subtype 1 channels expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Rivera-Acevedo, Ricardo E; Pless, Stephan Alexander; Schwarz, Stephan K W;

    2012-01-01

    expressed in Xenopus laevis oocytes, whereas the neutral local anesthetic, benzocaine, does not, suggesting that a titratable amine is required for high-affinity inhibition. Consistent with this possibility, extracellular tetraethylammonium (TEA) and tetramethylammonium application produces potent, voltage...

  3. Identification and comparative analyses of Siamois cluster genes in Xenopus laevis and tropicalis.

    Science.gov (United States)

    Haramoto, Yoshikazu; Saijyo, Tomohito; Tanaka, Toshiaki; Furuno, Nobuaki; Suzuki, Atsushi; Ito, Yuzuru; Kondo, Mariko; Taira, Masanori; Takahashi, Shuji

    2017-06-15

    Two siamois-related homeobox genes siamois (sia1) and twin (sia2), have been reported in Xenopus laevis. These genes are expressed in the blastula chordin- and noggin-expressing (BCNE) center and the Nieuwkoop center, and have complete secondary axis-inducing activity when over-expressed on the ventral side of the embryo. Using whole genome sequences of X. tropicalis and X. laevis, we identified two additional siamois-related genes, which are tandemly duplicated near sia1 and sia2 to form the siamois gene cluster. Four siamois genes in X. tropicalis are transcribed at blastula to gastrula stages. In X. laevis, the siamois gene cluster is present on both homeologous chromosomes, XLA3L and XLA3S. Transcripts from seven siamois genes (three on XLA3L and four on XLA3S) in X. laevis were detected at blastula to gastrula stages. A transcribed gene, sia1p. S, encodes an inactive protein without a homeodomain. When over-expressed ventrally, all siamois-related genes tested in this study except for sia1p. S induced a complete secondary axis, indicating that X. tropicalis and X. laevis have four and six active siamois-related genes, respectively. Of note, each gene required different amounts of mRNA for full activity. These results suggest the possibility that siamois cluster genes have functional redundancy to endow robustness and quickness to organizer formation in Xenopus species. Copyright © 2017. Published by Elsevier Inc.

  4. Environmentally relevant concentrations of ammonium perchlorate inhibit thyroid function and alter sex ratios in developing Xenopus laevis.

    Science.gov (United States)

    Goleman, Wanda L; Carr, James A; Anderson, Todd A

    2002-03-01

    Embryos and larvae of the South African frog Xenopus laevis were exposed to ammonium perchlorate (AP) or control medium for 70 d. The dosage levels (59 ppb, 14,140 ppb) bracketed a range of perchlorate concentrations measured in surface waters at the Longhorn Army Ammunition Plant (LHAAP) in Karnack, Texas, USA. The experiment also included a 28-d nontreatment recovery period to assess the reversibility of AP effects. There were no significant effects of AP on mortality or hatching success. There were no effects of AP on developmental abnormalities such as bent/asymmetric tails or edema. Ammonium perchlorate inhibited forelimb emergence, the percentage of animals completing tail resorption, and hindlimb development during the 70-d exposure period. Only the upper AP concentration reduced whole-body thyroxine content, whereas both concentrations caused significant hypertrophy of the thyroid follicular epithelium. Both concentrations of AP caused a skewed sex ratio, significantly reducing the percentage of males at metamorphosis. The effects of AP on metamorphosis and thyroid function were reversed during the 28-d nontreatment recovery period. We conclude that AP inhibits thyroid activity and alters gonadal differentiation in developing X. laevis. These effects were observed at concentrations at or below concentrations reported in surface waters contaminated with ammonium perchlorate, suggesting that this contaminant may pose a threat to normal development and growth in natural amphibian populations.

  5. Immune Defenses against Batrachochytrium dendrobatidis, a Fungus Linked to Global Amphibian Declines, in the South African Clawed Frog, Xenopus laevis▿

    Science.gov (United States)

    Ramsey, Jeremy P.; Reinert, Laura K.; Harper, Laura K.; Woodhams, Douglas C.; Rollins-Smith, Louise A.

    2010-01-01

    Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections. PMID:20584973

  6. Effects of GSM-like radiofrequency irradiation during the oogenesis and spermiogenesis of Xenopus laevis.

    Science.gov (United States)

    Boga, Ayper; Emre, Mustafa; Sertdemir, Yasar; Uncu, İbrahim; Binokay, Secil; Demirhan, Osman

    2016-07-01

    We aimed to evaluate the effect of GSM-like radiofrequency electromagnetic radiation (RF-EMR) on the oogenesis, and spermiogenesis of Xenopus laevis, and so the development of the embryos obtained from Normal Females+Normal Males (i.e. "N(F)+N(M)"); Normal Females+RF-exposed Males (i.e. "N(F)+RF(M)"); RF-exposed Female+Normal Male (i.e. "RF(F)+N(M)"); and RF-exposed Female+RF-exposed Male (i.e. "RF(F)+RF(M)". Various, assessments were performed to determine potential teratogenic effects and mortality, body growth and behavior on first generation embryos. After exposing adults frogs of both sexes to 900MHz RF-EMR (at 1.0W/kg) for 8h a day over a 5-week period, the embryos' specific energy absorption rate (SAR) was calculated. In our present study (control group; 2.2% abnormal, 0.0% dead); with the N(F)+RF(M) combination, the long-term exposure of adult males to GSM-like radiation at 900MHz (RF: 2W) for 5 week/8h/day resulted in normal, abnormal and dead embryo ratios of 88.3%, 3.3% and 8.3%, respectively (p<0.001). In the RF(F)+N(M) combination, long-term exposure (5 week/8h/day) of adult females led to normal, abnormal and dead embryo ratios of 76.7%, 11.7%, and 11.7%, respectively (p<0.001). And in the RF(F)+RF(M) combination, long-term exposure (5 week/8h/day) of both adult males and females led to normal, abnormal and dead embryo ratios of 73.3%, 11.7%, and 15%, respectively (p<0.001). With the exception RF(F)+RF(M) group (p<0.001), no significant changes were observed on body growth (lengths) in comparison to the control group. It was also observed that the offspring of female adult Xenopus exposed to RF-EMR during oogenesis exhibited a more aggressive behavior compared to the control group. Cell phones radiation can thus lead to detrimental effects in humans' male and female reproductive cells.

  7. Xenopus laevis neuronal cell adhesion molecule (nrcam): plasticity of a CAM in the developing nervous system.

    Science.gov (United States)

    Lokapally, Ashwin; Metikala, Sanjeeva; Hollemann, Thomas

    2017-01-01

    Neuron-glial-related cell adhesion molecule (NRCAM) is a neuronal cell adhesion molecule of the L1 immunoglobulin superfamily, which plays diverse roles during nervous system development including axon growth and guidance, synapse formation, and formation of the myelinated nerve. Perturbations in NRCAM function cause a wide variety of disorders, which can affect wiring and targeting of neurons, or cause psychiatric disorders as well as cancers through abnormal modulation of signaling events. In the present study, we characterize the Xenopus laevis homolog of nrcam. Expression of Xenopus nrcam is most abundant along the dorsal midline throughout the developing brain and in the outer nuclear layer of the retina.

  8. Generation of transgenic frogs.

    Science.gov (United States)

    Loeber, Jana; Pan, Fong Cheng; Pieler, Tomas

    2009-01-01

    The possibility of generating transgenic animals is of obvious advantage for the analysis of gene function in development and disease. One of the established vertebrate model systems in developmental biology is the amphibian Xenopus laevis. Different techniques have been successfully applied to create Xenopus transgenics; in this chapter, the so-called meganuclease method is described. This technique is not only technically simple, but also comparably efficient and applicable to both Xenopus laevis and Xenopus tropicalis. The commercially available endonuclease I-SceI (meganuclease) mediates the integration of foreign DNA into the frog genome after coinjection into fertilized eggs. Tissue-specific gene expression, as well as germline transmission, has been observed.

  9. Ni2+ treatment causes cement gland formation in ectoderm explants of Xenopus laevis embryo

    Institute of Scientific and Technical Information of China (English)

    HUANGYONG; XIAOYANDING

    1999-01-01

    We found T-type calcium channel blocker Ni2+ can efficiently induce the formation of cement gland in Xenopus laevis animal cap explants.Nother T-typer specific calcium channel blocker Amiloride can also induce the formation of cement gland,while L-type specific calcium channel blocker Nifedipine as no inductive effect.These results may offer us an new approach to study the differentiation of cement gland through the change of intracelluar calcium concentration.

  10. Specific degradation of keratin in Xenopus laevis egg extracts undergoing apoptosis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cytochrome c activates apoptosis specific protease XCPP32 when being added to Xenopus laevis egg extracts, and induces apoptosis in this cell-free system. During apoptosis, cyto-skeleton proteins in egg extracts are degraded. Western blot assay indicates that 42-ku acidic keratin in egg extracts has been degraded by XCPP32. The degradation of 42-ku keratin may be crucial in apoptosis.

  11. The effect of 900 and 1800 MHz GSM-like radiofrequency irradiation and nicotine sulfate administration on the embryonic development of Xenopus laevis.

    Science.gov (United States)

    Boga, Ayper; Emre, Mustafa; Sertdemir, Yasar; Akillioglu, Kubra; Binokay, Secil; Demirhan, Osman

    2015-03-01

    The aim of this study was to investigate the effects of GSM-like radiofrequency electromagnetic radiation (RF EMR) and nicotine sulfate (NS) exposure on Xenopus embryonic development.The developmental effects of GSM-like RF-EMR (900-1800 MHz, at a SAR value of 1W/kg and NS on Xenopus laevis embryos were investigated). Following the application of radiofrequency radiation and/or NS administration, the embryos were closely examined in order to determine their possible teratogenic effects. Xenopus frogs obtained from the Department of Physiology of the Cukurova University, in accordance described by the Standard Guide of the American Society for Testing and Materials (ASTM). Following the exposure of Xenopus embryos to RF-EMR at 900 and 1800 MHz (1.0W/kg) for 4, 6 and 8h; the whole body specific energy absorption rate (SAR) of the embryos was calculated. With the exception of irradiation at 1800 MHz no dramatic developmental anomalies were observed in the Xenopus embryos in association with RF-EMR applications. Combined RF-EMR and NS applications resulted in dramatic abnormalities and death among the Xenopus embryos. The study results indicated that GSM-like RF-EMR (e.g. radiation from cell phones) was not as harmful to Xenopus embryos as might have been expected. However, the combined effects of GSM-like RF-EMR and NS on Xenopus embryos were more severe than the effect of RF-EMR or NS alone. In conclusion, the study results appear to suggest that the combined use of nicotine and cell phones might result in more pronounced detrimental effects on the health of smokers.

  12. Evaluation of the toxicity and teratogenity of six commercial textile dyes using the frog embryo teratogenesis assay-Xenopus.

    Science.gov (United States)

    Birhanli, Ayse; Ozmen, Murat

    2005-01-01

    Potential developmental toxicities of six different textile dyes were evaluated using the frog embryo teratogenesis assay-Xenopus (FETAX). Xenopus laevis embryos were exposed to astrazon red FBL, astrazon blue FGRL, remazol red RR, remazol turquoise blue G-A, cibacron red FN-3G, and cibacron blue FN-R from stage 8 to 11 for a 96-h exposure period in static renewal test conditions. A minimum of 17 concentration-response tests were performed with tested dyes, excluding a control group for each dye. Median lethal concentration (LC50), malformation (EC50), non observed adverse effect concentration (NOAEC), and lowest observed adverse effect concentration (LOAEC) were calculated. Also, teratogenic index (TI), minimum concentration to inhibit growth (MCIG), and MCIG/LC50 values were determined for each of the tested dyes. Characteristic abnormalities induced by a given test material were determined by the relationship between concentration and dye in the study. Results from these studies suggested that each tested dye is teratogenic for X. laevis embryos. The lowest LC50 was determined for astrazon red exposure corresponding to a value of 4.73 mg/L. The LC50 value was similar for this dye and astrazon blue; the highest TI was calculated for astrazon blue exposure. Tests with X. laevis indicated that each of the tested compounds possessed teratogenic potential with varying degrees of potency: astrazon blue FGRL > remazol turquoise blue G-A > astrazon red FBL > cibacron blue FN-R > cibacron red FN-3G > remazol red RR. Different types of malformations occurred in the embryos, depending on concentration and dye. From these results, we can suggest that astrazon blue is the most toxic compound, but that the others are also highly toxic and teratogenic substances for X. laevis embryos. Results of the study confirmed that the FETAX assay can be useful in an integrated biological hazard assesment for the preliminary screening of textile dye stuff.

  13. Egg jelly proteins stimulate directed motility in Xenopus laevis sperm.

    Science.gov (United States)

    Burnett, Lindsey A; Sugiyama, Hitoshi; Bieber, Allan L; Chandler, Douglas E

    2011-06-01

    Previously we have shown that extracts from Xenopus egg jelly (egg water) increase the passage of sperm through a porous membrane in a dose-dependent manner. Although this assay has shown that sperm accumulation occurs only in the presence of an egg water gradient, it has not revealed the dynamic features of how Xenopus sperm swim in such gradients. Here, we use video microscopic observations to trace sperm trajectories in a Zigmond chamber. Our results show that Xenopus sperm swim in linear and gently curving paths and only infrequently perform turns. In the presence of an egg water gradient, however, the percent of sperm swimming up the gradient axis and the net distance traveled by each sperm along this axis was increased significantly. There was no change in curvilinear velocity. Rather, the orientation of sperm travel was shifted to more closely match that of the gradient axis. In addition, using a porous filter assay, we demonstrate that the egg water protein allurin, in both purified and recombinant forms, stimulates directed motility of sperm. Finally, we use Oregon Green 488-conjugated allurin to show that this protein binds primarily to the sperm midpiece; binding of allurin to the entire head was observed in a minor subpopulation of sperm. Dose dependence of allurin binding occurred over the 0-1 µg/ml range and correlated well with previously published dose-dependent sperm attraction data. Binding was rapid with a half-time of about 10 sec. These data suggest that egg water proteins bind to sperm and modify sperm-orienting behavior.

  14. The roles of Bcl-xL in modulating apoptosis during development of Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Calderon-Segura Maria

    2005-09-01

    Full Text Available Abstract Background Apoptosis is a common and essential aspect of development. It is particularly prevalent in the central nervous system and during remodelling processes such as formation of the digits and in amphibian metamorphosis. Apoptosis, which is dependent upon a balance between pro- and anti-apoptotic factors, also enables the embryo to rid itself of cells damaged by gamma irradiation. In this study, the roles of the anti-apoptotic factor Bcl-xL in protecting cells from apoptosis were examined in Xenopus laevis embryos using transgenesis to overexpress the XR11 gene, which encodes Bcl-xL. The effects on developmental, thyroid hormone-induced and γ-radiation-induced apoptosis in embryos were examined in these transgenic animals. Results Apoptosis was abrogated in XR11 transgenic embryos. However, the transgene did not prevent the apoptotic response of tadpoles to thyroid hormone during metamorphosis. Post-metamorphic XR11 frogs were reared to sexual maturity, thus allowing us to produce second-generation embryos and enabling us to distinguish between the maternal and zygotic contributions of Bcl-xL to the γ-radiation apoptotic response. Wild-type embryos irradiated before the mid-blastula transition (MBT underwent normal cell division until reaching the MBT, after which they underwent massive, catastrophic apoptosis. Over-expression of Bcl-xL derived from XR11 females, but not males, provided partial protection from apoptosis. Maternal expression of XR11 was also sufficient to abrogate apoptosis triggered by post-MBT γ-radiation. Tolerance to post-MBT γ-radiation from zygotically-derived XR11 was acquired gradually after the MBT in spite of abundant XR11 protein synthesis. Conclusion Our data suggest that Bcl-xL is an effective counterbalance to proapoptotic factors during embryonic development but has no apparent effect on the thyroid hormone-induced apoptosis that occurs during metamorphosis. Furthermore, post-MBT apoptosis

  15. Calcium signalling during neural induction in Xenopus laevis embryos.

    Science.gov (United States)

    Moreau, Marc; Néant, Isabelle; Webb, Sarah E; Miller, Andrew L; Leclerc, Catherine

    2008-04-12

    In Xenopus, experiments performed with isolated ectoderm suggest that neural determination is a 'by default' mechanism, which occurs when bone morphogenetic proteins (BMPs) are antagonized by extracellular antagonists, BMP being responsible for the determination of epidermis. However, Ca(2+) imaging of intact Xenopus embryos reveals patterns of Ca(2+) transients which are generated via the activation of dihydropyridine-sensitive Ca(2+) channels in the dorsal ectoderm but not in the ventral ectoderm. These increases in the concentration of intracellular Ca(2+)([Ca(2+)]i) appear to be necessary and sufficient to orient the ectodermal cells towards a neural fate as increasing the [Ca(2+)]i artificially results in neuralization of the ectoderm. We constructed a subtractive cDNA library between untreated and caffeine-treated ectoderms (to increase [Ca(2+)]i) and then identified early Ca(2+)-sensitive target genes expressed in the neural territories. One of these genes, an arginine methyltransferase, controls the expression of the early proneural gene, Zic3. Here, we discuss the evidence for the existence of an alternative model to the 'by default' mechanism, where Ca(2+) plays a central regulatory role in the expression of Zic3, an early proneural gene, and in epidermal determination which only occurs when the Ca(2+)-dependent signalling pathways are inactive.

  16. Pattern and morphogenesis of presumptive superficial mesoderm in two closely related species, Xenopus laevis and Xenopus tropicalis.

    Science.gov (United States)

    Shook, David R; Majer, Christina; Keller, Ray

    2004-06-01

    The mesoderm, comprising the tissues that come to lie entirely in the deep layer, originates in both the superficial epithelial and the deep mesenchymal layers of the early amphibian embryo. Here, we characterize the mechanisms by which the superficial component of the presumptive mesoderm ingresses into the underlying deep mesenchymal layer in Xenopus tropicalis and extend our previous findings for Xenopus laevis. Fate mapping the superficial epithelium of pregastrula stage embryos demonstrates ingression of surface cells into both paraxial and axial mesoderm (including hypochord), in similar patterns and amounts in both species. Superficial presumptive notochord lies medially, flanked by presumptive hypochord and both overlie the deep region of the presumptive notochord. These tissues are flanked laterally by superficial presumptive somitic mesoderm, the anterior tip of which also appears to overlay the presumptive deep notochord. Time-lapse recordings show that presumptive somitic and notochordal cells move out of the roof of the gastrocoel and into the deep region during neurulation, whereas hypochordal cells ingress after neurulation. Scanning electron microscopy at the stage and position where ingression occurs suggests that superficial presumptive somitic cells in X. laevis ingress into the deep region as bottle cells whereas those in X. tropicalis ingress by "relamination" (e.g., [Dev. Biol. 174 (1996) 92]). In both species, the superficially derived presumptive somitic cells come to lie in the medial region of the presumptive somites during neurulation. By the early tailbud stages, these cells lie at the horizontal myoseptum of the somites. The morphogenic pathway of these cells strongly resembles that of the primary slow muscle pioneer cells of the zebrafish. We present a revised fate map of Xenopus, and we discuss the conservation of superficial mesoderm within amphibians and across the chordates and its implications for the role of this tissue in

  17. Survival fraction and phenotype alterations of Xenopus laevis embryos at 3 Gy, 150 kV X-ray irradiation.

    Science.gov (United States)

    Carotenuto, Rosa; Tussellino, Margherita; Mettivier, Giovanni; Russo, Paolo

    2016-11-25

    To determine the radiosensitivity of Xenopus laevis embryos, aquatic organism model, for toxicity studies utilizing X-rays at acute high dose levels, by analysing its survival fraction and phenotype alterations under one-exposure integral dose. We used the standard Frog Embryo Teratogenesis Assay Xenopus test during the early stages of X. laevis development. The embryos were harvested until st. 46 when they were irradiated. The radiation effects were checked daily for a week and the survival, malformations and growth inhibition were assessed. Sibling tadpoles as control organisms were used. Statistical analysis was performed to assess the extent of any damage. Irradiation was performed with an X-ray tube operated at 150 kV. The tube containing the tadpoles was exposed to an air kerma of 3 Gy as measured in air with an in-beam ionization chamber. After one week, survival fraction of irradiated embryos was 58%, while for control embryos it was 81%. Hence, irradiation with 150 kV, 3 Gy X-rays produced a 23% decrease of survival in regard to unirradiated embryos. The 70% of the irradiated embryos showed an altered distribution of the skin pigmentation, in particular on the dorsal area and in the olfactory pits, where the pigment concentration increased by a factor 2. In conclusion exposure of X. laevis to 3 Gy, 150 kV X-rays induced a reduction of embryos survival and a significant modification of pigmentation. The authors think that X. laevis embryos, at st 46, is a suitable biological model for large scale investigations on the effects of ionizing radiation.

  18. COMPARATIVE TOXICITY OF DIURON ON SURVIVAL AND GROWTH OF PACIFIC TREEFROG, BULLFROG, RED-LEGGED FROG, AND AFRICAN CLAWED FROG EMBRYOS AND TADPOLES

    Science.gov (United States)

    The effects of the herbicide diuron on survival and growth of Pacific treefrog (Pseudacris regilla),bullfrog(Rana catesbeiana), red-legged frog(Rana aurora),and African clawed frog(Xenopus laevis)embryos and tadpoles were determined in static-renewal tests. P.regilla and X.laevis...

  19. Combining different mRNA capture methods to analyze the transcriptome: analysis of the Xenopus laevis transcriptome.

    Directory of Open Access Journals (Sweden)

    Michael D Blower

    Full Text Available mRNA sequencing (mRNA-seq is a commonly used technique to survey gene expression from organisms with fully sequenced genomes. Successful mRNA-seq requires purification of mRNA away from the much more abundant ribosomal RNA, which is typically accomplished by oligo-dT selection. However, mRNAs with short poly-A tails are captured poorly by oligo-dT based methods. We demonstrate that combining mRNA capture via oligo-dT with mRNA capture by the 5' 7-methyl guanosine cap provides a more complete view of the transcriptome and can be used to assay changes in mRNA poly-A tail length on a genome-wide scale. We also show that using mRNA-seq reads from both capture methods as input for de novo assemblers provides a more complete reconstruction of the transcriptome than either method used alone. We apply these methods of mRNA capture and de novo assembly to the transcriptome of Xenopus laevis, a well-studied frog that currently lacks a finished sequenced genome, to discover transcript sequences for thousands of mRNAs that are currently absent from public databases. The methods we describe here will be broadly applicable to many organisms and will provide insight into the transcriptomes of organisms with sequenced and unsequenced genomes.

  20. Do Nanoparticle Physico-Chemical Properties and Developmental Exposure Window Influence Nano ZnO Embryotoxicity in Xenopus laevis?

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    Patrizia Bonfanti

    2015-07-01

    Full Text Available The growing global production of zinc oxide nanoparticles (ZnONPs suggests a realistic increase in the environmental exposure to such a nanomaterial, making the knowledge of its biological reactivity and its safe-by-design synthesis mandatory. In this study, the embryotoxicity of ZnONPs (1–100 mg/L specifically synthesized for industrial purposes with different sizes, shapes (round, rod and surface coatings (PEG, PVP was tested using the frog embryo teratogenesis assay-Xenopus (FETAX to identify potential target tissues and the most sensitive developmental stages. The ZnONPs did not cause embryolethality, but induced a high incidence of malformations, in particular misfolded gut and abdominal edema. Smaller, round NPs were more effective than the bigger, rod ones, and PEGylation determined a reduction in embryotoxicity. Ingestion appeared to be the most relevant exposure route. Only the embryos exposed from the stomodeum opening showed anatomical and histological lesions to the intestine, mainly referable to a swelling of paracellular spaces among enterocytes. In conclusion, ZnONPs differing in shape and surface coating displayed similar toxicity in X. laevis embryos and shared the same target organ. Nevertheless, we cannot exclude that the physico-chemical characteristics may influence the severity of such effects. Further research efforts are mandatory to ensure the synthesis of safer nano-ZnO-containing products.

  1. Do Nanoparticle Physico-Chemical Properties and Developmental Exposure Window Influence Nano ZnO Embryotoxicity in Xenopus laevis?

    Science.gov (United States)

    Bonfanti, Patrizia; Moschini, Elisa; Saibene, Melissa; Bacchetta, Renato; Rettighieri, Leonardo; Calabri, Lorenzo; Colombo, Anita; Mantecca, Paride

    2015-07-28

    The growing global production of zinc oxide nanoparticles (ZnONPs) suggests a realistic increase in the environmental exposure to such a nanomaterial, making the knowledge of its biological reactivity and its safe-by-design synthesis mandatory. In this study, the embryotoxicity of ZnONPs (1-100 mg/L) specifically synthesized for industrial purposes with different sizes, shapes (round, rod) and surface coatings (PEG, PVP) was tested using the frog embryo teratogenesis assay-Xenopus (FETAX) to identify potential target tissues and the most sensitive developmental stages. The ZnONPs did not cause embryolethality, but induced a high incidence of malformations, in particular misfolded gut and abdominal edema. Smaller, round NPs were more effective than the bigger, rod ones, and PEGylation determined a reduction in embryotoxicity. Ingestion appeared to be the most relevant exposure route. Only the embryos exposed from the stomodeum opening showed anatomical and histological lesions to the intestine, mainly referable to a swelling of paracellular spaces among enterocytes. In conclusion, ZnONPs differing in shape and surface coating displayed similar toxicity in X. laevis embryos and shared the same target organ. Nevertheless, we cannot exclude that the physico-chemical characteristics may influence the severity of such effects. Further research efforts are mandatory to ensure the synthesis of safer nano-ZnO-containing products.

  2. Migratory and adhesive properties of Xenopus laevis primordial germ cells in vitro

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    Aliaksandr Dzementsei

    2013-11-01

    The directional migration of primordial germ cells (PGCs to the site of gonad formation is an advantageous model system to study cell motility. The embryonic development of PGCs has been investigated in different animal species, including mice, zebrafish, Xenopus and Drosophila. In this study we focus on the physical properties of Xenopus laevis PGCs during their transition from the passive to the active migratory state. Pre-migratory PGCs from Xenopus laevis embryos at developmental stages 17–19 to be compared with migratory PGCs from stages 28–30 were isolated and characterized in respect to motility and adhesive properties. Using single-cell force spectroscopy, we observed a decline in adhesiveness of PGCs upon reaching the migratory state, as defined by decreased attachment to extracellular matrix components like fibronectin, and a reduced adhesion to somatic endodermal cells. Data obtained from qPCR analysis with isolated PGCs reveal that down-regulation of E-cadherin might contribute to this weakening of cell-cell adhesion. Interestingly, however, using an in vitro migration assay, we found that movement of X. laevis PGCs can also occur independently of specific interactions with their neighboring cells. The reduction of cellular adhesion during PGC development is accompanied by enhanced cellular motility, as reflected in increased formation of bleb-like protrusions and inferred from electric cell-substrate impedance sensing (ECIS as well as time-lapse image analysis. Temporal alterations in cell shape, including contraction and expansion of the cellular body, reveal a higher degree of cellular dynamics for the migratory PGCs in vitro.

  3. High-sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus Embryo

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    Camille Lombard-Banek

    2016-10-01

    Full Text Available Direct measurement of protein expression with single-cell resolution promises to deepen the understanding of basic molecular processes during normal and impaired development. High-resolution mass spectrometry provides detailed coverage of the proteomic composition of large numbers of cells. Here we discuss recent mass spectrometry developments based on single-cell capillary electrophoresis that extend discovery proteomics to sufficient sensitivity to enable the measurement of proteins in single cells. The single-cell mass spectrometry system is used to detect a large number of proteins in single embryonic cells in blastomeres in the 16-cell embryo of the South African clawed frog (Xenopus laevis that give rise to distinct tissue types. Single-cell measurements of protein expression provide complementary information on gene transcription during early development of the vertebrate embryo, raising a potential to understand how differential gene expression coordinates normal cell heterogeneity during development.

  4. Single-Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16-Cell Frog (Xenopus) Embryo.

    Science.gov (United States)

    Lombard-Banek, Camille; Moody, Sally A; Nemes, Peter

    2016-02-12

    We advance mass spectrometry from a cell population-averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh-resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE-μESI-HRMS enabled the identification of 500-800 nonredundant protein groups by measuring 20 ng, or frog (Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.

  5. Influence of 50-Hz Electromagnetic Field on Anurian (Xenopus laevis Metamorphosis

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    S. Grimaldi

    2004-01-01

    Full Text Available In this study, we show the effect of a 1-mT magnetic field AC at 50 Hz on Xenopus laevis tadpole populations. In the course of a 65-day exposure to the field, tadpole survival showed a small, but significant, decrease (p < 0.0004, together with a striking parallel 6-day shift in tadpole maturation frequency and a significant impairment of their metamorphosis. Particularly, metamorphosis was successful for 85% of individuals in the unirradiated tadpole population and for 45% of individuals in the irradiated tadpole population, respectively.

  6. Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

    OpenAIRE

    Tian, Jingdong; Gong, Hui; Thomsen, Gerald H.; Lennarz, William J.

    1997-01-01

    A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-depe...

  7. Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell—free extracts of Xenopus laevis eggs

    Institute of Scientific and Technical Information of China (English)

    LIUXIAOLING; YANSHEN

    2000-01-01

    Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation,nuclear envelope assembly,and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle.The assembled nuclei,being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii.However,incubation of dinoflagellate Cyrthecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.

  8. Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell-free extracts of Xenopus laevis eggs

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.

  9. Atmospheric pressure plasma accelerates tail regeneration in tadpoles Xenopus laevis

    Science.gov (United States)

    Rivie, A.; Martus, K.; Menon, J.

    2017-08-01

    Atmospheric pressure plasma is a partially ionized gas composed of neutral and charged particles, including electrons and ions, as well as reactive oxygen species (ROS). Recently, it is utilized as possible therapy in oncology, sterilization, skin diseases, wound healing and tissue regeneration. In this study we focused on effect of plasma exposure on tail regeneration of tadpoles, Xenopus leavis with special emphasis on role of ROS, antioxidant defenses and morphological features of the regenerate. When amputated region of the tail was exposed to the helium plasma it resulted in a faster rate of growth, elevated ROS and increase in antioxidant enzymes in the regenerate compared to that of untreated control. An increase in nitric oxide (free radical) as well as activity of nitric oxide synthase(s) were observed once the cells of the regeneration blastema - a mass of proliferating cells are ready for differentiation. Microscopically the cells of the regenerate of plasma treated tadpoles show altered morphology and characteristics of cellular hypoxia and oxidative stress. We summarize that plasma exposure accelerates the dynamics of wound healing and tail regeneration through its effects on cell proliferation and differentiation as well as angiogenesis mediated through ROS signaling.

  10. Differences in mobility at the range edge of an expanding invasive population of Xenopus laevis in the west of France.

    Science.gov (United States)

    Louppe, Vivien; Courant, Julien; Herrel, Anthony

    2017-01-15

    Theoretical models predict that spatial sorting at the range edge of expanding populations should favor individuals with increased mobility relative to individuals at the center of the range. Despite the fact that empirical evidence for the evolution of locomotor performance at the range edge is rare, data on cane toads support this model. However, whether this can be generalized to other species remains largely unknown. Here, we provide data on locomotor stamina and limb morphology in individuals from two sites: one from the center and one from the periphery of an expanding population of the clawed frog Xenopus laevis in France where it was introduced about 30 years ago. Additionally, we provide data on the morphology of frogs from two additional sites to test whether the observed differences can be generalized across the range of this species in France. Given the known sexual size dimorphism in this species, we also test for differences between the sexes in locomotor performance and morphology. Our results show significant sexual dimorphism in stamina and morphology, with males having longer legs and greater stamina than females. Moreover, in accordance with the predictions from theoretical models, individuals from the range edge had a greater stamina. This difference in locomotor performance is likely to be driven by the significantly longer limb segments observed in animals in both sites sampled in different areas along the range edge. Our data have implications for conservation because spatial sorting on the range edge may lead to an accelerated increase in the spread of this invasive species in France.

  11. The history and development of FETAX (ASTM standard guide, E-1439 on conducting the frog embryo teratogenesis Assay-Xenopus)

    Science.gov (United States)

    Dumont, J.N.; Bantle, J.A.; Linder, G.; ,

    2003-01-01

    The energy crisis of the 1970's and 1980's prompted the search for alternative sources of fuel. With development of alternate sources of energy, concerns for biological resources potentially adversely impacted by these alternative technologies also heightened. For example, few biological tests were available at the time to study toxic effects of effluents on surface waters likely to serve as receiving streams for energy-production facilities; hence, we began to use Xenopus laevis embryos as test organisms to examine potential toxic effects associated with these effluents upon entering aquatic systems. As studies focused on potential adverse effects on aquatic systems continued, a test procedure was developed that led to the initial standardization of FETAX. Other .than a limited number of aquatic toxicity tests that used fathead minnows and cold-water fishes such as rainbow trout, X. laevis represented the only other aquatic vertebrate test system readily available to evaluate complex effluents. With numerous laboratories collaborating, the test with X. laevis was refined, improved, and developed as ASTM E-1439, Standard Guide for the Conducting Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Collabrative work in the 1990s yielded procedural enhancements, for example, development of standard test solutions and exposure methods to handle volatile organics and hydrophobic compounds. As part of the ASTM process, a collaborative interlaboratory study was performed to determine the repeatability and reliability of FETAX. Parallel to these efforts, methods were also developed to test sediments and soils, and in situ test methods were developed to address "lab-to-field extrapolation errors" that could influence the method's use in ecological risk assessments. Additionally, a metabolic activation system composed of rat liver microsomes was developed which made FETAX more relevant to mammalian studies.

  12. Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis.

    Science.gov (United States)

    Oka, Tomohiro; Tooi, Osamu; Mitsui, Naoko; Miyahara, Maki; Ohnishi, Yuta; Takase, Minoru; Kashiwagi, Akihiko; Shinkai, Tadashi; Santo, Noriaki; Iguchi, Taisen

    2008-05-30

    There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1-100 ppb atrazine or 0.27 ppb (1 nM) 17beta-estradiol (E(2)) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E(2), but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1-100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.

  13. Xenopus laevis as a novel model to study long bone critical-size defect repair by growth factor-mediated regeneration.

    Science.gov (United States)

    Feng, Liang; Milner, Derek J; Xia, Chunguang; Nye, Holly L D; Redwood, Patrick; Cameron, Jo Ann; Stocum, David L; Fang, Nick; Jasiuk, Iwona

    2011-03-01

    We used the tarsus of an adult Xenopus laevis frog as an in vivo load-bearing model to study the regeneration of critical-size defects (CSD) in long bones. We found the CSD for this bone to be about 35% of the tarsus length. To promote regeneration, we implanted biocompatible 1,6 hexanediol diacrylate scaffolds soaked with bone morphogenetic proteins-4 and vascular endothelial growth factors. In contrast to studies that use scaffolds as templates for bone formation, we used scaffolds as a growth factor delivery vehicle to promote cartilage-to-bone regeneration. Defects in control frogs were filled with scaffolds lacking growth factors. The limbs were harvested at a series of time points ranging from 3 weeks to 6 months after implantation and evaluated using micro-computed tomography and histology. In frogs treated with growth factor-loaded scaffolds, we observed a cartilage-to-bone regeneration in the skeletal defect. Five out of eight defects were completely filled with cartilage by 6 weeks. Blood vessels had invaded the cartilage, and bone was beginning to form in ossifying centers. By 3 months, these processes were well advanced, and extensive ossification was observed in 6-month samples. In contrast, the defects in control frogs showed only formation of fibrous scar tissue. This study demonstrates the utility of a Xenopus model system for tissue engineering research and that the normal in vivo mechanism of endochondral bone development and fracture repair can be mimicked in the repair of CSD with scaffolds used as growth factor delivery mechanisms.

  14. Effect of antikeratin microinjection on the embryonic development of Xenopus laevis

    Institute of Scientific and Technical Information of China (English)

    YUHAOJIAN; JINGWUXIE; 等

    1993-01-01

    Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis.,and its effects on embryonic development were studied.Survival rate of the antikeratin-injected embryos was much lower(only 35.67% at gastrula)than that of the control(74.85% at gastrula),in which embryos were injected with mouse IgG.Most of survivors in the experimental series showed aberrant external appearance.On the other hand,in cleavage stage,ie 2-7h after fertilization,immunohistochemical staining of embryos showed that the expermental embryos were mostly keratin negative,while embryos of the control ones were keratin positive.When introducing this antikeratin into one cell of a 2-cell embryo,only the uninjected half of the embryo continued its development while the other half could not develop at all.These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.

  15. Effect of light on expression of clock genes in Xenopus laevis melanophores.

    Science.gov (United States)

    Magalhães Moraes, Maria Nathália de Carvalho; de Oliveira Poletini, Maristela; Ribeiro Ramos, Bruno Cesar; de Lima, Leonardo Henrique Ribeiro Graciani; de Lauro Castrucci, Ana Maria

    2014-01-01

    Light-dark cycles are considered important cues to entrain biological clocks. A feedback loop of clock gene transcription and translation is the molecular basis underlying the mechanism of both central and peripheral clocks. Xenopus laevis embryonic melanophores respond to light with melanin granule dispersion, response possibly mediated by the photopigment melanopsin. To test whether light modulates clock gene expression in Xenopus melanophores, we used qPCR to evaluate the relative mRNA levels of Per1, Per2, Clock and Bmal1 in cultured melanophores exposed to light-dark (LD) cycle or constant darkness (DD). LD cycles elicited temporal changes in the expression of Per1, Per2 and Bmal1. A 10-min pulse of blue light was able to increases the expression of Per1 and Per2. Red light had no effect on the expression of these clock genes. These data suggest the participation of a blue-wavelength sensitive pigment in the light-dark cycle-mediated oscillation of the endogenous clock. Our results add an important contribution to the emerging field of peripheral clocks, which in nonmammalian vertebrates have been mostly studied in Drosophila and Danio rerio. Within this context, we show that X. laevis melanophores, which have already led to melanopsin discovery, represent an ideal model to understanding circadian rhythms.

  16. Genetics, Morphology, Advertisement Calls, and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog (Xenopus, Pipidae) from West and Central Africa.

    Science.gov (United States)

    Evans, Ben J; Carter, Timothy F; Greenbaum, Eli; Gvoždík, Václav; Kelley, Darcy B; McLaughlin, Patrick J; Pauwels, Olivier S G; Portik, Daniel M; Stanley, Edward L; Tinsley, Richard C; Tobias, Martha L; Blackburn, David C

    2015-01-01

    African clawed frogs, genus Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon) to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana) and three species groups within the subgenus Xenopus (amieti, laevis, and muelleri species groups).

  17. Genetics, Morphology, Advertisement Calls, and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog (Xenopus, Pipidae from West and Central Africa.

    Directory of Open Access Journals (Sweden)

    Ben J Evans

    Full Text Available African clawed frogs, genus Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana and three species groups within the subgenus Xenopus (amieti, laevis, and muelleri species groups.

  18. Swimming kinematics and respiratory behaviour of Xenopus laevis larvae raised in altered gravity

    Science.gov (United States)

    Fejtek, M.; Souza, K.; Neff, A.; Wassersug, R.

    1998-01-01

    We examined the respiratory behaviours and swimming kinematics of Xenopus laevis tadpoles hatched in microgravity (Space Shuttle), simulated microgravity (clinostat) and hypergravity (3 g centrifuge). All observations were made in the normal 1 g environment. Previous research has shown that X. laevis raised in microgravity exhibit abnormalities in their lungs and vestibular system upon return to 1 g. The tadpoles raised in true microgravity exhibited a significantly lower tailbeat frequency than onboard 1 g centrifuge controls on the day of landing (day0), but this behaviour normalized within 9 days. The two groups did not differ significantly in buccal pumping rates. Altered buoyancy in the space-flight microgravity tadpoles was indicated by an increased swimming angle on the day after landing (day1). Tadpoles raised in simulated microgravity differed to a greater extent in swimming behaviours from their 1 g controls. The tadpoles raised in hypergravity showed no substantive effects on the development of swimming or respiratory behaviours, except swimming angle. Together, these results show that microgravity has a transient effect on the development of locomotion in X. laevis tadpoles, most notably on swimming angle, indicative of stunted lung development. On the basis of the behaviours we studied, there is no indication of neuromuscular retardation in amphibians associated with embryogenesis in microgravity.

  19. Excitatory and depressant effects of dieldrin and aldrin-transdiol in the spinal cord of the toad (Xenopus laevis)

    NARCIS (Netherlands)

    Akkermans, L.M.A.; Bercken, J. van den; Versluijs-Helder, M.

    An investigation was made into the action of the insecticide dieldrin and one of its metabolites, aldrin-transdiol, on the isolated spinal cord of the toad, Xenopus laevis. Conventional electrophysiological techniques were used for stimulating and recording of dorsal and ventral spinal roots. An

  20. Characterization and regulation of voltage-operated Ca2+ channels in neuroendocrine melanotrope cells of Xenopus laevis

    NARCIS (Netherlands)

    Zhang, H.Y.

    2006-01-01

    To investigate the way by which multiple incoming neurochemical messengers control second messenger systems to evoke an unambiguous neuronal output, melanotrope neuroendocrine cells in the pars intermedia of the pituitary gland of the South African clawed toad Xenopus laevis were studied. Researches

  1. Diurnal variation of tight junction integrity associates inversely with matrix metalloproteinase expression in Xenopus laevis corneal epithelium: implications for circadian regulation of homeostatic surface cell desquamation.

    Directory of Open Access Journals (Sweden)

    Allan F Wiechmann

    Full Text Available The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium.Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2, membrane type 1-MMP (MT1-MMP and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime.MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface

  2. Pescadillo homologue 1 and Peter Pan function during Xenopus laevis pronephros development.

    Science.gov (United States)

    Tecza, Aleksandra; Bugner, Verena; Kühl, Michael; Kühl, Susanne J

    2011-10-01

    pes1 (pescadillo homologue 1) and ppan (Peter Pan) are multifunctional proteins involved in ribosome biogenesis, cell proliferation, apoptosis, cell migration and regulation of gene expression. Both proteins are required for early neural development in Xenopus laevis, as previously demonstrated. We show that the expression of both genes in the developing pronephros depends on wnt4 and fzd3 (frizzled homologue 3) function. Loss of pes1 or ppan by MO (morpholino oligonucleotide)-based knockdown approaches resulted in strong malformations during pronephric tubule formation. Defects were already notable during specification of pronephric progenitor cells, as shown by lhx1 expression. Moreover, we demonstrated that Xenopus pes1 and ppan interact physically and functionally and that pes1 and ppan can cross-rescue the loss of function phenotype of one another. Interference with rRNA synthesis, however, did not result in a similar early pronephros phenotype. These results demonstrate that pes1 and ppan are required for Xenopus pronephros development and indicate that their function in the pronephros is independent of their role in ribosome biosynthesis.

  3. Expresión de canales de potasio voltaje dependientes en ovocitos de Xenopus laevis (Amphibia Voltage gated potassium channels expressed in Xenopus laevis(AMPHIBIA oocytes

    Directory of Open Access Journals (Sweden)

    Clavijo Carlos

    2003-06-01

    Full Text Available La expresión en sistemas heterólogos ha sido una herramienta ampliamente utilizada enlos últimos años para el estudio funcional y estructural de proteínas. Para la carac-terización de las propiedades biofísicas de canales, bombas y transportadores engeneral su expresión en ovocitos de Xenopus laevis, ha sido fundamental. Este estudioreporta la expresión de dos canales de potasio voltaje dependientes, Kv1.1y Shakerenovocitos de X. laevisusando un protocolo ajustado a las condiciones de latitud y altitudde Bogotá para la extracción, aislamiento, cultivo y microinyección de éstas células.Heterologous expression has been an important tool for structural and functionalcharacterization of proteins. The study of biophysical properties of ion channels,pumps and transporters has been possible thanks to their expression in Xenopuslaevisoocytes. Here we report the expression of two voltage gated channels, Kv1.1and Shaker, in X. laevisoocytes using a method for oocyte extraction, isolation, cul-ture, and microinjection adapted to the latitude and altitude conditions of Bogotá,Colombia.

  4. Developmental toxicity, uptake and distribution of sodium chromate assayed by frog embryo teratogenesis assay-Xenopus(FETAX)

    Energy Technology Data Exchange (ETDEWEB)

    Bosisio, Stefano [Via Giuseppe Garibaldi, 21, 21020, Casciago (Italy); Fortaner, Salvador, E-mail: salvador.fortaner@jrc.it [European Commission, ECVAM Unit, Institute for Health and Consumer Protection, Joint Research Centre, via Fermi 2749, 21027, Ispra (Italy); Bellinetto, Sonia [Via Gisora, 5, 21039, Bedero Valcuvia (Italy); Farina, Massimo; Del Torchio, Riccardo [European Commission, ECVAM Unit, Institute for Health and Consumer Protection, Joint Research Centre, via Fermi 2749, 21027, Ispra (Italy); Prati, Mariangela; Gornati, Rosalba; Bernardini, Giovanni [Department of Biotechnology and Molecular Sciences, University of Insubria, Via Dunant, 3, 21100, Varese (Italy); Sabbioni, Enrico [CeSI, Ageing Research Center, ' G. d' Annunzio' University Foundation, Via Colle dell' Ara, 66100, Chieti (Italy)

    2009-09-01

    The embryotoxicity and teratogenicity of Cr(VI) on the survival and morphology of the anuran Xenopus laevis have been assessed by frog embryo teratogenesis assay-Xenopus (FETAX). The lethal median (LC{sub 50}) and teratogenic median (TC{sub 50}) concentration values of Cr(VI) were 890 {mu}M and 260 {mu}M, respectively. The calculated teratogenic index (TI) value was 3.42, suggesting that hexavalent chromium has a teratogenic potential. Malformations of embryos included lifting of the body, coiling of the tail and body oedema. Furthermore, the chromium salt caused significant growth retardation at 25 {mu}M exposure concentrations. The use of radiolabelled {sup 51}Cr(VI) allowed the determination of the time course uptake of Cr in Xenopus exposed to concentrations ranging from 0.025 to 500 {mu}M. The evaluation of its distribution into the body (head-abdomen-tail) was evaluated at different exposure times. Chromium is taken up at 24 h by Xenopus embryos for all concentrations tested. At 48 h post fertilization (stage of larva) the amount of Cr accumulated by the two-day-old larva ranged from 0.42 to 580 pg mg{sup -1} wet weight at 0.025 and 500 {mu}M respectively. These amounts were lower than those at 24 h (2.77 to 11016 pg mg{sup -1} wet weight embryo) reaching values of the same order of magnitude at 120 h (five-days-old larva). Since at 48 h Xenopus development leads to a swimming embryo, the observed uptake at 24 h could be the result of the binding of Cr to jelly coat compounds surrounding the embryo body as confirmed by gel filtration experiments on {sup 51}Cr-jelly coat. The interaction of Cr with jelly coat is in agreement with the role of jelly coat in protecting the embryo against pathogen and chemical toxins to ensure fertilization. This work further supports the hypothesis that Cr contamination of surface waters could contribute to explain the reported worldwide depletion of frog population.

  5. Developmental toxicity, uptake and distribution of sodium chromate assayed by frog embryo teratogenesis assay-Xenopus(FETAX).

    Science.gov (United States)

    Bosisio, Stefano; Fortaner, Salvador; Bellinetto, Sonia; Farina, Massimo; Del Torchio, Riccardo; Prati, Mariangela; Gornati, Rosalba; Bernardini, Giovanni; Sabbioni, Enrico

    2009-09-01

    The embryotoxicity and teratogenicity of Cr(VI) on the survival and morphology of the anuran Xenopus laevis have been assessed by frog embryo teratogenesis assay-Xenopus (FETAX). The lethal median (LC(50)) and teratogenic median (TC(50)) concentration values of Cr(VI) were 890 microM and 260 microM, respectively. The calculated teratogenic index (TI) value was 3.42, suggesting that hexavalent chromium has a teratogenic potential. Malformations of embryos included lifting of the body, coiling of the tail and body oedema. Furthermore, the chromium salt caused significant growth retardation at 25 microM exposure concentrations. The use of radiolabelled (51)Cr(VI) allowed the determination of the time course uptake of Cr in Xenopus exposed to concentrations ranging from 0.025 to 500 microM. The evaluation of its distribution into the body (head-abdomen-tail) was evaluated at different exposure times. Chromium is taken up at 24 h by Xenopus embryos for all concentrations tested. At 48 h post fertilization (stage of larva) the amount of Cr accumulated by the two-day-old larva ranged from 0.42 to 580 pg mg(-1) wet weight at 0.025 and 500 microM respectively. These amounts were lower than those at 24 h (2.77 to 11016 pg mg(-1) wet weight embryo) reaching values of the same order of magnitude at 120 h (five-days-old larva). Since at 48 h Xenopus development leads to a swimming embryo, the observed uptake at 24 h could be the result of the binding of Cr to jelly coat compounds surrounding the embryo body as confirmed by gel filtration experiments on (51)Cr-jelly coat. The interaction of Cr with jelly coat is in agreement with the role of jelly coat in protecting the embryo against pathogen and chemical toxins to ensure fertilization. This work further supports the hypothesis that Cr contamination of surface waters could contribute to explain the reported worldwide depletion of frog population.

  6. Species-specific loss of sexual dimorphism in vocal effectors accompanies vocal simplification in African clawed frogs (Xenopus).

    Science.gov (United States)

    Leininger, Elizabeth C; Kitayama, Ken; Kelley, Darcy B

    2015-03-01

    Phylogenetic studies can reveal patterns of evolutionary change, including the gain or loss of elaborate courtship traits in males. Male African clawed frogs generally produce complex and rapid courtship vocalizations, whereas female calls are simple and slow. In a few species, however, male vocalizations are also simple and slow, suggesting loss of male-typical traits. Here, we explore features of the male vocal organ that could contribute to loss in two species with simple, slow male calls. In Xenopus boumbaensis, laryngeal morphology is more robust in males than in females. Larynges are larger, have a more complex cartilaginous morphology and contain more muscle fibers. Laryngeal muscle fibers are exclusively fast-twitch in males but are both fast- and slow-twitch in females. The laryngeal electromyogram, a measure of neuromuscular synaptic strength, shows greater potentiation in males than in females. Male-specific physiological features are shared with X. laevis, as well as with a species of the sister clade, Silurana tropicalis, and thus are likely ancestral. In X. borealis, certain aspects of laryngeal morphology and physiology are sexually monomorphic rather than dimorphic. In both sexes, laryngeal muscle fibers are of mixed-twitch type, which limits the production of muscle contractions at rapid intervals. Muscle activity potentiation and discrete tension transients resemble female rather than male X. boumbaensis. The de-masculinization of these laryngeal features suggests an alteration in sensitivity to the gonadal hormones that are known to control the sexual differentiation of the larynx in other Xenopus and Silurana species.

  7. Light conditions affect the roll-induced vestibuloocular reflex in Xenopus laevis tadpoles

    Science.gov (United States)

    El-Yamany, Nabil A.

    2008-12-01

    In Xenopus laevis tadpoles, effects of asymmetrical light conditions on the roll-induced vestibuloocular reflex (rVOR) were tested for the developmental period between stage 47 and 49. For comparison, the rVOR was tested in dim- and high-symmetrical light environments. Test parameters were the rVOR gain and rVOR amplitude. Under all light conditions, the rVOR increased from tadpole stage 47 to 49. For all stages, the asymmetrical light field induced the strongest response, the dim light field the weakest one. The response for the left and right eye was identical, even if the tadpoles were tested under asymmetrical light conditions. The experiments can be considered as hints (1) for an age-dependent light sensitivity of vestibular neurons, and (2) for the existence of control systems for coordinated eye movements that has its origin in the proprioceptors of the extraocular eye muscles.

  8. Transmembrane Signal Transduction in Oocyte Maturation and Fertilization: Focusing on Xenopus laevis as a Model Animal

    Directory of Open Access Journals (Sweden)

    Ken-ichi Sato

    2014-12-01

    Full Text Available Fertilization is a cell biological phenomenon of crucial importance for the birth of new life in a variety of multicellular and sexual reproduction species such as algae, animal and plants. Fertilization involves a sequence of events, in which the female gamete “egg” and the male gamete “spermatozoon (sperm” develop, acquire their functions, meet and fuse with each other, to initiate embryonic and zygotic development. Here, it will be briefly reviewed how oocyte cytoplasmic components are orchestrated to undergo hormone-induced oocyte maturation and sperm-induced activation of development. I then review how sperm-egg membrane interaction/fusion and activation of development in the fertilized egg are accomplished and regulated through egg coat- or egg plasma membrane-associated components, highlighting recent findings and future directions in the studies using Xenopus laevis as a model experimental animal.

  9. FUNCION DE SOX2 EN LA REGENERACION DE LA MEDULA ESPINAL DE XENOPUS LAEVIS

    OpenAIRE

    2010-01-01

    Los mamíferos presentan una limitada capacidad de regeneración frente a una lesión de la médula espinal, lo que impide una correcta recuperación locomotriz. Sin embargo, los renacuajos de Xenopus laevis sí son capaces de regenerar la médula espinal, pero se desconocen los mecanismos involucrados en este proceso. Es posible que esta regeneración proceda a través de la activación de células madre y progenitores neurales. Sox2 es un factor de transcripción que se expresa en las células mad...

  10. Nuclear reconstitution of demembranated Orychophragmus violaceus sperm in Xenopus laevis egg extracts

    Institute of Scientific and Technical Information of China (English)

    LU; Ping(卢萍); REN; Min(任民); ZHAI; Zhonghe(翟中和)

    2002-01-01

    The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon- densation and pronuclear formation from demembranated plant (Orychophragmus violaceus)sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.

  11. Cadmium but not lead exposure affects Xenopus laevis fertilization and embryo cleavage.

    Science.gov (United States)

    Slaby, Sylvain; Lemière, Sébastien; Hanotel, Julie; Lescuyer, Arlette; Demuynck, Sylvain; Bodart, Jean-François; Leprêtre, Alain; Marin, Matthieu

    2016-08-01

    Among the toxicological and ecotoxicological studies, few have investigated the effects on germ cells, gametes or embryos, while an impact at these stages will result in serious damage at a population level. Thus, it appeared essential to characterize consequences of environmental contaminant exposures at these stages. Therefore, we proposed to assess the effects of exposure to cadmium and lead ions, alone or in a binary mixture, on early stages of Xenopus laevis life cycle. Fertilization and cell division during segmentation were the studied endpoints. Cadmium ion exposures decreased in the fertilization rates in a concentration-dependent manner, targeting mainly the oocytes. Exposure to this metal ions induced also delays or blockages in the embryonic development. For lead ion exposure, no such effect was observed. For the exposure to the mixture of the two metal ions, concerning the fertilization success, we observed results similar to those obtained with the highest cadmium ion concentration.

  12. Biochemical response to exposure to six textile dyes in early developmental stages of Xenopus laevis.

    Science.gov (United States)

    Güngördü, Abbas; Birhanli, Ayse; Ozmen, Murat

    2013-01-01

    The present study was undertaken to determine the toxic effect of a lethal concentration of six different commercially used textile dyes on the 46th stage of Xenopus laevis tadpoles. The tadpoles were exposed to Astrazon Red FBL, Astrazon Blue FGRL, Remazol Red RR, Remazol Turquoise Blue G-A, Cibacron Red FN-3G, and Cibacron Blue FN-R for 168 h in static test conditions, and thus, 168-h median lethal concentrations (LC(50)s) of each dye were determined to be 0.35, 0.13, 112, 7, 359, and 15.8 mg/L, respectively. Also, to evaluate the sublethal effects of each dye, tadpoles were exposed to different concentrations of dyes (with respect to 168-h LC(50)s) for 24 h. The alteration of selected enzyme activities was tested. For this aim, glutathione S-transferase (GST), carboxylesterase, and lactate dehydrogenase (LDH) were assayed. After dye exposure, the GST induction or inhibition and LDH induction indicated some possible mechanisms of oxidative stress and deterioration in aerobic respiration processes induced by the tested dyes. Findings of the study suggest that selected biomarker enzymes are useful in understanding the toxic mechanisms of these dyes in X. laevis tadpoles as early warning indicators. Therefore, these selected biomarkers may evaluate the effect of environmental factors, such as textile dye effluents and other industrial pollutants, on amphibians in biomonitoring studies.

  13. Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Bindesh Shrestha

    Full Text Available Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI, an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen, were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.

  14. Overexpression of Fyn tyrosine kinase causes abnormal development of primary sensory neurons in Xenopus laevis embryos.

    Science.gov (United States)

    Saito, R; Fujita, N; Nagata, S

    2001-06-01

    The expression and function of the Src family protein tyrosine kinase Fyn in Xenopus laevis embryos have been examined. In situ hybridization analysis demonstrated nervous system-specific expression of Fyn mRNA in tail-bud embryos. However, a class of primary sensory neurons; that is, Rohon-Beard (RB) neurons, which is positive for immunoglobulin superfamily cell adhesion molecules (CAM), neural cell adhesion molecule (N-CAM) and contactin, is devoid of Fyn expression. Injection of Fyn mRNA into one of the blastomeres at the 2-cell stage led to overexpression of Fyn in the injected half of the tail-bud embryos. Immunolabeling of the embryos with anti-HNK-1 antibody revealed that the peripheral axons of RB neurons were partially misguided and bound to each other to form abnormal subcutaneous fascicles. Similar abnormality was induced by injection of the Fyn overexpression vector. The incidence of abnormality appeared dose-dependent, being 68-92% of the injected embryos at 50-400 pg of mRNA. Co-injection of the contactin antisense vector depleted contactin mRNA accumulation without affecting Fyn overexpression and reduced the incidence of the abnormal RB-cell phenotype. However, the N-CAM antisense was ineffective in reducing this abnormality. These results suggest that Fyn can modify signals regulating axonal guidance or fasciculation in the developing X. laevis nervous system and that contactin may affect this action of Fyn.

  15. Proteomics analysis of Xenopus laevis gonad tissue following chronic exposure to atrazine.

    Science.gov (United States)

    Chen, Xiuping; Wang, Jiamei; Zhu, Haojun; Ding, Jiatong; Peng, Yufa

    2015-08-01

    Atrazine is the most commonly detected pesticide contaminant in ground and surface water. Previous studies have shown that atrazine is an endocrine disruptor owing to its adverse effects on the male reproductive system in several vertebrates, but very few molecular mechanisms for these effects have been revealed. In the present study, Xenopus laevis were exposed to 100 ppb of atrazine for 120 d, and then the isobaric tags for relative and absolute quantitation (iTRAQ) technique was used to detect global changes in protein profiles of the testes and ovaries. The results showed that 100 ppb of atrazine exposure adversely affected the growth of X. laevis and did not induce hermaphroditism but delayed or prevented the development of male seminiferous tubules. Proteomic analysis showed that atrazine altered expression of 143 and 121 proteins in the testes and ovaries, respectively, and most of them are involved in cellular and metabolic processes and biological regulation based on their biological processes. In addition, apoptosis, tight junctions, and metabolic pathways were significantly altered in the atrazine-treated gonads. Based on the above results, it is postulated that the reproductive toxicity of atrazine may be the result of disruption of tight junctions and metabolic signaling pathways and/or induction of apoptosis in germ cells. © 2015 SETAC.

  16. Effects of perfluorooctanesulfonate and perfluorobutanesulfonate on the growth and sexual development of Xenopus laevis.

    Science.gov (United States)

    Lou, Qin-Qin; Zhang, Yin-Feng; Zhou, Zhen; Shi, Ya-Li; Ge, Ya-Nan; Ren, Dong-Kai; Xu, Hai-Ming; Zhao, Ya-Xian; Wei, Wu-Ji; Qin, Zhan-Fen

    2013-09-01

    Perfluorobutanesulfonate (PFBS), as a substitute for perfluorooctanesulfonate (PFOS), is widespread in the environment and biotic samples as well as PFOS. To investigate effects of PFOS and PFBS on the growth and sexual development of amphibians, we exposed Xenopus laevis tadpoles at a series of concentrations of PFOS and PFBS (0.1; 1; 100; 1,000 μg/l) as well as 17-beta-estradiol (E2, 100 ng/l) and 5 alpha-androstan-17-beta-ol-3-one (DHT, 100 ng/l) from stage 46/47 to 2 months postmetamorphosis. We found that neither PFOS nor PFBS had a significant effect on the survival and growth. However, they caused hepatohistological impairment at higher concentrations (100; 1,000 μg/l). Unlike E2, PFOS at all concentrations did not alter the sex ratio and induce intersex, but caused degeneration of spermatogonia in testes except for the lowest concentration. PFBS had no effect on the sex ratio and gonadal histology. PFOS and PFBS promoted expression of estrogen receptor (ER) and androgen receptor (AR), but not affected aromatase expression in the brain. The increase in expression of ER and AR suggests an increase in the responsiveness to the corresponding sex hormone and potential effects on sexual development. Our results show that PFBS as well as PFOS have adverse effects on hepato-histology and sexual development on X. laevis. Also, PFOS- and PFBS-induced increase in ER and AR expression highlights the need to further study effects of PFOS and PFBS on subsequently gonadal development, sexual dimorphism, and secondary sex characteristics in X. laevis. It is debatable that PFBS is widely used as a substitute of PFOS.

  17. The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

    Energy Technology Data Exchange (ETDEWEB)

    Bluhm, A.P.C.; Obeid, N.N.; Castrucci, A.M.L.; Visconti, M.A. [Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-05-25

    Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

  18. An integumentary mucin (FIM-B.1) from Xenopus laevis homologous with von Willebrand factor.

    Science.gov (United States)

    Probst, J C; Gertzen, E M; Hoffmann, W

    1990-07-03

    We present a new protein from X. laevis skin termed "frog integumentary mucin B.1" (FIM-B.1) with a general structure similar to FIM-A.1 (formerly "spasmolysin"). The central region consisting of tandem repeats of 11 amino acid residues is probably a target for extensive O-glycosylation, whereas the C-terminal cysteine-rich domain shows pronounced homology with the C1-C2 domains and the C-terminal end of von Willebrand factor. Furthermore, we describe homology with antistasin, an anticoagulant peptide from a leech. We also discuss some implications concerning the evolutionary origin of von Willebrand factor. In situ hybridization studies revealed the expression of FIM-B.1 exclusively in mucous glands of the skin. This is comparable with FIM-A.1 but is in contrast to all other physiologically active peptides, which are synthesized in granular glands.

  19. Analysis of neural progenitors from embryogenesis to juvenile adult in Xenopus laevis reveals biphasic neurogenesis and continuous lengthening of the cell cycle

    Directory of Open Access Journals (Sweden)

    Raphaël Thuret

    2015-12-01

    Full Text Available Xenopus laevis is a prominent model system for studying neural development, but our understanding of the long-term temporal dynamics of neurogenesis remains incomplete. Here, we present the first continuous description of neurogenesis in X. laevis, covering the entire period of development from the specification of neural ectoderm during gastrulation to juvenile frog. We have used molecular markers to identify progenitors and neurons, short-term bromodeoxyuridine (BrdU incorporation to map the generation of newborn neurons and dual pulse S-phase labelling to characterise changes in their cell cycle length. Our study revealed the persistence of Sox3-positive progenitor cells from the earliest stages of neural development through to the juvenile adult. Two periods of intense neuronal generation were observed, confirming the existence of primary and secondary waves of neurogenesis, punctuated by a period of quiescence before metamorphosis and culminating in another period of quiescence in the young adult. Analysis of multiple parameters indicates that neural progenitors alternate between global phases of differentiation and amplification and that, regardless of their behaviour, their cell cycle lengthens monotonically during development, at least at the population level.

  20. Yolk proteins from nematodes, chickens, and frogs bind strongly and preferentially to left-handed Z-DNA

    National Research Council Canada - National Science Library

    P Krishna; B P Kennedy; J H van de Sande; J D McGhee

    1988-01-01

    Yolk proteins purified from the nematode Caenorhabditis elegans, from the frog Xenopus laevis, and from chicken eggs all have the unexpected property of binding strongly and preferentially to a left...

  1. Expression and functional characterization of Xhmg-at-hook genes in Xenopus laevis.

    Directory of Open Access Journals (Sweden)

    Simone Macrì

    Full Text Available High Mobility Group A proteins (HMGA1 and HMGA2 are architectural nuclear factors involved in development, cell differentiation, and cancer formation and progression. Here we report the cloning, developmental expression and functional analysis of a new multi-AT-hook factor in Xenopus laevis (XHMG-AT-hook that exists in three different isoforms. Xhmg-at-hook1 and 3 isoforms, but not isoform 2, are expressed throughout the entire development of Xenopus, both in the maternal and zygotic phase. Localized transcripts are present in the animal pole in the early maternal phase; during the zygotic phase, mRNA can be detected in the developing central nervous system (CNS, including the eye, and in the neural crest. We show evidence that XHMG-AT-hook proteins differ from typical HMGA proteins in terms of their properties in DNA binding and in protein/protein interaction. Finally, we provide evidence that they are involved in early CNS development and in neural crest differentiation.

  2. Effect of intercalating agents on RNA polymerase I promoter selection in Xenopus laevis.

    Science.gov (United States)

    Pruitt, S C; Reeder, R H

    1984-01-01

    We have analyzed the effect of DNA intercalating agents on the transcription signals from two different Xenopus laevis RNA polymerase I promoters. The transcription signal from the promoter for the 7.5-kilobase rRNA precursor (the gene promoter) is unaffected over a large range of intercalating agent concentrations regardless of whether the template is injected plasmid DNA in oocytes, the amplified endogenous nucleoli of oocytes, or the endogenous chromosomes of cultured Xenopus kidney cells. The transcription signal from a closely related promoter located in the spacer DNA between genes (the spacer promoter) ranges between undetectable to equivalent to the gene promoter signal on different templates. The transcription signal from the spacer promoter is also differentially affected by intercalating agents relative to the gene promoter. Depending on the template, these agents can either increase or decrease the transcription signal from the spacer promoter. Fusions between the gene and spacer promoters demonstrate that intercalating agents affect transcription initiation. One explanation for these results is that the degree of supercoiling of the template DNA can differentially inhibit transcription from the spacer promoters. The different effects of intercalating agents on transcription from the spacer promoters of various templates could then be explained as differences in the degree of supercoiling present on these templates initially. Images PMID:6543244

  3. High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos

    Directory of Open Access Journals (Sweden)

    Ken-ichi T. Suzuki

    2013-03-01

    Recently, gene editing with transcription activator-like effector nucleases (TALENs has been used in the life sciences. TALENs can be easily customized to recognize a specific DNA sequence and efficiently introduce double-strand breaks at the targeted genomic locus. Subsequent non-homologous end-joining repair leads to targeted gene disruption by base insertion, deletion, or both. Here, to readily evaluate the efficacy of TALENs in Xenopus laevis embryos, we performed the targeted gene disruption of tyrosinase (tyr and pax6 genes that are involved in pigmentation and eye formation, respectively. We constructed TALENs targeting tyr and pax6 and injected their mRNAs into fertilized eggs at the one-cell stage. Expectedly, introduction of tyr TALEN mRNA resulted in drastic loss of pigmentation with high efficiency. Similarly, for pax6, TALENs led to deformed eyes in the injected embryos. We confirmed mutations of the target alleles by restriction enzyme digestion and sequence analyses of genomic PCR products. Surprisingly, not only biallelic but also paralogous, gene disruption was observed. Our results demonstrate that targeted gene disruption by TALENs provides a method comparable to antisense morpholinos in analyzing gene function in Xenopus F0 embryos, but also applies beyond embryogenesis to any life stage.

  4. Prepatterning and patterning of the thalamus along embryonic development of Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Sandra eBandín

    2015-08-01

    Full Text Available Previous developmental studies of the thalamus (alar part of the diencephalic prosomere p2 have defined the molecular basis for the acquisition of the thalamic competence (preparttening, the subsequent formation of the secondary organizer in the zona limitans intrathalamica, and the early specification of two anteroposterior domains (rostral and caudal progenitor domains in response to inducing activities and that are shared in birds and mammals. In he present study we have analyzed the embryonic development of the thalamus in the anuran Xenopus laevis to determine conserved or specific features in the amphibian diencephalon. From early embryonic stages to the beginning of the larval period, the expression patterns of 22 markers were analyzed by means of combined in situ hybridization and immunohistochemical techniques. The early genoarchitecture observed in the diencephalon allowed us to discern the boundaries of the thalamus with the prethalamus, pretectum, and epithalamus. Common molecular features were observed in the thalamic prepatterning among vertebrates in which Wnt3a, Fez, Pax6 and Xiro1 expression were of particular importance in Xenopus. The formation of the zona limitans intrathalamica was observed, as in other vertebrates, by the progressive expression of Shh. The largely conserved expressions of Nkx2.2 in the rostral thalamic domain versus Gbx2

  5. Adrenocorticotropin receptors: Functional expression from rat adrenal mRNA in Xenopus laevis oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Mertz, L.M.; Catt, K.J. (National Inst. of Health, Bethesda, MD (United States))

    1991-10-01

    The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A){sup +} RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. Size fractionation of rat adrenal poly(A){sup +}RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1-to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNA in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.

  6. Biosensor, ELISA, and frog embryo teratogenesis assay: Xenopus (FETAX) analysis of water associated with frog malformations in Minnesota

    Science.gov (United States)

    Garber, Eric A. E.; Erb, Judith L.; Downward, James G.; Priuska, Eric M.; Wittliff, James L.; Feng, Wenke; Magner, Joseph; Larsen, Gerald L.

    2001-03-01

    Between 1995 and 1997 over 62% of the counties in Minnesota reported the presence of malformed frogs. While most sites have recently shown a decline in malformed frog populations, one site in northeastern Minnesota with no prior history of containing malformed frogs was recently discovered to contain > 67% malformed Rana pipiens (northern leopard frogs). As part of an effort to study the presence of hormonally active agents in fresh water sources, water samples were collected from lakes in Minnesota containing malformed frogs and analyzed for the presence of hormonally active compounds using a novel evanescent field fluorometric biosensor and the frog embryo teratogenesis assay: Xenopus (FETAX) bioassay. The waveguide based biosensor developed by ThreeFold Sensors (TFS biosensor, Ann Arbor, MI) detects the presence of estrogenic compounds capable of interacting with free human ER-a and by inhibiting binding to an immobilized estrogen. The FETAX bioassay is a developmental assay, which measures teratogenicity, mortality, and inhibition of growth during the first 96 hours of organogenesis and thereby provides a universal screen for endocrine disruptors. TFS biosensor and FETAX screening of the water samples suggest a relationship between estrogenic activity, mineral supplementation, and the occurrence of malformed frogs.

  7. Protein arginine methyltransferase Prmt5-Mep50 methylates histones H2A and H4 and the histone chaperone nucleoplasmin in Xenopus laevis eggs.

    Science.gov (United States)

    Wilczek, Carola; Chitta, Raghu; Woo, Eileen; Shabanowitz, Jeffrey; Chait, Brian T; Hunt, Donald F; Shechter, David

    2011-12-01

    Histone proteins carry information contained in post-translational modifications. Eukaryotic cells utilize this histone code to regulate the usage of the underlying DNA. In the maturing oocytes and eggs of the frog Xenopus laevis, histones are synthesized in bulk in preparation for deposition during the rapid early developmental cell cycles. During this key developmental time frame, embryonic pluripotent chromatin is established. In the egg, non-chromatin-bound histones are complexed with storage chaperone proteins, including nucleoplasmin. Here we describe the identification and characterization of a complex of the protein arginine methyltransferase 5 (Prmt5) and the methylosome protein 50 (Mep50) isolated from Xenopus eggs that specifically methylates predeposition histones H2A/H2A.X-F and H4 and the histone chaperone nucleoplasmin on a conserved motif (GRGXK). We demonstrate that nucleoplasmin (Npm), an exceedingly abundant maternally deposited protein, is a potent substrate for Prmt5-Mep50 and is monomethylated and symmetrically dimethylated at Arg-187. Furthermore, Npm modulates Prmt5-Mep50 activity directed toward histones, consistent with a regulatory role for Npm in vivo. We show that H2A and nucleoplasmin methylation appears late in oogenesis and is most abundant in the laid egg. We hypothesize that these very abundant arginine methylations are constrained to pre-mid blastula transition events in the embryo and therefore may be involved in the global transcriptional repression found in this developmental time frame.

  8. 氟吗啉对非洲爪蟾胚胎发育的影响%Effects of the Developmental Toxicity of Flumorph on Xenopus laevis Embryos

    Institute of Scientific and Technical Information of China (English)

    朱新萍; 蔡磊明; 蒋平安

    2007-01-01

    简述了FETAX(Frog Embryo Teratogenesis Assay-Xenopus)试验的发展,并以非洲爪蟾(Xenopus laevis)处于8~11阶段的胚胎为试验材料,对杀菌剂氟吗啉进行了发育毒性的研究,通过对受试胚胎96 h死亡数、半数致死浓度[LC50(96 h)]、半数效应浓度[EC50(96 h)]、致畸类型的统计,得出LC 50(96 h)值为83.15 mg/L,EC50(96 h)值为76.65 mg/L,TI(LC50/EC50)为1.01≤1.5.结果表明,氟吗啉对非洲爪蟾的胚胎发育不具有致畸影响,但表现出一定的发育毒性.在氟吗啉质量浓度大于20 mg/L时,可明显的抑制胚胎的孵化和体长生长的发育,表现的中毒症状有脊索弯曲、弯尾、眼部畸形、腹部水肿等.

  9. APPLICATION OF FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS TO ECOLOGICAL RISK ASSESSMENT

    Science.gov (United States)

    An expert workshop recently was convened to consider the FETAX (frog embryo teratogenesis assay-Xenopus) as a screening method for identifying the potential development toxicity of single chemicals and chemical mixtures. Comparison of FETAX data with acute toxicity data from tes...

  10. Investigation of Blood Flow and the Effect of Vasoactive Substances in Cutaneous Blood Vessels of "Xenopus Laevis"

    Science.gov (United States)

    Škorjanc, Aleš; Belušic, Gregor

    2015-01-01

    In the present study, a preparation of frog skin was presented, which can be used to demonstrate the basic concepts of blood flow regulation in a very clear and attractive way to high school and university students. In a freshly euthanized "Xenopus," a patch of abdominal skin was exposed from the internal side and viewed with a USB…

  11. Mechanical, electrical, and morphological characteristics of skeletal muscle fibers from Xenopus and other species of frogs.

    Science.gov (United States)

    Oba, T; Yamamoto, M; Aoki, T; Hotta, K

    1983-01-01

    Mechanical, electrical, and morphological properties of iliofibularis or semitendinosus of Xenopus laevis, Rana catesbeiana, and Rana nigromaculata were investigated in an attempt to find out the differences between them which will give the basic knowledge for the study of excitation-contraction coupling. With application of electrical stimulation, a single muscle fiber from Xenopus contracted at a faster rate of rise than did the other muscles tested. The maximum rate of rise (Tmax) of tension was in the order of Xenopus, R. catesbeiana, and R. nigromaculata. Ca2+ sensitivity and Tmax of mechanically skinned fibers of Xenopus resembled those of R. catesbeiana. Xenopus muscle had a small cross-sectional area of T-tubule compared with that in other species and the action potential exhibited a small positive-going hump. The volume density of the terminal cisternae of sarcoplasmic reticulum (SR) to the myofibril was the largest in the Xenopus muscle, with a statistically significant difference. Therefore, the Xenopus muscle appears to be good material for investigation of mechanisms related to Ca2+ release from SR, as elicited by the excitation of T-tubules.

  12. How Xenopus laevis embryos replicate reliably: investigating the random-completion problem

    CERN Document Server

    Yang, Scott Cheng-Hsin

    2008-01-01

    DNA synthesis in \\textit{Xenopus} frog embryos initiates stochastically in time at many sites (origins) along the chromosome. Stochastic initiation implies fluctuations in the time to complete and may lead to cell death if replication takes longer than the cell cycle time ($\\approx 25$ min). Surprisingly, although the typical replication time is about 20 min, \\textit{in vivo} experiments show that replication fails to complete only about 1 in 300 times. How is replication timing accurately controlled despite the stochasticity? Biologists have proposed two solutions to this "random-completion problem." The first solution uses randomly located origins but increases their rate of initiation as S phase proceeds, while the second uses regularly spaced origins. In this paper, we investigate the random-completion problem using a type of model first developed to describe the kinetics of first-order phase transitions. Using methods from the field of extreme-value statistics, we derive the distribution of replication-c...

  13. Membrane Type-1 Matrix Metalloproteinases and Tissue Inhibitor of Metalloproteinases-2 RNA Levels Mimic Each Other during Xenopus laevis Metamorphosis

    OpenAIRE

    Walsh, Logan A.; Deanna A Carere; Cooper, Colin A.; Sashko Damjanovski

    2007-01-01

    Matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs (tissue inhibitors of MMPs), are two protein families that work together to remodel the extracellular matrix (ECM). TIMPs serve not only to inhibit MMP activity, but also aid in the activation of MMPs that are secreted as inactive zymogens. Xenopus laevis metamorphosis is an ideal model for studying MMP and TIMP expression levels because all tissues are remodeled under the control of one molecule, thyroid hormone. Here, us...

  14. PARTICIPACION DE PROTEINAS-G EN EL PROCESO DE MADURACION INDUCIDO POR PROGESTERONA EN OVOCITOS DE XENOPUS LAEVIS

    OpenAIRE

    ROMO MARTY, XIMENA CAROLINA

    2005-01-01

    La hormona progesterona induce el proceso de maduración meiótica del ovocito de Xenopus laevis a través de un mecanismo de acción no genómico, el cual se caracteriza por ser un evento rápido en el tiempo y que involucra la inhibición del sistema efector a 134p.

  15. Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development.

    Science.gov (United States)

    Sheets, Michael D; Fox, Catherine A; Dowdle, Megan E; Blaser, Susanne Imboden; Chung, Andy; Park, Sookhee

    2017-01-01

    The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented

  16. Dose-Dependent Early Life Stage Toxicities in Xenopus laevis Exposed In Ovo to Selenium.

    Science.gov (United States)

    Massé, Anita J; Muscatello, Jorgelina R; Janz, David M

    2015-11-17

    Selenium (Se) is a developmental toxicant in oviparous vertebrates. The adverse reproductive effects of Se toxicity have been predominantly investigated in fishes and birds with only a few studies focusing on amphibians. The objective of this study was to determine tissue-based toxicity thresholds for early life stage Se toxicities in Xenopus laevis as a consequence of in ovo exposure through maternal transfer of dietary Se. Following a 68-day dietary exposure to food augmented with l-selenomethionine (SeMet) at measured concentrations of 0.7 (control), 10.9, 30.4, or 94.2 μg Se/g dry mass (d.m.), adult female X. laevis were bred with untreated males, and resulting embryos were incubated until 5 days postfertilization (dpf). The measured Se concentrations in eggs were 1.6, 10.8, 28.1, and 81.7 μg Se/g d.m., respectively. No biologically significant effects were observed on fertilization success, hatchability, or mortality in offspring. Frequency and severity of morphological abnormalities were significantly greater in 5 dpf tadpoles from the highest exposure group when compared to the control, with eye lens abnormalities being the most prominent of all abnormalities. The estimated EC10 value for frequency of total early life stage abnormalities was 44.9 μg Se/g egg d.m., which suggests that this amphibian species is less sensitive to in ovo Se exposure than most of the fish species studied to date.

  17. Functional and Structural Effects of Amyloid-β Aggregate on Xenopus laevis Oocytes

    Science.gov (United States)

    Parodi, Jorge; la Paz, Lenin Ochoa-de; Miledi, Ricardo; Martínez-Torres, Ataúlfo

    2012-01-01

    Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of “spontaneous” blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca2+ was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca2+-dependent Cl− currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (Tout) and the serum-activated, oscillatory Cl− currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca2+-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication. PMID:23104436

  18. Ion Currents Induced by ATP and Angiotensin II in Cultured Follicular Cells of Xenopus laevis

    Science.gov (United States)

    Montiel-Herrera, Marcelino; Zaske, Ana María; García-Colunga, Jesús; Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2011-01-01

    Xenopus laevis oocytes are commonly used to study the biophysical and pharmacological properties of foreign ion channels and receptors, but little is known about those endogenously expressed in their enveloping layer of follicular cells (FCs). Whole-cell recordings and the perforated patch-clamp technique in cultured FCs held at -60 mV revealed that ATP (20-250 μM) generates inward currents of 465 ± 93 pA (mean ± standard error) in ∼60% of the FCs studied, whereas outward currents of 317 ± 100 pA were found in ∼5% of the cells. The net effect of ATP on the FCs was to activate both mono- and biphasic inward currents, with an associated increase in membrane chloride conductance. Two-microelectrode voltage-clamp recordings of nude oocytes held at -60 mV disclosed that ATP elicited biphasic inward currents, corresponding to the well-known Fin and Sin-like currents. ATP receptor antagonists like suramin, TNP-ATP, and RB2 did not inhibit any of these responses. On the other hand, when using wholecell recordings, 1 μM Ang II yielded smooth inward currents of 157 ± 45 pA in ∼16% of the FC held at -60 mV. The net Ang II response, mediated by the activation of the AT1 receptor, was a chloride current inhibited by 10 nM ZD7155. This study will help to better understand the roles of ATP and Ang II receptors in the physiology of X. laevis oocytes. PMID:22083304

  19. Exposure to butachlor causes thyroid endocrine disruption and promotion of metamorphosis in Xenopus laevis.

    Science.gov (United States)

    Li, Shuying; Li, Meng; Wang, Qiangwei; Gui, Wenjun; Zhu, Guonian

    2016-06-01

    Butachlor is extensively applied in rice paddy ecosystem in china, and has been widespread contaminant in the aquatic environment. Here, Xenopus laevis was used for the evaluation of teratogenesis developmental toxicity, and disruption of thyroid system when exposure to different concentrations of butachlor by window phase exposure. Acute toxicity investigation shown that 96 h-LC50 value of butachlor was 1.424 mg L(-1) and 0.962 mg L(-1) for tadpoles (starting from stages 46/47) and embryos (starting from stages 8/9), respectively. Exposure to butachlor caused malformation, including abnormal eye, pericardial edema, enlarged proctodaeum and bent tail. Window phase exposure test indicated that butachlor significantly promote the contents of whole-body thyroid hormones (THs, T3 and T4) at higher levels, indicating thyroid endocrine disruption. At 7 days, exposure to butachlor up-regulated the mRNA expression of genes involved in THs synthesis and metabolism (tshα, tg, tpo and dio1) and THs receptors (trα and trβ). At 14 days, up-regulation of the mRNA expression of genes related to THs synthesis and metabolism (tshα, tshβ, tg, tpo, dio1, dio2 and ttr) and THs receptors (trβ) were also observed after the exposure to butachlor. At 21 days, butachlor up-regulated the mRNA expression of tshα, tg, tpo genes and down-regulated the mRNA expression of tshβ, tg, dio1, ttr and trα genes. These results showed that butachlor could change the mRNA expression of genes involved in the HPT axis and increase whole-body thyroid hormones levels of X. laevis tadpoles in a dose- and time-dependent manner, causing thyroid endocrine disruption and developmental toxicity.

  20. Low concentrations of metal mixture exposures have adverse effects on selected biomarkers of Xenopus laevis tadpoles

    Energy Technology Data Exchange (ETDEWEB)

    Yologlu, Ertan, E-mail: ertanyologlu82@gmail.com [Adiyaman University, Faculty of Education, Department of Science Education, 02040 Adiyaman (Turkey); Ozmen, Murat [Inonu University, Laboratory of Environmental Toxicology, Department of Biology, Faculty of Arts & Science, 44280 Malatya (Turkey)

    2015-11-15

    Highlights: • Selected metal mixtures were evaluated for toxicity of safety limit concentrations. • Xenopus laevis tadpoles were used as model test organism. • Combinations of LC{sub 50} and LC{sub 50}/2 caused 100% lethality for some metals. • Metals did not change metallothionein levels in low concentrations. • Selected enzyme activities showed induction after low concentration exposures. - Abstract: Polluted ecosystems may contain mixtures of metals, such that the combinations of metals, even in low concentrations, may cause adverse effects. In the present study, we focused on toxic effects of mixtures of selected metals, the LC{sub 50} values, and also their safety limit in aquatic systems imposed by the European legislation using a model organism. Xenopus laevis tadpoles were used as test organisms. They were exposed to metals or their combinations due to 96-h LC{sub 50} values. Glutathione S-transferase (GST), glutathione reductase (GR), acetylcholinesterase (AChE), carboxylesterase (CaE), glutathione peroxidase (GPx), and catalase (CAT) levels were evaluated. Metallothionein concentrations were also determined. The LC{sub 50}s for Cd, Pb, and Cu were calculated as 5.81 mg AI/L, 123.05 mg AI/L, and 0.85 mg AI/L, respectively. Low lethality ratios were observed with unary exposure of each metal in lower concentrations. Double or triple combinations of LC{sub 50} and LC{sub 50}/2 concentrations caused 100% lethality with Cd + Cu and Pb + Cd + Cu mixtures, while the Pb + Cu mixture also caused high lethal ratios. The selected enzyme activities were significantly affected by metals or mixtures, and dose-related effects were determined. The metallothionein levels generally increased as related to concentration in unary metals and mixtures. Acceptable limit values of unary metals and mixtures did not significantly change metallothionein levels. The results suggest that oxidative stress-related mechanisms are involved in the toxicity induced by selected

  1. DEADSouth protein localizes to germ plasm and is required for the development of primordial germ cells in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Takeshi Yamaguchi

    2012-11-01

    DEADSouth mRNA is a component of germ plasm in Xenopus laevis and encodes a DDX25 DEAD-box RNA helicase. To determine the intracellular localization of DEADSouth protein, we injected mRNA encoding DEADSouth tagged with mCherry fluorescent protein into fertilized eggs from transgenic Xenopus expressing EGFP fused with a mitochondrial targeting signal. The DEADSouth-mCherry fusion protein was localized to the germ plasm, a mitochondria-rich region in primordial germ cells (PGCs. DEADSouth overexpression resulted in a reduction of PGC numbers after stage 20. Conversely, DEADSouth knockdown using an antisense locked nucleic acid gapmer inhibited movement of the germ plasm from the cortex to the perinuclear region, resulting in inhibition of PGC division at stage 12 and a decrease in PGC numbers at later stages. The knockdown phenotype was rescued by intact DEADSouth mRNA, but not mutant mRNA encoding inactive DEADSouth helicase. Surprisingly, it was also rescued by mouse vasa homolog and Xenopus vasa-like gene 1 mRNAs that encode DDX4 RNA helicases. The rescue was dependent on the 3′ untranslated region (3′UTR of DEADSouth mRNA, which was used for PGC-specific expression. The 3′UTR contributed to localization of the injected mRNA to the germ plasm, resulting in effective localization of DEADSouth protein. These results demonstrate that localization of DEADSouth helicase to the germ plasm is required for proper PGC development in Xenopus laevis.

  2. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization

    Science.gov (United States)

    Bates, Ryan C.; Fees, Colby P.; Holland, William L.; Winger, Courtney C.; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J.

    2014-01-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC- γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca]i). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 minute after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca]i and other fertilization events. As compared to 14 other lipids, PA strongly bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca]i, PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca]i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca]i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  3. Hargreaves does not evaluate nociception following a surgical laparotomy in Xenopus leavis frogs.

    Science.gov (United States)

    Vachon, P

    2014-10-01

    The present study was performed to determine the effectiveness of the Hargreaves test for the evaluation of nociception in frogs, more precisely to determine if cutaneous thresholds to a radiant heat stimulus would increase with analgesics following an abdominal laparotomy performed under general anaesthesia. Non breeding female Xenopus leavis frogs (3 groups (non-anaesthetized, anaesthetized with tricaine methanesulfonate (MS222), with or without an abdominal laparotomy) were used to evaluate the effectiveness of the Hargreaves test. Cutaneous thresholds were evaluated at baseline and following anaesthetic recovery (over 8 h) at six different body locations. Increased reaction times were observed in the gular area only at 1 h post-recovery following a MS222 bath immersion in frogs with (p leavis frogs.

  4. Brain-derived neurotrophic factor (BDNF) expression in normal and regenerating olfactory epithelium of Xenopus laevis.

    Science.gov (United States)

    Frontera, Jimena Laura; Cervino, Ailen Soledad; Jungblut, Lucas David; Paz, Dante Agustín

    2015-03-01

    Olfactory epithelium has the capability to continuously regenerate olfactory receptor neurons throughout life. Adult neurogenesis results from proliferation and differentiation of neural stem cells, and consequently, olfactory neuroepithelium offers an excellent opportunity to study neural regeneration and the factors involved in the maintenance and regeneration of all their cell types. We analyzed the expression of BDNF in the olfactory system under normal physiological conditions as well as during a massive regeneration induced by chemical destruction of the olfactory epithelium in Xenopus laevis larvae. We described the expression and presence of BDNF in the olfactory epithelium and bulb. In normal physiological conditions, sustentacular (glial) cells and a few scattered basal (stem) cells express BDNF in the olfactory epithelium as well as the granular cells in the olfactory bulb. Moreover, during massive regeneration, we demonstrated a drastic increase in basal cells expressing BDNF as well as an increase in BDNF in the olfactory bulb and nerve. Together these results suggest an important role of BDNF in the maintenance and regeneration of the olfactory system.

  5. Changes in oscillatory dynamics in the cell cycle of early Xenopus laevis embryos.

    Directory of Open Access Journals (Sweden)

    Tony Y-C Tsai

    2014-02-01

    Full Text Available During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min and the subsequent 11 cycles are short (∼30 min and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development.

  6. Inhibition of the thyroid hormone pathway in Xenopus laevis by 2-mercaptobenzothiazole

    Energy Technology Data Exchange (ETDEWEB)

    Tietge, Joseph E., E-mail: tietge.joe@epa.gov [US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804 (United States); Degitz, Sigmund J., E-mail: degitz.sigmund@epa.gov [US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804 (United States); Haselman, Jonathan T., E-mail: haselman.jon@epa.gov [US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804 (United States); Butterworth, Brian C., E-mail: butterworth.brian@epa.gov [US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804 (United States); Korte, Joseph J., E-mail: korte.joe@epa.gov [US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804 (United States); Kosian, Patricia A., E-mail: kosian.pat@epa.gov [US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804 (United States); Lindberg-Livingston, Annelie J., E-mail: lind1020@d.umn.edu [US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804 (United States); and others

    2013-01-15

    Determining the effects of chemicals on the thyroid system is an important aspect of evaluating chemical safety from an endocrine disrupter perspective. Since there are numerous chemicals to test and limited resources, prioritizing chemicals for subsequent in vivo testing is critical. 2-Mercaptobenzothiazole (MBT), a high production volume chemical, was tested and shown to inhibit thyroid peroxidase (TPO) enzyme activity in vitro, a key enzyme necessary for the synthesis of thyroid hormone. To determine the thyroid disrupting activity of MBT in vivo, Xenopus laevis larvae were exposed using 7- and 21-day protocols. The 7-day protocol used 18-357 {mu}g/L MBT concentrations and evaluated: metamorphic development, thyroid histology, circulating T4, circulating thyroid stimulating hormone, thyroidal sodium-iodide symporter gene expression, and thyroidal T4, T3, and related iodo-amino acids. The 21-day protocol used 23-435 {mu}g/L MBT concentrations and evaluated metamorphic development and thyroid histology. Both protocols demonstrated that MBT is a thyroid disrupting chemical at the lowest concentrations tested. These studies complement the in vitro study used to identify MBT as a high priority for in vivo testing, supporting the utility/predictive potential of a tiered approach to testing chemicals for TPO activity inhibition. The 7-day study, with more comprehensive, sensitive, and diagnostic endpoints, provides information at intermediate biological levels that enables linking various endpoints in a robust and integrated pathway for thyroid hormone disruption associated with TPO inhibition.

  7. Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes.

    Science.gov (United States)

    Bassez, T; Paris, J; Omilli, F; Dorel, C; Osborne, H B

    1990-11-01

    The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.

  8. Cadmium but not lead exposure affects Xenopus laevis fertilization and embryo cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Slaby, Sylvain [Univ. Lille Nord de France, EA 4515 – LGCgE – Laboratoire Génie Civil et géo-Environnement, Université de Lille 1, Cité scientifique, SN3, F-59655 Villeneuve d’Ascq (France); Univ. Lille, CNRS, INRA, UMR 8576 – UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille (France); Lemière, Sébastien [Univ. Lille Nord de France, EA 4515 – LGCgE – Laboratoire Génie Civil et géo-Environnement, Université de Lille 1, Cité scientifique, SN3, F-59655 Villeneuve d’Ascq (France); Hanotel, Julie; Lescuyer, Arlette [Univ. Lille, CNRS, INRA, UMR 8576 – UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille (France); Demuynck, Sylvain [Univ. Lille Nord de France, EA 4515 – LGCgE – Laboratoire Génie Civil et géo-Environnement, Université de Lille 1, Cité scientifique, SN3, F-59655 Villeneuve d’Ascq (France); Bodart, Jean-François [Univ. Lille, CNRS, INRA, UMR 8576 – UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille (France); and others

    2016-08-15

    Highlights: • First embryonic steps were studied. • Fertilization success was impacted by cadmium exposures. • Oocytes were most affected instead of spermatozoa by cadmium exposures. • First embryonic cleavages were slown down or stopped by cadmium exposures. • Lead exposures did not affected fertilization and segmentation. - Abstract: Among the toxicological and ecotoxicological studies, few have investigated the effects on germ cells, gametes or embryos, while an impact at these stages will result in serious damage at a population level. Thus, it appeared essential to characterize consequences of environmental contaminant exposures at these stages. Therefore, we proposed to assess the effects of exposure to cadmium and lead ions, alone or in a binary mixture, on early stages of Xenopus laevis life cycle. Fertilization and cell division during segmentation were the studied endpoints. Cadmium ion exposures decreased in the fertilization rates in a concentration-dependent manner, targeting mainly the oocytes. Exposure to this metal ions induced also delays or blockages in the embryonic development. For lead ion exposure, no such effect was observed. For the exposure to the mixture of the two metal ions, concerning the fertilization success, we observed results similar to those obtained with the highest cadmium ion concentration.

  9. Expression of functional G protein-coupled receptors in photoreceptors of transgenic Xenopus laevis.

    Science.gov (United States)

    Zhang, Li; Salom, David; He, Jianhua; Okun, Alex; Ballesteros, Juan; Palczewski, Krzysztof; Li, Ning

    2005-11-08

    G protein-coupled receptors (GPCRs) constitute the largest superfamily of transmembrane signaling proteins; however, the only known GPCR crystal structure is that of rhodopsin. This disparity reflects the difficulty in generating purified GPCR samples of sufficient quantity and quality. Rhodopsin, the light receptor of retinal rod neurons, is produced in large amounts of homogeneous quality in the vertebrate retina. We used transgenic Xenopus laevis to convert these retina rod cells into bioreactors to successfully produce 20 model GPCRs. The receptors accumulated in rod outer segments and were homogeneously glycosylated. Ligand and [(35)S]GTPgammaS binding assays of the 5HT(1A) and EDG(1) GPCRs confirmed that they were properly folded and functional. 5HT(1A)R was highly purified by taking advantage of the rhodopsin C-terminal immunoaffinity tag common to all GPCR constructs. We have also developed an automated system that can generate hundreds of transgenic tadpoles per day. This expression approach could be extended to other animal model systems and become a general method for the production of large numbers of GPCRs and other membrane proteins for pharmacological and structural studies.

  10. Developmental changes in head movement kinematics during swimming in Xenopus laevis tadpoles.

    Science.gov (United States)

    Hänzi, Sara; Straka, Hans

    2017-01-15

    During the post-embryonic developmental growth of animals, a number of physiological parameters such as locomotor performance, dynamics and behavioural repertoire are adjusted to match the requirements determined by changes in body size, proportions and shape. Moreover, changes in movement parameters also cause changes in the dynamics of self-generated sensory stimuli, to which motion-detecting sensory systems have to adapt. Here, we examined head movements and swimming kinematics of Xenopus laevis tadpoles with a body length of 10-45 mm (developmental stage 46-54) and compared these parameters with fictive swimming, recorded as ventral root activity in semi-intact in vitro preparations. Head movement kinematics was extracted from high-speed video recordings of freely swimming tadpoles. Analysis of these locomotor episodes indicated that the swimming frequency decreased with development, along with the angular velocity and acceleration of the head, which represent self-generated vestibular stimuli. In contrast, neither head oscillation amplitude nor forward velocity changed with development despite the ∼3-fold increase in body size. The comparison between free and fictive locomotor dynamics revealed very similar swimming frequencies for similarly sized animals, including a comparable developmental decrease of the swimming frequency. Body morphology and the motor output rhythm of the spinal central pattern generator therefore develop concurrently. This study thus describes development-specific naturalistic head motion profiles, which form the basis for more natural stimuli in future studies probing the vestibular system. © 2017. Published by The Company of Biologists Ltd.

  11. Triclosan and anuran metamorphosis: no effect on thyroid-mediated metamorphosis in Xenopus laevis.

    Science.gov (United States)

    Fort, Douglas J; Rogers, Robert L; Gorsuch, Joseph W; Navarro, Lisa T; Peter, Robert; Plautz, James R

    2010-02-01

    Nieuwkoop and Faber stage 51 Xenopus laevis larvae were exposed for 21 days to four different concentrations of triclosan (TCS): <0.2 (control), 0.6, 1.5, 7.2, or 32.3 microg TCS/l. Primary endpoints were survival, hind limb length, body length (whole; snout to vent), developmental stage, wet whole body weight, and thyroid histology. Thyroid hormone (TH) concentrations were determined in whole thyroid and plasma samples from stage-matched exposure day 21 specimens. TH receptor-beta (TRbeta) expression was measured in stage-matched tail fin tissue samples collected at exposure days 0 and 21. Reduced larval growth occurred at exposure day 21 with 1.5 microg/l treatment. Larval developmental stage at exposure day 21 was not significantly different from controls based on observed parameters. Thyroid histology was not affected by TCS, and thyroxine (T4) levels in thyroid glands or plasma were not different from controls. A concentration-dependent increase in TRbeta expression in exposure day 21 larvae was not detected. However, increased expression was found in stage-matched larvae exposed to 1.5 or 7.2 microg TCS/l. Our study indicates that environmentally relevant TCS concentrations do not alter the normal course of thyroid-mediated metamorphosis in this standard anuran model.

  12. Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

    Science.gov (United States)

    Raymond Delpech, Valérie; Ihara, Makoto; Coddou, Claudio; Matsuda, Kazuhiko; Sattelle, David B

    2003-11-01

    Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken alpha7, chicken alpha4beta2 and the Drosophila melanogaster/chicken hybrid receptors SAD/beta2 and ALS/beta2. No agonist action of NTX (0.1-100 microM) was observed on chicken alpha7, chicken alpha4beta2 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (alpha7) or heteromeric (alpha4beta2) nicotinic AChRs. Block by NTX of the chicken alpha7, chicken alpha4beta2 and the SAD/beta2 and ALS/beta2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.

  13. Wound healing ability of Xenopus laevis embryos. I. Rapid wound closure achieved by bisectional half embryos.

    Science.gov (United States)

    Yoshii, Yasuko; Noda, Masahiro; Matsuzaki, Takashi; Ihara, Setsunosuke

    2005-10-01

    We examined wound closure in 'half embryos' produced by the transverse bisection of Xenopus laevis embryos at the primary eye vesicle stage. Both the anterior- and posterior-half embryos survived for more than 6 days, and grew into 'half tadpoles'. Histology and videomicroscopy revealed that the open wound in the half embryo was rapidly closed by an epithelial sheet movement in the wound marginal zone. The time-course of wound closure showed a downward convex curve: the wound area decreased to one-fifth of the original area within 30 min, and the wound continued to contract slowly thereafter. The rapidity of closure of the epidermis as well as the absence of inflammatory cells are typical features of an embryonic type of wound healing. There was a dorso-ventral polarity in the motility of the epidermis: the wound was predominantly closed by the ventral and lateral epidermis. The change in the contour of the wound edge with time suggested a complex mechanism involved in the wound closure that could not be explained only by the purse-string theory. The present experimental system would be a unique and useful model for analyses of cellular movements in the embryonic epithelia.

  14. EFFECTS OF WATER QUALITY ON DEVELOPMENT OF XENOPUS LAEVIS: A FETAX ASSESSMENT OF SURFACE WATER ASSOCIATED WITH MALFORMATIONS IN NATIVE ANURANS

    Science.gov (United States)

    The purpose of this work was to determine if surface water from a site in Minnesota with malformed anurans was able to elicit adverse developmental effects in the Frog Embryo Teratogenesis Assay: Xenopus (FETAX)...

  15. Species-specific loss of sexual dimorphism in vocal effectors accompanies vocal simplification in African clawed frogs (Xenopus)

    Science.gov (United States)

    Leininger, Elizabeth C.; Kitayama, Ken; Kelley, Darcy B.

    2015-01-01

    ABSTRACT Phylogenetic studies can reveal patterns of evolutionary change, including the gain or loss of elaborate courtship traits in males. Male African clawed frogs generally produce complex and rapid courtship vocalizations, whereas female calls are simple and slow. In a few species, however, male vocalizations are also simple and slow, suggesting loss of male-typical traits. Here, we explore features of the male vocal organ that could contribute to loss in two species with simple, slow male calls. In Xenopus boumbaensis, laryngeal morphology is more robust in males than in females. Larynges are larger, have a more complex cartilaginous morphology and contain more muscle fibers. Laryngeal muscle fibers are exclusively fast-twitch in males but are both fast- and slow-twitch in females. The laryngeal electromyogram, a measure of neuromuscular synaptic strength, shows greater potentiation in males than in females. Male-specific physiological features are shared with X. laevis, as well as with a species of the sister clade, Silurana tropicalis, and thus are likely ancestral. In X. borealis, certain aspects of laryngeal morphology and physiology are sexually monomorphic rather than dimorphic. In both sexes, laryngeal muscle fibers are of mixed-twitch type, which limits the production of muscle contractions at rapid intervals. Muscle activity potentiation and discrete tension transients resemble female rather than male X. boumbaensis. The de-masculinization of these laryngeal features suggests an alteration in sensitivity to the gonadal hormones that are known to control the sexual differentiation of the larynx in other Xenopus and Silurana species. PMID:25788725

  16. Lymphoid tumors of Xenopus laevis with different capacities for growth in larvae and adults.

    Science.gov (United States)

    Robert, J; Guiet, C; Du Pasquier, L

    1994-01-01

    Three new lymphoid tumors offering an assortment of variants in terms of MHC class I expressions, MHC class II expression, and Ig gene transcription have been discovered in the amphibian Xenopus. One was developed in an individual of the isogenic LG15 clone (LG15/0), one in a frog of the LG15/40 clone (derived from a small egg recombinant of LG15), and one (ff-2) in a male ff sib of the individual in which MAR1, the first lymphoid tumor in Xenopus was found 2 years ago. These tumors developed primarily as thymus outgrowths and were transplantable in histocompatible tadpoles but not in nonhistocompatible hosts. Whereas LG15/0 and LG15/40 tumor cells also grow in adult LG15 frogs, the ff-2 tumor, like the MAR1 cell line, is rejected by adult ff animals. Using flow cytometry with fluorescence-labeled antibodies and immunoprecipitation analysis, we could demonstrate that, like MAR1, these three new tumors express on their cell surface lymphopoietic markers recognized by mAbs F1F6 and RC47, as well as T-cell lineage markers recognized by mAbs AM22 (CD8-like) and X21.2, but not by immunologobulin (Ig) nor MHC class II molecules. Another lymphocyte-specific marker AM15 is expressed by 15/0 and 15/40 but not ff-2 tumor cells. The ff-2 tumor cell expresses MHC class I molecule in association with beta 2-microglobulin on the surface, 15/40 cells contain cytoplasmic class I alpha chain that is barely detected at the cell surface by fluocytometry, and 15/0 cells do not synthesize class I alpha chain at all. The three new tumors all produce large amounts of IgM mRNA of two different sizes but no Ig protein on the membrane nor in the cytoplasm. All tumor cell types synthesize large amount of Myc mRNA and MHC class I-like transcripts considered to be non classical.

  17. Reproductive Maturation of the Tropical Clawed Frog, Xenopus tropicalis

    Science.gov (United States)

    The model species Xenopus tropicalis is being widely used in developmental biology and amphibian toxicology studies. In order to increase our understanding of the role of steroid hormones in maturation in this species, we collected baseline reproductive data from metamorphosis t...

  18. Thyroid hormone-dependent development in Xenopus laevis: a sensitive screen of thyroid hormone signaling disruption by municipal wastewater treatment plant effluent.

    Science.gov (United States)

    Searcy, Brian T; Beckstrom-Sternberg, Stephen M; Beckstrom-Sternberg, James S; Stafford, Phillip; Schwendiman, Angela L; Soto-Pena, Jenifer; Owen, Michael C; Ramirez, Claire; Phillips, Joel; Veldhoen, Nik; Helbing, Caren C; Propper, Catherine R

    2012-05-01

    Because thyroid hormones (THs) are conserved modulators of development and physiology, identification of compounds adversely affecting TH signaling is critical to human and wildlife health. Anurans are an established model for studying disruption of TH signaling because metamorphosis is dependent upon the thyroid system. In order to strengthen this model and identify new gene transcript biomarkers for TH disruption, we performed DNA microarray analysis of Xenopus laevis tadpole tail transcriptomes following treatment with triiodothyronine (T(3)). Comparison of these results with previous studies in frogs and mammals identified 36 gene transcripts that were TH-sensitive across clades. We then tested molecular biomarkers for sensitivity to disruption by exposure to wastewater effluent (WWE). X. laevis tadpoles, exposed to WWE from embryo through metamorphosis, exhibited an increased developmental rate compared to controls. Cultured tadpole tails showed dramatic increases in levels of four TH-sensitive gene transcripts (thyroid hormone receptor β (TRβ), deiodinase type II (DIO2), and corticotropin releasing hormone binding protein (CRHBP), fibroblast activation protein α (FAPα)) when exposed to T(3) and WWE extracts. TRβ, DIO2, and CRHBP were identified as TH sensitive in other studies, while FAPα mRNA transcripts were highly TH sensitive in our array. The results validate the array and demonstrate TH-disrupting activity by WWE. Our findings demonstrate the usefulness of cross-clade analysis for identification of gene transcripts that provide sensitivity to endocrine disruption. Further, the results suggest that development is disrupted by exposure to complex mixes of compounds found in WWE possibly through interference with TH signaling.

  19. Progressively restricted expression of a new homeobox-containing gene during Xenopus laevis embryogenesis.

    Science.gov (United States)

    Su, M W; Suzuki, H R; Solursh, M; Ramirez, F

    1991-04-01

    We have isolated cDNAs encoding a novel Xenopus homeodomain-containing protein homologous to the mouse Hox-7.1 and the Drosophila muscle segment homebox (msh). Northern blot and RNAase protection experiments established that transcripts of the frog gene, termed Xhox-7.1, first appear at about the beginning of gastrulation. After a rapid increase, mRNA levels plateau between the neurula and middle-tailbud stages, and decrease steadily thereafter. In situ hybridization localized the Xhox-7.1 message to the dorsal mesodermal mantle of gastrula stage embryos. Comparison of the hybridization patterns of progressively more anterior cross-section of tailbud stage embryos localized the signal to the dorsal neural tube and neural crest, to specific regions of the lateral plate mesoderm, and to the cardiogenic region. By the tadpole stage, the Xhox-7.1 message appears only at specific sites in the central nervous system, such as in the dorsal hindbrain. Thus, during embryonic development levels of Xhox-7.1 expression decrease as the transcript becomes more progressively localized. Finally, evidence is presented of a distinct msh-like transcript (provisionally termed Xhox-7.1') which begins to accumulate at early-gastrula stage, as well.

  20. Effect of allyl isothiocyanate on developmental toxicity in exposed Xenopus laevis embryos

    Directory of Open Access Journals (Sweden)

    John Russell Williams

    2015-01-01

    Full Text Available The pungent natural compound allyl isothiocyanate isolated from the seeds of Cruciferous (Brassica plants such as mustard is reported to exhibit numerous beneficial health-promoting antimicrobial, antifungal, anticarcinogenic, cardioprotective, and neuroprotective properties. Because it is also reported to damage DNA and is toxic to aquatic organisms, the objective of the present study was to determine whether it possesses teratogenic properties. The frog embryo teratogenesis assay-Xenopus (FETAX was used to determine the following measures of developmental toxicity of the allyl isothiocyanate: (a 96-h LC50, defined as the median concentration causing 50% embryo lethality; (b 96-h EC50, defined as the median concentration causing 50% malformations of the surviving embryos; and (c teratogenic malformation index (TI, equal to 96-h LC50/96-h EC50. The quantitative results and the photographs of embryos before and after exposure suggest that allyl isothiocyanate seems to exhibit moderate teratogenic properties. The results also indicate differences in the toxicity of allyl isothiocyanate toward exposed embryos observed in the present study compared to reported adverse effects of allyl isothiocyanate in fish, rodents, and humans. The significance of the results for food safety and possible approaches to protect against adverse effects of allyl isothiocyanate are discussed.

  1. How Xenopus laevis embryos replicate reliably: Investigating the random-completion problem

    Science.gov (United States)

    Yang, Scott Cheng-Hsin; Bechhoefer, John

    2008-10-01

    DNA synthesis in Xenopus frog embryos initiates stochastically in time at many sites (origins) along the chromosome. Stochastic initiation implies fluctuations in the time to complete and may lead to cell death if replication takes longer than the cell cycle time (≈25min) . Surprisingly, although the typical replication time is about 20min , in vivo experiments show that replication fails to complete only about 1 in 300 times. How is replication timing accurately controlled despite the stochasticity? Biologists have proposed two solutions to this “random-completion problem.” The first solution uses randomly located origins but increases their rate of initiation as S phase proceeds, while the second uses regularly spaced origins. In this paper, we investigate the random-completion problem using a type of model first developed to describe the kinetics of first-order phase transitions. Using methods from the field of extreme-value statistics, we derive the distribution of replication-completion times for a finite genome. We then argue that the biologists’ first solution to the problem is not only consistent with experiment but also nearly optimizes the use of replicative proteins. We also show that spatial regularity in origin placement does not alter significantly the distribution of replication times and, thus, is not needed for the control of replication timing.

  2. Comparison of diazinon toxicity to embryos of Xenopus laevis and Danio rerio; degradation of diazinon in water.

    Science.gov (United States)

    Modra, Helena; Vrskova, Dagmar; Macova, Stanislava; Kohoutkova, Jana; Hajslova, Jana; Haluzova, Ivana; Svobodova, Zdenka

    2011-06-01

    The aim of this study was to determine the toxic effect of diazinon (organophosphate insecticide) to embryos of Xenopus laevis and Danio rerio. The 96-h LC₅₀ values showed higher toxicity of diazinon for X. leavis in standard solution (9.84 mg/L) compared to the pond water (12.64 mg/L). Teratogenic index for diazinon was 1.3 and 1.6, respectively. The 96-h LC₅₀ diazinon values demonstrated similar sensitivity of embryos D. rerio (8.21-9.34 mg/L) and X. laevis in standard test solutions. Our results reflect that direct application of diazinon into the water can be associated with significant risks to aquatic organisms.

  3. Functional expression and characterization of a Xenopus laevis peptidylglycine alpha-amidating monooxygenase, AE-II, in insect-cell culture.

    Science.gov (United States)

    Suzuki, K; Ohta, M; Okamoto, M; Nishikawa, Y

    1993-04-01

    The alpha-amidating reaction of peptide hormones is a two-step process which is catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N-C lyase (PHL). There are three types of mRNA for these amidating enzymes in Xenopus laevis, namely AE-I, AE-II and AE-III. AE-I encodes only PHM and AE-III encodes both PHM and PHL. AE-II seems to encode subtypes of both PHM and PHL. While AE-II mRNA is present in high amounts in frog skin, the actual enzymes originating from AE-II have not been detected. When we expressed AE-II in cultured insect-cells using the baculovirus expression vector system, the expressed enzyme was specifically localized to the membrane fraction due to its hydrophobic transmembrane domain. Alternatively, when the transmembrane-domain-deleted AE-II (Met1-Ile731) was expressed, the enzyme was secreted into the culture medium; this secreted enzyme was purified to homogeneity by a simple two-step procedure. We have verified that the reaction product of the purified enzyme was the amidated peptide, indicating that AE-II has the ability to catalyze the entire amidating reaction.

  4. Cloning and characterization of Xen-dorphin prohormone from Xenopus laevis: a new opioid-like prohormone distinct from proenkephalin and prodynorphin.

    Science.gov (United States)

    Pattee, Patrick; Ilie, Alina-Elena; Benyhe, Sandor; Toth, Geza; Borsodi, Anna; Nagalla, Srinivasa R

    2003-12-26

    Opioid-like peptides mediate analgesia and induce behavioral effects such as tolerance and dependence by ligand-receptor-mediated mechanisms. The classical opioid prohormones can generate several bioactive peptides, and these divergent families of prohormones share a common well conserved ancestral opioid motif (Tyr-Gly-Gly-Phe). Evidence from pharmacological and molecular cloning studies indicates the presence of multiple isoforms of opioid ligands and receptors that are as yet uncharacterized. To identify potential new members we used the opioid motif as an anchor sequence and isolated two distinct isoforms (Xen-dorphins A and B) of an opioid prohormone from Xenopus laevis brain cDNA library. Xen-dorphin prohormones can generate multiple novel opioid ligands distinct from the known members of this family. Both isoforms are present in a wide variety of tissues including the brain. Two potential bioactive peptides, Xen-dorphin-1A and -1B, that were chemically synthesized showed opioid agonist activity in frog and rat brain membranes using a [35S]GTPgammaS assay. Initial radioligand binding experiments demonstrated that Xen-dorphin-1B binds with high affinity to opioid receptor(s) and with potential preference to the kappa-opioid receptor subtype. Cloning of the Xen-dorphin prohormone provides new evidence for the potential presence of other members in the opioid peptide superfamily.

  5. Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

    Science.gov (United States)

    Sutton, F.; Paul, S. S.; Wang, X. Q.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

  6. Tone and call responses of units in the auditory nerve and dorsal medullary nucleus of Xenopus laevis

    DEFF Research Database (Denmark)

    Elliott, Taffeta M.; Christensen-Dalsgaard, Jakob; Kelley, Darcy B.

    2007-01-01

    those in partially terrestrial anurans. Broad tuning exists across characteristic frequencies (CFs). Threshold minima are -101 dB re 1 mm/s at 675 Hz; -87 dB at 1,600 Hz; and -61 dB at 3,000 Hz (-90, -77, and -44 dB re 1 Pa, respectively), paralleling the peak frequency of vocalizations at 1.2-1.6 k......The clawed frog Xenopus laevis produces vocalizations consisting of distinct patterns of clicks. This study provides the first description of spontaneous, pure-tone and communication-signal evoked discharge properties of auditory nerve (n.VIII) fibers and dorsal medullary nucleus (DMN) cells...... in an obligatorily aquatic anuran. Responses of 297 n.VIII and 253 DMN units are analyzed for spontaneous rates (SR), frequency tuning, rate-intensity functions, and firing rate adaptation, with a view to how these basic characteristics shape responses to recorded call stimuli. Response properties generally resemble...

  7. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration.

    Directory of Open Access Journals (Sweden)

    Kazumasa Mitogawa

    Full Text Available Axolotls (Ambystoma mexicanum can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity.

  8. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration.

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    Mitogawa, Kazumasa; Makanae, Aki; Satoh, Ayano; Satoh, Akira

    2015-01-01

    Axolotls (Ambystoma mexicanum) can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity.

  9. Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis.

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    Tietge, Joseph E; Butterworth, Brian C; Haselman, Jonathan T; Holcombe, Gary W; Hornung, Michael W; Korte, Joseph J; Kosian, Patricia A; Wolfe, Marilyn; Degitz, Sigmund J

    2010-06-01

    Thyroid axis disruption is an important consideration when evaluating risks associated with chemicals. Bioassay methods that include thyroid-related endpoints have been developed in a variety of species, including amphibians, whose metamorphic development is thyroid hormone (TH)-dependent. Inhibition of TH synthesis in these species leads to developmental delay, and assays designed to capture these effects take several weeks to complete. In an effort to develop a shorter term approach, the early responses of various endpoints were evaluated in Xenopus laevis throughout 8d of exposure to three TH synthesis inhibitors: methimazole (100mg/L), 6-propylthiouracil (6-PTU) (20mg/L), and perchlorate (4 mg/L). Endpoints included thyroid gland histology and cell numbers, circulating TH concentrations, and thyroidal TH and associated iodo-compounds. Thyroidal 3,5-diodo-L-tyrosine (DIT) and thyroxine (T4) were significantly reduced from day 2 onward by all three chemicals, while 3-monoiodo-L-tyrosine (MIT) was significantly reduced by methimazole and perchlorate, but not by 6-PTU. These reductions were the earliest indicators of TH synthesis inhibition. Histological effects were apparent on day 4 and became more exaggerated through day 8. However, reductions in circulating T4 and increases in thyroid gland cell numbers were not apparent until day 6. Reductions of thyroidal MIT, DIT, and T4 and circulating T4 are indicative of inhibitory effects of the chemicals on TH synthesis. Changes in thyroid histology and cell number represent compensatory effects modulated by circulating TSH. These observations establish a basis for the development of short term amphibian-based methods to evaluate thyroid axis effects using a suite of diagnostic endpoints.

  10. Twitch and tetanic tension during culture of mature Xenopus laevis single muscle fibres.

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    Jaspers, R T; Feenstra, H M; Lee- de Groot, M B; Huijing, P A; van der Laarse, W J

    2001-12-01

    Investigation of the mechanisms of muscle adaptation requires independent control of the regulating factors. The aim of the present study was to develop a serum-free medium to culture mature single muscle fibres of Xenopus laevis. As an example, we used the culture system to study adaptation of twitch and tetanic force characteristics, number of sarcomeres in series and fibre cross-section. Fibres dissected from m. iliofibularis (n = 10) were kept in culture at a fibre mean sarcomere length of 2.3 microm in a culture medium without serum. Twitch and tetanic tension were determined daily. Before and after culture the number of sarcomeres was determined by laser diffraction and fibre cross-sectional area (CSA) was determined by microscopy. For five fibres twitch tension increased during culture and tetanic tension was stable for periods varying from 8 to 14 days ('stable fibres'), after which fibres were removed from culture for analysis. Fibre CSA and the number of sarcomeres in series remained constant during culture. Five other fibres showed a substantial reduction in twitch and tetanic tension within the first five days of culture ('unstable fibres'). After 7-9 days of culture, three of these fibres died. For two of the unstable fibres, after the substantial force reduction, twitch and tetanic tension increased again. Finally at day 14 and 18 of culture, respectively, the tensions attained values higher than their original values. For stable fibres, twitch contraction time, twitch half-relaxation time and tetanus 10%-relaxation time increased during culture. For unstable fibres these parameters fluctuated. For all fibres the stimulus threshold fluctuated during the first two days, and then remained constant, even for the fibres that were cultured for at least two weeks. It is concluded that the present culture system for mature muscle fibres allows long-term studies within a well-defined medium. Unfortunately, initial tetanic and twitch force are poor predictors

  11. Characterization of the hypothalamus of Xenopus laevis during development. I. The alar regions.

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    Domínguez, Laura; Morona, Ruth; González, Agustín; Moreno, Nerea

    2013-03-01

    The patterns of expression of a set of conserved developmental regulatory transcription factors and neuronal markers were analyzed in the alar hypothalamus of Xenopus laevis throughout development. Combined immunohistochemical and in situ hybridization techniques were used for the identification of subdivisions and their boundaries. The alar hypothalamus was located rostral to the diencephalon in the secondary prosencephalon and represents the rostral continuation of the alar territories of the diencephalon and brainstem, according to the prosomeric model. It is composed of the supraoptoparaventricular (dorsal) and the suprachiasmatic (ventral) regions, and limits dorsally with the preoptic region, caudally with the prethalamic eminence and the prethalamus, and ventrally with the basal hypothalamus. The supraoptoparaventricular area is defined by the orthopedia (Otp) expression and is subdivided into rostral and caudal portions, on the basis of the Nkx2.2 expression only in the rostral portion. This region is the source of many neuroendocrine cells, primarily located in the rostral subdivision. The suprachiasmatic region is characterized by Dll4/Isl1 expression, and was also subdivided into rostral and caudal portions, based on the expression of Nkx2.1/Nkx2.2 and Lhx1/7 exclusively in the rostral portion. Both alar regions are mainly connected with subpallial areas strongly implicated in the limbic system and show robust intrahypothalamic connections. Caudally, both regions project to brainstem centers and spinal cord. All these data support that in terms of topology, molecular specification, and connectivity the subdivisions of the anuran alar hypothalamus possess many features shared with their counterparts in amniotes, likely controlling similar reflexes, responses, and behaviors. Copyright © 2012 Wiley Periodicals, Inc.

  12. Developmental segregation of spinal networks driving axial- and hindlimb-based locomotion in metamorphosing Xenopus laevis.

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    Combes, D; Merrywest, S D; Simmers, J; Sillar, K T

    2004-08-15

    Amphibian metamorphosis includes a complete reorganization of an organism's locomotory system from axial-based swimming in larvae to limbed propulsion in the young adult. At critical stages during this behavioural switch, larval and adult motor systems operate in the same animal, commensurate with a gradual and dynamic reconfiguration of spinal locomotor circuitry. To study this plasticity, we have developed isolated preparations of the spinal cord and brainstem from pre- to post-metamorphic stages of the amphibian Xenopus laevis, in which spinal motor output patterns expressed spontaneously or in the presence of NMDA correlate with locomotor behaviour in the freely swimming animal. Extracellular ventral root recordings along the spinal cord of pre-metamorphic tadpoles revealed motor output corresponding to larval axial swimming, whereas postmetamorphic animals expressed motor patterns appropriate for bilaterally synchronous hindlimb flexion-extension kicks. However, in vitro recordings from metamorphic climax stages, with the tail and the limbs both functional, revealed two distinct motor patterns that could occur either independently or simultaneously, albeit at very different frequencies. Activity at 0.5-1 Hz in lumbar ventral roots corresponded to bipedal extension-flexion cycles, while the second, faster pattern (2-5 Hz) recorded from tail ventral roots corresponded to larval-like swimming. These data indicate that at intermediate stages during metamorphosis separate networks, one responsible for segmentally organized axial locomotion and another for more localized appendicular rhythm generation, coexist in the spinal cord and remain functional after isolation in vitro. These preparations now afford the opportunity to explore the cellular basis of locomotor network plasticity and reconfiguration necessary for behavioural changes during development.

  13. Characterization of the hypothalamus of Xenopus laevis during development. II. The basal regions.

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    Domínguez, Laura; González, Agustín; Moreno, Nerea

    2014-04-01

    The expression patterns of conserved developmental regulatory transcription factors and neuronal markers were analyzed in the basal hypothalamus of Xenopus laevis throughout development by means of combined immunohistochemical and in situ hybridization techniques. The connectivity of the main subdivisions was investigated by in vitro tracing techniques with dextran amines. The basal hypothalamic region is topologically rostral to the basal diencephalon and is composed of the tuberal (rostral) and mammillary (caudal) subdivisions, according to the prosomeric model. It is dorsally bounded by the optic chiasm and the alar hypothalamus, and caudally by the diencephalic prosomere p3. The tuberal hypothalamus is defined by the expression of Nkx2.1, xShh, and Isl1, and rostral and caudal portions can be distinguished by the distinct expression of Otp rostrally and Nkx2.2 caudally. In the mammillary region the xShh/Nkx2.1 combination defined the rostral mammillary area, expressing Nkx2.1, and the caudal retromammillary area, expressing xShh. The expression of xLhx1, xDll4, and Otp in the mammillary area and Isl1 in the tuberal region highlights the boundary between the two basal hypothalamic territories. Both regions are strongly connected with subpallial regions, especially those conveying olfactory/vomeronasal information, and also possess abundant intrahypothalamic connections. They show reciprocal connections with the diencephalon (mainly the thalamus), project to the midbrain tectum, and are bidirectionally related to the rhombencephalon. These results illustrate that the basal hypothalamus of anurans shares many features of specification, regionalization, and hodology with amniotes, reinforcing the idea of a basic bauplan in the organization of this prosencephalic region in all tetrapods. Copyright © 2013 Wiley Periodicals, Inc.

  14. Regional expression of Pax7 in the brain of Xenopus laevis during embryonic and larval development

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    Sandra eBandín

    2013-12-01

    Full Text Available Pax7 is a member of the highly conserved Pax gene family that is expressed in restricted zones of the central nervous system during development, being involved in early brain regionalization and the maintenance of the regional identity. Using sensitive immunohistochemical techniques we have analyzed the spatiotemporal pattern of Pax7 expression in the brain of the anuran amphibian Xenopus laevis, during development. Pax7 expression was first detected in early embryos in the basal plate of prosomere 3, roof and alar plates of prosomere 1 and mesencephalon, and the alar plate of rhombomere 1. As development proceeded, Pax7 cells were observed in the hypothalamus close to the catecholaminergic population of the mammillary region. In the diencephalon, Pax7 was intensely expressed in a portion of the basal plate of prosomere 3, in the roof plate and in scattered cells of the thalamus in prosomere 2, throughout the roof of prosomere 1, and in the commissural and juxtacommissural domains of the pretectum. In the mesencephalon, Pax7 cells were localized in the optic tectum and, to a lesser extent, in the torus semicircularis. The rostral portion of the alar part of rhombomere 1, including the ventricular layer of the cerebellum, expressed Pax7 and, gradually, some of these dorsal cells were observed to populate ventrally the interpeduncular nucleus and the isthmus (rhombomere 0. Additionally, Pax7 positive cells were found in the ventricular zone of the ventral part of the alar plate along the rhombencephalon and the spinal cord. The findings show that the strongly conserved features of Pax7 expression through development shared by amniote vertebrates are also present in the anamniote amphibians as a common characteristic of the brain organization of tetrapods.

  15. Differential regulation of potassium currents by FGF-1 and FGF-2 in embryonic Xenopus laevis myocytes.

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    Chauhan-Patel, R; Spruce, A E

    1998-10-01

    1. Fibroblast growth factors (FGFs) are involved in the regulation of many aspects of muscle development. This study investigated their role in regulating voltage-dependent K+ currents in differentiating Xenopus laevis myocytes. Both FGF-1 and FGF-2 are expressed by developing muscle cells, so their actions were compared. Experiments were performed on cultured myocytes isolated from stage 15 embryos. 2. Long-term exposure of the embryonic myocytes to FGF-1 downregulated inward rectifier K+ current (IK(IR)) density as well as both sustained and inactivating voltage-dependent outward K+ currents (IK,S and IK,I, respectively) and their densities. In contrast, FGF-2 upregulated these currents, although, because of an increase in capacitance caused by FGF-2, current density did not change with this factor. 3. The regulation of IK(IR) by FGF-1 was prevented by the cytoplasmic tyrosine kinase inhibitor herbimycin A, but that of IK,S and IK,I was unaffected, indicating that FGF-1 achieves its regulatory effects on electrical development via separate signalling pathways. The receptor tyrosine kinase inhibitor genistein in isolation suppressed K+ currents, but this may have occurred through a channel-blocking mechanism. 4. In many cells, IK, S was found to be composed of two components with differing voltage dependencies of activation. The FGFs brought about an alteration in the amount of total IK,S by equal effects on each component. Conversely, herbimycin A increased the proportion of low voltage-activated current without affecting total current amplitude. Therefore, we suggest that a single species of channel whose voltage dependence is shifted by tyrosine phosphorylation generates IK,S. 5. In summary, FGF-1 and FGF-2 exert opposite effects on voltage-dependent K+ currents in embryonic myocytes and, furthermore, FGF-1 achieves its effects on different K+ currents via separate second messenger pathways.

  16. Prolonged vestibular stimulation induces homeostatic plasticity of the vestibulo-ocular reflex in larval Xenopus laevis.

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    Dietrich, Haike; Straka, Hans

    2016-07-01

    Vestibulo-ocular reflexes (VOR) stabilise retinal images during head/body motion in vertebrates by generating spatio-temporally precise extraocular motor commands for corrective eye movements. While VOR performance is generally robust with a relatively stable gain, cerebellar circuits are capable of adapting the underlying sensory-motor transformation. Here, we studied cerebellum-dependent VOR plasticity by recording head motion-induced lateral rectus and superior oblique extraocular motor discharge in semi-intact preparations of Xenopus laevis tadpoles. In the absence of visual feedback, prolonged sinusoidal rotation caused either an increase or decrease of the VOR gain depending on the motion stimulus amplitude. The observed changes in extraocular motor discharge gradually saturated after 20 min of constant rotation and returned to baseline in the absence of motion stimulation. Furthermore, plastic changes in lateral rectus and superior oblique motor commands were plane-specific for horizontal and vertical rotations, respectively, suggesting that alterations are restricted to principal VOR connections. Comparison of multi- and single-unit activity indicated that plasticity occurs in all recorded units of a given extraocular motor nucleus. Ablation of the cerebellum abolished motoneuronal gain changes and prevented the induction of plasticity, thus demonstrating that both acquisition and retention of this type of plasticity require an intact cerebellar circuitry. In conclusion, the plane-specific and stimulus intensity-dependent modification of the VOR gain through the feed-forward cerebellar circuitry represents a homeostatic plasticity that likely maintains an optimal working range for the underlying sensory-motor transformation.

  17. Induction of cardiac muscle differentiation in isolated animal pole explants of Xenopus laevis embryos.

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    Logan, M; Mohun, T

    1993-07-01

    We have isolated a cDNA fragment encoding a portion of the myosin heavy chain alpha-isoform (XMHC alpha) in the amphibian, Xenopus laevis. The XMHC alpha transcript is highly enriched in adult heart RNA and is expressed exclusively in embryonic heart tissue. It therefore provides a tissue-specific marker for cardiac muscle differentiation during early embryogenesis. Using an RNAase protection assay, we can detect the onset of cardiac muscle differentiation in an anterior, ventral region of tailbud embryos, many hours before the appearance of a beating heart. Whole-mount in situ RNA hybridisation indicates that expression of the XMHC alpha gene is restricted to the developing heart primordium. XMHC alpha gene expression can also be induced in isolated animal pole explants of blastulae by treatment with the growth factor, activin A. Induction is dose-dependent, requiring high doses of the growth factor compared with that required for myotomal (skeletal) muscle differentiation. In contrast, no XMHC alpha transcripts are detected in explants incubated with basic FGF, despite the induction of myotomal muscle differentiation. Activin-induced explants show a similar temporal pattern of XMHC alpha gene expression to that found in normal embryogenesis. Furthermore, cells expressing this gene appear clustered in one or two foci within fused explant aggregates, which often show regular, spontaneous contractions after several days in culture. These results show that terminal differentiation of cardiac muscle can occur in growth factor-induced explants and may be distinguished from skeletal muscle differentiation by the dose and nature of the inducing factor.

  18. CFTR channel in oocytes from Xenopus laevis and its regulation by xShroom1 protein.

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    Palma, Alejandra G; Galizia, Luciano; Kotsias, Basilio A; Marino, Gabriela I

    2016-05-01

    Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in Xenopus laevis oocytes, and it is required for the expression of the epithelial sodium channel (ENaC). As there is a close relationship between ENaC and the cystic fibrosis transmembrane regulator (CFTR), we examined the action of xShroom1 on CFTR expression and activity. Biotinylation was used to measure CFTR surface expression, and currents were registered with voltage clamp when stimulated with forskolin and 3-isobutyl-1-methylxanthine. Oocytes were coinjected with CFTR complementary RNAs (cRNAs) and xShroom1 sense or antisense oligonucleotides. We observed an increment in CFTR currents and CFTR surface expression in oocytes coinjected with CFTR and xShroom1 antisense oligonucleotides. MG-132, a proteasome inhibitor, did not prevent the increment in currents when xShroom1 was suppressed by antisense oligonucleotides. In addition, we inhibited the delivery of newly synthesized proteins to the plasma membrane with BFA and we found that the half-life of plasma membrane CFTR was prolonged when coinjected with the xShroom1 antisense oligonucleotides. Chloroquine, an inhibitor of the late endosome/lysosome, did not significantly increase CFTR currents when xShroom1 expression was inhibited. The higher expression of CFTR when xShroom1 is suppressed is in concordance with the functional studies suggesting that the suppression of the xShroom1 protein resulted in an increment in CFTR currents by promoting the increase of the half-life of CFTR in the plasma membrane. The role of xShroom1 in regulating CFTR expression could be relevant in the understanding of the channel malfunction in several diseases.

  19. Early changes in the ultrastructure of the pars intermedia of the pituitary of Xenopus laevis after change of background color.

    Science.gov (United States)

    Volcanes, B D; Weatherhead, B

    1976-01-01

    Stereological analysis of the secretory cells of the pars intermedia of Xenopus laevis over a period of 3 days following the transfer of animals from a white to a black background has revealed that significant alterations in the ultrastructural appearance of these cells can be detected 8 h after the transfer. In particular, changes in the secretory granules and the rough endoplasmic reticulum were found to correlate well with previous reports concerning the melanocyte-stimulating hormone (MSH) content and the capacity for protein synthesis of the pars intermedia.

  20. Transcription of the 5S rRNA heterochromatic genes is epigenetically controlled in Arabidopsis thaliana and Xenopus laevis.

    Science.gov (United States)

    Douet, J; Tourmente, S

    2007-07-01

    5S ribosomal DNA is a highly conserved tandemly repeated multigenic family. As suggested for a long time, we have shown that only a fraction of the 5S rRNA genes are expressed in Arabidopsis thaliana. In Xenopus laevis, there is a developmental control of the expression of the 5S rRNA genes with only one of the two 5S rDNA families expressed during oogenesis. For both Arabidopsis and Xenopus, the strongest transcription of 5S rRNA, respectively in the seed and during oogenesis is correlated with heterogeneity in the transcribed 5S rRNAs. Epigenetic mechanisms such as modification of the chromatin structure are involved in the transcriptional regulation of the 5S rRNA genes in both organisms. In Arabidopsis, two silencing pathways, methylation-dependent (RNAi) and methylation-independent (MOM pathway), are involved in the silencing of a 5S rDNA fraction.

  1. Cdc42 Effector Protein 2 (XCEP2 is required for normal gastrulation and contributes to cellular adhesion in Xenopus laevis

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    Nelson Richard W

    2004-10-01

    Full Text Available Abstract Background Rho GTPases and their downstream effector proteins regulate a diverse array of cellular processes during embryonic development, including reorganization of cytoskeletal architecture, cell adhesion, and transcription. Changes in the activation state of Rho GTPases are converted into changes in cellular behavior by a diversity of effector proteins, which are activated in response to changes in the GTP binding state of Rho GTPases. In this study we characterize the expression and function of one such effector, XCEP2, that is present during gastrulation stages in Xenopus laevis. Results In a search for genes whose expression is regulated during early stages of embryonic development in Xenopus laevis, a gene encoding a Rho GTPase effector protein (Xenopus Cdc42 effector protein 2, or XCEP2 was isolated, and found to be highly homologous, but not identical, to a Xenopus sequence previously submitted to the Genbank database. These two gene sequences are likely pseudoalleles. XCEP2 mRNA is expressed at constant levels until mid- to late- gastrula stages, and then strongly down-regulated at late gastrula/early neurula stages. Injection of antisense morpholino oligonucleotides directed at one or both pseudoalleles resulted in a significant delay in blastopore closure and interfered with normal embryonic elongation, suggesting a role for XCEP2 in regulating gastrulation movements. The morpholino antisense effect could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA. Antisense morpholino oligonucleotides were found to have no effect on mesodermal induction, suggesting that the observed effects were due to changes in the behavior of involuting cells, rather than alterations in their identity. XCEP2 antisense morpholino oligonucleotides were also observed to cause complete disaggregation of cells composing animal cap explants, suggesting a specific role of XCEP2 in maintenance or regulation of cell

  2. Organophosphate pesticides induce morphological abnormalities and decrease locomotor activity and heart rate in Danio rerio and Xenopus laevis.

    Science.gov (United States)

    Watson, Fiona L; Schmidt, Hayden; Turman, Zackery K; Hole, Natalie; Garcia, Hena; Gregg, Jonathan; Tilghman, Joseph; Fradinger, Erica A

    2014-06-01

    Organophosphate pesticides (OPs), a class of acetylcholinesterase inhibitors, are used widely in agriculture to reduce insect populations. Because of the conservation of acetylcholinesterase between invertebrates and vertebrates, OPs also can adversely affect nontarget species, such as aquatic and terrestrial animals. This study used uniform conditions to analyze the morphological and physiological effects caused by developmental exposure to 3 commonly used OPs-chlorpyrifos, dichlorvos, and diazinon-on 2 aquatic vertebrate species, Danio rerio (zebrafish) and Xenopus laevis. Survival, locomotor activity, heart rate, and gross anatomical abnormalities, including kyphosis and edema, were observed over a 5-d period in response to OP concentrations ranging from 0 µM to 1000 µM. Both zebrafish and Xenopus showed decreased survival for all 3 OPs at higher concentrations. However, Xenopus showed higher mortality than zebrafish at lower chlorpyrifos and dichlorvos concentrations. Both models showed a dose-dependent decrease in heart rate and free-swimming larval activity in response to chlorpyrifos and dichlorvos. In addition, kyphosis and decreased spine length were prominent in Xenopus in response to 10 µM of chlorpyrifos and 0.1 µM dichlorvos. Although diazinon induced no effects on skeletal and cardiac motor activity in either species, it did induce cardiac edemas in zebrafish. Differences in the biological actions of OPs and their differential effects in these 2 vertebrate models demonstrate the importance of using common protocols and multiple models to evaluate the ecotoxicology of OPs.

  3. Transcription factor COUP-TFII is indispensable for venous and lymphatic development in zebrafish and Xenopus laevis

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    Aranguren, Xabier L., E-mail: xabier.lopezaranguren@med.kuleuven.be [Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Campus Gasthuisberg, Onderwijs and Navorsing 1, Herestraat 49, B-3000 Leuven (Belgium); Beerens, Manu, E-mail: manu.beerens@med.kuleuven.be [Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Campus Gasthuisberg, Onderwijs and Navorsing 1, Herestraat 49, B-3000 Leuven (Belgium); Vandevelde, Wouter, E-mail: woutervandevelde@gmail.com [Vesalius Research Center, VIB, Campus Gasthuisberg, Onderwijs and Navorsing 1, Herestraat 49, B-3000 Leuven (Belgium); Vesalius Research Center, Katholieke Universiteit Leuven, Campus Gasthuisberg, Onderwijs and Navorsing 1, Herestraat 49, B-3000 Leuven (Belgium); Dewerchin, Mieke, E-mail: mieke.dewerchin@vib-kuleuven.be [Vesalius Research Center, VIB, Campus Gasthuisberg, Onderwijs and Navorsing 1, Herestraat 49, B-3000 Leuven (Belgium); Vesalius Research Center, Katholieke Universiteit Leuven, Campus Gasthuisberg, Onderwijs and Navorsing 1, Herestraat 49, B-3000 Leuven (Belgium); Carmeliet, Peter, E-mail: peter.carmeliet@vib-kuleuven.be [Vesalius Research Center, VIB, Campus Gasthuisberg, Onderwijs and Navorsing 1, Herestraat 49, B-3000 Leuven (Belgium); Vesalius Research Center, Katholieke Universiteit Leuven, Campus Gasthuisberg, Onderwijs and Navorsing 1, Herestraat 49, B-3000 Leuven (Belgium); Luttun, Aernout, E-mail: aernout.luttun@med.kuleuven.be [Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Campus Gasthuisberg, Onderwijs and Navorsing 1, Herestraat 49, B-3000 Leuven (Belgium)

    2011-06-24

    Highlights: {yields} COUP-TFII deficiency in zebrafish affects arterio-venous EC specification. {yields} COUP-TFII is indispensable for lymphatic development in zebrafish. {yields} COUP-TFII knockdown in Xenopus disrupts lymphatic EC differentiation and migration. {yields} COUP-TFII's role in EC fate decisions is evolutionary conserved. -- Abstract: Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment of venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.

  4. A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity.

    Science.gov (United States)

    Wang, Yu-Wei; Tan, Ji-Min; Du, Can-Wei; Luan, Ning; Yan, Xiu-Wen; Lai, Ren; Lu, Qiu-Min

    2015-08-01

    Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.

  5. The B-subdomain of the Xenopus laevis XFIN KRAB-AB domain is responsible for its weaker transcriptional repressor activity compared to human ZNF10/Kox1.

    Directory of Open Access Journals (Sweden)

    Nadine Born

    Full Text Available The Krüppel-associated box (KRAB domain interacts with the nuclear hub protein TRIM28 to initiate or mediate chromatin-dependent processes like transcriptional repression, imprinting or suppression of endogenous retroviruses. The prototype KRAB domain initially identified in ZNF10/KOX1 encompasses two subdomains A and B that are found in hundreds of zinc finger transcription factors studied in human and murine genomes. Here we demonstrate for the first time transcriptional repressor activity of an amphibian KRAB domain. After sequence correction, the updated KRAB-AB domain of zinc finger protein XFIN from the frog Xenopus laevis was found to confer transcriptional repression in reporter assays in Xenopus laevis A6 kidney cells as well as in human HeLa, but not in the minnow Pimephales promelas fish cell line EPC. Binding of the XFIN KRAB-AB domain to human TRIM28 was demonstrated in a classical co-immunoprecipitation approach and visualized in a single-cell compartmentalization assay. XFIN-AB displayed reduced potency in repression as well as lower strength of interaction with TRIM28 compared to ZNF10 KRAB-AB. KRAB-B subdomain swapping between the two KRAB domains indicated that it was mainly the KRAB-B subdomain of XFIN that was responsible for its lower capacity in repression and binding to human TRIM28. In EPC fish cells, ZNF10 and XFIN KRAB repressor activity could be partially restored to low levels by adding exogenous human TRIM28. In contrast to XFIN, we did not find any transcriptional repression activity for the KRAB-like domain of human PRDM9 in HeLa cells. PRDM9 is thought to harbor an evolutionary older domain related to KRAB whose homologs even occur in invertebrates. Our results support the notion that functional bona fide KRAB domains which confer transcriptional repression and interact with TRIM28 most likely co-evolved together with TRIM28 at the beginning of tetrapode evolution.

  6. Regulation of Melanopsins and Per1 by α-MSH and Melatonin in Photosensitive Xenopus laevis Melanophores

    Directory of Open Access Journals (Sweden)

    Maria Nathália de Carvalho Magalhães Moraes

    2014-01-01

    Full Text Available α-MSH and light exert a dispersing effect on pigment granules of Xenopus laevis melanophores; however, the intracellular signaling pathways are different. Melatonin, a hormone that functions as an internal signal of darkness for the organism, has opposite effects, aggregating the melanin granules. Because light functions as an important synchronizing signal for circadian rhythms, we further investigated the effects of both hormones on genes related to the circadian system, namely, Per1 (one of the clock genes and the melanopsins, Opn4x and Opn4m (photopigments. Per1 showed temporal oscillations, regardless of the presence of melatonin or α-MSH, which slightly inhibited its expression. Melatonin effects on melanopsins depend on the time of application: if applied in the photophase it dramatically decreased Opn4x and Opn4m expressions, and abolished their temporal oscillations, opposite to α-MSH, which increased the melanopsins’ expressions. Our results demonstrate that unlike what has been reported for other peripheral clocks and cultured cells, medium changes or hormones do not play a major role in synchronizing the Xenopus melanophore population. This difference is probably due to the fact that X. laevis melanophores possess functional photopigments (melanopsins that enable these cells to primarily respond to light, which triggers melanin dispersion and modulates gene expression.

  7. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

    Science.gov (United States)

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-03-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.

  8. Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.

    Science.gov (United States)

    Sakane, Yuto; Sakuma, Tetsushi; Kashiwagi, Keiko; Kashiwagi, Akihiko; Yamamoto, Takashi; Suzuki, Ken-Ichi T

    2014-01-01

    Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis.

  9. A perchlorate sensitive iodide transporter in frogs

    OpenAIRE

    Carr, Deborah L.; James A. Carr; Ray E. Willis; Pressley, Thomas A.

    2008-01-01

    Nucleotide sequence comparisons have identified a gene product in the genome database of African clawed frogs (Xenopus laevis) as a probable member of the solute carrier family of membrane transporters. To confirm its identity as a putative iodide transporter, we examined the function of this sequence after heterologous expression in mammalian cells. A green monkey kidney cell line transfected with the Xenopus nucleotide sequence had significantly greater 125I uptake than sham-transfected con...

  10. Multiscale computational analysis of Xenopus laevis morphogenesis reveals key insights of systems-level behavior

    Directory of Open Access Journals (Sweden)

    DeSimone Douglas W

    2007-10-01

    Full Text Available Abstract Background Tissue morphogenesis is a complex process whereby tissue structures self-assemble by the aggregate behaviors of independently acting cells responding to both intracellular and extracellular cues in their environment. During embryonic development, morphogenesis is particularly important for organizing cells into tissues, and although key regulatory events of this process are well studied in isolation, a number of important systems-level questions remain unanswered. This is due, in part, to a lack of integrative tools that enable the coupling of biological phenomena across spatial and temporal scales. Here, we present a new computational framework that integrates intracellular signaling information with multi-cell behaviors in the context of a spatially heterogeneous tissue environment. Results We have developed a computational simulation of mesendoderm migration in the Xenopus laevis explant model, which is a well studied biological model of tissue morphogenesis that recapitulates many features of this process during development in humans. The simulation couples, via a JAVA interface, an ordinary differential equation-based mass action kinetics model to compute intracellular Wnt/β-catenin signaling with an agent-based model of mesendoderm migration across a fibronectin extracellular matrix substrate. The emergent cell behaviors in the simulation suggest the following properties of the system: maintaining the integrity of cell-to-cell contact signals is necessary for preventing fractionation of cells as they move, contact with the Fn substrate and the existence of a Fn gradient provides an extracellular feedback loop that governs migration speed, the incorporation of polarity signals is required for cells to migrate in the same direction, and a delicate balance of integrin and cadherin interactions is needed to reproduce experimentally observed migratory behaviors. Conclusion Our computational framework couples two different

  11. cis- and trans-acting elements of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis.

    Science.gov (United States)

    Wahli, W; Martinez, E; Corthésy, B; Cardinaux, J R

    1989-01-01

    Vitellogenin genes are expressed under strict estrogen control in the liver of female oviparous vertebrates. Gene transfer experiments using estrogen-responsive cells have shown that the 13 bp perfect palindromic element GGTCACTGTGACC found upstream of the Xenopus laevis vitellogenin gene A2 promoter mediates hormonal stimulation and thus, was called the estrogen-responsive element (ERE). In the Xenopus vitellogenin genes B1 and B2 there are two closely adjacent EREs with one or more base substitutions when compared to the consensus ERE GGTCANNNTGACC. On their own, these degenerated elements have only a low or no regulatory capacity at all but act together synergistically to form an estrogen-responsive unit (ERU) with the same strength as the perfect palindromic 13 bp element. Analysis of estrogen receptor binding to the gene B1 ERU revealed a cooperative interaction of receptor dimers to the two adjacent imperfect EREs which most likely explains the synergistic stimulation observed in vivo. Furthermore, a promoter activator element located between positions --113 and --42 of the gene B1 and functional in the human MCF-7 and the Xenopus B3.2 cells has been identified and shown to be involved in the high level of induced transcription activity when the ERE is placed at a distance from the promoter. Finally, a hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to characterize two additional novel cis-acting elements within the vitellogenin gene B1 promoter. One of them, a negative regulatory element (NRE), is responsible for repression of promoter activity in the absence of hormone. The second is related to the NF-I binding site and is required, together with the ERE, to mediate hormonal induction. Moreover, we detected three trans-acting activities in Xenopus liver nuclear extracts that interact with these regions and demonstrated that they participate in the regulation of the expression of the vitellogenin

  12. Multiple noggins in vertebrate genome: cloning and expression of noggin2 and noggin4 in Xenopus laevis.

    Science.gov (United States)

    Eroshkin, Fedor M; Ermakova, Galina V; Bayramov, Andrey V; Zaraisky, Andrey G

    2006-01-01

    Noggin is a neural inducer secreted by cells of the Spemann organizer. A single noggin gene was identified until very recently in all tested vertebrates. The only exception was zebrafish, in which two close homologs of noggin, named noggin1 and noggin3, and one gene more diverged from them, noggin2, were cloned. Nevertheless, finding of three zebrafish noggins was attributed exclusively to specific genomic duplications in the fish evolutionary branch. However, very recently it was shown that Xenopus tropicalis have additional noggin homolog, called noggin2 [Fletcher, R.B., Watson, A.L., Harland, R.M. (2004). Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod. Gene Expr. Patterns 5, 225-230], which indicates at least two independent noggin genes in vertebrate phylum. Now we report identification of two novel noggin homologs in each of so evolutionary distant species as Xenopus laevis, chicken and fugu. One of these noggins is ortholog of the X. tropicalis and zebrafish noggin2, whereas another, named noggin4, was not known previously. In the X. laevis embryos, the expression of noggin2 very resembles that of its counterpart in X. tropicalis: it begins with neurulation at the anterior margin of the neural plate and, afterward, continues mainly in the forebrain and dorsal hindbrain. At the same time, noggin4 is expressed starting from the beginning of gastrulation, throughout the ectoderm, with a local expression maximum in the prospective anterior neurectoderm. Later, it is widely expressed on the dorsal side of embryo, including neural tube, eyes, otic vesicles, cranial placodes, branchial arches, and somites. The data presented here demonstrate that the vertebrate phylum contains at least three distinct noggin genes.

  13. Frog embryo teratogenesis assay on Xenopus and predictivity compared with in vivo mammalian studies.

    Science.gov (United States)

    Leconte, Isabelle; Mouche, Isabelle

    2013-01-01

    Frog embryo teratogenesis assay on Xenopus (FETAX) has been routinely used in our laboratory for the last 12 years as a routine developmental toxicity screening test for pharmaceutical candidate compounds. To date, out of more than 400 candidates tested in FETAX, around 60 have also been evaluated in mammalian embryotoxicity studies according to standard ICH protocols.Compound teratogenic potential in both FETAX and mammalian embryotoxicity studies is determined after analysis of the developmental toxicity characterized by embryotoxicity, growth delay, and/or potential to induce malformations. Based on this experience, the predictivity of FETAX is 81% with a minimal proportion of false positive results.

  14. Transformation of frog embryos with a rabbit beta-globin gene.

    OpenAIRE

    Rusconi, S; Schaffner, W

    1981-01-01

    In order to study the fate and possible expression of foreign DNA during embryogenesis of the frog Xenopus laevis, we have injected a rabbit beta-globin gene into fertilized Xenopus eggs. Frog embryo DNA was extracted at various stages of development, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filters, and hybridized to labeled beta-globin recombinant plasmid DNA. It was found that the injected DNA replicated extrachromosomally, reaching, at gastrula stage, a l...

  15. Extracts from plants used in Mexican traditional medicine activate Ca(2+)-dependent chloride channels in Xenopus laevis oocytes.

    Science.gov (United States)

    Rojas, A; Mendoza, S; Moreno, J; Arellano, R O

    2003-01-01

    The two-electrode voltage-clamp technique was employed to investigate the effects of chloroform-methanol (1:1) extracts derived from five medicinal plants on Xenopus laevis oocytes. When evaluated at concentrations of 1 to 500 microg/ml, the extracts prepared from the aerial parts of Baccharis heterophylla H.B.K (Asteraceae), Chenopodium murale L. (Chenopodiaceae), Desmodium grahami Gray (Leguminosae) and Solanum rostratum Dun (Solanaceae) produced concentration-dependent oscillatory inward currents in the oocytes, while the extract of Gentiana spathacea did not induce any response. The reversal potential of the currents elicited by the active extracts was -17 +/- 2 mV and was similar to the chloride equilibrium potential in oocytes. These ionic responses were independent of extracellular calcium. However, they were eliminated by overnight incubation with BAPTA-AM (10 microM), suggesting that the currents were dependent on intracellular Ca2+ increase. Thus the plant extracts activate the typical oscillatory Ca(2+)-dependent Cl- currents generated in the Xenopus oocyte membrane more probably via a mechanism that involves release of Ca2+ from intracellular reservoirs. These observations suggest that Xenopus oocyte electrophysiological recording constitutes a suitable assay for the study of the mechanisms of action of herbal medicines.

  16. A comparative analysis of frog early development.

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    del Pino, Eugenia M; Venegas-Ferrín, Michael; Romero-Carvajal, Andrés; Montenegro-Larrea, Paola; Sáenz-Ponce, Natalia; Moya, Iván M; Alarcón, Ingrid; Sudou, Norihiro; Yamamoto, Shinji; Taira, Masanori

    2007-07-17

    The current understanding of Xenopus laevis development provides a comparative background for the analysis of frog developmental modes. Our analysis of development in various frogs reveals that the mode of gastrulation is associated with developmental rate and is unrelated to egg size. In the gastrula of the rapidly developing embryos of the foam-nesting frogs Engystomops coloradorum and Engystomops randi, archenteron and notochord elongation overlapped with involution at the blastopore lip, as in X. laevis embryos. In embryos of dendrobatid frogs and in the frog without tadpoles Eleutherodactylus coqui, which develop somewhat more slowly than X. laevis, involution and archenteron elongation concomitantly occurred during gastrulation; whereas elongation of the notochord and, therefore, dorsal convergence and extension, occurred in the postgastrula. In contrast, in the slow developing embryos of the marsupial frog Gastrotheca riobambae, only involution occurred during gastrulation. The processes of archenteron and notochord elongation and convergence and extension were postgastrulation events. We produced an Ab against the homeodomain protein Lim1 from X. laevis as a tool for the comparative analysis of development. By the expression of Lim1, we were able to identify the dorsal side of the G. riobambae early gastrula, which otherwise was difficult to detect. Moreover, the Lim1 expression in the dorsal lip of the blastopore and notochord differed among the studied frogs, indicating variation in the timing of developmental events. The variation encountered gives evidence of the modular character of frog gastrulation.

  17. Xenopus laevis: an ideal experimental model for studying the developmental dynamics of neural network assembly and sensory-motor computations.

    Science.gov (United States)

    Straka, Hans; Simmers, John

    2012-04-01

    The amphibian Xenopus laevis represents a highly amenable model system for exploring the ontogeny of central neural networks, the functional establishment of sensory-motor transformations, and the generation of effective motor commands for complex behaviors. Specifically, the ability to employ a range of semi-intact and isolated preparations for in vitro morphophysiological experimentation has provided new insights into the developmental and integrative processes associated with the generation of locomotory behavior during changing life styles. In vitro electrophysiological studies have begun to explore the functional assembly, disassembly and dynamic plasticity of spinal pattern generating circuits as Xenopus undergoes the developmental switch from larval tail-based swimming to adult limb-based locomotion. Major advances have also been made in understanding the developmental onset of multisensory signal processing for reactive gaze and posture stabilizing reflexes during self-motion. Additionally, recent evidence from semi-intact animal and isolated CNS experiments has provided compelling evidence that in Xenopus tadpoles, predictive feed-forward signaling from the spinal locomotor pattern generator are engaged in minimizing visual disturbances during tail-based swimming. This new concept questions the traditional view of retinal image stabilization that in vertebrates has been exclusively attributed to sensory-motor transformations of body/head motion-detecting signals. Moreover, changes in visuomotor demands associated with the developmental transition in propulsive strategy from tail- to limb-based locomotion during metamorphosis presumably necessitates corresponding adaptive alterations in the intrinsic spinoextraocular coupling mechanism. Consequently, Xenopus provides a unique opportunity to address basic questions on the developmental dynamics of neural network assembly and sensory-motor computations for vertebrate motor behavior in general.

  18. RNA-seq in the tetraploid Xenopus laevis enables genome-wide insight in a classic developmental biology model organism.

    Science.gov (United States)

    Amin, Nirav M; Tandon, Panna; Osborne Nishimura, Erin; Conlon, Frank L

    2014-04-01

    Advances in sequencing technology have significantly advanced the landscape of developmental biology research. The dissection of genetic networks in model and non-model organisms has been greatly enhanced with high-throughput sequencing technologies. RNA-seq has revolutionized the ability to perform developmental biology research in organisms without a published genome sequence. Here, we describe a protocol for developmental biologists to perform RNA-seq on dissected tissue or whole embryos. We start with the isolation of RNA and generation of sequencing libraries. We further show how to interpret and analyze the large amount of sequencing data that is generated in RNA-seq. We explore the abilities to examine differential expression, gene duplication, transcript assembly, alternative splicing and SNP discovery. For the purposes of this article, we use Xenopus laevis as the model organism to discuss uses of RNA-seq in an organism without a fully annotated genome sequence. Copyright © 2013. Published by Elsevier Inc.

  19. Astrocytes phagocytose focal dystrophies from shortening myelin segments in the optic nerve of Xenopus laevis at metamorphosis.

    Science.gov (United States)

    Mills, Elizabeth A; Davis, Chung-ha O; Bushong, Eric A; Boassa, Daniela; Kim, Keun-Young; Ellisman, Mark H; Marsh-Armstrong, Nicholas

    2015-08-18

    Oligodendrocytes can adapt to increases in axon diameter through the addition of membrane wraps to myelin segments. Here, we report that myelin segments can also decrease their length in response to optic nerve (ON) shortening during Xenopus laevis metamorphic remodeling. EM-based analyses revealed that myelin segment shortening is accomplished by focal myelin-axon detachments and protrusions from otherwise intact myelin segments. Astrocyte processes remove these focal myelin dystrophies using known phagocytic machinery, including the opsonin milk fat globule-EGF factor 8 (Mfge8) and the downstream effector ras-related C3 botulinum toxin substrate 1 (Rac1). By the end of metamorphic nerve shortening, one-quarter of all myelin in the ON is enwrapped or internalized by astrocytes. As opposed to the removal of degenerating myelin by macrophages, which is usually associated with axonal pathologies, astrocytes selectively remove large amounts of myelin without damaging axons during this developmental remodeling event.

  20. Some effects of the venom of the Chilean spider Latrodectus mactans on endogenous ion-currents of Xenopus laevis oocytes.

    Science.gov (United States)

    Parodi, Jorge; Romero, Fernando; Miledi, Ricardo; Martínez-Torres, Ataúlfo

    2008-10-31

    A study was made of the effects of the venom of the Chilean spider Latrodectus mactans on endogenous ion-currents of Xenopus laevis oocytes. 1 microg/ml of the venom made the resting plasma membrane potential more negative in cells voltage-clamped at -60 mV. The effect was potentially due to the closure of one or several conductances that were investigated further. Thus, we determined the effects of the venom on the following endogenous ionic-currents: (a) voltage-activated potassium currents, (b) voltage-activated chloride-currents, and (c) calcium-dependent chloride-currents (Tout). The results suggest that the venom exerts its action mainly on a transient outward potassium-current that is probably mediated by a Kv channel homologous to shaker. Consistent with the electrophysiological evidence we detected the expression of the mRNA coding for xKv1.1 in the oocytes.

  1. In Situ Microprobe Single-Cell Capillary Electrophoresis Mass Spectrometry: Metabolic Reorganization in Single Differentiating Cells in the Live Vertebrate (Xenopus laevis) Embryo.

    Science.gov (United States)

    Onjiko, Rosemary M; Portero, Erika P; Moody, Sally A; Nemes, Peter

    2017-07-05

    Knowledge of single-cell metabolism would provide a powerful look into cell activity changes as cells differentiate to all the tissues of the vertebrate embryo. However, single-cell mass spectrometry technologies have not yet been made compatible with complex three-dimensional changes and rapidly decreasing cell sizes during early development of the embryo. Here, we bridge this technological gap by integrating capillary microsampling, microscale metabolite extraction, and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) to enable direct metabolic analysis of identified cells in the live frog embryo (Xenopus laevis). Microprobe CE-ESI-MS of cell content allowed us to detect ∼230 different molecular features (positive ion mode), including 70 known metabolites, in single dorsal and ventral cells in 8-to-32-cell embryos. Relative quantification followed by multivariate and statistical analysis of the data found that microsampling enhanced detection sensitivity compared to whole-cell dissection by minimizing chemical interferences and ion suppression effects from the culture media. In addition, higher glutathione/oxidized glutathione ratios suggested that microprobed cells exhibited significantly lower oxidative stress than those dissected from the embryo. Fast (5 s/cell) and scalable microsampling with minimal damage to cells in the 8-cell embryo enabled duplicate and triplicate metabolic analysis of the same cell, which surprisingly continued to divide to the 16-cell stage. Last, we used microprobe single-cell CE-ESI-MS to uncover previously unknown reorganization of the single-cell metabolome as the dorsal progenitor cell from the 8-cell embryo formed the neural tissue fated clone through divisions to the 32-cell embryo, peering, for the first time, into the formation of metabolic single-cell heterogeneity during early development of a vertebrate embryo.

  2. Effects of Endocrine Disruptors Ethinylestradiol and Procloraz on the vocal system of the frog Xenopus tropicalis

    DEFF Research Database (Denmark)

    Brande-Lavridsen, Nanna; Nørum, Ulrik; Korsgaard, Bodil

    2009-01-01

      Prochloraz masculinizes the larynx of female Xenopus tropicalis   Endogenous sex steroids are not only important for sexual differentiation in amphibians, but also for the development of secondary sex characteristics. The advertisement call of male frogs is used to attract females and in male...

  3. Symmetry breakage in the frog Xenopus: role of Rab11 and the ventral-right blastomere.

    Science.gov (United States)

    Tingler, Melanie; Ott, Tim; Tözser, Janos; Kurz, Sabrina; Getwan, Maike; Tisler, Matthias; Schweickert, Axel; Blum, Martin

    2014-06-01

    Vertebrates display asymmetric arrangements of inner organs such as heart and stomach. The Nodal signaling cascade in the left lateral plate mesoderm in all cases directs asymmetric morphogenesis and placement during organogenesis. Mechanisms that lead up to left-asymmetric Nodal induction seem to differ between the vertebrates. Cilia produce a leftward extracellular fluid flow in zebrafish, medaka, mouse, rabbit, and Xenopus embryos during neurulation. In Xenopus, earlier asymmetric cues were described. Some, such as Rab11, apparently act in the zygote. Others were efficiently manipulated in ventral-right cells at the four-cell stage, a lineage presumably independent of the ciliated gastrocoel roof plate (GRP) during neurulation. Here, we show that one- and four-cell manipulations of Rab11 showed equal low efficiencies of left-right disturbances. We also reevaluated the lineage of the GRP. By tracing back future ciliated cells from the gastrula to the four-cell stage, we show that ventral cells contribute to ciliated sensory cells at the border of the GRP. Knockdown of the Nodal inhibitor Coco in the ventral right lineage resulted in embryos with ectopic right-sided Nodal and Pitx2c expression. Together, these experiments support a cilia-based mechanism of symmetry breakage in the frog Xenopus.

  4. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos.

    Science.gov (United States)

    Yu, Shuangying; Tang, Song; Mayer, Gregory D; Cobb, George P; Maul, Jonathan D

    2015-02-01

    Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 μg/L endosulfan or 0, 2.5, and 5.0 μg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides increased transcript abundance of CSA and MUTL. In addition, mRNA abundance of HSP70 and GADD45α were increased by endosulfan and mRNA abundance of XPG was increased by α-cypermethrin. XPC, HR23B, XPG, and GADD45α exhibited elevated mRNA concentrations whereas there was a reduction in MUTL transcript concentrations in UVB-alone treatments. It appeared that even

  5. Differential expression of two TEF-1 (TEAD) genes during Xenopus laevis development and in response to inducing factors.

    Science.gov (United States)

    Naye, François; Tréguer, Karine; Soulet, Fabienne; Faucheux, Corinne; Fédou, Sandrine; Thézé, Nadine; Thiébaud, Pierre

    2007-01-01

    Transcription enhancer factors 1 (TEF-1 or TEAD) make a highly conserved family of eukaryotic DNA binding proteins that activate not only viral regulatory elements but muscle specific genes and are involved in several developmental processes. In this study, we report the identification and the expression pattern of NTEF-1 (TEAD1) and DTEF-1 (TEAD3), two members of this family in Xenopus laevis. Both X. laevis NTEF-1 (XNTEF-1 or XTEAD1) and DTEF-1 (XDTEF-1 or XTEAD3) possess a 72 amino acid TEA domain characteristic of TEF-1 proteins. XNTEF-1 is a 426 amino acid protein that has 96% identity with the avian or the mammalian NTEF-1 proteins while XDTEF-1 is a 433 amino acid protein with 77 to 80% identity with the avian and mammalian DTEF-1 sequences respectively. Temporal expression analysis by RT-PCR indicated that the two genes are expressed maternally and throughout embryonic development. In the adult, the two genes are broadly expressed although they showed differences of expression between tissues. Spatial expression analysis by whole mount in situ hybridization showed that the XNTEF-1 and XDTEF-1 mRNAS were predominantly detected in eye, embryonic brain, somites and heart. In animal cap assay, the two genes are activated by bFGF but are differently regulated by BMP4, and the muscle regulatory factor Mef2d.

  6. Environmental signals: synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis.

    Science.gov (United States)

    Lutz, Ilka; Jie, Zhang; Opitz, Robert; Kloas, Werner; Ying, Xu; Menzel, Ralph; Steinberg, Christian E W

    2005-12-01

    According to outdated paradigms humic substances (HS) are considered to be refractory or inert that do not directly interact with aquatic organisms. However, they are taken up and induce biotransformation activities and may act as hormone-like substances. In the present study, we tested whether HS can interfere with endocrine regulation in the amphibian Xenopus laevis. In order to exclude contamination with phyto-hormones, which may occur in environmental isolates, the artificial HS1500 was applied. The in vivo results showed that HS1500 causes significant estrogenic effects on X. laevis during its larval development and results of semi-quantitative RT-PCR revealed a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, preliminary RT-PCR results showed that the thyroid-stimulating hormone (TSHbeta-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Hence, HS may have estrogenic and anti-thyroidal effects on aquatic animals, and therefore may influence the structure of aquatic communities and they may be considered environmental signaling chemicals.

  7. Peter Pan functions independently of its role in ribosome biogenesis during early eye and craniofacial cartilage development in Xenopus laevis.

    Science.gov (United States)

    Bugner, Verena; Tecza, Aleksandra; Gessert, Susanne; Kühl, Michael

    2011-06-01

    The Xenopus oocyte possesses a large maternal store of ribosomes, thereby uncoupling early development from the de novo ribosome biosynthesis required for cell growth. Brix domain-containing proteins, such as Peter Pan (PPan), are essential for eukaryotic ribosome biogenesis. In this study, we demonstrate that PPan is expressed maternally as well as in the eye and cranial neural crest cells (NCCs) during early Xenopus laevis development. Depletion of PPan and interference with rRNA processing using antisense morpholino oligonucleotides resulted in eye and cranial cartilage malformations. Loss of PPan, but not interference with rRNA processing, led to an early downregulation of specific marker genes of the eye, including Rx1 and Pax6, and of NCCs, such as Twist, Slug and FoxD3. We found that PPan protein is localized in the nucleoli and mitochondria and that loss of PPan results in increased apoptosis. These findings indicate a novel function of PPan that is independent of its role in ribosome biogenesis.

  8. In Vivo Study of Dynamics and Stability of Dendritic Spines on Olfactory Bulb Interneurons in Xenopus laevis Tadpoles.

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    Yu-Bin Huang

    Full Text Available Dendritic spines undergo continuous remodeling during development of the nervous system. Their stability is essential for maintaining a functional neuronal circuit. Spine dynamics and stability of cortical excitatory pyramidal neurons have been explored extensively in mammalian animal models. However, little is known about spiny interneurons in non-mammalian vertebrate models. In the present study, neuronal morphology was visualized by single-cell electroporation. Spiny neurons were surveyed in the Xenopus tadpole brain and observed to be widely distributed in the olfactory bulb and telencephalon. DsRed- or PSD95-GFP-expressing spiny interneurons in the olfactory bulb were selected for in vivo time-lapse imaging. Dendritic protrusions were classified as filopodia, thin, stubby, or mushroom spines based on morphology. Dendritic spines on the interneurons were highly dynamic, especially the filopodia and thin spines. The stubby and mushroom spines were relatively more stable, although their stability significantly decreased with longer observation intervals. The 4 spine types exhibited diverse preferences during morphological transitions from one spine type to others. Sensory deprivation induced by severing the olfactory nerve to block the input of mitral/tufted cells had no significant effects on interneuron spine stability. Hence, a new model was established in Xenopus laevis tadpoles to explore dendritic spine dynamics in vivo.

  9. Effect of ammodytin L from the venom of Vipera ammodytes on Xenopus laevis differentiated muscle fibres and regenerating limbs.

    Science.gov (United States)

    Bernardini, S; Cannata, S; Filoni, S; Luly, P; Rufini, S

    1996-01-01

    Ammodytin L is a non-catalytic, phospholipase-like snake venom toxin from Vipera ammodytes, which shows a cytotoxic activity on differentiated myotubes when tested in vitro. In the range of concentrations in which ammodytin L induced necrosis of myogenic cells in culture, other cell types (erythrocytes, platelets, fibroblasts) did not appear to be affected. To test the in vivo toxicity and the effective cytolytic specificity of ammodytin L we have followed the morphological changes in muscle tissue of Xenopus laevis limbs after intramuscular toxin injection. Only muscular cells were affected by ammodytin L, and the toxin did not induce any morphological change in other cell types. Further evidence of the muscle-specific action of the toxin was obtained from experiments carried out using the Xenopus kidney cell line B3.2 in culture. Ammodytin L was unable to affect parameters of cell viability such as lactate dehydrogenase leakage, [3H]thymidine incorporation, growth curves and morphological changes. Moreover, direct ammodytin L application to cultured regenerative limbs did not provoke alterations in undifferentiated myoblasts. These data suggest that ammodytin L, like other phospholipase-like toxins, exerts its toxicity by selectively damaging differentiated muscle fibres.

  10. Potential protective effect of L-cysteine against the toxicity of acrylamide and furan in exposed Xenopus laevis embryos: an interaction study

    Science.gov (United States)

    The embryo toxicities of two food-processing-induced toxic compounds, acrylamide and furan, with and without added L-cysteine were examined individually and in mixtures using the frog embryo teratogenesis assay-Xenopus (FETAX). The following measures of developmental toxicity were used (a) 96-h LC5...

  11. Canonical and Alternative Pathways in Cyclin-Dependent Kinase 1/Cyclin B Inactivation upon M-Phase Exit in Xenopus laevis Cell-Free Extracts

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    Jacek Z. Kubiak

    2011-01-01

    Full Text Available Cyclin-Dependent Kinase 1 (CDK1 is the major M-phase kinase known also as the M-phase Promoting Factor or MPF. Studies performed during the last decade have shown many details of how CDK1 is regulated and also how it regulates the cell cycle progression. Xenopus laevis cell-free extracts were widely used to elucidate the details and to obtain a global view of the role of CDK1 in M-phase control. CDK1 inactivation upon M-phase exit is a primordial process leading to the M-phase/interphase transition during the cell cycle. Here we discuss two closely related aspects of CDK1 regulation in Xenopus laevis cell-free extracts: firstly, how CDK1 becomes inactivated and secondly, how other actors, like kinases and phosphatases network and/or specific inhibitors, cooperate with CDK1 inactivation to assure timely exit from the M-phase.

  12. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Shuangying, E-mail: shuangying.yu@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Tang, Song, E-mail: song.tang@usask.ca [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Mayer, Gregory D., E-mail: greg.mayer@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Cobb, George P., E-mail: george_cobb@baylor.edu [Department of Environmental Science, Baylor University, One Bear Place #97266, Waco, TX 76798 (United States); Maul, Jonathan D., E-mail: jonathan.maul@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States)

    2015-02-15

    Highlights: • Interactive effects of UVB radiation-pesticide co-exposures were examined in frogs. • Responses included induction of DNA photo-adducts and DNA damage and repair genes. • Elevated DNA adduct levels occurred for co-exposures compared to UVB alone. • One mechanism is that pesticides may alter nuclear excision repair gene expression. - Abstract: Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 μg/L endosulfan or 0, 2.5, and 5.0 μg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides

  13. Developmental toxicology of potato alkaloids in the frog embryo teratogenesis assay--Xenopus (FETAX).

    Science.gov (United States)

    Friedman, M; Rayburn, J R; Bantle, J A

    1991-08-01

    Potatoes frequently contain growth inhibitors and toxic compounds including digestive enzyme inhibitors, lectins and glycoalkaloids. The literature suggests that Solanum alkaloids have the ability to induce neurological damage such as spina bifida and other malformations. As part of a programme of improvement in the safety of potatoes using molecular plant genetics and parallel food safety evaluation, we evaluated the effect of several potato glycoalkaloids and aglycones in the frog embryo teratogenesis assay--Xenopus (FETAX) with and without metabolic activation by Aroclor 1254-induced rat liver microsomes. The data suggest that the glycoalkaloid alpha-chaconine is teratogenic and more embryotoxic than alpha-solanine, in terms of the median lethal concentration (LC50) after 96 hr of exposure, the concentration inducing gross terata in 50% of the surviving frog embryos (96-hr EC50, malformation), and the minimum concentration needed to inhibit the growth of the embryos. Since these two compounds differ only in the nature of the carbohydrate side chain attached to the 3-OH group of solanidine, the side chain appears to be an important factor in governing teratogenicity. The aglycones demissidine, solanidine and solasodine were less toxic than the glycosides alpha-chaconine and alpha-solanine. The in vitro teratogenesis assay should be useful for: (a) predicting the teratogenic potential of solanaceae alkaloids, glycoalkaloids and related natural products; and (b) facilitating experimental approaches to suppress plant genes and enzymes that control the biosynthesis of the most toxic compounds.

  14. The synthetic gestagen levonorgestrel directly affects gene expression in thyroid and pituitary glands of Xenopus laevis tadpoles.

    Science.gov (United States)

    Lorenz, Claudia; Opitz, Robert; Trubiroha, Achim; Lutz, Ilka; Zikova, Andrea; Kloas, Werner

    2016-08-01

    The synthetic gestagen levonorgestrel (LNG) was previously shown to perturb thyroid hormone-dependent metamorphosis in Xenopus laevis. However, so far the mechanisms underlying the anti-metamorphic effects of LNG remained unknown. Therefore, a series of in vivo and ex vivo experiments was performed to identify potential target sites of LNG action along the pituitary-thyroid axis of X. laevis tadpoles. Prometamorphic tadpoles were treated in vivo with LNG (0.01-10nM) for 72h and brain-pituitary and thyroid tissue was analyzed for marker gene expression. While no treatment-related changes were observed in brain-pituitary tissue, LNG treatment readily affected thyroidal gene expression in tadpoles including decreased slc5a5 and iyd mRNA expression and a strong induction of dio2 and dio3 expression. When using an ex vivo organ explant culture approach, direct effects of LNG on both pituitary and thyroid gland gene expression were detecTable Specifically, treatment of pituitary explants with 10nM LNG strongly stimulated dio2 expression and concurrently suppressed tshb expression. In thyroid glands, ex vivo LNG treatment induced dio2 and dio3 mRNA expression in a thyrotropin-independent manner. When thyroid explants were cultured in thyrotropin-containing media, LNG caused similar gene expression changes as seen after 72h in vivo treatment including a very strong repression of thyrotropin-induced slc5a5 expression. Concerning the anti-thyroidal activity of LNG as seen under in vivo conditions, our ex vivo data provide clear evidence that LNG directly affects expression of genes important for thyroidal iodide handling as well as genes involved in negative feedback regulation of pituitary tshb expression. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Thyroid disruption by Di-n-butyl phthalate (DBP and mono-n-butyl phthalate (MBP in Xenopus laevis.

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    Ouxi Shen

    Full Text Available BACKGROUND: Di-n-butyl phthalate (DBP, a chemical widely used in many consumer products, is estrogenic and capable of producing seriously reproductive and developmental effects in laboratory animals. However, recent in vitro studies have shown that DBP and mono-n-butyl phthalate (MBP, the major metabolite of DBP, possessed thyroid hormone receptor (TR antagonist activity. It is therefore important to consider DBP and MBP that may interfere with thyroid hormone system. METHODOLOGY/PRINCIPAL FINDINGS: Nieuwkoop and Faber stage 51 Xenopus laevis were exposed to DBP and MBP (2, 10 or 15 mg/L separately for 21 days. The two test chemicals decelerated spontaneous metamorphosis in X. laevis at concentrations of 10 and 15 mg/L. Moreover, MBP seemed to possess stronger activity. The effects of DBP and MBP on inducing changes of expression of selected thyroid hormone response genes: thyroid hormone receptor-beta (TRβ, retinoid X receptor gamma (RXRγ, alpha and beta subunits of thyroid-stimulating hormone (TSHα and TSHβ were detected by qPCR at all concentrations of the compounds. Using mammalian two-hybrid assay in vitro, we found that DBP and MBP enhanced the interactions between co-repressor SMRT (silencing mediator for retinoid and thyroid hormone receptors and TR in a dose-dependent manner, and MBP displayed more markedly. In addition, MBP at low concentrations (2 and 10 mg/L caused aberrant methylation of TRβ in head tissue. CONCLUSIONS: The current findings highlight potential disruption of thyroid signalling by DBP and MBP and provide data for human risk assessment.

  16. Lbx1 expression and frog limb development.

    Science.gov (United States)

    Sabo, Michelle C; Nath, Kimberly; Elinson, Richard P

    2009-12-01

    In order to identify prospective limb muscle cells in a frog, we cloned Lbx1 from the direct developing frog Eleutherodactylus coqui. Like in embryos of the frog Xenopus laevis but unlike in other vertebrates, EcLbx1 is expressed in all trunk somites. Like in embryos of chick, mouse, and zebrafish, cells expressing EcLbx1 are then found in limb buds, consistent with migration of those cells from somites. EcLbx1 is also expressed in the dorsal spinal cord as in other vertebrates.

  17. Single-cell mass spectrometry with multi-solvent extraction identifies metabolic differences between left and right blastomeres in the 8-cell frog (Xenopus) embryo.

    Science.gov (United States)

    Onjiko, Rosemary M; Morris, Sydney E; Moody, Sally A; Nemes, Peter

    2016-06-21

    Single-cell metabolic mass spectrometry enables the discovery (untargeted) analysis of small molecules in individual cells. Using single-cell capillary electrophoresis high-resolution mass spectrometry (CE-HRMS), we recently uncovered small-molecule differences between embryonic cells located along the animal-vegetal and dorsal-ventral axes of the 16-cell frog (Xenopus laevis) embryo, raising the question whether metabolic cell heterogeneity also exists along the left-right body axis. To address this question, we here advance single-cell CE-HRMS for identifying and quantifying metabolites in higher analytical sensitivity, and then use the methodology to compare metabolite production between left and right cells. Our strategy utilizes multiple solvents with complementary physicochemical properties to extract small molecules from single cells and improve electrophoretic separation, increasing metabolite ion signals for quantification and tandem HRMS. As a result, we were able to identify 55 different small molecules in D1 cells that were isolated from 8-cell embryos. To quantify metabolite production between left and right cells, we analyzed n = 24 different D1 cells in technical duplicate-triplicate measurements. Statistical and multivariate analysis based on 80 of the most repeatedly quantified compounds revealed 10 distinct metabolites that were significantly differentially accumulated in the left or right cells (p embryo.

  18. Functional interaction between CFTR and the sodium-phosphate co-transport type 2a in Xenopus laevis oocytes.

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    Naziha Bakouh

    Full Text Available BACKGROUND: A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR. CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes. METHODOLOGY/FINDINGS: NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression. CONCLUSION/PERSPECTIVES: We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in

  19. A concentration gradient of retinoids in the early Xenopus laevis embryo.

    Science.gov (United States)

    Chen, Y; Huang, L; Solursh, M

    1994-01-01

    Previous studies have postulated that Xenopus embryos contain an endogenous retinoic acid (RA) concentration gradient from posterior to anterior during the process of primary axis formation, since RA is able to alter profoundly primary axis formation in Xenopus embryos, to increase the expression of some posterior markers, and to inhibit the expression of some anterior markers, including homeobox-containing genes. Here, we provide direct evidence for this hypothesis. By using a reporter cell system, we demonstrate that the endogenous biologically active retinoid concentration in whole Xenopus embryos increases 3-fold from the two-cell stage to the neurula stage, and that the active retinoid concentration in the dorsal marginal zone, a region wherein the Spemann's organizer is located, increases about 5-fold from the early gastrula to late gastrula stages, suggesting the developmental regulation of the retinoid levels. In the early neurula stage (stage 13-14), endogenous active retinoids are present in a concentration gradient with the highest level at the posterior end, about 10-fold higher than that at the anterior end, of the embryo. This concentration gradient may be established during gastrulation and may provide positional cues for primary axis formation.

  20. A developmental biological study of aldolase gene expression in Xenopus laevis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source, aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism.We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that its promoter (ca. 2 kb)contains regions necessary for tissue-specific expression and also a GC rich region which is essential for basal transcriptional activity.

  1. The Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD on the Mortality and Growth of Two Amphibian Species (Xenopus laevis and Pseudacris triseriata

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    Alex Collier

    2008-12-01

    Full Text Available We observed a slight drop in the growth of Xenopus laevis and Pseudacris triseriata larvae following acute exposure (24-48 h during egg development to three concentrations of TCDD (0.3, 3.0, 30.0 μg/l. Our exposure protocol was modeled on a previous investigation that was designed to mimic the effects of maternal deposition of TCDD. The doses selected were consistent with known rates of maternal transfer between mother and egg using actual adult body burdens from contaminated habitats. Egg and embryonic mortality immediately following exposure increased only among 48 h X. laevis treatments. Control P. triseriata and X. laevis completed metamorphosis more quickly than TCDDtreated animals. The snout-vent length of recently transformed P. triseriata did not differ between treatments although controls were heavier than high-dosed animals. Likewise, the snout-vent length and weight of transformed X. laevis did not differ between control and TCDD treatments. These findings provide additional evidence that amphibians, including P. triseriata and X. laevis are relatively insensitive to acute exposure to TCDD during egg and embryonic development. Although the concentrations selected for this study were relatively high, they were not inconsistent with our current understanding of bioaccumulation via maternal transfer.

  2. Membrane type-1 matrix metalloproteinases and tissue inhibitor of metalloproteinases-2 RNA levels mimic each other during Xenopus laevis metamorphosis.

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    Logan A Walsh

    Full Text Available Matrix metalloproteinases (MMPs and their endogenous inhibitors TIMPs (tissue inhibitors of MMPs, are two protein families that work together to remodel the extracellular matrix (ECM. TIMPs serve not only to inhibit MMP activity, but also aid in the activation of MMPs that are secreted as inactive zymogens. Xenopus laevis metamorphosis is an ideal model for studying MMP and TIMP expression levels because all tissues are remodeled under the control of one molecule, thyroid hormone. Here, using RT-PCR analysis, we examine the metamorphic RNA levels of two membrane-type MMPs (MT1-MMP, MT3-MMP, two TIMPs (TIMP-2, TIMP-3 and a potent gelatinase (Gel-A that can be activated by the combinatory activity of a MT-MMP and a TIMP. In the metamorphic tail and intestine the RNA levels of TIMP-2 and MT1-MMP mirror each other, and closely resemble that of Gel-A as all three are elevated during periods of cell death and proliferation. Conversely, MT3-MMP and TIMP-3 do not have similar RNA level patterns nor do they mimic the RNA levels of the other genes examined. Intriguingly, TIMP-3, which has been shown to have anti-apoptotic activity, is found at low levels in tissues during periods of apoptosis.

  3. Influence of inorganic phosphate and pH on sarcoplasmic reticular ATPase in skinned muscle fibres of Xenopus laevis.

    Science.gov (United States)

    Stienen, G J; Papp, Z; Zaremba, R

    1999-08-01

    1. The influence of 30 mM inorganic phosphate (Pi) and pH (6.2-7.4) on the rate of ATP utilization was determined in mechanically skinned bundles of myofibrils from the iliofibularis muscle of Xenopus laevis at approximately 5 C. 2. BDM (2,3-butanedione monoxime; 10 mM) depressed isometric force production and actomyosin (AM) ATPase activity equally. Therefore sarcoplasmic reticular (SR) ATPase activity could be determined by extrapolation of the total ATPase activity to zero force. 3. The SR ATPase activity without added Pi at pH 7.1 was 42 +/- 2 % of the total ATPase activity. Addition of 30 mM Pi reduced SR ATPase activity slightly, by 9 +/- 5 %, and depressed force by 62 +/- 2 % and AM ATPase activity by 21 +/- 6 %. 4. At pH 6.2, force, SR ATPase activity and AM ATPase activity were reduced by 21 +/- 5, 61 +/- 5 and 10 +/- 4 % of their respective values at pH 7.1. 5. The SR ATPase activity at 30 mM Pi and pH 6.2 was reduced markedly to 20 +/- 6 % of the value under control conditions, suggesting that the maximum rate of Ca2+ uptake during muscle fatigue was strongly depressed. This reduction was larger than expected on the basis of the effects of Pi and pH alone.

  4. Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis

    Energy Technology Data Exchange (ETDEWEB)

    Mouchet, Florence [Laboratoire d' Ecologie fonctionnelle - EcoLab, Universite Paul Sabatier, UMR UPS INPT CNRS 5245, Campus INP-ENSAT, Avenue de l' Agrobiopole, 31326 Auzeville-Tolosane (France)], E-mail: florence.mouchet@cict.fr; Landois, Perine [CIRIMAT, Universite Paul Sabatier, UMR UPS INPT CNRS 5085, Bat. 2R1, 118 Route de Narbonne 31062 Toulouse cedex 9 (France); Sarremejean, Elodie; Bernard, Guillaume [Laboratoire d' Ecologie fonctionnelle - EcoLab, Universite Paul Sabatier, UMR UPS INPT CNRS 5245, Campus INP-ENSAT, Avenue de l' Agrobiopole, 31326 Auzeville-Tolosane (France); Puech, Pascal [CEMES, 29 rue Jeanne Marvig, BP 94347, 31055 Toulouse cedex 4 (France); Pinelli, Eric [Laboratoire d' Ecologie fonctionnelle - EcoLab, Universite Paul Sabatier, UMR UPS INPT CNRS 5245, Campus INP-ENSAT, Avenue de l' Agrobiopole, 31326 Auzeville-Tolosane (France); Flahaut, Emmanuel [CIRIMAT, Universite Paul Sabatier, UMR UPS INPT CNRS 5085, Bat. 2R1, 118 Route de Narbonne 31062 Toulouse cedex 9 (France); Gauthier, Laury [Laboratoire d' Ecologie fonctionnelle - EcoLab, Universite Paul Sabatier, UMR UPS INPT CNRS 5245, Campus INP-ENSAT, Avenue de l' Agrobiopole, 31326 Auzeville-Tolosane (France)

    2008-04-28

    Because of their outstanding properties, carbon nanotubes (CNTs) are being assessed for inclusion in many manufactured products. Due to their massive production and growing number of potential applications, the impact of CNTs on the environment must be taken into consideration. The present investigation evaluates the ecotoxicological potential of double-walled carbon nanotubes (DWNTs) in the amphibian larvae Xenopus laevis at a large range of concentrations in water (from 10 to 500 mg L{sup -1}). Acute toxicity and genotoxicity were analysed after 12 days of static exposure in laboratory conditions. Acute toxicity was evaluated according to the mortality and the growth of larvae. The genotoxic effects were analysed by scoring the micronucleated erythrocytes of the circulating blood of larvae according to the International Standard micronucleus assay. Moreover, histological preparations of larval intestine were prepared after 12 days of exposure for observation using optical and transmission electron microscopy (TEM). Finally, the intestine of an exposed larva was prepared on a slide for analyse by Raman imaging. The results showed no genotoxicity in erythrocytes of larvae exposed to DWNTs in water, but acute toxicity at every concentration of DWNTs studied which was related to physical blockage of the gills and/or digestive tract. Indeed, black masses suggesting the presence of CNTs were observed inside the intestine using optical microscopy and TEM, and confirmed by Raman spectroscopy analysis. Assessing the risks of CNTs requires better understanding, especially including mechanistic and environmental investigations.

  5. Effects of 4-ter-Octylphenol on Xenopus tropicalis in a Long Term Exposure

    Science.gov (United States)

    2011-03-17

    tubules of control animals were lined with spermatocysts containing spermatogonia, spermatocytes and spermatids at various stages of spermatogenesis ...sloughing was seen in 1 µg/L and 36 µg/LOP exposed frogs, indicating that OP exposure may be disrupting spermatogenesis , but is not sufficient to reduce...disrupts spermatogenesis in Xenopus laevis frogs. Toxicol.Sci 84, 394-407. Lei, B., Huang, S., Zhou, Y., Wang, D., and Wang, Z., 2009. Levels of six

  6. Electron microscopy of the amphibian model systems Xenopus laevis and Ambystoma mexicanum.

    Science.gov (United States)

    Kurth, Thomas; Berger, Jürgen; Wilsch-Bräuninger, Michaela; Kretschmar, Susanne; Cerny, Robert; Schwarz, Heinz; Löfberg, Jan; Piendl, Thomas; Epperlein, Hans H

    2010-01-01

    In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy.

  7. Identification of Genes Expressed in the Migrating Primitive Myeloid Lineage of Xenopus laevis

    Science.gov (United States)

    Agricola, Zachary N.; Jagpal, Amrita K.; Allbee, Andrew W.; Prewitt, Allison R.; Shifley, Emily T.; Rankin, Scott A.; Zorn, Aaron M.; Kenny, Alan P.

    2017-01-01

    Background During primitive hematopoiesis in Xenopus, cebpa and spib expressing myeloid cells emerge from the anterior ventral blood island. Primitive myeloid cells migrate throughout the embryo and are critical for immunity, healing, and development. Although definitive hematopoiesis has been studied extensively, molecular mechanisms leading to the migration of primitive myelocytes remain poorly understood. We hypothesized these cells have specific extracellular matrix modifying and cell motility gene expression. Results In situ hybridization screens of transcripts expressed in Xenopus foregut mesendoderm at stage 23 identified seven genes with restricted expression in primitive myeloid cells: destrin; coronin actin binding protein, 1a; formin-like 1; ADAM metallopeptidase domain 28; cathepsin S; tissue inhibitor of metalloproteinase-1; and protein tyrosine phosphatase nonreceptor 6. A detailed in situ hybridization analysis revealed these genes are initially expressed in the aVBI but become dispersed throughout the embryo as the primitive myeloid cells become migratory, similar to known myeloid markers. Morpholino-mediated loss-of-function and mRNA-mediated gain-of-function studies revealed the identified genes are downstream of Spib.a and Cebpa, key transcriptional regulators of the myeloid lineage. Conclusions We have identified genes specifically expressed in migratory primitive myeloid progenitors, providing tools to study how different gene networks operate in these primitive myelocytes during development and immunity. PMID:26264370

  8. Biophysics of underwater hearing in the clawed frog, Xenopus laevis

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, J; Elepfandt, A

    1995-01-01

    the larynx with metal or tissue glue. This shows that the sound-induced vibrations of the lungs are probably coupled to the middle ear cavities via the larynx. Also, anatomical observations show that the two middle ear cavities and the larynx are connected in an air-filled recess in submerged animals...... of the air bubble in the middle ear cavity. Filling the middle ear cavity with water lowered the disk vibrations by 10-30 dB in the frequency range of 0.5-3 kHz. Inflating the lungs shifted the low-frequency peak downwards, but did not change the high-frequency peak. Thus, the disk vibrations...... in the frequency range of the mating call (main energy at 1.7-1.9 kHz) were mainly caused by pulsations of the air in the middle ear cavity; sound transmission via the lungs was more important at low frequencies (below 1 kHz). Furthermore, the low-frequency peak could be reversibly reduced in amplitude by loading...

  9. Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis)

    Science.gov (United States)

    Hayes, Tyrone B.; Khoury, Vicky; Narayan, Anne; Nazir, Mariam; Park, Andrew; Brown, Travis; Adame, Lillian; Chan, Elton; Buchholz, Daniel; Stueve, Theresa; Gallipeau, Sherrie

    2010-01-01

    The herbicide atrazine is one of the most commonly applied pesticides in the world. As a result, atrazine is the most commonly detected pesticide contaminant of ground, surface, and drinking water. Atrazine is also a potent endocrine disruptor that is active at low, ecologically relevant concentrations. Previous studies showed that atrazine adversely affects amphibian larval development. The present study demonstrates the reproductive consequences of atrazine exposure in adult amphibians. Atrazine-exposed males were both demasculinized (chemically castrated) and completely feminized as adults. Ten percent of the exposed genetic males developed into functional females that copulated with unexposed males and produced viable eggs. Atrazine-exposed males suffered from depressed testosterone, decreased breeding gland size, demasculinized/feminized laryngeal development, suppressed mating behavior, reduced spermatogenesis, and decreased fertility. These data are consistent with effects of atrazine observed in other vertebrate classes. The present findings exemplify the role that atrazine and other endocrine-disrupting pesticides likely play in global amphibian declines. PMID:20194757

  10. Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Grunnet, M; Angelo, K;

    2002-01-01

    will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells...... and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual......-function vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels....

  11. Generation of stable Xenopus laevis transgenic lines expressing a transgene controlled by weak promoters.

    Science.gov (United States)

    L'hostis-Guidet, Anne; Recher, Gaëlle; Guillet, Brigitte; Al-Mohammad, Abdulrahim; Coumailleau, Pascal; Tiaho, François; Boujard, Daniel; Madigou, Thierry

    2009-10-01

    Combining two existing protocols of trangenesis, namely the REMI and the I-SceI meganuclease methods, we generated Xenopus leavis expressing a transgene under the control of a promoter that presented a restricted pattern of activity and a low level of expression. This was realized by co-incubating sperm nuclei, the I-SceI enzyme and the transgene prior to transplantation into unfertilized eggs. The addition of the woodchuck hepatitis virus posttranscriptional regulatory element in our constructs further enhanced the expression of the transgene without affecting the tissue-specificity of the promoter activity. Using this combination of methods we produced high rates of fully transgenic animals that stably transmitted the transgene to the next generations with a transmission rate of 50% indicating a single integration event.

  12. E-cadherin is required for cranial neural crest migration in Xenopus laevis.

    Science.gov (United States)

    Huang, Chaolie; Kratzer, Marie-Claire; Wedlich, Doris; Kashef, Jubin

    2016-03-15

    The cranial neural crest (CNC) is a highly motile and multipotent embryonic cell population, which migrates directionally on defined routes throughout the embryo, contributing to facial structures including cartilage, bone and ganglia. Cadherin-mediated cell-cell adhesion is known to play a crucial role in the directional migration of CNC cells. However, migrating CNC co-express different cadherin subtypes, and their individual roles have yet to be fully explored. In previous studies, the expression of individual cadherin subtypes has been analysed using different methods with varying sensitivities, preventing the direct comparison of expression levels. Here, we provide the first comprehensive and comparative analysis of the expression of six cadherin superfamily members during different phases of CNC cell migration in Xenopus. By applying a quantitative RT-qPCR approach, we can determine the copy number and abundance of each expressed cadherin through different phases of CNC migration. Using this approach, we show for the first time expression of E-cadherin and XB/C-cadherin in CNC cells, adding them as two new members of cadherins co-expressed during CNC migration. Cadherin co-expression during CNC migration in Xenopus, in particular the constant expression of E-cadherin, contradicts the classical epithelial-mesenchymal transition (EMT) model postulating a switch in cadherin expression. Loss-of-function experiments further show that E-cadherin is required for proper CNC cell migration in vivo and also for cell protrusion formation in vitro. Knockdown of E-cadherin is not rescued by co-injection of other classical cadherins, pointing to a specific function of E-cadherin in mediating CNC cell migration. Finally, through reconstitution experiments with different E-cadherin deletion mutants in E-cadherin morphant embryos, we demonstrate that the extracellular domain, but not the cytoplasmic domain, of E-cadherin is sufficient to rescue CNC cell migration in vivo.

  13. Inbreeding Ratio and Genetic Relationships among Strains of the Western Clawed Frog, Xenopus tropicalis.

    Directory of Open Access Journals (Sweden)

    Takeshi Igawa

    Full Text Available The Western clawed frog, Xenopus tropicalis, is a highly promising model amphibian, especially in developmental and physiological research, and as a tool for understanding disease. It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s. The major strains thus far known include the Nigerian and Ivory Coast strains. However, due to its short history as an experimental animal, the genetic relationship among the various strains has not yet been clarified, and establishment of inbred strains has not yet been achieved. Since 2003 the Institute for Amphibian Biology (IAB, Hiroshima University has maintained stocks of multiple X. tropicalis strains and conducted consecutive breeding as part of the National BioResource Project. In the present study we investigated the inbreeding ratio and genetic relationship of four inbred strains at IAB, as well as stocks from other institutions, using highly polymorphic microsatellite markers and mitochondrial haplotypes. Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains. The Ivory Coast strains clearly differed from the Nigerian strains genetically, and three subgroups were identified within both the Nigerian and Ivory Coast strains. It is noteworthy that the Ivory Coast strains have an evolutionary divergent genetic background. Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts.

  14. Temperature dependence of locomotor performance in the tropical clawed frog, Xenopus tropicalis.

    Science.gov (United States)

    Herrel, Anthony; Bonneaud, Camille

    2012-07-15

    Amphibians are ideal taxa with which to investigate the effects of climate change on physiology, dispersal capacity and distributional ranges as their physiological performance and fitness is highly dependent on temperature. Moreover, amphibians are among the most endangered vertebrate taxa. Here we use the tropical clawed frog, Xenopus tropicalis, as a model system to explore effects of temperature on locomotor performance. Our analyses show that locomotion is thermally sensitive, as illustrated by significant effects of temperature on terrestrial exertion capacity (time until exhaustion) and aquatic burst speed (maximal burst swimming velocity and maximal burst swimming acceleration capacity). Exertion performance measures had relatively lower temperature optima and narrower performance breadth ranges than measures of burst speed. The narrow 80% performance breadths confirm predictions that animals from stable environments should display high thermal sensitivity and, combined with the divergent temperature optima for exertion capacity and burst speed, underscore the vulnerability of tropical species such as X. tropicalis to even relatively small temperature changes. The temperature sensitivity of locomotor performance traits in X. tropicalis suggests that tropical ectotherms may be impacted by predicted changes in climate.

  15. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

    Science.gov (United States)

    Nakajima, Keisuke; Yaoita, Yoshio

    2015-01-16

    Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3'UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3'UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 3'UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  16. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Keisuke Nakajima

    2015-01-01

    Full Text Available Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3′UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3′UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf. In contrast, TALEN mRNAs without this 3′UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  17. Acute effects of Fe₂O₃, TiO₂, ZnO and CuO nanomaterials on Xenopus laevis.

    Science.gov (United States)

    Nations, Shawna; Wages, Mike; Cañas, Jaclyn E; Maul, Jonathan; Theodorakis, Chris; Cobb, George P

    2011-05-01

    Metal oxide nanomaterials have exhibited toxicity to a variety of aquatic organisms, especially microbes and invertebrates. To date, few studies have evaluated the toxicity of metal oxide nanomaterials on aquatic vertebrates. Therefore, this study examined effects of ZnO, TiO(2), Fe(2)O(3), and CuO nanomaterials (20-100 nm) on amphibians utilizing the Frog Embryo Teratogenesis Assay Xenopus (FETAX) protocol, a 96 h exposure with daily solution exchanges. Nanomaterials were dispersed in reconstituted moderately hard test medium. These exposures did not increase mortality in static renewal exposures containing up to 1,000 mg L(-1) for TiO(2), Fe(2)O(3), CuO, and ZnO, but did induce developmental abnormalities. Gastrointestinal, spinal, and other abnormalities were observed in CuO and ZnO nanomaterial exposures at concentrations as low as 3.16 mg L(-1) (ZnO). An EC(50) of 10.3 mg L(-1) ZnO was observed for total malformations. The minimum concentration to inhibit growth of tadpoles exposed to CuO or ZnO nanomaterials was 10 mg L(-1). The results indicate that select nanomaterials can negatively affect amphibians during development. Evaluation of nanomaterial exposure on vertebrate organisms are imperative to responsible production and introduction of nanomaterials in everyday products to ensure human and environmental safety.

  18. The cytoskeletal protein Zyxin inhibits Shh signaling during the CNS patterning in Xenopus laevis through interaction with the transcription factor Gli1.

    Science.gov (United States)

    Martynova, Natalia Y; Ermolina, Ludmila V; Ermakova, Galina V; Eroshkin, Fedor M; Gyoeva, Fatima K; Baturina, Natalia S; Zaraisky, Andrey G

    2013-08-01

    Zyxin is a cytoskeletal protein that controls cell movements by regulating actin filaments assembly, but it can also modulate gene expression owing to its interactions with the proteins involved in signaling cascades. Therefore, identification of proteins that interact with Zyxin in embryonic cells is a promising way to unravel mechanisms responsible for coupling of two major components of embryogenesis: morphogenetic movements and cell differentiation. Now we show that in Xenopus laevis embryos Zyxin can bind to and suppress activity of the primary effector of Sonic hedgehog (Shh) signaling cascade, the transcription factor Gli1. By using loss- and gain-of-function approaches, we demonstrate that Zyxin is essential for reduction of Shh signaling within the dorsal part of the neural tube of X. laevis embryo. Thus, our finding discloses a novel function of Zyxin in fine tuning of the central neural system patterning which is based on the ventral-to-dorsal gradient of Shh signaling.

  19. Characterization of recombinant Xenopus laevis type I iodothyronine deiodinase: Substitution of a proline residue in the catalytic center by serine (Pro132Ser) restores sensitivity to 6-propyl-2-thiouracil

    NARCIS (Netherlands)

    G.G.J.M. Kuiper (George); W. Klootwijk (Willem); G.M. Dubois (Ghislaine Morvan); O. Destree (Olivier); V.M. Darras (Veerle); S. van der Geyten (Serge); B.A. Demeneix (Barbara); T.J. Visser (Theo)

    2006-01-01

    textabstractIn frogs such as Rana and Xenopus, metamorphosis does not occur in the absence of a functional thyroid gland. Previous studies indicated that coordinated development in frogs requires tissue and stage-dependent type II and type III iodothyronine deiodinase expression patterns to obtain r

  20. Efficacy of tricaine methanesulfonate (MS-222 as an anesthetic agent for blocking sensory-motor responses in Xenopus laevis tadpoles.

    Directory of Open Access Journals (Sweden)

    Carlana Ramlochansingh

    Full Text Available Anesthetics are drugs that reversibly relieve pain, decrease body movements and suppress neuronal activity. Most drugs only cover one of these effects; for instance, analgesics relieve pain but fail to block primary fiber responses to noxious stimuli. Alternately, paralytic drugs block synaptic transmission at neuromuscular junctions, thereby effectively paralyzing skeletal muscles. Thus, both analgesics and paralytics each accomplish one effect, but fail to singularly account for all three. Tricaine methanesulfonate (MS-222 is structurally similar to benzocaine, a typical anesthetic for anamniote vertebrates, but contains a sulfate moiety rendering this drug more hydrophilic. MS-222 is used as anesthetic in poikilothermic animals such as fish and amphibians. However, it is often argued that MS-222 is only a hypnotic drug and its ability to block neural activity has been questioned. This prompted us to evaluate the potency and dynamics of MS-222-induced effects on neuronal firing of sensory and motor nerves alongside a defined motor behavior in semi-intact in vitro preparations of Xenopus laevis tadpoles. Electrophysiological recordings of extraocular motor discharge and both spontaneous and evoked mechanosensory nerve activity were measured before, during and after administration of MS-222, then compared to benzocaine and a known paralytic, pancuronium. Both MS-222 and benzocaine, but not pancuronium caused a dose-dependent, reversible blockade of extraocular motor and sensory nerve activity. These results indicate that MS-222 as benzocaine blocks the activity of both sensory and motor nerves compatible with the mechanistic action of effective anesthetics, indicating that both caine-derivates are effective as single-drug anesthetics for surgical interventions in anamniotes.

  1. Ontogenetic distribution of the transcription factor Nkx2.2 in the developing forebrain of Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Laura eDominguez

    2011-03-01

    Full Text Available The expression of the Nkx2.2 gene is involved in the organization of the alar-basal boundary in the forebrain of vertebrates. Its expression in different diencephalic and telencephalic regions, helped to define distinct progenitor domains in mouse and chick. Here we investigated the pattern of Nkx2.2 protein distribution throughout the development of the forebrain of the anuran amphibian, Xenopus laevis. We used immunohistochemical and in situ hybridization techniques for its detection in combination with other essential territorial markers in the forebrain. No expression was observed in the telencephalon. In the alar hypothalamus, Nkx2.2 positive cells were scattered in the suprachiasmatic territory, but also in the supraoptoparaventricular area, as defined by the expression of the transcription factor Otp and the lack of xDll4. In the basal hypothalamus Nkx2.2 expressing cells were localized in the tuberal region, with the exception of the arcuate nucleus, rich in Otp expressing cells. In the diencephalon it was expressed in all three prosomeres (P1-P3 and not in the zona limitans intrathalamica. The presence of Nkx2.2 expressing cells in P3 was restricted to the alar portion, as well as in prosomere P2, whereas in P1 the Nkx2.2 expressing cells were located in the basal plate and identified the alar/basal boundary. These results showed that Nkx2.2 and Sonic hedgehog are expressed in parallel adjacent stripes along the anterior-posterior axis. The results of this study showed a conserved distribution pattern of Nkx2.2 among vertebrates, crucial to recognize subdivisions that are otherwise indistinct, and supported the relevance of this transcription factor in the organization of the forebrain, particularly in the delineation of the alar/basal boundary of the forebrain.

  2. Allosteric activation of the Ca2+ receptor expressed in Xenopus laevis oocytes by NPS 467 or NPS 568.

    Science.gov (United States)

    Hammerland, L G; Garrett, J E; Hung, B C; Levinthal, C; Nemeth, E F

    1998-06-01

    The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the concentration of extracellular Ca2+. In this study, two novel phenylalkylamine compounds, NPS 467 and NPS 568, were examined for effects on Xenopus laevis oocytes expressing the bovine or human parathyroid Ca2+ receptors. Increases in chloride current (ICl) were elicited in oocytes expressing the bovine Ca2+ receptor when the extracellular Ca2+ concentration was raised above 1.5 mM, whereas Ca2+ concentrations > 3 mM were generally necessary to elicit responses in oocytes expressing the human Ca2+ receptor. NPS 467 and NPS 568 potentiated the activation of ICl by extracellular Ca2+ in oocytes expressing either Ca2+ receptor homolog, and this resulted in a leftward shift of the Ca2+ concentration-response curve. Neither compound was active in the absence of extracellular Ca2+. Certain inorganic and organic cations known to activate the Ca2+ receptor were substituted for elevated levels of extracellular Ca2+ to increase ICl and the effects of these agonists were also potentiated by NPS 568 or NPS 467. The effects of NPS 568 were stereoselective and the R-enantiomer was about 10-fold more potent than the corresponding S-enantiomer. Neither NPS 467 nor 568 affected ICl in water-injected oocytes or in oocytes expressing the substance K receptor or the metabotropic glutamate receptor 1a. These results provide compelling evidence that NPS 467 and NPS 568 act directly upon the parathyroid Ca2+ receptor to increase its sensitivity to activation by extracellular Ca2+. This activity suggests that these compounds are positive allosteric modulators of the Ca2+ receptor. As such, these compounds define a new class of pharmacological agents with potent and selective actions on the Ca2+ receptor.

  3. ATP formation and ATP hydrolysis during fatiguing, intermittent stimulation of different types of single muscle fibres from Xenopus laevis.

    Science.gov (United States)

    Nagesser, A S; Van der Laarse, W J; Elzinga, G

    1993-12-01

    This report describes changes of the rate of ATP hydrolysis in single, intact muscle fibres during the development of fatigue induced by intermittent tetanic stimulation. High (type 3) and low (type 1) oxidative muscle fibres dissected from the iliofibularis muscle of Xenopus laevis were studied at 20 degrees C. The rate of ATP hydrolysis was calculated during different time intervals from changes in the content of nucleotides, creatine compounds and lactate, as well as lactate efflux and oxygen uptake. During the first phase of intermittent stimulation, phosphocreatine is fully reduced while the rate of oxygen consumption increases to its maximum, the lactate content increases to a maximum level, and a small amount of IMP is formed; the rate of ATP hydrolysis in type 3 fibres is constant while force decreases, whereas the rate decreases approximately in proportion to force in type 1 fibres. After the first phase, the rate of ATP hydrolysis in type 3 fibres decreases slightly and the fibres reach a steady metabolic state in which the rates of ATP formation and hydrolysis are equal; in type 1 fibres a drastic change of the rate of ATP hydrolysis occurs and a steady metabolic state is not reached. On the basis of the time courses of the metabolic changes, it is concluded that the rate of ATP hydrolysis in type 3 fibres is reduced by acidification and/or a reduced calcium efflux from the sarcoplasmic reticulum, whereas in type 1 fibres inorganic phosphate and/or acidification inhibit the rate initially and ADP is a likely candidate to explain the drastic fall of the rate of ATP hydrolysis during late phases of fatiguing stimulation.

  4. Relation between force and calcium ion concentration in different fibre types of the iliofibularis muscle of Xenopus laevis.

    Science.gov (United States)

    Stienen, G J; van der Laarse, W J; Diegenbach, P C; Elzinga, G

    1987-01-01

    Calcium activated isometric force was measured in segments of single muscle fibres of the iliofibularis muscle of Xenopus laevis skinned by freeze-drying. A subdivision in five different fibre types was made, based on the location of the fibres inside the muscle, fibre diameter and a quantitative histochemical assay for succinate dehydrogenase activity. The Ca2+ sensitivity was characterized by fitting a Hill curve to the force levels reached at different Ca2+ concentrations. The parameter n of this equation indicates the steepness and pK the midpoint of this force-pCa relation. A considerable variability in the Ca2+ sensitivity characteristics was found between different fibres. The parameter n varied between 1.1 and 4.2 while pK varied between 5.5 and 6.6. The distribution of the data indicates the presence of three groups with different Ca2+ sensitivity; a group of fibres with low Ca2+ sensitivity but with considerable variation of the steepness of the Ca2+ sensitivity curves (type 1 fibres), an intermediate group (type 2, 3 and 4 fibres) with also considerable variation in steepness of the Ca2+ sensitivity curves, in which the lowest values for n are found in type 3 and 4 fibres and a group with high Ca2+ sensitivity and low n containing at least one tonic (type 5) fibre. At sub-saturating Ca2+ concentrations occasionally a transient decrease of the rate of force development was found which resembled the force oscillation reported for some mammalian muscle fibres.

  5. Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation and convergent extension.

    Science.gov (United States)

    Davidson, L A; Keller, R E

    1999-10-01

    We have characterized the cell movements and prospective cell identities as neural folds fuse during neural tube formation in Xenopus laevis. A newly developed whole-mount, two-color fluorescent RNA in situ hybridization method, visualized with confocal microscopy, shows that the dorsal neural tube gene xpax3 and the neural-crest-specific gene xslug are expressed far lateral to the medial site of neural fold fusion and that expression moves medially after fusion. To determine whether cell movements or dynamic changes in gene expression are responsible, we used low-light videomicroscopy followed by fluorescent in situ and confocal microscopy. These methods revealed that populations of prospective neural crest and dorsal neural tube cells near the lateral margin of the neural plate at the start of neurulation move to the dorsal midline using distinctive forms of motility. Before fold fusion, superficial neural cells apically contract, roll the neural plate into a trough and appear to pull the superficial epidermal cell sheet medially. After neural fold fusion, lateral deep neural cells move medially by radially intercalating between other neural cells using two types of motility. The neural crest cells migrate as individual cells toward the dorsal midline using medially directed monopolar protrusions. These movements combine the two lateral populations of neural crest into a single medial population that form the roof of the neural tube. The remaining cells of the dorsal neural tube extend protrusions both medially and laterally bringing about radial intercalation of deep and superficial cells to form a single-cell-layered, pseudostratified neural tube. While ours is the first description of medially directed cell migration during neural fold fusion and re-establishment of the neural tube, these complex cell behaviors may be involved during cavitation of the zebrafish neural keel and secondary neurulation in the posterior axis of chicken and mouse.

  6. Insulin-like growth factor 1 regulation of proliferation and differentiation of Xenopus laevis myogenic cells in vitro.

    Science.gov (United States)

    Miyata, Sairi; Yada, Tomotaka; Ishikawa, Natsuko; Taheruzzaman, Kazi; Hara, Ryohei; Matsuzaki, Takashi; Nishikawa, Akio

    2017-03-01

    To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1's cell differentiation promotion, suggesting the possibility that IGF-1's differentiation-promotion effect is an indirect effect via IGF-1's cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100-500 ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-L-thyronine (T3) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T3. In larval tail cells, cell count was 76% lower in the presence of T3, and IGF-1 did not promote proliferation and differentiation in T3-containing medium. In larval dorsal cells, cell count was also lower in the presence of T3, but IGF-1 enhanced proliferation and differentiation in T3-containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T3 and helps accelerate dorsal muscle remodeling during metamorphosis.

  7. Transplantation of Xenopus laevis tissues to determine the ability of motor neurons to acquire a novel target.

    Directory of Open Access Journals (Sweden)

    Karen L Elliott

    Full Text Available The evolutionary origin of novelties is a central problem in biology. At a cellular level this requires, for example, molecularly resolving how brainstem motor neurons change their innervation target from muscle fibers (branchial motor neurons to neural crest-derived ganglia (visceral motor neurons or ear-derived hair cells (inner ear and lateral line efferent neurons. Transplantation of various tissues into the path of motor neuron axons could determine the ability of any motor neuron to innervate a novel target. Several tissues that receive direct, indirect, or no motor innervation were transplanted into the path of different motor neuron populations in Xenopus laevis embryos. Ears, somites, hearts, and lungs were transplanted to the orbit, replacing the eye. Jaw and eye muscle were transplanted to the trunk, replacing a somite. Applications of lipophilic dyes and immunohistochemistry to reveal motor neuron axon terminals were used. The ear, but not somite-derived muscle, heart, or liver, received motor neuron axons via the oculomotor or trochlear nerves. Somite-derived muscle tissue was innervated, likely by the hypoglossal nerve, when replacing the ear. In contrast to our previous report on ear innervation by spinal motor neurons, none of the tissues (eye or jaw muscle was innervated when transplanted to the trunk. Taken together, these results suggest that there is some plasticity inherent to motor innervation, but not every motor neuron can become an efferent to any target that normally receives motor input. The only tissue among our samples that can be innervated by all motor neurons tested is the ear. We suggest some possible, testable molecular suggestions for this apparent uniqueness.

  8. Thyroid Hormone-Induced Activation of Notch Signaling is Required for Adult Intestinal Stem Cell Development During Xenopus Laevis Metamorphosis.

    Science.gov (United States)

    Hasebe, Takashi; Fujimoto, Kenta; Kajita, Mitsuko; Fu, Liezhen; Shi, Yun-Bo; Ishizuya-Oka, Atsuko

    2016-11-21

    In Xenopus laevis intestine during metamorphosis, the larval epithelial cells are removed by apoptosis, and the adult epithelial stem (AE) cells appear concomitantly. They proliferate and differentiate to form the adult epithelium (Ep). Thyroid hormone (TH) is well established to trigger this remodeling by regulating the expression of various genes including Notch receptor. To study the role of Notch signaling, we have analyzed the expression of its components, including the ligands (DLL and Jag), receptor (Notch), and targets (Hairy), in the metamorphosing intestine by real-time reverse transcription-polymerase chain reaction and in situ hybridization or immunohistochemistry. We show that they are up-regulated during both natural and TH-induced metamorphosis in a tissue-specific manner. Particularly, Hairy1 is specifically expressed in the AE cells. Moreover, up-regulation of Hairy1 and Hairy2b by TH was prevented by treating tadpoles with a γ-secretase inhibitor (GSI), which inhibits Notch signaling. More importantly, TH-induced up-regulation of LGR5, an adult intestinal stem cell marker, was suppressed by GSI treatment. Our results suggest that Notch signaling plays a role in stem cell development by regulating the expression of Hairy genes during intestinal remodeling. Furthermore, we show with organ culture experiments that prolonged exposure of tadpole intestine to TH plus GSI leads to hyperplasia of secretory cells and reduction of absorptive cells. Our findings here thus provide evidence for evolutionarily conserved role of Notch signaling in intestinal cell fate determination but more importantly reveal, for the first time, an important role of Notch pathway in the formation of adult intestinal stem cells during vertebrate development. Stem Cells 2016.

  9. Light modulates the melanophore response to a-MSH in Xenopus laevis: an analysis of the signal transduction crosstalk mechanisms involved.

    OpenAIRE

    Isoldi,Mauro César; Provencio, Ignacio; Castrucci, Ana Maria de Lauro

    2010-01-01

    Melanin granule (melanosome) dispersion within Xenopus laevis melanophores is evoked either by light or a-MSH. We have previously demonstrated that the initial biochemical steps of light and a-MSH signaling are distinct, since the increase in cAMP observed in response to a-MSH was not seen after light exposure. cAMP concentrations in response to a-MSH were significantly lower in cells pre-exposed to light as compared to the levels in dark-adapted melanophores. Here we demonstrate the presence...

  10. Quantitative analysis of orofacial development and median clefts in Xenopus laevis.

    Science.gov (United States)

    Kennedy, Allyson E; Dickinson, Amanda J

    2014-05-01

    Xenopus has become a useful tool to study the molecular mechanisms underlying orofacial development. However, few quantitative analyses exist to describe the anatomy of this region. In this study we combine traditional facial measurements with geometric morphometrics to describe anatomical changes in the orofacial region during normal and abnormal development. Facial measurements and principal component (PC) analysis indicate that during early tadpole development the face expands primarily in the midface region accounting for the development of the upper jaw and primary palate. The mouth opening correspondingly becomes flatter and wider as it incorporates the jaw elements. A canonical variate analysis of orofacial and mouth opening shape emphasized that changes in the orofacial shape occur gradually. Orofacial anatomy was quantified after altered levels of retinoic acid using all-trans retinoic acid or an inhibitor of retinoic acid receptors or by injecting antisense oligos targeting RALDH2. Such perturbations resulted in major decreases in the width of the midface and the mouth opening illustrated in facial measurements and a PC analysis. The mouth opening shape also had a gap in the primary palate resulting in a median cleft in the mouth opening that was only illustrated quantitatively in the morphometric analysis. Finally, canonical and discriminant function analysis statistically distinguished the orofacial and mouth opening shape changes among the different modes used to alter retinoic acid signaling levels. By combining quantitative analyses with molecular studies of orofacial development we will be better equipped to understand the complex morphogenetic processes involved in palate development and clefting.

  11. Mass-spectrometric identification of binding proteins of Mr 25,000 protein, a part of vitellogenin B1, detected in particulate fraction of Xenopus laevis oocytes.

    Science.gov (United States)

    Sugimoto, Isamu; Li, Zhijun; Yoshitome, Satoshi; Ito, Susumu; Hashimoto, Eikichi

    2004-10-01

    A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. Our previous report showed the existence of several binding proteins of pp25 in the particulate fraction of Xenopus oocytes. In an attempt to elucidate the function of pp25, two of these binding proteins were purified, analyzed by mass-spectrometry, and identified as ribosomal proteins S13 and S14. Other binding proteins in the particulate fraction mostly corresponded to those derived from purified 40S and 60S ribosomal subunits, as shown by the overlay assay method. However, pp25 did not show any effect on protein synthesis in the rabbit reticulocyte lysate system. A model in which pp25 connects a type of serpin (serine protease inhibitor), the only pp25-binding protein detected in the cytoplasm, to the endoplasmic reticulum through two serine clusters is proposed to explain a possible function of this protein.

  12. Shorter exposures to harder X-rays trigger early apoptotic events in Xenopus laevis embryos.

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    JiaJia Dong

    Full Text Available BACKGROUND: A long-standing conventional view of radiation-induced apoptosis is that increased exposure results in augmented apoptosis in a biological system, with a threshold below which radiation doses do not cause any significant increase in cell death. The consequences of this belief impact the extent to which malignant diseases and non-malignant conditions are therapeutically treated and how radiation is used in combination with other therapies. Our research challenges the current dogma of dose-dependent induction of apoptosis and establishes a new parallel paradigm to the photoelectric effect in biological systems. METHODOLOGY/PRINCIPAL FINDINGS: We explored how the energy of individual X-ray photons and exposure time, both factors that determine the total dose, influence the occurrence of cell death in early Xenopus embryo. Three different experimental scenarios were analyzed and morphological and biochemical hallmarks of apoptosis were evaluated. Initially, we examined cell death events in embryos exposed to increasing incident energies when the exposure time was preset. Then, we evaluated the embryo's response when the exposure time was augmented while the energy value remained constant. Lastly, we studied the incidence of apoptosis in embryos exposed to an equal total dose of radiation that resulted from increasing the incoming energy while lowering the exposure time. CONCLUSIONS/SIGNIFICANCE: Overall, our data establish that the energy of the incident photon is a major contributor to the outcome of the biological system. In particular, for embryos exposed under identical conditions and delivered the same absorbed dose of radiation, the response is significantly increased when shorter bursts of more energetic photons are used. These results suggest that biological organisms display properties similar to the photoelectric effect in physical systems and provide new insights into how radiation-mediated apoptosis should be understood and

  13. Taxonomy Icon Data: African clawed frog [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available g Xenopus_laevis_NL.png Xenopus_laevis_S.png Xenopus_laevis_NS.png http://biosciencedbc.jp/taxonomy_icon/ico...n.cgi?i=Xenopus+laevis&t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Xenopus+laevis&t=NL http://biosciencedbc.jp/taxonomy..._icon/icon.cgi?i=Xenopus+laevis&t=S http://biosciencedbc.jp/taxonomy..._icon/icon.cgi?i=Xenopus+laevis&t=NS http://togodb.biosciencedbc.jp/togodb/view/taxonomy_icon_comment_en?species_id=11 ...

  14. Comparative evaluation of genotoxicity of captan in amphibian larvae (Xenopus laevis and Pleurodeles waltl) using the comet assay and the micronucleus test.

    Science.gov (United States)

    Mouchet, F; Gauthier, L; Mailhes, C; Ferrier, V; Devaux, A

    2006-06-01

    Captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) is a fungicide used to inhibit the growth of many types of fungi on plants used as foodstuffs. The toxic and genotoxic potentials of captan were evaluated with the micronucleus test (MNT; AFNOR,2000) and the comet assay (CA) using amphibian larvae (Xenopus laevis and Pleurodeles waltl). Acute toxicity results showed that captan was toxic (1) to Xenopus larvae exposed to from 2 mg/L to 125 or 62.5 microg/L, depending on the nature of the water [reconstituted water containing mineral salts or mineral water (MW; Volvic, Danone, France)] and (2) to Pleurodeles exposed to from 2 mg/L to 125 microg/L in both types of water. The MNT results obtained in MW showed that captan (62.5 microg/L) was genotoxic to Xenopus but not genotoxic to Pleurodeles at all concentrations tested. CA established that the genotoxicity of captan to Xenopus and Pleurodeles larvae depended on the concentration, the exposure times, and the comet parameters (tail DNA, TEM, OTM, and TL). The CA and MNT results were compared for their ability to detect DNA damage at the concentrations of captan and the exposure times applied. CA showed captan to be genotoxic from the first day of exposure. In amphibians, CA appears to be a sensitive and suitable method for detecting genotoxicity such as that caused by captan. Copyright 2006 Wiley Periodicals, Inc.

  15. Expression of two nonallelic type II procollagen genes during Xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts.

    Science.gov (United States)

    Su, M W; Suzuki, H R; Bieker, J J; Solursh, M; Ramirez, F

    1991-10-01

    The pattern of type II collagen expression during Xenopus laevis embryogenesis has been established after isolating specific cDNA and genomic clones. Evidence is presented suggesting that in X. laevis there are two transcriptionally active copies of the type II procollagen gene. Both genes are activated at the beginning of neurula stage and steady-state mRNA levels progressively increase thereafter. Initially, the transcripts are localized to notochord, somites, and the dorsal region of the lateral plate mesoderm. At later stages of development and parallel to increased mRNA accumulation, collagen expression becomes progressively more confined to chondrogenic regions of the tadpole. During the early period of mRNA accumulation, there is also a transient pattern of expression in localized sites that will later not undergo chondrogenesis, such as the floor plate in the ventral neural tube. At later times and coincident with the appearance of chondrogenic tissues in the developing embryo, expression of the procollagen genes is characterized by the production of an additional, alternatively spliced transcript. The alternatively spliced sequences encode the cysteine-rich globular domain in the NH2-propeptide of the type II procollagen chain. Immunohistochemical analyses with a type II collagen monoclonal antibody documented the deposition of the protein in the extracellular matrix of the developing embryo. Type II collagen expression is therefore temporally regulated by tissue-specific transcription and splicing factors directing the synthesis of distinct molecular forms of the precursor protein in the developing Xenopus embryo.

  16. Exposure to atrazine affects the expression of key genes in metabolic pathways integral to energy homeostasis in Xenopus laevis tadpoles

    Energy Technology Data Exchange (ETDEWEB)

    Zaya, Renee M., E-mail: renee.zaya@wmich.edu [Great Lakes Environmental and Molecular Sciences Center, Department of Biological Sciences, 3425 Wood Hall, Western Michigan University, 1903 West Michigan Avenue, Kalamazoo, MI 49008 (United States); Amini, Zakariya, E-mail: zakariya.amini@wmich.edu [Great Lakes Environmental and Molecular Sciences Center, Department of Biological Sciences, 3425 Wood Hall, Western Michigan University, 1903 West Michigan Avenue, Kalamazoo, MI 49008 (United States); Whitaker, Ashley S., E-mail: ashley.s.whitaker@wmich.edu [Great Lakes Environmental and Molecular Sciences Center, Department of Biological Sciences, 3425 Wood Hall, Western Michigan University, 1903 West Michigan Avenue, Kalamazoo, MI 49008 (United States); Ide, Charles F., E-mail: charles.ide@wmich.edu [Great Lakes Environmental and Molecular Sciences Center, Department of Biological Sciences, 3425 Wood Hall, Western Michigan University, 1903 West Michigan Avenue, Kalamazoo, MI 49008 (United States)

    2011-08-15

    In our laboratory, Xenopus laevis tadpoles exposed throughout development to 200 or 400 {mu}g/L atrazine, concentrations reported to periodically occur in puddles, vernal ponds and runoff soon after application, were smaller and had smaller fat bodies (the tadpole's lipid storage organ) than controls. It was hypothesized that these changes were due to atrazine-related perturbations of energy homeostasis. To investigate this hypothesis, selected metabolic responses to exposure at the transcriptional and biochemical levels in atrazine-exposed tadpoles were measured. DNA microarray technology was used to determine which metabolic pathways were affected after developmental exposure to 400 {mu}g/L atrazine. From these data, genes representative of the affected pathways were selected for assay using quantitative real time polymerase chain reaction (qRT-PCR) to measure changes in expression during a 2-week exposure to 400 {mu}g/L. Finally, ATP levels were measured from tadpoles both early in and at termination of exposure to 200 and 400 {mu}g/L. Microarray analysis revealed significant differential gene expression in metabolic pathways involved with energy homeostasis. Pathways with increased transcription were associated with the conversion of lipids and proteins into energy. Pathways with decreased transcription were associated with carbohydrate metabolism, fat storage, and protein synthesis. Using qRT-PCR, changes in gene expression indicative of an early stress response to atrazine were noted. Exposed tadpoles had significant decreases in acyl-CoA dehydrogenase (AD) and glucocorticoid receptor protein (GR) mRNA after 24 h of exposure, and near-significant (p = 0.07) increases in peroxisome proliferator-activated receptor {beta} (PPAR-{beta}) mRNA by 72 h. Decreases in AD suggested decreases in fatty acid {beta}-oxidation while decreases in GR may have been a receptor desensitization response to a glucocorticoid surge. Involvement of PPAR-{beta}, an energy

  17. Identification and quantification in single muscle fibers of four isoforms of parvalbumin in the iliofibularis muscle of Xenopus laevis.

    Science.gov (United States)

    Simonides, W S; van Hardeveld, C

    1989-10-05

    The major parvalbumins present in the iliofibularis muscle of Xenopus laevis were identified and the total parvalbumin content of different types of single fibers of this muscle was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS). The criteria used in the identification of proteins as parvalbumins were: a relative molecular mass (Mr) between 10,000 and 14,000, an isoelectric point (pI) between 4.0 and 5.0, and a Ca2+-dependent mobility when run on a polyacrylamide gel in the absence of SDS. Four proteins were thus identified as parvalbumins: PA1, Mr 14,000, pI 4.90; PA2, Mr 11,000, pI 4.90; PA3, Mr 11,000, pI 4.95; and PA4, Mr 11,000, pI 4.25. An ultraviolet absorbance spectrum characteristic of parvalbumins was recorded for a purified preparation of these four proteins. Because the apparent Mr of rabbit parvalbumin in the gel system used was 14,000, whereas the true value is 12,100, it is not excluded that the Mr of component PA1 of 14,000 is an overestimation. The total parvalbumin content of muscles and single muscle fibers was determined using the supernatant obtained after centrifugation of tissue homogenates. Analysis of the protein pattern after electrophoresis in the presence of SDS of this fraction indicated that the Mr 14,000 and 11,000 protein bands contained virtually only parvalbumin. Quantification of the total parvalbumin content of relatively fast (type 1) and slow (type 2) contracting and relaxing single muscle fibers, using laser densitometric analysis of minigels, yielded mean values (mg protein/g wet wt., +/- S.D.) of 5.2 +/- 0.8 for nine type 1 fibers, and 1.9 +/- 1.0 for five type 2 fibers. Both fiber types contained about 2.5-times as much of the Mr 14,000 isoform relative to the combined Mr 11,000 isoforms.

  18. The acrylamide (S-2 as a positive and negative modulator of Kv7 channels expressed in Xenopus laevis oocytes.

    Directory of Open Access Journals (Sweden)

    Sigrid Marie Blom

    Full Text Available BACKGROUND: Activation of voltage-gated potassium channels of the Kv7 (KCNQ family reduces cellular excitability. These channels are therefore attractive targets for treatment of diseases characterized by hyperexcitability, such as epilepsy, migraine and neuropathic pain. Retigabine, which opens Kv7.2-5, is now in clinical trial phase III for the treatment of partial onset seizures. One of the main obstacles in developing Kv7 channel active drugs has been to identify compounds that can discriminate between the neuronal subtypes, a feature that could help diminish side effects and increase the potential of drugs for particular indications. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we have made a thorough investigation of the Bristol-Myers Squibb compound (S-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1], [4]oxazin-6-yl-ethyl]-3-(2-fluoro-phenyl-acrylamide [(S-2] on human Kv7.1-5 channels expressed in Xenopus laevis oocytes. We found that the compound was a weak inhibitor of Kv7.1. In contrast, (S-2 efficiently opened Kv7.2-5 by producing hyperpolarizing shifts in the voltage-dependence of activation and enhancing the maximal current amplitude. Further, it reduced inactivation, accelerated activation kinetics and slowed deactivation kinetics. The mechanisms of action varied between the subtypes. The enhancing effects of (S-2 were critically dependent on a tryptophan residue in S5 also known to be crucial for the effects of retigabine, (S-1 and BMS-204352. However, while (S-2 did not at all affect a mutant Kv7.4 with a leucine in this position (Kv7.4-W242L, a Kv7.2 with the same mutation (Kv7.2-W236L was inhibited by the compound, showing that (S-2 displays a subtype-selective interaction with in the Kv7 family. CONCLUSIONS/SIGNIFICANCE: These results offer further insight into pharmacological activation of Kv7 channels, add to the understanding of small molecule interactions with the channels and may contribute to the design of

  19. δβγ-ENaC is inhibited by CFTR but stimulated by cAMP in Xenopus laevis oocytes.

    Science.gov (United States)

    Rauh, Robert; Hoerner, Christian; Korbmacher, Christoph

    2017-02-01

    The epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel critically regulate airway surface liquid by driving fluid absorption and secretion, respectively. Their functional interplay is complex and incompletely understood. ENaC is a heteromeric channel with three well-characterized subunits (α, β, and γ). In humans, an additional δ-ENaC subunit exists in lung and several other tissues, where it may replace the α-subunit to form δβγ-ENaC. Little is known about the physiological role of δβγ-ENaC and its possible interaction with CFTR. The aim of the present study was to investigate the effect of human CFTR on human δβγ-ENaC heterologously expressed in Xenopus laevis oocytes. In oocytes coexpressing δβγ-ENaC and CFTR the ENaC-mediated amiloride-sensitive whole cell current (ΔIami) was reduced by ~50% compared with that measured in oocytes expressing δβγ-ENaC alone. Moreover, basal level of proteolytic ENaC activation was reduced in the presence of CFTR. The inhibitory effect of CFTR on δβγ-ENaC was due to a combination of decreased average open probability (Po) and reduced channel expression at the cell surface. Interestingly, in oocytes expressing δβγ-ENaC, increasing intracellular [cAMP] by IBMX and forskolin increased ΔIami by ~50%. This stimulatory effect was not observed for human and rat αβγ-ENaC and was independent of CFTR coexpression and coactivation. Experiments with a mutant channel (δβS520Cγ-ENaC) which can be converted to a channel with a Po of nearly 1 suggested that cAMP activates δβγ-ENaC by increasing Po In conclusion, our results demonstrate that δβγ-ENaC is inhibited by CFTR but activated by cAMP.

  20. Clotrimazole exposure modulates aromatase activity in gonads and brain during gonadal differentiation in Xenopus tropicalis frogs.

    Science.gov (United States)

    Gyllenhammar, Irina; Eriksson, Hanna; Söderqvist, Anneli; Lindberg, Richard H; Fick, Jerker; Berg, Cecilia

    2009-01-31

    Clotrimazole is a pharmaceutical used for treatment of fungal infections. It has been found in surface waters outside municipal wastewater treatment plants but data are scarce regarding its effects on aquatic organisms. It is known that clotrimazole and other imidazole fungicides are inhibitors of the enzyme aromatase (CYP 19). Aromatase converts androgens into estrogens and is suggested to be involved in the sex differentiation in amphibians. The aim of the present study was to evaluate effects of larval exposure to clotrimazole on aromatase activity in brain and gonads, and on gonadal differentiation in Xenopus tropicalis frogs. Another purpose was to determine if larval exposure to ethynylestradiol (EE(2)), at a concentration known to cause male-to-female sex reversal, affects aromatase activity in brain and gonads during gonadal differentiation. Tadpoles were exposed from shortly after hatching (Nieuwkoop and Faber developmental stages 47-48) until complete metamorphosis (NF stage 66) to 6, 41, and 375 nM clotrimazole or 100 nM (nominal) EE(2). Aromatase activity was measured in the brain and gonad/kidney complex of tadpoles during gonadal differentiation (NF stage 56) and, in the clotrimazole experiment, also at metamorphosis. In clotrimazole-exposed tadpoles gonadal aromatase activity increased over exposure time in the 41 and 375 nM groups but did not differ significantly from the control group. Gonadal aromatase activity was increased in both sexes exposed to 41 and 375 nM clotrimazole at metamorphosis. Brain aromatase activity was decreased in tadpoles (NF stage 56) exposed to 375 nM clotrimazole, but at metamorphosis no differences were seen between groups or between sexes. No effects of clotrimazole on sex ratio or gonadal histology were noted at completed metamorphosis. EE(2)-exposed tadpoles had a slightly decreased gonadal aromatase activity, though not significantly different from control group, and there was no effect of EE(2) on brain aromatase

  1. Jumping performance in the highly aquatic frog, Xenopus tropicalis: sex-specific relationships between morphology and performance

    Directory of Open Access Journals (Sweden)

    Anthony Herrel

    2014-11-01

    Full Text Available Frogs are characterized by a morphology that has been suggested to be related to their unique jumping specialization. Yet, the functional demands associated with jumping and swimming may not be that different as suggested by studies with semi-aquatic frogs. Here, we explore whether features previously identified as indicative of good burst swimming performance also predict jumping performance in a highly aquatic frog, Xenopus tropicalis. Moreover, we test whether the morphological determinants of jumping performance are similar in the two sexes and whether jumping performance differs in the two sexes. Finally we test whether jumping capacity is positively associated with burst swimming and terrestrial endurance capacity in both sexes. Our results show sex-specific differences in jumping performance when correcting for differences in body size. Moreover, the features determining jumping performance are different in the two sexes. Finally, the relationships between different performance traits are sex-dependent as well with females, but not males, showing a trade-off between peak jumping force and the time jumped to exhaustion. This suggests that different selective pressures operate on the two sexes, with females being subjected to constraints on locomotion due to their greater body mass and investment in reproductive capacity. In contrast, males appear to invest more in locomotor capacity giving them higher performance for a given body size compared to females.

  2. Assessment of lead ecotoxicity in water using the amphibian larvae (Xenopus laevis) and preliminary study of its immobilization in meat and bone meal combustion residues.

    Science.gov (United States)

    Mouchet, F; Cren, S; Cunienq, C; Deydier, E; Guilet, R; Gauthier, L

    2007-04-01

    Lead (Pb) is a major chemical pollutant of the environment. It has been associated with human activities for the last 6000 years. Quite rightly, it remains a public health concern today. The present investigation evaluates the toxic potential of Pb in larvae of the toad Xenopus laevis after 12 days exposure in lab conditions. Acute toxicity, genotoxicity and Pb bioaccumulation were analyzed. The genotoxic effects were analyzed in the circulating blood from the levels of micronucleus induction according to the French standard micronucleus assay (AFNOR 2000 Association française de normalization. Norme NFT 90-325. Qualité de l'Eau. Evaluation de la génotoxicité au moyen de larves d'amphibien (Xenopus laevis, Pleurodeles waltl)). Lead bioaccumulation was analyzed in the liver of larvae at the end of exposure. Moreover, the toxic potential of lead, in aquatic media, was investigated in the presence of meat and bone meal combustion residues (MBMCR) known to be rich in phosphates and a potential immobiliser of lead. Previously, acute toxicity and genotoxicity of MBMCR alone were evaluated using Xenopus larvae. The results obtained in the present study demonstrated: (i) that lead is acutely toxic and genotoxic to amphibian larvae from 1 mg Pb/l and its bioaccumulation is significant in the liver of larvae from the lowest concentration of exposure (1 microg Pb/l), (ii) MBMCR were not acutely toxic nor genotoxic in Xenopus larvae, (iii) lead in presence of MBMCR induced inhibition or reduction of the toxic and genotoxic potential of lead in water at concentrations that do not exceed the capacity of MBMCR of Pb-binding (iv) Pb accumulation in larvae exposed to lead with MBMCR in water was lower than Pb-accumulation in larvae exposed to lead alone except at the concentration of 0.01 mg Pb/l suggesting complex mechanisms of MBMCR interaction in organisms. The results confirm the high toxicity and genotoxicity of lead in the aquatic compartment and suggest the potential

  3. Quick-freeze, deep-etch, rotary-shadow views of the extracellular matrix and cortical cytoskeleton of Xenopus laevis eggs.

    Science.gov (United States)

    Larabell, C A; Chandler, D E

    1989-11-01

    The quick-freeze, deep-etch, rotary-shadow technique provides a powerful tool to study the structural dynamics of extracellular matrices. Using this technique, we show that the extracellular investments of the Xenopus laevis egg are multilayered and securely anchored to the egg surface. The cortical cytoskeleton within the egg contains embedded cortical granules with surrounding endoplasmic reticulum and is capped by a thin reticular sheet that contacts the inner surface of the plasma membrane. The extracellular matrix undergoes three distinct changes at fertilization: a) formation of a "smooth" layer below the vitelline envelope (VE), b) transformation of the VE itself to an altered VE composed of concentric fibrous sheets, and c) formation of a dense, "briar-patch"-like fertilization layer at the upper surface of the VE.

  4. Regression of blood vessels in the ventral velum of Xenopus laevis Daudin during metamorphosis: light microscopic and transmission electron microscopic study.

    Science.gov (United States)

    Bartel, H; Lametschwandtner, A

    2000-08-01

    Structural changes of the ventral velum of Xenopus laevis tadpoles from late prometamorphosis (stage 58) to the height of metamorphic climax (stage 62) were examined by light and transmission electron microscopy. Special emphasis was given to the blood vessel regression. Early changes of velar capillaries were formation of luminal and abluminal endothelial cell processes, vacuolation, and cytoplasmic and nuclear chromatin condensation. At the height of metamorphic climax, transmission electron microscopy revealed apoptotic endothelial cells with nuclear condensation and fragmentation, intraluminal bulging of rounded endothelial cells which narrowed or even plugged the capillary, and different stages of endothelial cell detachment ('shedding') into the vessel lumen. These changes explain the 'miniaturisation' of the velar microvascular bed as well as the typical features found in resin-casts of regressing velar vessels which have been observed in a previous scanning electron microscopy study of the ventral velum.

  5. The role of brain-derived neurotrophic factor in the regulation of cell growth and gene expression in melanotrope cells of Xenopus laevis.

    Science.gov (United States)

    Jenks, Bruce G; Kuribara, Miyuki; Kidane, Adhanet H; Kramer, Bianca M R; Roubos, Eric W; Scheenen, Wim J J M

    2012-07-01

    Brain-derived neurotrophic factor (BDNF) is, despite its name, also found outside the central nervous system (CNS), but the functional significance of this observation is largely unknown. This review concerns the expression of BDNF in the pituitary gland. While the presence of the neurotrophin in the mammalian pituitary gland is well documented its functional significance remains obscure. Studies on the pars intermedia of the pituitary of the amphibian Xenopus laevis have shown that BDNF is produced by the neuroendocrine melanotrope cells, its expression is physiologically regulated, and the melanotrope cells themselves express receptors for the neurotrophin. The neurotrophin has been shown to act as an autocrine factor on the melanotrope to promote cell growth and regulate gene expression. In doing so BDNF supports the physiological function of the cell to produce and release α-melanophore-stimulating hormone for the purpose of adjusting the animal's skin color to that of its background.

  6. A novel short anionic antibacterial peptide isolated from the skin of Xenopus laevis with broad antibacterial activity and inhibitory activity against breast cancer cell.

    Science.gov (United States)

    Li, Siming; Hao, Linlin; Bao, Wanguo; Zhang, Ping; Su, Dan; Cheng, Yunyun; Nie, Linyan; Wang, Gang; Hou, Feng; Yang, Yang

    2016-07-01

    A vastarray of bioactive peptides from amphibian skin secretions is attracting increasing attention due to the growing problem of bacteria resistant to conventional antibiotics. In this report, a small molecular antibacterial peptide, named Xenopus laevis antibacterial peptide-P1 (XLAsp-P1), was isolated from the skin of Xenopus laevis using reversed-phase high-performance liquid chromatography. The primary structure of XLAsp-P1, which has been proved to be a novel peptide by BLAST search in AMP database, was DEDDD with a molecular weight of 607.7 Da analysed by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). The highlight of XLAsp-P1 is the strong in vitro potency against a variety of Gram-positive and Gram-negative bacteria with minimum inhibitory concentrations (MICs) starting at 10 μg/mL and potent inhibitory activity against breast cancer cell at tested concentrations from 5 to 50 μg/mL. In addition, only 6.2 % of red blood cells was haemolytic when incubated with 64 μg/mL (higher than MICs of all bacterial strain) of XLAsp-P1. The antimicrobial mechanism for this novel peptide was the destruction of the cell membrane investigated by transmission electron microscopy. All these showed that XLAsp-P1 is a novel short anionic antibacterial peptide with broad antibacterial activity and inhibitory activity against breast cancer cell.

  7. Examination of KNK437- and quercetin-mediated inhibition of heat shock-induced heat shock protein gene expression in Xenopus laevis cultured cells.

    Science.gov (United States)

    Manwell, Laurie A; Heikkila, John J

    2007-11-01

    We examined the effect of quercetin (3,3',4',5,7-pentahydroxyflavon) and KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolactam), a benzylidene lactam compound, on heat-induced heat shock protein (hsp) gene expression in Xenopus laevis A6 kidney epithelial cells. In previous studies, both quercetin and KNK437 inhibited heat shock factor activity resulting in a repression of hsp mRNA and protein accumulation in human cultured cells. In this first study of the effect of these hsp gene expression inhibitors in a non-mammalian cell line, we report that both quercetin and KNK437 reduced the heat shock-induced accumulation of hsp30, hsp47 and hsp70 mRNA in X. laevis cultured cells. However, these inhibitors had no effect on the relative level of a non-heat shock protein mRNA, ef1alpha, in either control or heat shocked cells. Western blot and immunocytochemical analyses revealed that quercetin partially inhibited HSP30 protein accumulation. In contrast, HSP30 protein was not detectable in KNK437-treated cells. Finally, treatment of A6 cells with KNK437 inhibited the heat shock-induced acquisition of thermotolerance, as determined by preservation of actin filaments and cellular morphology using immunocytochemistry and laser scanning confocal microscopy.

  8. A perchlorate sensitive iodide transporter in frogs.

    Science.gov (United States)

    Carr, Deborah L; Carr, James A; Willis, Ray E; Pressley, Thomas A

    2008-03-01

    Nucleotide sequence comparisons have identified a gene product in the genome database of African clawed frogs (Xenopus laevis) as a probable member of the solute carrier family of membrane transporters. To confirm its identity as a putative iodide transporter, we examined the function of this sequence after heterologous expression in mammalian cells. A green monkey kidney cell line transfected with the Xenopus nucleotide sequence had significantly greater (125)I uptake than sham-transfected control cells. The uptake in carrier-transfected cells was significantly inhibited in the presence of perchlorate, a competitive inhibitor of mammalian Na(+)/iodide symporter. Tissue distributions of the sequence were also consistent with a role in iodide uptake. The mRNA encoding the carrier was found to be expressed in the thyroid gland, stomach, and kidney of tadpoles from X. laevis, as well as the bullfrog Rana catesbeiana. The ovaries of adult X. laevis also were found to express the carrier. Phylogenetic analysis suggested that the putative X. laevis iodide transporter is orthologous to vertebrate Na(+)-dependent iodide symporters. We conclude that the amphibian sequence encodes a protein that is indeed a functional Na(+)/iodide symporter in X. laevis, as well as R. catesbeiana.

  9. JAK‐STAT pathway activation in response to spinal cord injury in regenerative and non‐regenerative stages of Xenopus laevis

    Science.gov (United States)

    Tapia, Victor S.; Herrera‐Rojas, Mauricio

    2017-01-01

    Abstract Xenopus laevis tadpoles can regenerate the spinal cord after injury but this capability is lost during metamorphosis. Comparative studies between pre‐metamorphic and metamorphic Xenopus stages can aid towards understanding the molecular mechanisms of spinal cord regeneration. Analysis of a previous transcriptome‐wide study suggests that, in response to injury, the JAK‐STAT pathway is differentially activated in regenerative and non‐regenerative stages. We characterized the activation of the JAK‐STAT pathway and found that regenerative tadpoles have an early and transient activation. In contrast, the non‐regenerative stages have a delayed and sustained activation of the pathway. We found that STAT3 is activated in response to injury mainly in Sox2/3+ ependymal cells, motoneurons and sensory neurons. Finally, to study the role of temporal activation we generated a transgenic line to express a constitutively active version of STAT3. The sustained activation of the JAK‐STAT pathway in regenerative tadpoles reduced the expression of pro‐neurogenic genes normally upregulated in response to spinal cord injury, suggesting that activation of the JAK‐STAT pathway modulates the fate of neural progenitors. PMID:28316792

  10. NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

    Science.gov (United States)

    Baur, Tina; Ramadan, Kristijan; Schlundt, Andreas; Kartenbeck, Jürgen; Meyer, Hemmo H

    2007-08-15

    Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.

  11. IRE1α影响非洲爪蟾胰腺发育%The effects of IRE1α on pancreas development in Xenopus laevies

    Institute of Scientific and Technical Information of China (English)

    李昕昕; 冯娇娇; 殷晨阳; 徐校佩; 王璐璐; 郭静; 王学军; 王宁; 袁栎

    2012-01-01

    目的:探讨肌醇依赖性激酶1α(inositol requiring enzyme 1α,IRE1α)对非洲爪蟾胰腺发育的影响.方法:通过显微注射基因特异性反义寡核苷酸实现基因敲降;利用整体胚胎原位杂交方法检测基因表达.结果:IRE1α表达于爪蟾发育中的胰腺;利用基因特异性反义寡核苷酸敲降IRE1α后,非洲爪蟾肠道明显异常,胰腺内外分泌标志性基因胰岛素、淀粉酶表达显著减少;胰腺前体细胞的标志基因pdx1和p48表达明显减少;敲降IRE1 α下游基因X-盒结合蛋白1(X-box binding protein 1,XBP1)后,非洲爪蟾的胰岛素、淀粉酶表达也明显减少.结论:IRE1α影响非洲爪蟾胰腺发育,可能通过XBP1发挥作用.%Objective:To investigate the effects of inositol requiring enzyme la (IRE1α) on pancreas development of Xenopus laevies. Methods:The gene-specific antisense oligonucleotides,morpholino (MO)s,were microinjected to knockdown IRE1α and XBP1. The whole mount in situ hybridization of Xenopus embryos was used to detect the gene expression. Resuits:IRE1α was expressed in developing pancreas in Xenopus. There were serious defects in gut development observed in IREla knockdown embryos of Xenopus. The expression of the endocrine marker gene(insulin) and the exocrine pancreas marker gene(amylase) was undetectable during tadpole stages of development in IRE1α knockdown embryos compared to the control embryos. The expression of the pancreas progenitor cell marker genes (pdx1 and p48) was repressed significantly. And after knockdown of XBP1 ,the expression of insulin and amylase decreased significantly and can't be detectable. Conclusion:IRE1α plays an essential role in pancreas development of Xenopus and may be through XBP1 dependent pathway.

  12. Use of the enhanced frog embryo teratogenesis assay-Xenopus (FETAX) to determine chemically-induced phenotypic effects.

    Science.gov (United States)

    Hu, Lingling; Zhu, Jingmin; Rotchell, Jeanette M; Wu, Lijiao; Gao, Jinjuan; Shi, Huahong

    2015-03-01

    The frog embryo teratogenesis assay-Xenopus (FETAX) is an established method for the evaluation of the developmental toxicities of chemicals. To develop an enhanced FETAX that is appropriate for common environmental contaminants, we exposed Xenopus tropicalis embryos to eight compounds, including tributyltin, triphenyltin, CdCl2, pyraclostrobin, picoxystrobin, coumoxystrobin, all-trans-retinoic acid and 9-cis-retinoic acid. Multiple malformations were induced in embryos particularly following exposure to tributyltin, triphenyltin and pyraclostrobin at environmentally relevant concentrations. Based on the range of observed malformations, we proposed a phenotypic assessment method with 20 phenotypes and a 0-5 scoring system. This derived index exhibited concentration-dependent relationships for all of the chemicals tested. Furthermore, the phenotype profiles were characteristic of the different tested chemicals. Our results indicate that malformation phenotypes can be quantitatively integrated with the primary endpoints in conventional FETAX assessments to allow for increased sensitivity and measurement of quantitative effects and to provide indicative mechanistic information for each tested chemical.

  13. Friend of GATA (FOG interacts with the nucleosome remodeling and deacetylase complex (NuRD to support primitive erythropoiesis in Xenopus laevis.

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    Mizuho S Mimoto

    Full Text Available Friend of GATA (FOG plays many diverse roles in adult and embryonic hematopoiesis, however the mechanisms by which it functions and the roles of potential interaction partners are not completely understood. Previous work has shown that overexpression of FOG in Xenopus laevis causes loss of blood suggesting that in contrast to its role in mammals, FOG might normally function to repress erythropoiesis in this species. Using loss-of-function analysis, we demonstrate that FOG is essential to support primitive red blood cell (RBC development in Xenopus. Moreover, we show that it is specifically required to prevent excess apoptosis of circulating primitive RBCs and that in the absence of FOG, the pro-apoptotic gene Bim-1 is strongly upregulated. To identify domains of FOG that are essential for blood development and, conversely, to begin to understand the mechanism by which overexpressed FOG represses primitive erythropoiesis, we asked whether FOG mutants that are unable to interact with known co-factors retain their ability to rescue blood formation in FOG morphants and whether they repress erythropoiesis when overexpressed in wild type embryos. We find that interaction of FOG with the Nucleosome Remodeling and Deacetylase complex (NuRD, but not with C-terminal Binding Protein, is essential for normal primitive RBC development. In contrast, overexpression of all mutant and wild type constructs causes a comparable repression of primitive erythropoiesis. Together, our data suggest that a requirement for FOG and its interaction with NuRD during primitive erythropoiesis are conserved in Xenopus and that loss of blood upon FOG overexpression is due to a dominant-interfering effect.

  14. Diclofenac can exhibit estrogenic modes of action in male Xenopus laevis, and affects the hypothalamus-pituitary-gonad axis and mating vocalizations.

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    Efosa, Norah Johanna; Kleiner, Wibke; Kloas, Werner; Hoffmann, Frauke

    2017-04-01

    Diclofenac (DCF) is a non-steroidal analgesic and antiphlogistic. Due to its tremendous use, DCF can be found in the environment, especially in sewage, but also surface waters, ground and drinking water. Previous studies indicated that DCF can modulate the reproductive physiology of fish by altering the expression of important key enzymes of the hypothalamus-pituitary-gonad-axis (HPG-axis) and might act as an estrogenic endocrine disrupting chemical (EDC). Other studies, however, demonstrated that DCF does not exhibit any estrogenicity. Thus, in the present study we investigated whether an exposure to DCF can affect reproductive behavior and physiology of adult male X. laevis by analyzing DCF effects on the mate calling behavior of the frogs and on gene expression patterns of key biomarkers of the HPG-axis. In addition, plasma sex steroid levels were determined to gain detailed insights into the mechanisms of DCF action. We could demonstrate that DCF can act as EDC by exhibiting slight estrogenic modes of action. In addition, pharmacological impacts on gonadal steroidogenesis could be revealed leading to imbalances in sex steroid levels and ratios. DCF furthermore altered the calling behavior of exposed males, potentially reducing the mating and reproductive success of the frogs, possibly leading to severe population effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Investigations of the effects of the antimalarial drug dihydroartemisinin (DHA) using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX).

    Science.gov (United States)

    Longo, Monica; Zanoncelli, Sara; Della Torre, Paola; Rosa, Francesco; Giusti, AnnaMaria; Colombo, Paolo; Brughera, Marco; Mazué, Guy; Olliaro, Piero

    2008-08-01

    Artemisinin derivatives are effective and safe drugs for treating malaria, but they are not recommended during the first trimester of pregnancy because of resorptions and abnormalities observed in animal reproduction studies. Previous studies in rats showed that artemisinin embryotoxicity derives from the depletion of primitive red blood cells (RBCs) over a narrow critical time window (gestation Days 9-14). In order to further investigate the susceptibility of primitive RBCs to artemisinins and to establish whether this susceptibility is species-specific or inherent to the compound, we studied dihydroartemisinin (DHA), both a drug in its own right and the main metabolite of current artemisinin derivatives in use, in the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). This model readily allows investigation and monitoring of primitive and definitive RBCs. Effects on frog larvae exposed to DHA for 48 h during early embryonic development, starting from 24 h post fertilization, were similar to those on rat embryos in terms of reduction in the number of primitive RBCs (clonally produced within the ventral blood island). In contrast, RBCs of older larvae (stage 47, produced at the definitive sites of hematopoiesis) were affected minimally and subsequently recovered. Compared to rat embryos, the frog larvae had no areas of necrosis but they shared similar heart defects. The mitochondrion appeared to be the main subcellular target, similar to observations in Plasmodium. These results implicate artemisinin-induced embryotoxicity through perturbation of metabolically active RBCs; whereas this mode of action does not appear to be species-specific, the stages of susceptibility varied between different species. The window of susceptibility and duration of exposure must be considered to evaluate the clinical relevance of these findings.

  16. High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Esther K. Kieserman, Chanjae Lee, Ryan S. Gray, Tae Joo Park and John B. Wallingford Corresponding author ([]()). ### INTRODUCTION Embryos of the frog *Xenopus laevis* are an ideal model system for in vivo imaging of dynamic biological processes, from the inner workings of individual cells to the reshaping of tissues during embryogenesis. Their externally developing embryos are more amenable to in vivo analysis than in...

  17. Further Development and Validation of the Frog Embryo Teratogenesis Assay-Xenopus (FETAX).

    Science.gov (United States)

    1992-11-23

    first used in studying the hollow blistula stage of a few hundred cells developmental toxicity of selenium (Browne and (Figure 1) to a free-swimming...ONTROLS pigmentation, locomotion and hatchability are described in the ASTM New Standard Guide It is necessary to use appropriate controls in (Bantle and...Dumnont IN: Toxicity of selenium to acety enic alcohols on embryos and larvae of developin Xeno usr kawvs embryos. J Xenous laevis. In: Aquatic

  18. 4-Bromo-2,5-dimethoxyphenethylamine (2C-B) and structurally related phenylethylamines are potent 5-HT2A receptor antagonists in Xenopus laevis oocytes

    Science.gov (United States)

    Villalobos, Claudio A; Bull, Paulina; Sáez, Patricio; Cassels, Bruce K; Huidobro-Toro, J Pablo

    2004-01-01

    We recently described that several 2-(2,5-dimethoxy-4-substituted phenyl)ethylamines (PEAs), including 4-I=2C-I, 4-Br=2C-B, and 4-CH3=2C-D analogs, are partial agonists at 5-HT2C receptors, and show low or even negligible intrinsic efficacy at 5-HT2A receptors. These results raised the proposal that these drugs may act as 5-HT2 antagonists. To test this hypothesis, Xenopus laevis oocytes were microinjected with the rat clones for 5-HT2A or 5-HT2C receptors. The above-mentioned PEAs and its 4-H analog (2C-H) blocked the 5-HT-induced currents at 5-HT2A, but not at the 5-HT2C receptor, revealing 5-HT2 receptor subtype selectivity. The 5-HT2A receptor antagonism required a 2-min preincubation to attain maximum inhibition. All PEAs tested shifted the 5-HT concentration–response curves to the right and downward. Their potencies varied with the nature of the C(4) substituent; the relative rank order of their 5-HT2A receptor antagonist potency was 2C-I>2C-B>2C-D>2C-H. The present results demonstrate that in X. laevis oocytes, a series of 2,5-dimethoxy-4-substituted PEAs blocked the 5-HT2A but not the 5-HT2C receptor-mediated responses. As an alternative hypothesis, we suggest that the psychostimulant activity of the PEAs may not be exclusively associated with partial or full 5-HT2A receptor agonism. PMID:15006903

  19. Binding of adrenergic ligands to liver plasma membrane preparations from the axolotl, Ambystoma mexicanum; the toad, Xenopus laevis; and the Australian lungfish, Neoceratodus forsteri.

    Science.gov (United States)

    Janssens, P A; Grigg, J A

    1988-09-01

    The beta-adrenergic ligand iodocyanopindolol (ICP) bound specifically to hepatic plasma membrane preparations from the axolotl, Ambystoma mexicanum (Bmax, 40 fmol/mg protein (P) at free concentration above 140 pM; KD, 42 pM); the toad, Xenopus laevis (Bmax, 200 fmol/mg P at 1 nM; KD, 300 pM); and the Australian lungfish, Neoceratodus forsteri (Bmax, 100 fmol/mg P at 5 nM). For the lungfish, the Scatchard plot was curved showing two classes of binding site with KD's of 20 and 500 pM. Neither the alpha 1-adrenergic ligand prazosin nor the alpha 2-adrenergic ligand yohimbine bound specifically to hepatic membrane preparations from any of the three species. Several adrenergic ligands displaced ICP from hepatic membrane preparations of all three species with KD's of Axolotl--propranolol, 50 nM; isoprenaline, 600 nM; adrenaline, 10 microM; phenylephrine, 20 microM; noradrenaline, 40 microM; and phentolamine, greater than 100 microM; X. laevis--propranolol, 30 nM; isoprenaline, 100 microM; adrenaline, 200 microM; noradrenaline, 300 microM; phenylephrine, 1 mM; and phentolamine, greater than 1 mM; N. forsteri,--propranolol, 25 nM; isoprenaline, 1 microM; adrenaline, 20 microM; phenylephrine, 35 microM; noradrenaline, 600 microM; and phentolamine, 400 microM. These findings suggest that alpha-adrenergic receptors are not present in hepatic plasma membrane preparations from these three species and that the hepatic actions of catecholamines are mediated via beta-adrenergic receptors. The order of binding of the beta-adrenergic ligands suggests that the receptors are of the beta 2 type.

  20. The gastrocoel roof plate in embryos of different frogs.

    Science.gov (United States)

    Sáenz-Ponce, Natalia; Santillana-Ortiz, Juan-Diego; del Pino, Eugenia M

    2012-02-01

    The morphology of the gastrocoel roof plate and the presence of cilia in this structure were examined in embryos of four species of frogs. Embryos of Ceratophrys stolzmanni (Ceratophryidae) and Engystomops randi (Leiuperidae) develop rapidly, provide comparison for the analysis of gastrocoel roof plate development in the slow-developing embryos of Epipedobates machalilla (Dendrobatidae) and Gastrotheca riobambae (Hemiphractidae). Embryos of the analyzed frogs develop from eggs of different sizes, and display different reproductive and developmental strategies. In particular, dorsal convergence and extension and archenteron elongation begin during gastrulation in embryos of rapidly developing frogs, as in Xenopus laevis. In contrast, cells that involute during gastrulation are stored in the large circumblastoporal collar that develops around the closed blastopore in embryos of slow-developing frogs. Dorsal convergence and extension only start after blastopore closure in slow-developing frog embryos. However, in the neurulae, a gastrocoel roof plate develops, despite the accumulation of superficial mesodermal cells in the circumblastoporal collar. Embryos of all four species develop a ciliated gastrocoel roof plate at the beginning of neurulation. Accordingly, fluid-flow across the gastrocoel roof plate is likely the mechanism of left-right asymmetry patterning in these frogs, as in X. laevis and other vertebrates. A ciliated gastrocoel roof plate, with a likely origin as superficial mesoderm, is conserved in frogs belonging to four different families and with different modes of gastrulation.

  1. The Xenopus laevis Atg4B Protease: Insights into Substrate Recognition and Application for Tag Removal from Proteins Expressed in Pro- and Eukaryotic Hosts.

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    Steffen Frey

    Full Text Available During autophagy, members of the ubiquitin-like Atg8 protein family get conjugated to phosphatidylethanolamine and act as protein-recruiting scaffolds on the autophagosomal membrane. The Atg4 protease produces mature Atg8 from C-terminally extended precursors and deconjugates lipid-bound Atg8. We now found that Xenopus laevis Atg4B (xAtg4B is ideally suited for proteolytic removal of N-terminal tags from recombinant proteins. To implement this strategy, an Atg8 cleavage module is inserted in between tag and target protein. An optimized xAtg4B protease fragment includes the so far uncharacterized C-terminus, which crucially contributes to recognition of the Xenopus Atg8 homologs xLC3B and xGATE16. xAtg4B-mediated tag cleavage is very robust in solution or on-column, efficient at 4°C and orthogonal to TEV protease and the recently introduced proteases bdSENP1, bdNEDP1 and xUsp2. Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification. We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae. In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps.

  2. The Xenopus laevis Atg4B Protease: Insights into Substrate Recognition and Application for Tag Removal from Proteins Expressed in Pro- and Eukaryotic Hosts.

    Science.gov (United States)

    Frey, Steffen; Görlich, Dirk

    2015-01-01

    During autophagy, members of the ubiquitin-like Atg8 protein family get conjugated to phosphatidylethanolamine and act as protein-recruiting scaffolds on the autophagosomal membrane. The Atg4 protease produces mature Atg8 from C-terminally extended precursors and deconjugates lipid-bound Atg8. We now found that Xenopus laevis Atg4B (xAtg4B) is ideally suited for proteolytic removal of N-terminal tags from recombinant proteins. To implement this strategy, an Atg8 cleavage module is inserted in between tag and target protein. An optimized xAtg4B protease fragment includes the so far uncharacterized C-terminus, which crucially contributes to recognition of the Xenopus Atg8 homologs xLC3B and xGATE16. xAtg4B-mediated tag cleavage is very robust in solution or on-column, efficient at 4°C and orthogonal to TEV protease and the recently introduced proteases bdSENP1, bdNEDP1 and xUsp2. Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification. We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae. In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps.

  3. Prx-1 expression in Xenopus laevis scarless skin-wound healing and its resemblance to epimorphic regeneration.

    Science.gov (United States)

    Yokoyama, Hitoshi; Maruoka, Tamae; Aruga, Akio; Amano, Takanori; Ohgo, Shiro; Shiroishi, Toshihiko; Tamura, Koji

    2011-12-01

    Despite a strong clinical need for inducing scarless wound healing, the molecular factors required to accomplish it are unknown. Although skin-wound healing in adult mammals often results in scarring, some amphibians can regenerate injured body parts, even an amputated limb, without it. To understand the mechanisms of perfect skin-wound healing in regenerative tetrapods, we studied the healing process in young adult Xenopus "froglets" after experimental skin excision. We found that the excision wound healed completely in Xenopus froglets, without scarring. Mononuclear cells expressing a homeobox gene, prx1, accumulated under the new epidermis of skin wounds on the limb and trunk and at the regenerating limb. In transgenic Xenopus froglets expressing a reporter for the mouse prx1 limb-specific enhancer, activity was seen in the healing skin and in the regenerating limb. Comparable activity did not accompany skin-wound healing in adult mice. Our results suggest that scarless skin-wound healing may require activation of the prx1 limb enhancer, and competence to activate the enhancer is probably a prerequisite for epimorphic regeneration, such as limb regeneration. Finally, the induction of this prx1 enhancer activity may be useful as a reliable marker for therapeutically induced scarless wound healing in mammals.

  4. Expression of odorant receptor family, type 2 OR in the aquatic olfactory cavity of amphibian frog Xenopus tropicalis.

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    Tosikazu Amano

    Full Text Available Recent genome wide in silico analyses discovered a new family (type 2 or family H of odorant receptors (ORs in teleost fish and frogs. However, since there is no evidence of the expression of these novel OR genes in olfactory sensory neurons (OSN, it remains unknown if type 2 ORs (OR2 function as odorant receptors. In this study, we examined expression of OR2 genes in the frog Xenopus tropicalis. The overall gene expression pattern is highly complex and differs depending on the gene and developmental stage. RT-PCR analysis in larvae showed that all of the OR2η genes we identified were expressed in the peripheral olfactory system and some were detected in the brain and skin. Whole mount in situ hybridization of the larval olfactory cavity confirmed that at least two OR2η genes so far tested are expressed in the OSN. Because tadpoles are aquatic animals, OR2η genes are probably involved in aquatic olfaction. In adults, OR2η genes are expressed in the nose, brain, and testes to different degrees depending on the genes. OR2η expression in the olfactory system is restricted to the medium cavity, which participates in the detection of water-soluble odorants, suggesting that OR2ηs function as receptors for water-soluble odorants. Moreover, the fact that several OR2ηs are significantly expressed in non-olfactory organs suggests unknown roles in a range of biological processes other than putative odorant receptor functions.

  5. Pain perception and anaesthesia in research frogs.

    Science.gov (United States)

    Guénette, Sarah Annie; Giroux, Marie-Chantal; Vachon, Pascal

    2013-01-01

    Frogs possess pain receptors and pathways that support processing and perception of noxious stimuli however the level of organization is less well structured compared to mammals. It was long believed that the experience of pain was limited to 'higher' phylums of the animal kingdom. However, it is now commonly accepted that amphibians possess neuro-anatomical pathways conductive of a complete nociceptive experience. Xenopus laevis frogs have been one of the most popular aquatic research models for developmental studies and genetic research. These frogs have been extensively use in research for their eggs, that can be collected following hormonal stimulation either naturally or by surgical intervention. Many anaesthetics have been used in amphibians such as bath solutions of MS-222, benzocaine and eugenol as well as systemic injections of ketamine or tiletamine, barbiturates, propofol and gas administrations of methoxyflurane, halothane and isoflurane. Most of these anaesthetic drugs produce variability in depth and duration of anaesthesia. MS-222 appears to be one of the most reliable anaesthetics. This review will focus on the evidence of pain perception in frogs and will compare the effectiveness and limitations of different anaesthetics used in Xenopus leavis frogs.

  6. Are Fish and Standardized FETAX Assays Protective Enough for Amphibians? A Case Study on Xenopus laevis Larvae Assay with Biologically Active Substances Present in Livestock Wastes

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    Federica Martini

    2012-01-01

    Full Text Available Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim and essential metals (zinc, copper, manganese, and selenium that may be found in livestock wastes were used for the larvae exposure. Lethal (LC50 and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians.

  7. Are Fish and Standardized FETAX Assays Protective Enough for Amphibians? A Case Study on Xenopus laevis Larvae Assay with Biologically Active Substances Present in Livestock Wastes

    Science.gov (United States)

    Martini, Federica; Tarazona, José V.; Pablos, M. Victoria

    2012-01-01

    Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim) and essential metals (zinc, copper, manganese, and selenium) that may be found in livestock wastes were used for the larvae exposure. Lethal (LC50) and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians. PMID:22629159

  8. Protein-Carbohydrate Interaction between Sperm and the Egg-Coating Envelope and Its Regulation by Dicalcin, a Xenopus laevis Zona Pellucida Protein-Associated Protein.

    Science.gov (United States)

    Miwa, Naofumi

    2015-05-22

    Protein-carbohydrate interaction regulates multiple important processes during fertilization, an essential biological event where individual gametes undergo intercellular recognition to fuse and generate a zygote. In the mammalian female reproductive tract, sperm temporarily adhere to the oviductal epithelium via the complementary interaction between carbohydrate-binding proteins on the sperm membrane and carbohydrates on the oviductal cells. After detachment from the oviductal epithelium at the appropriate time point following ovulation, sperm migrate and occasionally bind to the extracellular matrix, called the zona pellucida (ZP), which surrounds the egg, thereafter undergoing the exocytotic acrosomal reaction to penetrate the envelope and to reach the egg plasma membrane. This sperm-ZP interaction also involves the direct interaction between sperm carbohydrate-binding proteins and carbohydrates within the ZP, most of which have been conserved across divergent species from mammals to amphibians and echinoderms. This review focuses on the carbohydrate-mediated interaction of sperm with the female reproductive tract, mainly the interaction between sperm and the ZP, and introduces the fertilization-suppressive action of dicalcin, a Xenopus laevis ZP protein-associated protein. The action of dicalcin correlates significantly with a dicalcin-dependent change in the lectin-staining pattern within the ZP, suggesting a unique role of dicalcin as an inherent protein that is capable of regulating the affinity between the lectin and oligosaccharides attached on its target glycoprotein.

  9. Protein-Carbohydrate Interaction between Sperm and the Egg-Coating Envelope and Its Regulation by Dicalcin, a Xenopus laevis Zona Pellucida Protein-Associated Protein

    Directory of Open Access Journals (Sweden)

    Naofumi Miwa

    2015-05-01

    Full Text Available Protein-carbohydrate interaction regulates multiple important processes during fertilization, an essential biological event where individual gametes undergo intercellular recognition to fuse and generate a zygote. In the mammalian female reproductive tract, sperm temporarily adhere to the oviductal epithelium via the complementary interaction between carbohydrate-binding proteins on the sperm membrane and carbohydrates on the oviductal cells. After detachment from the oviductal epithelium at the appropriate time point following ovulation, sperm migrate and occasionally bind to the extracellular matrix, called the zona pellucida (ZP, which surrounds the egg, thereafter undergoing the exocytotic acrosomal reaction to penetrate the envelope and to reach the egg plasma membrane. This sperm-ZP interaction also involves the direct interaction between sperm carbohydrate-binding proteins and carbohydrates within the ZP, most of which have been conserved across divergent species from mammals to amphibians and echinoderms. This review focuses on the carbohydrate-mediated interaction of sperm with the female reproductive tract, mainly the interaction between sperm and the ZP, and introduces the fertilization-suppressive action of dicalcin, a Xenopus laevis ZP protein-associated protein. The action of dicalcin correlates significantly with a dicalcin-dependent change in the lectin-staining pattern within the ZP, suggesting a unique role of dicalcin as an inherent protein that is capable of regulating the affinity between the lectin and oligosaccharides attached on its target glycoprotein.

  10. Multiple ING1 and ING2 genes in Xenopus laevis and evidence for differential association of thyroid hormone receptors and ING proteins to their promoters.

    Science.gov (United States)

    Wagner, Mary J; Helbing, Caren C

    2008-03-01

    ING (INhibitor of Growth) tumor suppressor proteins are epigenetic factors involved in numerous cellular processes including apoptosis in species ranging from yeast to humans. We recently isolated ING1 and ING2 transcript variants in Xenopus laevis and showed that these transcripts were differentially regulated by thyroid hormone (TH) during postembryonic development. However, no information exists regarding ING gene structure and how it relates to these differential responses to TH. To further investigate the regulation of ING genes by TH, we isolated ING1 and ING2 gene sequences and demonstrated that there are at least duplicate genes for each. The relationship between transcript variants and their responsiveness to TH were examined through promoter sequence and chromatin immunoprecipitation analyses on tail homogenates. Both TH receptors (TRs) differentially associated with ING1 and ING2 promoter regions with increased recruitment in the presence of TH. This occurred irrespective of gene transcript level response to this hormone. However, differential recruitment of RNA polymerase II corresponded well to transcript levels. ING proteins consistently associated with their own gene promoters except in the region generating the TH-inducible xING1b5 transcript. In this case, a substantial recruitment of TRbeta in the absence ING proteins occurred. These data establish the TH-dependent recruitment of transcription factors to ING promoter regions and suggest that differential TR recruitment in response to TH may not be a sufficient indicator for modulating the expression of ING in the tail.

  11. Post-transcriptional modification of the wobble nucleotide in anticodon-substituted yeast tRNAArgII after microinjection into Xenopus laevis oocytes.

    Science.gov (United States)

    Fournier, M; Haumont, E; de Henau, S; Gangloff, J; Grosjean, H

    1983-02-11

    An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAArgII by NCG trinucleotide (N = A, C, G or U) is described. Partial digestion with S1-nuclease and T1-RNAase provides fragments which, when annealed together, form an "anticodon-deprived" yeast tRNAArgII. A novel anticodon, phosphorylated with (32P) label on its 5' terminal residue, is then inserted using T4-RNA ligase. Such "anticodon-substituted" yeast tRNAArgII are microinjected into the cytoplasm of Xenopus laevis oocytes and shown to be able to interact with the anticodon maturation enzymes under in vivo conditions. Our results indicate that when adenosine occurs in the wobble position (A34) in yeast tRNAArgII it is efficiently modified into inosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5U. In contrast, when a cytosine (C34) or guanosine (G34) occurs, they are not modified. These results are at variance with those obtained previously under similar conditions with anticodon derivatives of yeast tRNAAsp harbouring A, C, G or U as the first anticodon nucleotide. In this case, guanosine and uridine were modified while adenosine and cytosine were not.

  12. Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors.

    Science.gov (United States)

    Music, Ena; Khan, Saad; Khamis, Imran; Heikkila, John J

    2014-11-01

    The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

  13. Evaluation of spin labels for in-cell EPR by analysis of nitroxide reduction in cell extract of Xenopus laevis oocytes

    Science.gov (United States)

    Azarkh, Mykhailo; Okle, Oliver; Eyring, Philipp; Dietrich, Daniel R.; Drescher, Malte

    2011-10-01

    Spin-label electron paramagnetic resonance (SL-EPR) spectroscopy has become a powerful and useful tool for studying structure and dynamics of biomacromolecules. However, utilizing these methods at physiological temperatures for in-cell studies is hampered by reduction of the nitroxide spin labels and thus short half-lives in the cellular environment. Consequently, reduction kinetics of two structurally different nitroxides was investigated in cell extracts of Xenopus laevis oocytes using rapid-scan cw-experiments at X-band. The five member heterocyclic ring nitroxide PCA (3-carboxy-2,2,5,5-tetramethylpyrrolidinyl-1-oxy) under investigation features much higher stability against intracellular reduction than the six member ring analog TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxilic acid) and is therefore a suitable spin label type for in-cell EPR. The kinetic data can be described according to the Michaelis-Menten model and thus suggest an enzymatic or enzyme-mediated reduction process.

  14. Uptake and caffeine-induced release of calcium in fast muscle fibers of Xenopus laevis: effects of MgATP and P(i).

    Science.gov (United States)

    Stienen, G J; van Graas, I A; Elzinga, G

    1993-09-01

    To elucidate the origin of the reduction in force during prolonged muscle fatigue, the dependency of Ca2+ uptake and release on MgATP and P(i) concentration was studied in saponin-skinned fast skeletal muscle fibers of the iliofibularis muscle of Xenopus laevis at 3 degrees C. The sarcoplasmic reticulum was loaded with Ca2+ for 5 min at pCa 7.0. The amount of Ca2+ released was derived from the area of the caffeine-induced force response. Ca2+ uptake increased with the MgATP concentration present during loading. It was half maximal at 20 microM and saturated at higher concentrations. The kinetics of Ca2+ release were affected for MgATP concentrations between 0.1 and 0.5 mM or less, but the amount of Ca2+ released by caffeine in ATP-free solutions was substantial. Phosphate (15 mM) only slightly reduced Ca2+ uptake when the loading period was short (1 min). It is unlikely, therefore, that the reduction in MgATP concentration contributes to the depression of Ca2+ released from the sarcoplasmic reticulum during fatigue. The increase in P(i) concentration could play a small role by reducing Ca2+ uptake.

  15. The 22 S cylinder particles of Xenopus laevis. II. Immunological characterization and localization of their proteins in tissues and cultured cells.

    Science.gov (United States)

    Hügle, B; Kleinschmidt, J A; Franke, W W

    1983-11-01

    Cylinder-shaped particles of 10 nm diameter were isolated from nuclei of Xenopus laevis oocytes and purified by sucrose gradient centrifugation and DEAE-Sephacel chromatography. Antibodies to protein constituents of these isolated particles were elicited in guinea pigs and examined by immunoblotting and immunoprecipitation techniques as well as by immunofluorescence microscopy. The antibodies reacted with only two out of the 12 constituent polypeptides characteristic for these particles when examined in the denatured state by the immunoblotting technique, including the largest component of Mr 30 000, but were able to precipitate the whole ensemble of these polypeptides in immunoprecipitation experiments, in agreement with the notion that these proteins form the 22 S particle complex. The antibodies displayed a rather narrow range of interspecies cross-reactivity, showing reaction with cells of other amphibia but not with avian and mammalian cells. In oocytes as well as in transcriptionally active somatic cells the antigen was localized in the nucleoplasm, excluding nucleoli, as well as in the cytoplasm, usually suggesting a higher concentration in the nucleoplasm. During mitosis, the proteins were dispersed throughout the cytoplasm whereas the chromosomes were negative. Inactive cells such as mature erythrocytes, spermatids and spermatozoa were negative. These immunolocalization findings support our conclusion based on fractionation studies that the cylindershaped particles and their protein constituents occur both in the nucleoplasm and the cytoplasm of a broad range of cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Readaptation of the vestibuloocular reflex to 1g-Condition in immature lower vertebrates ( Xenopus laevis) after micro- or hypergravity exposure

    Science.gov (United States)

    Sebastian, C.; Horn, E.; Eβeling, K.; Neubert, J.

    The effects of altered gravitational conditions (AGC) on the development of the static vestibulo-ocular reflex (VOR) and readaptation to 1g were investigated in the amphibian Xenopus laevis. Tadpoles were exposed to microgravity (μg) during the German Space Mission D-2 for 10 days, using the STATEX closed survival system, or to 3g for 9 days during earth-bound experiments. At the beginning of AGC, the tadpoles had not yet developed the static VOR. The main results were: (i) Tadpoles with ug- or 3g-experience had a lower gain of the static VOR than the 1g-controls during the 2nd and 5th post-AGC days, (ii) Readaptation to response levels of 1g-reared controls usually occurred during the following weeks, except in slowly developing tadpoles with 3g-experience. Readaptation was less pronounced if, during the acute VOR test, tadpoles were rolled from the inclined to the normal posture than in the opposite test situation. It is postulated that (i) gravity is necessarily involved in the development of the static VOR, but only during a period including the time before onset of the first behavioural response; and (ii) readaptation which is superimposed by the processes of VOR development depends on many factors including the velocity of development, the actual excitation level of the vestibular systems and the neuroplastic properties of its specific pathways.

  17. Fertilization competence of the egg-coating envelope is regulated by direct interaction of dicalcin and gp41, the Xenopus laevis ZP3.

    Science.gov (United States)

    Miwa, Naofumi; Ogawa, Motoyuki; Hanaue, Mayu; Takamatsu, Ken

    2015-08-05

    Fertilization begins with species-restricted interaction of sperm and the egg-coating envelope, which includes a three-dimensional meshwork of filaments composed of glycoproteins (called ZP proteins). Growing evidence has unveiled the molecular nature of ZP proteins; however, the structural property conferring fertilization competence to the egg-coating envelope remains unknown. Here, we show the molecular mechanism that mediates direct interaction between dicalcin, a novel fertilization-suppressive ZP protein-associated protein, and gp41, a Xenopus laevis ortholog of mammalian ZP3, and subsequently demonstrate the structural basis of the envelope for fertilization competence. The interactive regions between dicalcin and gp41 comprised five and nine amino acid residues within dicalcin and twenty-three within gp41 [corrected]. Synthetic peptides corresponding to these regions dramatically affected fertilization: treatment with dicalcin- or gp41-derived peptides decreased or increased fertilization rates, respectively. Prior application of these peptides caused distinct alterations in the in vivo lectin-staining pattern of the envelope as well. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide induced the formation of a well-organized meshwork, whereas the gp41-derived peptide caused the formation of a significantly disorganized meshwork. These findings indicated that the fertilization competence of the egg-coating envelope is crucially regulated by the direct interaction between dicalcin and gp41.

  18. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABAA receptors expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Hammer, Harriet; Ebert, Bjarke; Jensen, Henrik S.

    2015-01-01

    The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit...... different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences....... The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABAAR) subtypes α1β2γ2S, α2β2γ2S, α3β2γ2S, α5β2γ2S and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited...

  19. Altered development of Xenopus embryos in a hypogeomagnetic field.

    Science.gov (United States)

    Mo, Wei-Chuan; Liu, Ying; Cooper, Helen M; He, Rong-Qiao

    2012-04-01

    The hypogeomagnetic field (HGMF; magnetic fields HGMF exposure on living systems remains unclear. In this article, we examine the biological effects of HGMF on the embryonic development of Xenopus laevis (African clawed frog). A decrease in horizontal third cleavage furrows and abnormal morphogenesis were observed in Xenopus embryos growing in the HGMF. HGMF exposure at the two-cell stage, but no later than the four-cell stage, is enough to alter the third cleavage geometry pattern. Immunofluorescent staining for α-tubulin showed reorientation of the spindle of four-cell stage blastomeres. These results indicate that a brief (2-h) exposure to HGMF is sufficient to interfere with the development of Xenopus embryos at cleavage stages. Also, the mitotic spindle could be an early sensor to the deprivation of the geomagnetic field, which provides a clue to the molecular mechanism underlying the morphological and other changes observed in the developing and/or developed embryos.

  20. The impact of the goitrogen 6-propylthiouracil (PTU) on West-African clawed frog (Xenopus tropicalis) exposed during metamorphosis.

    Science.gov (United States)

    Carlsson, Gunnar; Norrgren, Leif

    2007-04-20

    This study investigated the suitability of using tadpoles of the West-African clawed frog (Xenopus tropicalis) for studying adverse effects on the thyroid hormone system after chemical exposure. Tadpoles were exposed to the thyroxine synthesis inhibitor 6-propylthiouracil (PTU) at concentrations between 2-75mg/L during 14 days. After 5 and 14 days of exposure the developmental stage, hind limb length, body length and weight were measured. Moreover, histological measurements of the thyroid glands were performed after 14 days of exposure. These measurements included maximum thyroid cross-section area, follicular area and epithelial cell height. Tadpoles in the 75mg/L treatment were less developed and had shorter hind limb length than the control group after 14 days of exposure. No effects were recorded on these parameters at lower PTU concentrations. The histological measurements revealed clear dose-response relationships in both follicular cross-section area and epithelial cell height, with lowest observed effect concentrations (LOECs) recorded at 2 and 5mg/L, respectively. This study shows that X. tropicalis is a suitable species for detection of thyroid disrupting chemicals. Further, histopathological measurements of thyroid glands are more sensitive parameters compared with apical endpoints when studying adverse effects on thyroid hormone system caused by PTU exposure in X. tropicalis.

  1. Development of the Larval Amphibian Growth and Development Assay: effects of chronic 4-tert-octylphenol or 17β-trenbolone exposure in Xenopus laevis from embryo to juvenile.

    Science.gov (United States)

    Haselman, Jonathan T; Kosian, Patricia A; Korte, Joseph J; Olmstead, Allen W; Iguchi, Taisen; Johnson, Rodney D; Degitz, Sigmund J

    2016-12-01

    The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized test guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of the Environment. The LAGDA was designed to evaluate apical effects of chronic chemical exposure on growth, thyroid-mediated amphibian metamorphosis and reproductive development. During the validation phase, two well-characterized endocrine-disrupting chemicals were tested to evaluate the performance of the initial assay design: xenoestrogen 4-tert-octylphenol (tOP) and xenoandrogen 17β-trenbolone (TB). Xenopus laevis embryos were exposed, in flow-through conditions, to tOP (nominal concentrations: 0.0, 6.25, 12.5, 25 and 50 µg l(-1) ) or TB (nominal concentrations: 0.0, 12.5, 25, 50 and 100 ng l(-1) ) until 8 weeks post-metamorphosis, at which time growth measurements were taken, and histopathology assessments were made of the gonads, reproductive ducts, liver and kidneys. There were no effects on growth in either study and no signs of overt toxicity, sex reversal or gonad dysgenesis. Exposure to tOP caused a treatment-related decrease in circulating thyroxine and an increase in thyroid follicular cell hypertrophy and hyperplasia (25 and 50 µg l(-1) ) during metamorphosis. Müllerian duct development was affected after exposure to both chemicals; tOP exposure caused dose-dependent maturation of oviducts in both male and female frogs, whereas TB exposure caused accelerated Müllerian duct regression in males and complete regression in >50% of the females in the 100 ng l(-1) treatment. Based on these results, the LAGDA performed adequately to evaluate apical effects of chronic exposure to two endocrine-active compounds and is the first standardized amphibian multiple life stage toxicity test to date. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  2. Cloning of interleukin-10 from African clawed frog (Xenopus tropicalis), with the Finding of IL-19/20 homologue in the IL-10 locus.

    Science.gov (United States)

    Qi, Zhitao; Zhang, Qihuan; Wang, Zisheng; Zhao, Weihong; Gao, Qian

    2015-01-01

    Interleukin-10 (IL-10) is a pleiotropic cytokine that plays an important role in immune system. In the present study, the IL-10 gene of African clawed frog (Xenopus tropicalis) was first cloned, and its expression pattern and 3D structure were also analyzed. The frog IL-10 mRNA encoded 172 amino acids which possessed several conserved features found in IL-10s from other species, including five-exon/four-intron genomic structure, conserved four cysteine residues, IL-10 family motif, and six α-helices. Real-time PCR showed that frog IL-10 mRNA was ubiquitous expressed in all examined tissues, highly in some immune related tissues including kidney, spleen, and intestine and lowly in heart, stomach, and liver. The frog IL-10 mRNA was upregulated at 24 h after LPS stimulation, indicating that it plays a part in the host immune response to bacterial infection. Another IL, termed as IL-20, was identified from the frog IL-10 locus, which might be the homologue of mammalian IL-19/20 according to the analysis results of the phylogenetic tree and the sequence identities.

  3. Swimming of Xenopus laevis sperm exhibits multiple gears and its duration is extended by egg jelly constituents.

    Science.gov (United States)

    Tholl, Nathan; Naqvi, Sumera; McLaughlin, Ericka; Boyles, Serenity; Bieber, Allan L; Chandler, Douglas E

    2011-06-01

    The motility of Xenopus sperm is initiated by the osmotic shock experienced when these cells are ejaculated into low-salinity pond water. Motility is brief and is required for the sperm to penetrate the jelly layers and fertilize the egg. In this study we demonstrate that extracts of egg jelly contain factors that extend the period of sperm motility as well as providing a chemoattractant activity as previously reported. Both activities are partially dependent on extracellular calcium. Time-lapse and video microscopy show that after activation of motility the number of motile sperm decreases rapidly, with a half-time of about 2 min. Addition of 10% v/v egg jelly extract ("egg water") increased the number of motile sperm 2-fold over controls at 20 s and about 4- to 10-fold over controls at 10 min after initiation of motility. Extension of motility lifetime was not mediated by a nonspecific protein or by allurin, the egg-water protein that has chemoattractant activity. The helical path of Xenopus sperm exhibited tight coupling between rotational and forward velocities in egg jelly, but coupling changed rapidly from moment to moment in low-salinity buffer. Our observations suggest that jelly-derived factors regulate both the longevity and directionality of sperm propulsion.

  4. Retinoic acid is a key regulatory switch determining the difference between lung and thyroid fates in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Wang Jean H

    2011-12-01

    Full Text Available Abstract Background The lung and thyroid are derived from the anterior endoderm. Retinoic acid and Fgf signalling are known to be essential for development of the lung in mouse but little is known on how the lung and thyroid are specified in Xenopus. Results If either retinoic acid or Fgf signalling is inhibited, there is no differentiation of the lung as assayed by expression of sftpb. There is no change in expression of thyroid gland markers when retinoic acid signalling is blocked after gastrulation and when Fgf signalling is inhibited there is a short window of time where pax2 expression is inhibited but expression of other markers is unaffected. If exogenous retinoic acid is given to the embryo between embryonic stages 20 and 26, the presumptive thyroid expresses sftpb and sftpc, specific markers of lung differentiation and expression of key thyroid transcription factors is lost. When the presumptive thyroid is transplanted into the posterior embryo, it also expresses sftpb, although pax2 expression is not blocked. Conclusions After gastrulation, retinoic acid is required for lung but not thyroid differentiation in Xenopus while Fgf signalling is needed for lung but only for early expression of pax2 in the thyroid. Exposure to retinoic acid can cause the presumptive thyroid to switch to a lung developmental program.

  5. Potential protective effect of L-cysteine against the toxicity of acrylamide and furan in exposed Xenopus laevis embryos: an interaction study.

    Science.gov (United States)

    Williams, John Russell; Rayburn, James R; Cline, George R; Sauterer, Roger; Friedman, Mendel

    2014-08-01

    The embryo toxicities of two food-processing-induced toxic compounds, acrylamide and furan, with and without added L-cysteine were examined individually and in mixtures using the frog embryo teratogenesis assay-Xenopus (FETAX). The following measures of developmental toxicity were used: (a) 96 h LC50, the median concentration causing 50% embryo lethality; (b) 96 h EC50, the median concentration causing 50% malformations of the surviving embryos; and (c) teratogenic index (96 h LC50/96 h EC50), an estimate of teratogenic risk. Calculations of toxic units (TU) were used to assess possible antagonism, synergism, or response addition of several mixtures. The evaluated compounds demonstrated counterintuitive effects. Furan had lower than expected toxicity in Xenopus embryos and, unlike acrylamide, does not seem to be teratogenic. However, the short duration of the tests may not show the full effects of furan if it is truly primarily genotoxic and carcinogenic. L-Cysteine showed unexpected properties in the delay of hatching of the embryos. The results from the interaction studies between combination of two or three components (acrylamide plus L-cysteine; furan plus L-cysteine; acrylamide plus furan; acrylamide plus furan and L-cysteine) show that furan and acrylamide seem to have less than response addition at 1:1 toxic unit ratio in lethality. Acrylamide and L-cysteine show severe antagonism even at low 19 acrylamide/1 L-cysteine TU ratios. Data from the mixture of acrylamide, furan, and L-cysteine show a slight antagonism, less than would have been expected from binary mixture exposures. Bioalkylation mechanisms and their prevention are discussed. There is a need to study the toxicological properties of mixtures of acrylamide and furan concurrently formed in heat-processed food.

  6. Four-dimensional imaging of cytoskeletal dynamics in Xenopus oocytes and eggs.

    Science.gov (United States)

    Bement, William M; Sokac, Anna M; Mandato, Craig A

    2003-12-01

    The Xenopus laevis (African clawed frog) system has long been popular for studies of both developmental and cell biology, based on a variety of its intrinsic features including the large size of Xenopus oocytes, eggs, and embryos, and the relative ease of manipulation. Unfortunately, the large size has also been considered a serious impediment for high-resolution light microscopy, as has the heavy pigmentation. However, the recent development and exploitation of 4D imaging approaches, and the fact that much of what is of most interest to cell and developmental biologists takes place near the cell surface, indicates that such concerns are no longer valid. Consequently, the Xenopus system in many respects is now as good as other model systems considered to be ideal for microscopy-based studies. Here, 4D imaging and its recent applications to cytoskeletal imaging in Xenopus oocytes and eggs are discussed.

  7. Differences in regulation of the first two M-phases in Xenopus laevis embryo cell-free extracts.

    Science.gov (United States)

    Chesnel, Franck; Vignaux, Françoise; Richard-Parpaillon, Laurent; Huguet, Antoine; Kubiak, Jacek Z

    2005-09-15

    The first embryonic M-phase is special, being the time when paternal and maternal chromosomes mix together for the first time. Reports from a variety of species suggest that the regulation of first M-phase has many particularities; however, no systematic comparative study of the biochemical aspects of first and the following M-phases has been previously undertaken. Here, we ask whether the regulation of the first embryonic M-phase is modified, using Xenopus cell-free extracts. We developed new types of extract specific for the first and the second M-phase obtained either from parthenogenetic or from in vitro fertilized embryos. Analyses of these extracts confirmed that the amplitude of histone H1 kinase activity reflecting CDK1/cyclin B (or MPF for M-phase Promoting Factor) activity is higher and persists longer than during the second M-phase, and that levels of cyclins B1 and B2 are correspondingly higher during the first than the second embryonic M-phase. Inhibition of protein synthesis shortly before M-phase entry reduced mitotic histone H1 kinase amplitude, shortened the period of mitotic phosphorylation of chosen marker proteins, and reduced cyclin B1 and B2 levels, suggesting a role of B-type cyclins in regulating the duration of mitotic events. Moreover, addition of exogenous cyclin B to the extract prior the second mitosis brought forward the activation of mitotic histone H1 kinase but prolonged the duration of this activity. We also confirmed that the inhibitory phosphorylation of CDK1 on tyrosine 15 oscillates between the first two embryonic M-phases, but is clearly more pronounced before the first than the second mitosis, while the MAP kinase ERK2 tended to show greater activation during the first embryonic M-phase but with a similar duration of activation. We conclude that discrete differences exist between the first two M-phases in Xenopus embryo and that higher CDK1/cyclin B activity and B-type cyclin levels could account for the different

  8. Vascular regression during amphibian metamorphosis--a scanning electron microscope study of vascular corrosion casts of the ventral velum in tadpoles of Xenopus laevis Daudin.

    Science.gov (United States)

    Aichhorn, H; Lametschwandtner, A

    1996-09-01

    We used scanning electron microscopy and vascular casting to study gross arterial supply, venous drainage, and microvascular patterns of the fully developed ventral velum of tadpoles of Xenopus laevis Daudin and analyzed changes of the velar vascular bed from prometamorphosis to metamorphic climax in a qualitative and quantitative manner. The multilayered, highly secretory ventral velum is supplied bilaterally by an anterior and a posterior velar artery, branches of the external carotid artery. Velar arterioles branch mainly dichotomously and form a flat two-dimensional capillary meshwork overlying the tops of filterplates I-IV. Thymopharyngeal veins, dorsal branches of the filter plates veins, and the internal jugular veins drain the velum toward the venous sinus of the heart. Location, architecture, and the drainage of the velar microvascular bed into the venous sinus make a significant contribution of the velar capillaries to gas exchange unlikely. Instead, velar capillaries rather serve the nutrition of the secretory epithelium. The overall morphology of velar vessels from prometamorphosis to metamorphic climax--deduced from vascular corrosion casts--points to atonic vessels with increased leakage indicated by adhering globular extravasations, and to obstructed or blind ending vessels evidenced by the tapered and/or rounded blind ending cast vessels. The significant decrease in the size of the ventral velum during the metamorphic cycle was paralleled by a miniaturization of the velar vascular bed. We hypothetize that this miniaturization occurs by a shortening and fusion of capillary mesh elements. Our findings in corrosion casts, particularly the miniaturization of the velar microvascular bed and the morphology of the regressing capillaries, point to profound morphologic and ultrastructural changes in velar vessels; a study on the fine structure of the microvascular bed of the ventral velum in metamorphic tadpoles is in progress.

  9. Chk1 is activated at the midblastula transition in Xenopus laevis embryos independently of DNA content and the cyclin E/Cdk2 developmental timer.

    Science.gov (United States)

    Adjerid, Nassiba; Wroble, Brian N; Sible, Jill C

    2008-04-15

    Cell cycle checkpoints that are engaged in response to damaged and unreplicated DNA may serve additional, constitutive functions. In the developing Xenopus laevis embryo, the checkpoint kinase Chk1 is transiently activated at the midblastula transition (MBT), a period of extensive cell cycle remodeling including the acquisition of cell cycle checkpoints. The timing of many cell cycle remodeling events at the MBT, such as the lengthening of cell cycles, depends upon a critical nucleocytoplasmic (N/C) ratio. However, other events, including the degradation of maternal cyclin E, do not depend upon the N/C ratio, and are regulated by an autonomous developmental timer. To better understand what regulates Chk1 activation at the MBT, embryos were treated with aphidicolin, at different developmental times and for different lengths of time, to reduce the DNA content at the MBT. Chk1 was activated at the MBT in these embryos establishing that Chk1 activation occurs independently of the N/C ratio. Cdc25A is normally phosphorylated by Chk1 at the MBT and then degraded. The degradation of Cdc25A demonstrated partial dependence on DNA content, suggesting that factors other than Chk1 regulate its degradation. When the cyclin E developmental timer was disrupted with the Cdk2 inhibitor delta34-Xic1, Chk1 was still activated at the MBT, indicating that activation of Chk1 at the MBT was not directly linked to the cyclin E timer. Conversely, unreplicated or damaged DNA, delayed the degradation of cyclin E at the MBT, indicating that the cyclin E/Cdk2 timer is sensitive to engagement of cell cycle checkpoints.

  10. Exposure of xenopus laevis tadpoles to cadmium reveals concentration-dependent bimodal effects on growth and monotonic effects on development and thyroid gland activity

    Science.gov (United States)

    Sharma, Bibek; Patino, R.

    2008-01-01

    Xenopus laevis were exposed to 0-855 ??g cadmium (Cd)/l (measured concentrations) in FETAX medium from fertilization to 47 days postfertilization. Measurements included embryonic survival and, at 47 days, tadpole survival, snout-vent length, tail length, total length, hindlimb length, weight, Nieuwkoop-Faber (NF) stage of development, initiation of metamorphic climax (??? NF 58), and thyroid follicle cell height. Embryonic and larval survival were unaffected by Cd. Relative to control tadpoles, reduced tail and total length were observed at 0.1- 8 and at 855 ??g Cd/l; and reduced snout-vent length, hindlimb length, and weight were observed at 0.1-1 and at 855 ??g Cd/l. Mean stage of development and rate of initiation of climax were unaffected by Cd at 0-84 ??g/l; however, none of the tadpoles exposed to 855 ??g Cd/l progressed beyond mid-premetamorphosis (NF 51). Thyroid glands with fully formed follicles were observed in all tadpoles ??? NF 49 examined. Follicle cell height was unaffected by Cd at 0-84 ??g/l but it was reduced at 855 ??g/l; in the latter, cell height was reduced even when compared with NF 49-51 tadpoles pooled from the 0 to 84 ??g Cd/l groups. In conclusion, (1) Cd affected tadpole growth in a bimodal pattern with the first and second inhibitory modes at concentrations below and above 84 ??g Cd/l, respectively; (2) exposure to high Cd concentrations (855 ??g/l) reduced thyroid activity and arrested tadpole development at mid-premetamorphosis; and (3) unlike its effect on growth, Cd inhibited tadpole development and thyroid function in a seemingly monotonic pattern.

  11. Effects of strain on contractile force and number of sarcomeres in series of Xenopus laevis single muscle fibres during long-term culture.

    Science.gov (United States)

    Jaspers, R T; Feenstra, H M; Verheyen, A K; van der Laarse, W J; Huijing, P A

    2004-01-01

    The aim of the present study is to test whether mechanical strain uniquely regulates muscle fibre atrophy/hypertrophy and adaptation of the number of sarcomeres in series within mature muscle fibres in vitro . Mature single muscle fibres from Xenopus laevis illiofibularis muscle were cultured (4-97 days) while kept at negative strain ( approximately 20% below passive slack length, 'short fibres') or at positive strain ( approximately 5% over passive slack length, 'long fibres'). Before and after culture the number of sarcomeres in series was determined using laser diffraction. During culture, twitch and tetanic force characteristics were measured every day. Survival time of long fibres was substantially less than that of short fibres. Of the long fibres 40% died or became inexcitable within 1 week, whereas this did not occur for short fibres. During culture, twitch and tetanic force of all short fibres increased substantially. Regression analysis showed that the post-culture number of sarcomeres in series was not significantly changed compared to the number before culture. It is concluded that culture at negative strain does not result in atrophy or a reduction of the number of sarcomeres in series, even after 97 days. For the long fibres we did not detect any hypertrophy as tetanic force remained stable or decreased slowly, while twitch force varied. Regression analysis of the change of the number of sarcomeres in series as a function of the culture time showed a positive slope ( P=0.054). Two out of four long fibres that were cultured for at least 2 weeks showed an increase in the number of sarcomeres of 4-5%. Compared with in vivo adaptation to mechanical stimuli this is much less than would be expected. The data suggest that strain may not be the only factor that regulates hypertrophy and the number of sarcomeres in series.

  12. Functional characterization of mouse urea transporters UT-A2 and UT-A3 expressed in purified Xenopus laevis oocyte plasma membranes.

    Science.gov (United States)

    Maciver, Bryce; Smith, Craig P; Hill, Warren G; Zeidel, Mark L

    2008-04-01

    Urea is a small solute synthesized by many terrestrial organisms as part of the catabolism of protein. In mammals it is transported across cellular membranes by specific urea transporter (UT) proteins that are the products of two separate, but closely related genes, referred to as UT-A and UT-B. Three major UT-A isoforms are found in the kidney, namely UT-A1, UT-A2, and UT-A3. UT-A2 is found in the thin, descending limb of the loop of Henle, whereas UT-A1 and UT-A3 are concentrated in the inner medullary collecting duct. UT-A2 and UT-A3 effectively represent two halves of the whole UT-A gene and, when joined together by 73 hydrophilic amino acids, constitute UT-A1. A biophysical characterization of mouse UT-A2 and UT-A3 was undertaken by expression in Xenopus laevis oocytes and subsequent preparation of highly enriched plasma membrane vesicles for use in stopped-flow fluorometry. Both isoforms were found to be highly specific for urea, and did not permeate water, ammonia, or other molecules closely related to urea (formamide, acetamide, methylurea, and dimethylurea). Single transporter flux rates of 46,000 +/- 10,000 and 59,000 +/- 15,000 (means +/- SE) urea molecules/s/channel for UT-A2 and UT-A3, respectively, were obtained. Overall, the UT-A2 and UT-A3 isoforms appear to have identical functional kinetics.

  13. Cryptochrome genes are highly expressed in the ovary of the African clawed frog, Xenopus tropicalis.

    Directory of Open Access Journals (Sweden)

    Yoko Kubo

    Full Text Available Cryptochromes (CRYs are flavoproteins sharing high homology with photolyases. Some of them have function(s including transcription regulation in the circadian clock oscillation, blue-light photoreception for resetting the clock phase, and light-dependent magnetoreception. Vertebrates retain multiple sets of CRY or CRY-related genes, but their functions are yet unclear especially in the lower vertebrates. Although CRYs and the other circadian clock components have been extensively studied in the higher vertebrates such as mice, only a few model species have been studied in the lower vertebrates. In this study, we identified two CRYs, XtCRY1 and XtCRY2 in Xenopus tropicalis, an excellent experimental model species. Examination of tissue specificity of their mRNA expression by real-time PCR analysis revealed that both the XtCRYs showed extremely high mRNA expression levels in the ovary. The mRNA levels in the ovary were about 28-fold (XtCry1 and 48-fold (XtCry2 higher than levels in the next abundant tissues, the retina and kidney, respectively. For the functional analysis of the XtCRYs, we cloned circadian positive regulator XtCLOCK and XtBMAL1, and found circadian enhancer E-box in the upstream of XtPer1 gene. XtCLOCK and XtBMAL1 exhibited strong transactivation from the XtPer1 E-box element, and both the XtCRYs inhibited the XtCLOCK:XtBMAL1-mediated transactivation, thereby suggesting this element to drive the circadian transcription. These results revealed a conserved main feedback loop in the X. tropicalis circadian clockwork and imply a possible physiological importance of CRYs in the ovarian functions such as synthesis of steroid hormones and/or control of estrus cycles via the transcription regulation.

  14. GABAergic transmission and chloride equilibrium potential are not modulated by pyruvate in the developing optic tectum of Xenopus laevis tadpoles.

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    Arseny S Khakhalin

    Full Text Available In the developing mammalian brain, gamma-aminobutyric acid (GABA is thought to play an excitatory rather than an inhibitory role due to high levels of intracellular Cl(- in immature neurons. This idea, however, has been questioned by recent studies which suggest that glucose-based artificial cerebrospinal fluid (ACSF may be inadequate for experiments on immature and developing brains. These studies suggest that immature neurons may require alternative energy sources, such as lactate or pyruvate. Lack of these other energy sources is thought to result in artificially high intracellular Cl(- concentrations, and therefore a more depolarized GABA receptor (GABAR reversal potential. Since glucose metabolism can vary widely among different species, it is important to test the effects of these alternative energy sources on different experimental preparations. We tested whether pyruvate affects GABAergic transmission in isolated brains of developing wild type Xenopus tadpoles in vitro by recording the responsiveness of tectal neurons to optic nerve stimulation, and by measuring currents evoked by local GABA application in a gramicidin perforated patch configuration. We found that, in contrast with previously reported results, the reversal potential for GABAR-mediated currents does not change significantly between developmental stages 45 and 49. Partial substitution of glucose by pyruvate had only minor effects on both the GABA reversal potential, and the responsiveness of tectal neurons at stages 45 and 49. Total depletion of energy sources from the ACSF did not affect neural responsiveness. We also report a strong spatial gradient in GABA reversal potential, with immature cells adjacent to the lateral and caudal proliferative zones having more positive reversal potentials. We conclude that in this experimental preparation standard glucose-based ACSF is an appropriate extracellular media for in vitro experiments.

  15. Evolution of Heat Sensors Drove Shifts in Thermosensation between Xenopus Species Adapted to Different Thermal Niches.

    Science.gov (United States)

    Saito, Shigeru; Ohkita, Masashi; Saito, Claire T; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

    2016-05-20

    Temperature is one of the most critical environmental factors affecting survival, and thus species that inhabit different thermal niches have evolved thermal sensitivities suitable for their respective habitats. During the process of shifting thermal niches, various types of genes expressed in diverse tissues, including those of the peripheral to central nervous systems, are potentially involved in the evolutionary changes in thermosensation. To elucidate the molecular mechanisms behind the evolution of thermosensation, thermal responses were compared between two species of clawed frogs (Xenopus laevis and Xenopus tropicalis) adapted to different thermal environments. X. laevis was much more sensitive to heat stimulation than X. tropicalis at the behavioral and neural levels. The activity and sensitivity of the heat-sensing TRPA1 channel were higher in X. laevis compared with those of X. tropicalis The thermal responses of another heat-sensing channel, TRPV1, also differed between the two Xenopus species. The species differences in Xenopus TRPV1 heat responses were largely determined by three amino acid substitutions located in the first three ankyrin repeat domains, known to be involved in the regulation of rat TRPV1 activity. In addition, Xenopus TRPV1 exhibited drastic species differences in sensitivity to capsaicin, contained in chili peppers, between the two Xenopus species. Another single amino acid substitution within Xenopus TRPV1 is responsible for this species difference, which likely alters the neural and behavioral responses to capsaicin. These combined subtle amino acid substitutions in peripheral thermal sensors potentially serve as a driving force for the evolution of thermal and chemical sensation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. An age-dependent sensitivity of the roll-induced vestibuloocular reflex to hypergravity exposure of several days in an amphibian ( Xenopus laevis)

    Science.gov (United States)

    Sebastian, C. E.; Pfau, K.; Horn, E. R.

    In tadpoles of the Southern Clawed Toad ( Xenopus laevis), the effects of an exposure to hypergravity of several days duration on the development of the roll-induced static vestibuloocular reflex (rVOR) were investigated. Special attention was given to the onset of the 9 or 12 days lasting 3G-period during early life. First recordings of the rVOR characteristics for complete 360 ° rolls of the tadpoles were performed 24 hrs after the end of the 3G-period. The rVOR peak-to-peak amplitudes as well as the VOR-gain for a roll angle of 15 ° from 3G-and 1G-samples recorded at the 2nd and 3rd day after 3G-termination agreed for the youngest group, but were reduced by approx. 30% in the older tadpoles. Long-term observations lasting up to 8 weeks after termination of the 3G-period, demonstrated (i) an early retardation of the development, and (ii) a developmental acceleration in all groups so that after 2 weeks in the stage 6/9- and 33/36-samples and after 8 weeks in the stage 45-tadpoles, the rVOR-amplitude as well as the rVOR-gain for a 15 ° roll were at the same level in both the 3G- and the 1G-samples. The results support the existence of a sensitive period for the rVOR development, and additionally demonstrate the importance of the period of the first appearance of the rVOR for the development of adaptive properties of the underlying neuronal network. They also demonstrate the dominant efficiency of genetic programs in the functional development of the vestibular system. Methodological approaches are discussed which will be useful in the further description of the critical period. They include studies on the neuronogenesis and synaptic maturation within the vestibular pathways as well as on the fundamentals of buoyancy control during swimming. A modular but closed mini-system for experimental use is described which allows survival periods lasting many weeks and multiple types of treatments of developing aquatic animals in orbit, controlled automatically.

  17. Proteomics reveals a switch in CDK1-associated proteins upon M-phase exit during the Xenopus laevis oocyte to embryo transition.

    Science.gov (United States)

    Marteil, Gaëlle; Gagné, Jean-Philippe; Borsuk, Ewa; Richard-Parpaillon, Laurent; Poirier, Guy G; Kubiak, Jacek Z

    2012-01-01

    Cyclin-dependent kinase 1 (CDK1) is a major M-phase kinase which requires the binding to a regulatory protein, Cyclin B, to be active. CDK1/Cyclin B complex is called M-phase promoting factor (MPF) for its key role in controlling both meiotic and mitotic M-phase of the cell cycle. CDK1 inactivation is necessary for oocyte activation and initiation of embryo development. This complex process requires both Cyclin B polyubiquitination and proteosomal degradation via the ubiquitin-conjugation pathway, followed by the dephosphorylation of the monomeric CDK1 on Thr161. Previous proteomic analyses revealed a number of CDK1-associated proteins in human HeLa cells. It is, however, unknown whether specific partners are involved in CDK1 inactivation upon M-phase exit. To better understand CDK1 regulation during MII-arrest and oocyte activation, we immunoprecipitated (IPed) CDK1 together with its associated proteins from M-phase-arrested and M-phase-exiting Xenopus laevis oocytes. A mass spectrometry (MS) analysis revealed a number of new putative CDK1 partners. Most importantly, the composition of the CDK1-associated complex changed rapidly during M-phase exit. Additionally, an analysis of CDK1 complexes precipitated with beads covered with p9 protein, a fission yeast suc1 homologue well known for its high affinity for CDKs, was performed to identify the most abundant proteins associated with CDK1. The screen was auto-validated by identification of: (i) two forms of CDK1: Cdc2A and B, (ii) a set of Cyclins B with clearly diminishing number of peptides identified upon M-phase exit, (iii) a number of known CDK1 substrates (e.g. peroxiredoxine) and partners (e.g. HSPA8, a member of the HSP70 family) both in IP and in p9 precipitated pellets. In IP samples we also identified chaperones, which can modulate CDK1 three-dimensional structure, as well as calcineurin, a protein necessary for successful oocyte activation. These results shed a new light on CDK1 regulation via a dynamic

  18. Force-dependent and force-independent heat production in single slow- and fast-twitch muscle fibres from Xenopus laevis.

    Science.gov (United States)

    Buschman, H P; van der Laarse, W J; Stienen, G J; Elzinga, G

    1996-10-15

    1. The origin of labile heat production, i.e. a heat component which rapidly decays after the onset of stimulation, and of stable (maintenance) heat production was investigated in intact single fast-twitch (type 1) and slow-twitch (type 3) iliofibularis muscle fibres from Xenopus laevis, at 20 degrees C, by varying stimulation frequency and by varying sarcomere length and the concentration of 2,3-butanedione 2-monoxime (BDM) added. 2. The labile heat produced consisted of a force-independent and a force-dependent part. The average parvalbumin (PA) content found in type 1 fibre bundles (0.84 +/- 0.08 mM; mean +/- S.E.M.; n = 5) and in type 3 fibre bundles (0.12 +/- 0.02 mM; n = 5) indicates that the force-independent labile heat is explained by Ca(2+)-Mg2+ exchange on PA, and amounts to a molar enthalpy change of -78 kJ (molPA)-1. 3. Force-dependent labile heat during fused contractions was similar to the calculated heat production resulting from the formation of force-generating cross-bridges, assuming an enthalpy change associated with cross-bridge formation of -30 kJ mol-1. 4. Activation heat, i.e. the part of the total stable heat that is not related to the contractile apparatus, and of which the calcium sequestration by the sarcoplasmic reticulum is the most important contributor, determined by varying sarcomere length or BDM concentration, was identical. For fused contractions the fraction activation heat of the stable maintenance rate of heat production was 34 +/- 4% (mean +/- S.E.M.; n = 13) in type 1 fibres, and 52 +/- 4% (n = 15) in type 3 fibres. In unfused contractions this was 48 +/- 5% (n = 13) in type 1 fibres, and 35 +/- 2% (n = 11) in type 3 fibres. 5. From the force-dependent stable rate of heat production the economy of cross-bridge cycling, expressed as the force-time integral for a single myosin head per ATP molecule hydrolysed, was calculated. It followed that cross-bridge interaction in type 3 fibres is more economical than in type 1 fibres

  19. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABA(A) receptors expressed in Xenopus laevis oocytes.

    Science.gov (United States)

    Hammer, Harriet; Ebert, Bjarke; Jensen, Henrik Sindal; Jensen, Anders A

    2015-01-01

    The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABA(A)R) subtypes α1β2γ(2S), α2β2γ(2S), α3β2γ(2S), α5β2γ(2S) and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α(1,2,3,5)β2γ(2S) GABA(A)Rs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold) as positive allosteric modulators at the α6β2δ GABA(A)R than at the α(1,2,3,5)β2γ(2S) receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non-selective modulation

  20. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABA(A receptors expressed in Xenopus laevis oocytes.

    Directory of Open Access Journals (Sweden)

    Harriet Hammer

    Full Text Available The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABA(AR subtypes α1β2γ(2S, α2β2γ(2S, α3β2γ(2S, α5β2γ(2S and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α(1,2,3,5β2γ(2S GABA(ARs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold as positive allosteric modulators at the α6β2δ GABA(AR than at the α(1,2,3,5β2γ(2S receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non

  1. Calcium Signaling and Meiotic Exit at Fertilization in Xenopus Egg

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    Alexander A. Tokmakov

    2014-10-01

    Full Text Available Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation.

  2. Calcium Signaling and Meiotic Exit at Fertilization in Xenopus Egg

    Science.gov (United States)

    Tokmakov, Alexander A.; Stefanov, Vasily E.; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

    2014-01-01

    Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation. PMID:25322156

  3. 非洲爪蟾胚胎用于发育神经毒性测试的方法%An assay for testing developmental neurotoxicity of chemicals using Xenopus laevis embryos

    Institute of Scientific and Technical Information of China (English)

    付旭锋; 李圆圆; 崔清华; 秦占芬

    2014-01-01

    Based on Frog Embryo Teratogenesis Assay-Xenopus ( FETAX ) of American Society for Testing and Materials, we aimed to establish an assay for evaluating developmental neurotoxicity of chemicals using body features, motoneuronal morphology and motor behavior as endpoints. Methylmercury chloride ( CH3 HgCl) was used as a model compound for developmental neurotoxicity. Following 3 d-exposure, the embryos exhibited weaker motor ability with increases in CH3 HgCl concentrations. After 4 d-exposure to CH3 HgCl, the embryos appeared shorter body lengths and motoneurons in 300 nmol·L-1 and 400 nmol·L-1 groups compared with the control. Seven day-exposure to CH3 HgCl resulted in a decrease in the swimming velocity of the tadpoles in a concentration-dependent manner. In conclusion, our results show that X. laevis embryos can be used to investigate developmental neurotoxicity of chemicals, and body features, motoneuronal morphology and motor behavior are sensitive endpoints.%在美国材料与测试协会( ASTM)的非洲爪蟾胚胎致畸试验( FETAX)的基础上,以已知具有发育神经毒性的氯化甲基汞为模式化合物,探索一种以体征、运动神经元形态和运动行为参数为终点指标的研究发育神经毒性的方法。非洲爪蟾胚胎暴露氯化甲基汞3 d时,观察到暴露组胚胎的运动能力随暴露浓度(100-400 nmol·L-1)的增加而减弱。暴露4 d发现300 nmol·L-1和400 nmol·L-1暴露组胚胎体长和运动神经元明显短于对照组。暴露持续7 d,通过行为分析软件对蝌蚪运动行为定量,发现暴露处理的蝌蚪的游泳速率明显小于对照组。以上结果显示,非洲爪蟾胚胎可用来研究化学品的发育神经毒性,胚胎的体征、运动神经元形态和运动行为可以作为相对敏感的评价指标。

  4. Regulation of ALF promoter activity in Xenopus oocytes.

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    Dan Li

    Full Text Available BACKGROUND: In this report we evaluate the use of Xenopus laevis oocytes as a matched germ cell system for characterizing the organization and transcriptional activity of a germ cell-specific X. laevis promoter. PRINCIPAL FINDINGS: The promoter from the ALF transcription factor gene was cloned from X. laevis genomic DNA using a PCR-based genomic walking approach. The endogenous ALF gene was characterized by RACE and RT-PCR for transcription start site usage, and by sodium bisulfite sequencing to determine its methylation status in somatic and oocyte tissues. Homology between the X. laevis ALF promoter sequence and those from human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis was relatively low, making it difficult to use such comparisons to identify putative regulatory elements. However, microinjected promoter constructs were very active in oocytes and the minimal promoter could be narrowed by PCR-mediated deletion to a region as short as 63 base pairs. Additional experiments using a series of site-specific promoter mutants identified two cis-elements within the 63 base pair minimal promoter that were critical for activity. Both elements (A and B were specifically recognized by proteins present in crude oocyte extracts based on oligonucleotide competition assays. The activity of promoter constructs in oocytes and in transfected somatic Xenopus XLK-WG kidney epithelial cells was quite different, indicating that the two cell types are not functionally equivalent and are not interchangeable as assay systems. CONCLUSIONS: Overall the results provide the first detailed characterization of the organization of a germ cell-specific Xenopus promoter and demonstrate the feasibility of using immature frog oocytes as an assay system for dissecting the biochemistry of germ cell gene regulation.

  5. High-throughput transgenesis in Xenopus using I-SceI meganuclease.

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    Ogino, Hajime; McConnell, William B; Grainger, Robert M

    2006-01-01

    In this report we describe an easy, highly efficient transgenesis method for Xenopus. The method is very simple; a commercially available meganuclease, I-SceI, is incubated with a transgene construct carrying its recognition sites, and is subsequently microinjected into fertilized eggs. Approximately 30% (in Xenopus tropicalis) or 20% (in Xenopus laevis) of injected embryos exhibit non-mosaic, promoter-dependent transgene expression, and transgenes from the founder animals are transmitted to offspring. The method is compatible with mRNA or antisense morpholino oligonucleotide injection, and these secondary reagents can be introduced simultaneously or sequentially with a transgene to test their interaction. This high-throughput transgenic technique will be a powerful tool for studying the complex wiring of regulatory networks at the genome-wide level, as well as for facilitating genetic studies in the rapidly breeding diploid frog, X. tropicalis.

  6. DDT exposure of frogs: A case study from Limpopo Province, South Africa.

    Science.gov (United States)

    Viljoen, Ignatius M; Bornman, Riana; Bouwman, Hindrik

    2016-09-01

    Amphibians are globally under pressure with environmental contaminants contributing to this. Despite caution aired more than 80 years ago of threats posed to amphibians by DDT spraying for disease vector control, no data have been published on concentrations or effects of DDT contamination in frogs from areas where DDT is actively sprayed to control the insect vectors of malaria. In this study, we sampled fat bodies of Xenopus laevis and Xenopus muelleri naturally occurring in an area where indoor residual spraying of DDT is employed and from adjacent, non-sprayed, areas. ΣDDT concentrations ranged between DDT in the eggs of the Grey Heron Ardea cinerea. This suggests that the DDT we found in frogs may have contributed to DDT loadings higher in the food web. These findings, combined with other studies from this area, support the need to reduce and eventually move away from DDT in malaria control safely and sustainably.

  7. Echocardiographic assessment of cardiac morphology and function in Xenopus.

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    Bartlett, Heather L; Escalera, Robert B; Patel, Sonali S; Wedemeyer, Elesa W; Volk, Kenneth A; Lohr, Jamie L; Reinking, Benjamin E

    2010-04-01

    Advances using Xenopus as a model permit valuable inquiries into cardiac development from embryo to adult. Noninvasive methods are needed to study cardiac function longitudinally. The objective of this study was to evaluate the feasibility of echocardiographic studies in Xenopus and establish normative data of adult cardiac structure and function. Doppler and 2D echocardiograms and electrocardiograms were acquired from adult Xenopus laevis and X. tropicalis. Frogs were exposed to either isoflurane or tricaine to discern the effect of sedating agents on cardiac function. Cardiac dimensions, morphology, flow velocities, and electrophysiologic intervals were measured and evaluated by using bivariate and regression analyses. Normal cardiac dimensions relative to body weight and species were established by echocardiography. Normal conduction intervals were determined by electrocardiography and did not vary by body weight or species. Anesthetic agent did not affect ejection fraction or flow velocity but did alter the QRS duration and QT interval. Echocardiographic and electrocardiographic studies in Xenopus provide information about cardiac anatomy and physiology and can readily be used for longitudinal analyses of developmental inquiries. Body weight, species, and anesthetic agent are factors that should be considered in experimental design and analyses.

  8. Functional gametes derived from explants of single blastomeres containing the “germ plasm” in Xenopus laevis: A genetic marker study

    NARCIS (Netherlands)

    Ikenishi, K.

    Single blastomeres containing the “germ plasm” were isolated from 32-cell embryos of Xenopus albino (ap/ap) or wild type and cultured in vitro until the corresponding normal control embryos reached the neurula stage. The resulting explants from albinos were implanted into wild-type host neurulae and

  9. Effects of the beta-cypermethrin on the survival and metamorphosis of Xenopus laevis tadpoles%高效氯氰菊酯对非洲爪蟾蝌蚪生存和变态发育影响

    Institute of Scientific and Technical Information of China (English)

    赛林霖; 薄存香; 李兰波; 谢琳; 郭启明

    2014-01-01

    Objective To investigate the effects of beta-cypermethrin on the survival and metamorphosis of Xenopus laevis tadpoles. Methods Xenopus laevis tadpoles at stage 51 were randomly selected and tested by acute and chronic toxicity experiments. In acute toxicity test,240 tadpoles were randomly divided into 8 groups according to the exposure dose of beta-cypermethrin,including 0. 00μg/L dose group( control group)and 0. 80,0. 96,1. 15,1. 38,1. 66,1. 99 and 2. 40μg/L dose groups,with 30 tadpoles in each group. Then the survival situation of Xenopus laevis tadpoles in 24,48,72 and 96 hours were observed,as well as the lethal concentrations of 50%( LC50 )in the above four time points. In chronic toxi-city test,450 tadpoles were randomly divided into 5 groups according to the exposure dose of beta-cypermethrin,including 0. 00 μg/L dose group( control group)and 0. 05,0. 10,0. 20 and 0. 40 μg/L dose groups. The survival situation,body weight,body length and the time of completed the allergy in the early stage of the abnormal peak( stage 58)and abnormal peak(stage 66)were all observed. Results In the acute toxicity test,there was no tadpoles died in the above four time points in the control group and 0. 80μg/L dose group,but all of the tadpoles were dead in the 24 hours of 2. 40μg/L dose group. The survival rate was decreased with the increasing of the infected doses(P0. 05 ). But in stage 66,the survival rate was decreased with the increasing of the infected doses( P0. 05),while the time of completing the allergy in 0. 20 and 0. 40 μg/L dose groups was longer than that of the con-trol group respectively[(92. 21 ± 2. 81)vs(89. 43 ± 2. 41),(95. 55 ± 5. 54)vs(89. 43 ± 2. 41),P0.05);在变态高峰期,5组非洲爪蟾蝌蚪存活率随染毒剂量的增加而降低(P0.05);0.20和0.40μg/L剂量组非洲爪蟾蝌蚪从第51期到完成变态反应所需时间长于对照组[(92.21±2.81)vs(89.43±2.41) d(,95.55±5.54)vs(89.43±2.41)d,P<0

  10. Analysis of gamma-aminobutyric acidB receptor function in the in vitro and in vivo regulation of alpha-melanotropin-stimulating hormone secretion from melanotrope cells of Xenopus laevis.

    Science.gov (United States)

    De Koning, H P; Jenks, B G; Roubos, E W

    1993-02-01

    The activity of many endocrine cells is regulated by gamma-aminobutyric acid (GABA). The effects of GABA are mediated by GABAA and/or GABAB receptors. While GABAB receptors in the central nervous system have now been extensively characterized, little is known of the function and pharmacology of GABAB receptors on endocrine cells. In the amphibian Xenopus laevis, GABA inhibits the release of alpha MSH from the endocrine melanotrope cells through both GABAA and GABAB receptors. We have investigated the following aspects of the GABAB receptor of the melanotrope cells of X. laevis: 1) the pharmacology of this receptor, using antagonists previously established to demonstrate GABAB receptors in the mammalian central nervous system; 2) the relative contribution to the regulation of hormone secretion by the GABAA and GABAB receptors on melanotrope cells in vitro; and 3) the role of the GABAB receptor with respect to the physiological function of the melanotrope cell in vivo, i.e. regulation of pigment dispersion in skin melanophores in relation to background color. Our results demonstrate that phaclofen, 2-hydroxysaclofen, and 4-aminobutylphosphonic acid dose-dependently blocked the inhibition of alpha MSH release by GABAB receptor activation, but not by GABAA receptor activation. The GABAB receptor antagonist delta-aminovaleric acid appeared to be a selective agonist on the GABAB receptor of melanotrope cells. The inhibitory secretory response to a low dose of GABA (10(-5) M) was not affected by bicuculline, but was significantly reduced by phaclofen, indicating that at a low GABA concentration, the GABAB receptor mechanism would dominate in inhibiting the melanotrope cells. Different thresholds of activation may form the basis for differential action of GABA through both GABA receptor types. The tonic inhibition of alpha MSH release in animals adapted to a white background was not affected by 4-aminobutylphosphonic acid, indicating that the GABAB receptor is not (solely

  11. Stimulation of circus movement by activin, bFGF and TGF-beta 2 in isolated animal cap cells of Xenopus laevis.

    Science.gov (United States)

    Minoura, I; Nakamura, H; Tashiro, K; Shiokawa, K

    1995-01-01

    Lobopodium is a hyaline cytoplasmic protrusion which rotates circumferencially around a cell. This movement is called circus movement, which is seen in dissociated cells of amphibian embryos. Relative abundance of the lobopodia-forming cells changes temporally and spatially within Xenopus embryos, reflecting stage-dependent difference of morphogenetic movements. The lobopodia-forming activity of dissociated animal cap cells was stimulated strongly by activin and bFGF, and weakly by TGF-beta 2. In addition, activin A was found to stimulate cellular attachment to the substratum when the cultivation lasted long. Thus, mesoderm-inducing growth factors stimulate lobopodia formation and cellular movements which may be necessary for gastrulation and neurulation in Xenopus early embryos.

  12. Xenopus tropicalis oocytes as an advantageous model system for the study of intracellular Ca(2+) signalling.

    Science.gov (United States)

    Marchant, J S; Parker, I

    2001-04-01

    1. The purpose of this study was to compare oocytes from the pipid frogs Xenopus tropicalis and Xenopus laevis, with respect to their utility for studying Ca(2+) signalling mechanisms and for expression of heterologous proteins. 2. We show that X. tropicalis oocytes possess an intracellular Ca(2+) store that is mobilized by inositol (1,4,5) trisphosphate (IP(3)). Ca(2+) signalling is activated by endogenous lysophosphatidic acid receptors and cytosolic Ca(2+) activates a plasma membrane chloride conductance. The spatiotemporal organization of cytosolic Ca(2+) signals, from the microscopic architecture of elementary Ca(2+) 'puffs' to the macroscopic patterns of Ca(2+) spiking are closely similar to the local and global patterns of Ca(2+) release previously characterized in oocytes from X. laevis. 3. By injecting X. tropicalis oocytes with cDNA encoding an ER-targeted fluorescent protein construct, we demonstrate the capacity of the X. tropicalis oocyte to readily express heterologous proteins. The organization of ER is polarized across the oocyte, with the IP(3)-releaseable store targeted within an approximately 8 microm wide band that circumscribes the cell. 4. We conclude that the X. tropicalis oocyte shares many of the characteristics that have made oocytes of X. laevis a favoured system for studying Ca(2+) signalling mechanisms. Moreover, X. tropicalis oocytes display further practical advantages in terms of imaging depth, Ca(2+) signal magnitude and electrical properties. These further enhance the appeal of X. tropicalis as an experimental system, in addition to its greater amenability to transgenic approaches.

  13. A revised model of Xenopus dorsal midline development: differential and separable requirements for Notch and Shh signaling.

    Science.gov (United States)

    Peyrot, Sara M; Wallingford, John B; Harland, Richard M

    2011-04-15

    The development of the vertebrate dorsal midline (floor plate, notochord, and hypochord) has been an area of classical research and debate. Previous studies in vertebrates have led to contrasting models for the roles of Shh and Notch signaling in specification of the floor plate, by late inductive or early allocation mechanisms, respectively. Here, we show that Notch signaling plays an integral role in cell fate decisions in the dorsal midline of Xenopus laevis, similar to that observed in zebrafish and chick. Notch signaling promotes floor plate and hypochord fates over notochord, but has variable effects on Shh expression in the midline. In contrast to previous reports in frog, we find that Shh signaling is not required for floor plate vs. notochord decisions and plays a minor role in floor plate specification, where it acts in parallel to Notch signaling. As in zebrafish, Shh signaling is required for specification of the lateral floor plate in the frog. We also find that the medial floor plate in Xenopus comprises two distinct populations of cells, each dependent upon different signals for its specification. Using expression analysis of several midline markers, and dissection of functional relationships, we propose a revised allocation mechanism of dorsal midline specification in Xenopus. Our model is distinct from those proposed to date, and may serve as a guide for future studies in frog and other vertebrate organisms.

  14. P-domains as shuffled cysteine-rich modules in integumentary mucin C.1 (FIM-C.1) from Xenopus laevis. Polydispersity and genetic polymorphism.

    Science.gov (United States)

    Hauser, F; Hoffmann, W

    1992-12-05

    A new frog integumentary mucin (FIM-C.1) has been discovered by molecular cloning. This mucin contains at least six typical P-domains as cysteine-rich modules. Shuffled P-domains have previously been detected in FIM-A.1, and they also form the basis of various P-domain peptides, which presumably have growth-modulating activities. Furthermore, FIM-C.1 contains at least three threonine-rich clusters, which consist of semi-repetitive cassettes. Various polydisperse transcripts have been characterized. They originate from two genes only and differ by deletions/insertions that are congruent with the semi-repetitive cassettes. Thus, polydispersities are probably generated by alternative splicing. Southern analysis revealed genetic polymorphism between different individuals. Furthermore, a specific antiserum was generated against a synthetic peptide deduced from the COOH-terminal end of FIM-C.1 and used for Western analysis.

  15. Alternative splicing of repetitive units is responsible for the polydispersities of integumentary mucin B.1 (FIM-B.1) from Xenopus laevis.

    Science.gov (United States)

    Joba, W; Hoffmann, W

    1996-10-01

    Frog integumentary mucin B.1 (FIM-B.1) represents a polymorphic extracellular mosaic protein which contains tandemly arranged serine/threonine-rich modules as well as cysteine-rich domains. The latter are probably important for oligomerization of FIM-B.1 and have also been found in many proteins of the complement cascade as well as regions homologous to von Willebrand factor. The repetitive modules are targets for extensive O-glycosylation. Previous cDNA cloning experiments clearly established polydispersities within the same individual, which originate from deletions/insertions in the repetitive domain. Here, we analyse part of the corresponding genomic region. Each repetitive unit as well as the cysteine-rich domain is encoded by an individual class 1-1 exon typical of shuffled modules. Alternative splicing of these multiple cassettes creates the polydisperse FIM-B.1 transcripts.

  16. Remodeling of the metabolome during early frog development.

    Directory of Open Access Journals (Sweden)

    Livia Vastag

    Full Text Available A rapid series of synchronous cell divisions initiates embryogenesis in many animal species, including the frog Xenopus laevis. After many of these cleavage cycles, the nuclear to cytoplasmic ratio increases sufficiently to somehow cause cell cycles to elongate and become asynchronous at the mid-blastula transition (MBT. We have discovered that an unanticipated remodeling of core metabolic pathways occurs during the cleavage cycles and the MBT in X. laevis, as evidenced by widespread changes in metabolite abundance. While many of the changes in metabolite abundance were consistently observed, it was also evident that different female frogs laid eggs with different levels of at least some metabolites. Metabolite tracing with heavy isotopes demonstrated that alanine is consumed to generate energy for the early embryo. dATP pools were found to decline during the MBT and we have confirmed that maternal pools of dNTPs are functionally exhausted at the onset of the MBT. Our results support an alternative hypothesis that the cell cycle lengthening at the MBT is triggered not by a limiting maternal protein, as is usually proposed, but by a decline in dNTP pools brought about by the exponentially increasing demands of DNA synthesis.

  17. Impact of mechanical stress on ion transport in native lung epithelium (Xenopus laevis): short-term activation of Na+, Cl (-) and K+ channels.

    Science.gov (United States)

    Bogdan, Roman; Veith, Christine; Clauss, Wolfgang; Fronius, Martin

    2008-09-01

    Epithelia, in general, and the lung epithelium, in particular, are exposed to mechanical forces, but little is known about their impact on pulmonary ion transport. In our present study, we employed transepithelial ion transport measurements on Xenopus lung preparations using custom-built Ussing chambers. Tissues were exposed to mechanical stress by increasing the water column (5 cm) at one side of the tissues. Apical exposure to hydrostatic pressure significantly decreased the short circuit current (I (SC): 24 +/- 1%, n = 152), slightly decreased the transepithelial resistance (R (T): 7 +/- 2%, n = 152), but increased the apical membrane capacitance (C (M): 16 +/- 6%, n = 9). The pressure-induced effect was sensitive to Na+ (amiloride), Cl(-) (DIDS, NFA, NPPB) and K+ channel blockers (Ba2+), glibenclamide). Further on, it was accompanied by increased extracellular ATP levels. The results show that mechanical stress leads to an activation of Na+, Cl(-), and K+ conductances in a native pulmonary epithelium resulting in a net decrease of ion absorption. This could be of considerable interest, since an altered ion transport may contribute to pathophysiological conditions, e.g., the formation of pulmonary edema during artificial ventilation.

  18. L-cysteine, N-acetyl-L-cysteine, and glutathione protect xenopus laevis embryos against acrylamide-induced malformations and mortality in the frog embryo teratogenesis assay (FETAX)

    Science.gov (United States)

    Dietary acrylamide is largely derived from heat-induced reactions between the amino group of the free amino acid asparagine and carbonyl groups of glucose and fructose during heat processing (baking, frying) of plant-derived foods such as potato fries and cereals. After consumption, acrylamide is a...

  19. Similarities of integumentary mucin B.1 from Xenopus laevis and prepro-von Willebrand factor at their amino-terminal regions.

    Science.gov (United States)

    Joba, W; Hoffmann, W

    1997-01-17

    Frog integumentary mucin B.1 (FIM-B.1) contains various cysteine-rich modules. In the past, a COOH-terminal "cystine knot" motif has been found that is similar to von Willebrand factor; this region is generally known to be responsible for dimerization processes. Furthermore, a "complement control protein" motif is present as an internal cysteine-rich domain in FIM-B.1. We characterize here the missing 75% toward the NH2 terminus of the FIM-B.1 precursor by molecular cloning. Analogous to prepro-von Willebrand factor, four elements with considerable similarity to D-domains are present (i.e. D1-D2-D'-D3). These domains have been described as essential for the multimerization of von Willebrand factor. Thus, the general structure of FIM-B.1 resembles that of the human mucin MUC2 as well as prepro-von Willebrand factor; these three molecules at least seem to share common structural elements allowing similar multimerization mechanisms.

  20. Taxonomy Icon Data: Western clawed frog [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available Western clawed frog Xenopus tropicalis Chordata/Vertebrata/Amphibia Xenopus_tropicalis_L.png Xenopus..._tropicalis_NL.png Xenopus_tropicalis_S.png Xenopus_tropicalis_NS.png http://biosciencedbc....jp/taxonomy_icon/icon.cgi?i=Xenopus+tropicalis&t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Xenopus+...tropicalis&t=NL http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Xenopus+tropical...is&t=S http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Xenopus+tropicalis&t=NS http://togodb.biosciencedbc.jp/togodb/view/taxonomy_icon_comment_en?species_id=137 ...

  1. Transposon transgenesis in Xenopus.

    Science.gov (United States)

    Yergeau, Donald A; Kelley, Clair M; Zhu, Haiqing; Kuliyev, Emin; Mead, Paul E

    2010-05-01

    Transposon-mediated integration strategies in Xenopus offer simple and robust methods for the generation of germline transgenic animals. Co-injection of fertilized one-cell embryos with plasmid DNA harboring a transposon transgene and synthetic mRNA encoding the cognate transposase enzyme results in mosaic integration of the transposon at early cleavage stages that are frequently passed through the germline in the adult animal. Micro-injection of fertilized embryos is a routine procedure used by many laboratories that use Xenopus as a developmental model and, as such, the transposon transgenesis method can be performed without additional equipment or specialized methodologies. The methods for injecting Xenopus embryos are well documented in the literature so here we provide a step-by-step guide to other aspects of transposon transgenesis, including screening mosaic founders for germline transmission of the transgene and general husbandry considerations related to management of populations of transgenic frogs.

  2. Transposon transgenesis in Xenopus

    Science.gov (United States)

    Yergeau, Donald A.; Kelley, Clair M.; Zhu, Haiqing; Kuliyev, Emin; Mead, Paul E.

    2010-01-01

    Transposon-mediated integration strategies in Xenopus offer simple and robust methods for the generation of germline transgenic animals. Co-injection of fertilized one-cell embryos with plasmid DNA harboring a transposon transgene and synthetic mRNA encoding the cognate transposase enzyme results in mosaic integration of the transposon at early cleavage stages that are frequently passed through the germline in the adult animal. Micro-injection of fertilized embryos is a routine procedure used by many laboratories that use Xenopus as a developmental model and, as such, the transposon transgenesis method can be performed without additional equipment or specialized methodologies. The methods for injecting Xenopus embryos are well documented in the literature so here we provide a step-by-step guide to other aspects of transposon transgenesis, including screening mosaic founders for germline transmission of the transgene and general husbandry considerations related to management of populations of transgenic frogs. PMID:20211730

  3. Hedgehog inhibition causes complete loss of limb outgrowth and transformation of digit identity in Xenopus tropicalis.

    Science.gov (United States)

    Stopper, Geffrey F; Richards-Hrdlicka, Kathryn L; Wagner, Günter P

    2016-03-01

    The study of the tetrapod limb has contributed greatly to our understanding of developmental pathways and how changes to these pathways affect the evolution of morphology. Most of our understanding of tetrapod limb development comes from research on amniotes, with far less known about mechanisms of limb development in amphibians. To better understand the mechanisms of limb development in anuran amphibians, we used cyclopamine to inhibit Hedgehog signaling at various stages of development in the western clawed frog, Xenopus tropicalis, and observed resulting morphologies. We also analyzed gene expression changes resulting from similar experiments in Xenopus laevis. Inhibition of Hedgehog signaling in X. tropicalis results in limb abnormalities including reduced digit number, missing skeletal elements, and complete absence of limbs. In addition, posterior digits assume an anterior identity by developing claws that are usually only found on anterior digits, confirming Sonic hedgehog's role in digit identity determination. Thus, Sonic hedgehog appears to play mechanistically separable roles in digit number specification and digit identity specification as in other studied tetrapods. The complete limb loss observed in response to reduced Hedgehog signaling in X. tropicalis, however, is striking, as this functional role for Hedgehog signaling has not been found in any other tetrapod. This changed mechanism may represent a substantial developmental constraint to digit number evolution in frogs. J. Exp. Zool. (Mol. Dev. Evol.) 9999B:XX-XX, 2016. © 2016 Wiley Periodicals, Inc.

  4. Expression of cyclin D1, cyclin D2, and N-myc in embryos of the direct developing frog Eleutherodactylus coqui, with a focus on limbs.

    Science.gov (United States)

    Nath, Kimberly; Fisher, Cara; Elinson, Richard P

    2013-01-01

    Species of frogs that develop directly have removed the tadpole from their ontogeny and form adult structures precociously. To see whether cell cycle regulators could be involved in this altered embryogenesis, we examined the expression of ccnd1, ccnd2, and mycn in embryos of the direct developing frog, Eleutherodactylus coqui. Notable differences compared to embryos of Xenopus laevis, a species with a tadpole, included prominent expression of ccnd2 in the midbrain and ccnd1 in the mandibular neural crest. The former may contribute to the precocious appearance of the adult-type visual system and the latter to the adult-type jaw. Large domains of ccnd2 and mycn presage the early appearance of limb buds, and ccnd1 and mycn are implicated in digit development.

  5. Tebuconazole disrupts steroidogenesis in Xenopus laevis

    DEFF Research Database (Denmark)

    2015-01-01

    or animals adapted to the xenobiotic, blood samples were collected 12 days into the study and at termination (day 27). After 12 days of exposure to 100 and 500μgL(-1) tebuconazole, plasma levels of testosterone (T) and dihydrotestosterone (DHT) were increased, while plasma 17β-estradiol (E2) concentrations...... were greatly reduced. Exposure to 0.1μgL(-1), on the other hand, resulted in decreased levels of T and DHT, with no effects observed for E2. After 27 days of exposure, effects were no longer observed in circulating androgen levels while the suppressive effect on E2 persisted in the two high......-exposure groups (100 and 500μgL(-1)). Furthermore, tebuconazole increased gonadal concentrations of T and DHT as well as expression of the enzyme CYP17 (500μgL(-1), 27 days). These results suggest that tebuconazole exposure may supress the action of CYP17 at the lowest exposure (0.1μgL(-1)), while CYP19...

  6. Germ plasm in Eleutherodactylus coqui, a direct developing frog with large eggs

    Directory of Open Access Journals (Sweden)

    Elinson Richard P

    2011-10-01

    Full Text Available Abstract Background RNAs for embryo patterning and for germ cell specification are localized to the vegetal cortex of the oocyte of Xenopus laevis. In oocytes of the direct developing frog Eleutherodactylus coqui, orthologous RNAs for patterning are not localized, raising the question as to whether RNAs and other components of germ plasm are localized in this species. Methods To identify germ plasm, E. coqui embryos were stained with DiOC6(3 or examined by in situ hybridization for dazl and DEADSouth RNAs. The cDNAs for the E. coqui orthologues were cloned by RT-PCR using degenerate primers. To examine activity of the E. coqui orthologues, RNAs, made from constructs of their 3'UTRs with mCherry, were injected into X. laevis embryos. Results Both DiOC6(3 and dazl and DEADSouth in situs identified many small islands at the vegetal surface of cleaving E. coqui embryos, indicative of germ plasm. Dazl was also expressed in primordial germ cells in the genital ridge. The 3'UTRs of E. coqui dazl and DEADSouth directed primordial germ cell specific protein synthesis in X. laevis. Conclusions E. coqui utilizes germ plasm with RNAs localized to the vegetal cortex to specify primordial germ cells. The large number of germ plasm islands suggests that an increase in the amount of germ plasm was important in the evolution of the large E. coqui egg.

  7. Evolution of advertisement calls in African clawed frogs

    Science.gov (United States)

    Tobias, Martha L.; Evans, Ben J.; Kelley, Darcy B.

    2014-01-01

    Summary For most frogs, advertisement calls are essential for reproductive success, conveying information on species identity, male quality, sexual state and location. While the evolutionary divergence of call characters has been examined in a number of species, the relative impacts of genetic drift or natural and sexual selection remain unclear. Insights into the evolutionary trajectory of vocal signals can be gained by examining how advertisement calls vary in a phylogenetic context. Evolution by genetic drift would be supported if more closely related species express more similar songs. Conversely, a poor correlation between evolutionary history and song expression would suggest evolution shaped by natural or sexual selection. Here, we measure seven song characters in 20 described and two undescribed species of African clawed frogs (genera Xenopus and Silurana) and four populations of X. laevis. We identify three call types — click, burst and trill — that can be distinguished by click number, call rate and intensity modulation. A fourth type is biphasic, consisting of two of the above. Call types vary in complexity from the simplest, a click, to the most complex, a biphasic call. Maximum parsimony analysis of variation in call type suggests that the ancestral type was of intermediate complexity. Each call type evolved independently more than once and call type is typically not shared by closely related species. These results indicate that call type is homoplasious and has low phylogenetic signal. We conclude that the evolution of call type is not due to genetic drift, but is under selective pressure. PMID:24723737

  8. Evolution of advertisement calls in African clawed frogs.

    Science.gov (United States)

    Tobias, Martha L; Evans, Ben J; Kelley, Darcy B

    2011-01-01

    For most frogs, advertisement calls are essential for reproductive success, conveying information on species identity, male quality, sexual state and location. While the evolutionary divergence of call characters has been examined in a number of species, the relative impacts of genetic drift or natural and sexual selection remain unclear. Insights into the evolutionary trajectory of vocal signals can be gained by examining how advertisement calls vary in a phylogenetic context. Evolution by genetic drift would be supported if more closely related species express more similar songs. Conversely, a poor correlation between evolutionary history and song expression would suggest evolution shaped by natural or sexual selection. Here, we measure seven song characters in 20 described and two undescribed species of African clawed frogs (genera Xenopus and Silurana) and four populations of X. laevis. We identify three call types - click, burst and trill - that can be distinguished by click number, call rate and intensity modulation. A fourth type is biphasic, consisting of two of the above. Call types vary in complexity from the simplest, a click, to the most complex, a biphasic call. Maximum parsimony analysis of variation in call type suggests that the ancestral type was of intermediate complexity. Each call type evolved independently more than once and call type is typically not shared by closely related species. These results indicate that call type is homoplasious and has low phylogenetic signal. We conclude that the evolution of call type is not due to genetic drift, but is under selective pressure.

  9. Induction of ovulation in Xenopus without hCG injection: the effect of adding steroids into the aquatic environment

    Science.gov (United States)

    2011-01-01

    Background The African clawed frog, Xenopus laevis, is widely used in studies of oogenesis, meiotic cell cycle and early embryonic development. However, in order to perform such studies, eggs are normally collected after the injection of hCG into the dorsal lymph sac of fully-grown female frogs following pre-injection of PMSF. Although this protocol is established and used as standard laboratory approach, there are some concerns over whether the injections could cause the transmission of deleterious microorganisms. Moreover, these injection protocols require a competent skilled worker to carry out the procedure efficiently. Methods Recently, we established a novel method to induce fish ovulation by simply adding the natural maturation-inducing hormone of teleosts, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-DHP), into the surrounding water. In the present study, we demonstrate how we can induce ovulation in frogs using the same methodology. Results In frogs, progesterone was effective in the induction of oocyte maturation in vitro. We then examined the ability of progesterone to induce ovulation in frogs. However treatment of frogs with progesterone alone only occasionally induced ovulation in vivo. The number of oocytes and the frequency of ovulation were significantly lower than that induced by hCG-injection. Thus, conditions were improved by using a combination of progesterone with estradiol and by pre-treating frogs with low concentrations of progesterone or estradiol. Finally, we established an efficient means of inducing ovulation in frogs which involved pre-treatment of frogs with salt solution followed by a mixture of estradiol and progesterone at high concentration. The frequency and numbers of oocytes obtained were identical to those resulting from PMSG-hCG induction. Fertilization rate of eggs ovulated by the new treatment method was comparable to eggs obtained by hCG-injection and juveniles developed normally. Conclusions To conclude, we

  10. Novel regulation of yolk utilization by thyroid hormone in embryos of the direct developing frog Eleutherodactylus coqui.

    Science.gov (United States)

    Singamsetty, Srikanth; Elinson, Richard P

    2010-01-01

    Thyroid hormone (TH) is required for metamorphosis of the long, coiled tadpole gut into the short frog gut. Eleutherodactylus coqui, a direct developing frog, lacks a tadpole. Its embryonic gut is a miniature adult form with a mass of yolky cells, called nutritional endoderm, attached to the small intestine. We tested the TH requirement for gut development in E. coqui. Inhibition of TH synthesis with methimazole arrested gut development in its embryonic form. Embryos treated with methimazole failed to utilize the yolk in their nutritional endoderm, and survived for weeks without further development. Conversely, methimazole and 3,3',5-tri-iodo-l-thyronine, the active form of TH, stimulated gut development and utilization and disappearance of the nutritional endoderm. In Xenopus laevis, the receptor for TH, TRβ, is upregulated in response to TH. Similarly, EcTRβ, the E. coqui ortholog, was upregulated by TH in the gut. EcTRβ expression was high in the nutritional endoderm, suggesting a direct role for TH in yolk utilization by these cells. An initial step in the breakdown of yolk in X. laevis is acidification of the yolk platelet. E. coqui embryos in methimazole failed to acidify their yolk platelets, but acidification was stimulated by TH indicating its role in an early step of yolk utilization. In addition to a conserved TH role in gut development, a novel regulatory role for TH in yolk utilization has evolved in these direct developers.

  11. Developing Xenopus embryos recover by compacting and expelling single wall carbon nanotubes.

    Science.gov (United States)

    Holt, Brian D; Shawky, Joseph H; Dahl, Kris Noel; Davidson, Lance A; Islam, Mohammad F

    2016-04-01

    Single wall carbon nanotubes are high aspect ratio nanomaterials being developed for use in materials, technological and biological applications due to their high mechanical stiffness, optical properties and chemical inertness. Because of their prevalence, it is inevitable that biological systems will be exposed to nanotubes, yet studies of the effects of nanotubes on developing embryos have been inconclusive and are lacking for single wall carbon nanotubes exposed to the widely studied model organism Xenopus laevis (African clawed frog). Microinjection of experimental substances into the Xenopus embryo is a standard technique for toxicology studies and cellular lineage tracing. Here we report the surprising finding that superficial (12.5 ± 7.5 µm below the membrane) microinjection of nanotubes dispersed with Pluronic F127 into one- to two-cell Xenopus embryos resulted in the formation and expulsion of compacted, nanotube-filled, punctate masses, at the blastula to mid-gastrula developmental stages, which we call "boluses." Such expulsion of microinjected materials by Xenopus embryos has not been reported before and is dramatically different from the typical distribution of the materials throughout the progeny of the microinjected cells. Previous studies of microinjections of nanomaterials such as nanodiamonds, quantum dots or spherical nanoparticles report that nanomaterials often induce toxicity and remain localized within the embryos. In contrast, our results demonstrate an active recovery pathway for embryos after exposure to Pluronic F127-coated nanotubes, which we speculate is due to a combined effect of the membrane activity of the dispersing agent, Pluronic F127, and the large aspect ratio of nanotubes.

  12. Genome-wide identification of Xenopus matrix metalloproteinases: conservation and unique duplications in amphibians

    Directory of Open Access Journals (Sweden)

    Mathew Smita

    2009-02-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are members of the superfamily of Zn2+ dependent extracellular or membrane-bound endopeptidases which have been implicated to play critical roles in vertebrate development and human pathogenesis. A number of MMP genes have been found to be upregulated in some or all organs during frog metamorphosis, suggesting that different MMPs may have different functions in various organs/tissues. The recent advances in EST (expressed sequence tag sequencing and the completion of the genome of Xenopus (X. tropicalis prompted us to systematically analyze the existence of MMPs in the Xenopus genome. Results We examined X. laevis and X. tropicalis ESTs and genomic sequences for MMPs and obtained likely homologs for 20 out of the 25 MMPs known in higher vertebrates. Four of the five missing MMPs, i.e. MMPs 8, 10, 12 and 27, were all encoded on human Chromosome 11 and the other missing MMP, MMP22 (a chicken MMP, was also absent in human genome. In addition, we identified several novel MMPs which appears to be derived from unique duplications over evolution, are present in the genomes of both Xenopus species. Conclusion We identified the homologs of most of the mammalian MMPs in Xenopus and discovered a number of novel MMPs. Our results suggest that MMP genes undergo dynamic changes over evolution. It will be of interest in the future to investigate whether MMP expression and functions during vertebrate development are conserved. The sequence information reported here should facilitate such an endeavor in the near future.

  13. Molecular footprinting of skeletal tissues in the catshark Scyliorhinus canicula and the clawed frog Xenopus tropicalis identifies conserved and derived features of vertebrate calcification

    Directory of Open Access Journals (Sweden)

    Sebastien eEnault

    2015-09-01

    Full Text Available Understanding the evolutionary emergence and subsequent diversification of the vertebrate skeleton requires a comprehensive view of the diverse skeletal cell types found in distinct developmental contexts, tissues and species. To date, our knowledge of the molecular nature of the shark calcified extracellular matrix, and its relationships with osteichthyan skeletal tissues, remain scarce. Here, based on specific combinations of expression patterns of the Col1a1, Col1a2 and Col2a1 fibrillar collagen genes, we compare the molecular footprint of endoskeletal elements from the chondrichthyan Scyliorhinus canicula and the tetrapod Xenopus tropicalis. We find that, depending on the anatomical location, Scyliorhinus skeletal calcification is associated to cell types expressing different subsets of fibrillar collagen genes, such as high levels of Col1a1 and Col1a2 in the neural arches, high levels of Col2a1 in the tesserae, or associated to a drastic Col2a1 downregulation in the centrum. We detect low Col2a1 levels in Xenopus osteoblasts, thereby revealing that the osteoblastic expression of this gene was significantly reduced in the tetrapod lineage. Finally, we uncover a striking parallel, from a molecular and histological perspective, between the vertebral cartilage calcification of both species and discuss the evolutionary origin of endochondral ossification.

  14. Thermal ecological physiology of native and invasive frog species: do invaders perform better?

    Science.gov (United States)

    Cortes, Pablo A; Puschel, Hans; Acuña, Paz; Bartheld, José L; Bozinovic, Francisco

    2016-01-01

    Biological invasions are recognized as an important biotic component of global change that threatens the composition, structure and functioning of ecosystems, resulting in loss of biodiversity and displacement of native species. Although ecological characteristics facilitating the establishment and spread of non-native species are widely recognized, little is known about organismal attributes underlying invasion success. In this study, we tested the effect of thermal acclimation on thermal tolerance and locomotor performance in the invasive Xenopus laevis and the Chilean native Calyptocephalella gayi. In particular, the maximal righting performance (μMAX), optimal temperature (TO), lower (CTmin) and upper critical thermal limits (CTmax), thermal breadth (Tbr) and the area under the performance curve (AUC) were studied after 6 weeks acclimation to 10 and 20°C. We observed higher values of μmax and AUC in X. laevis in comparison to C. gayi. On the contrary, the invasive species showed lower values of CTmin in comparison to the native one. In contrast, CTmax, TO and Tbr showed no inter-specific differences. Moreover, we found that both species have the ability to acclimate their locomotor performance and lower thermal tolerance limit at low temperatures. Our results demonstrate that X. laevis is a better performer than C. gayi. Although there were differences in CTmin, the invasive and native frogs did not differ in their thermal tolerance. Interestingly, in both species the lower and upper critical thermal limits are beyond the minimal and maximal temperatures encountered in nature during the coldest and hottest month, respectively. Overall, our findings suggest that both X. laevis and C. gayi would be resilient to climate warming expectations in Chile.

  15. Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease.

    Science.gov (United States)

    Ogino, Hajime; McConnell, William B; Grainger, Robert M

    2006-02-01

    In this study, we report a highly efficient transgenesis technique for Xenopus tropicalis based on a method described first for Medaka. This simple procedure entails co-injection of meganuclease I-SceI and a transgene construct flanked by two I-SceI sites into fertilized eggs. Approximately 30% of injected embryos express transgenes in a promoter-dependent manner. About 1/3 of such embryos show incorporation of the transgene at the one-cell stage and the remainder are 'half-transgenics' suggesting incorporation at the two-cell stage. Transgenes from both classes of embryos are shown to be transmitted and expressed in offspring. The procedure also works efficiently in Xenopus laevis. Because the needle injection procedure does not significantly damage embryos, a high fraction develop normally and can, as well, be injected with a second reagent, for example an mRNA or antisense morpholino oligonucleotide, thus allowing one to perform several genetic manipulations on embryos at one time. This simple and efficient technique will be a powerful tool for high-throughput transgenesis assays in founder animals, and for facilitating genetic studies in the fast-breeding diploid frog, X. tropicalis.

  16. Evaluating the Teratogenicity of Ritodrine and Nifedipine using a Frog Embryo Teratogenesis assay (FETAX).

    Science.gov (United States)

    Boğa Pekmezekmek, Ayper; Binokay, Uğur Seçil; Seçilmiş, Mehmet Ata; Kumcu, Eda; Şimşek, Erhan; Akillioğlu, Kübra; Sertdemir, Yaşar; Özaykan, Besim

    2015-01-01

    The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) was used to assess the teratogenic potential of two tocolytics. Embryos of the South African clawed frog, Xenopus laevis, were exposed to ritodrine or nifedipine. Exposure media were changed and monitored at 24-hour intervals. The 96-hour LC50 (Lethal concentration), the 96-hour EC50 (Malformation), and the No Observable Adverse Effect Concentrations (NOAEC) and the Lowest Observable Adverse Effect Concentration (LOAEC) for mortality, malformation and length were determined for each drug. Nifedipine was determined to be the more toxic and teratogenic than ritodrine, with a LC50 of 0.606 µg/L, an EC50 of 0.006 µg/L, and a teratogenicity Index (TI) value (LC50/EC50) of 101. On the other hand, the LC50 of ritodrine was 28.571 mg/L. In addition; the LC50, EC50 and TI values for nifedipine in the 5 mg/L ritodrine + nifedipine combination group were determined as 1.050 µg/L, 0.868 µg/L and 1.5 respectively. For ritodrine, the NOAEC and LOAEC values were determined as 2 mg/L and 4 mg/L, respectively. For the nifedipine and the ritodrine + nifedipine groups; while the LOAEC values of these groups were 0.0001 µg/L and 0.1 µg/L, respectively. NOAEC value couldn't be determined. Our results demonstrated that nifedipine administration was associated with higher levels of teratogenic and toxic effects. However, the ritodrine + nifedipine combination form reduced the toxic and teratogenic effects of nifedipine on Xenopus embryos. Further studies should be conducted in order to investigate the optimal combination concentrations of these substances for the treatment of preterm labor.

  17. Xenopus-FV3 host-pathogen interactions and immune evasion.

    Science.gov (United States)

    Jacques, Robert; Edholm, Eva-Stina; Jazz, Sanchez; Odalys, Torres-Luquis; Francisco, De Jesús Andino

    2017-11-01

    We first review fundamental insights into anti-ranavirus immunity learned with the Xenopus laevis/ranavirus FV3 model that are generally applicable to ectothermic vertebrates. We then further investigate FV3 genes involved in immune evasion. Focusing on FV3 knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD)-like protein (Δ64R-FV3), a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3), and an immediate-early18kDa protein (FV3-Δ18K), we assessed the involvement of these viral genes in replication, dissemination and interaction with peritoneal macrophages in tadpole and adult frogs. Our results substantiate the role of 64R and 52L as critical immune evasion genes, promoting persistence and dissemination in the host by counteracting type III IFN in tadpoles and type I IFN in adult frogs. Comparably, the substantial accumulation of genome copy numbers and exacerbation of type I and III IFN gene expression responses but deficient release of infectious virus suggests that 18K is a viral regulatory gene. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Targeted integration of genes in Xenopus tropicalis

    DEFF Research Database (Denmark)

    Shi, Zhaoying; Tian, Dandan; Xin, Huhu

    2017-01-01

    With the successful establishment of both targeted gene disruption and integration methods in the true diploid frog Xenopus tropicalis, this excellent vertebrate genetic model now is making a unique contribution to modelling human diseases. Here, we summarize our efforts on establishing homologous...

  19. Is the capacity for optic nerve regeneration related to continued retinal ganglion cell production in the frog?

    Science.gov (United States)

    Taylor, J S; Jack, J L; Easter, S S

    1989-01-01

    In the central nervous system of fish and frogs, some, but not all, axons can regenerate. Retinal ganglion cells are among those that can. The retinae of fish and frogs produce new retinal neurons, including ganglion cells, for months or years after hatching. We have evaluated the hypothesis that retinal axonal regeneration is obligatorily linked to continued production of new ganglion cells. We used bromodeoxyuridine immunocytochemistry to assess retinal neurogenesis in juvenile, yearling, and 10 year old Xenopus laevis. Retinal ganglion cell genesis was vigorous in the marginal retina of the juveniles, but in the yearlings and the 10 year olds, no new ganglion cells were produced there. Cellular proliferation in the central retina was evident at all three ages, but none of the cells produced centrally were in the ganglion cell layer. Regeneration was examined in vivo by cutting one optic nerve and then, weeks later, injecting the eye with tritiated proline. Autoradiographs of brain sections showed that the optic nerves of all three ages regenerated. Regeneration in vitro was assessed using retinal explants from frogs of all three ages. In all cases, the cultures produced neurites, with some age-specific differences in the patterns of outgrowth. We conclude that retinal axonal regeneration is not linked obligatorily to maintained neurogenesis.

  20. Development of a new approach for targeted gene editing in primordial germ cells using TALENs in Xenopus

    Directory of Open Access Journals (Sweden)

    Keisuke Nakajima

    2015-02-01

    Full Text Available A gene of interest can be efficiently modified using transcription activator-like effector nucleases (TALENs (Christian et al., 2010;Li et al., 2011. However, if a target gene is essential for development, growth and fertility, use of TALENs with high mutagenic activity in F0 frogs could result in developmental disorders or sterility, which would reduce the number of F1 progeny and make F1 phenotypical analysis difficult. We used the 3′ untranslated region of DEADSouth gene (DS-3′ of Xenopus tropicalis to solve this problem, because the addition of the DS-3′ to mRNA is known to induce primordial germ cell (PGC-specific expression and reduce the stability in somatic cells of mRNA in Xenopus laevis. At first, we inserted the X. tropicalis DS-3′ downstream of the EGFP termination codon and confirmed that the EGFP expression was specifically detected in PGCs for three weeks. Therefore, we inserted the DS-3′ downstream of the termination codon of the TALEN coding sequence. The tyrosinase gene was selected as the target gene for TALEN because the bi-allelic mutation of this gene is easily discernible by the albino phenotype. When fertilized eggs were microinjected with TALEN mRNAs fused to the DS-3′, their sperm and oocytes had a high rate (84–100% of target-gene modification in contrast to the lower rate (0–45% of nucleotide alteration observed in somatic cells.

  1. Dielectrospectroscopic monitoring of early embryogenesis in single frog embryos

    Science.gov (United States)

    Asami, Koji; Irimajiri, Akihiko

    2000-11-01

    Dielectric spectroscopy has been used to monitor the early embryogenesis of frog (Xenopus laevis) eggs. The dielectric spectra of a single egg in suspension over the frequency range 10 Hz to 10 MHz were collected at various stages of its development. The uncleaved egg showed a dielectric dispersion with a narrow distribution of relaxation times. After the first cleavage, the dielectric spectra were mainly composed of two subdispersions. In the cleavage process, up to the morula stage, changes in the spectra were quantitatively simulated by the `cell-aggregate' model in which the embryo is regarded as a concentrated suspension of shell-spheres that correspond to the blastomeres (i.e. the cells within the embryo). In the stages from the morula to the blastula, the changes in the dielectric spectra were explained as due to a reduction in the size of the blastomere accompanied by an expansion of the blastocoel (i.e. the central cavity in the embryo) using the `vesicle-inclusion' model that is a cell aggregate covered with a less conducting shell corresponding to the outermost layer of tightly interconnected cells.

  2. Coevolution of paired receptors in Xenopus carcinoembryonic antigen-related cell adhesion molecule families suggests appropriation as pathogen receptors.

    Science.gov (United States)

    Zimmermann, Wolfgang; Kammerer, Robert

    2016-11-16

    In mammals, CEACAM1 and closely related members represent paired receptors with similar extracellular ligand-binding regions and cytoplasmic domains with opposing functions. Human CEACAM1 and CEACAM3 which have inhibitory ITIM/ITSM and activating ITAM-like motifs, respectively, in their cytoplasmic regions are such paired receptors. Various bacterial pathogens bind to CEACAM1 on epithelial and immune cells facilitating both entry into the host and down-regulation of the immune response whereas interaction with granulocyte-specific CEACAM3 leads to their uptake and destruction. It is unclear whether paired CEACAM receptors also exist in other vertebrate clades. We identified more than 80 ceacam genes in Xenopus tropicalis and X. laevis. They consist of two subgroups containing one or two putative paired receptor pairs each. Analysis of genomic sequences of paired receptors provide evidence that their highly similar ligand binding domains were adjusted by recent gene conversion events. In contrast, selection for diversification is observed among inhibitory receptor orthologs of the two frogs which split some 60 million years ago. The allotetraploid X. laevis arose later by hybridization of two closely related species. Interestingly, despite the conservation of the genomic landscape surrounding the homeologous ceacam loci only one locus resembles the one found in X. tropicalis. From the second X. laevis locus more than 80 % of the ceacam genes were lost including 5 of the 6 paired receptor genes. This suggests that once the gene for one of the paired receptors is lost the remaining gene cluster degrades rapidly probably due to lack of selection pressure exerted by pathogens. The presence of paired receptors and selection for diversification suggests that also in amphibians CEACAM1-related inhibitory proteins are or were used as pathogen receptors.

  3. 爪蟾胚胎致畸实验(FETAX)在检测污染物发育毒性中的应用%Application of Frog Embryo Teratogenesis Assay-Xenopus in measuring the developmental toxicity of contaminants

    Institute of Scientific and Technical Information of China (English)

    钱丽娟; 刘青坡; 郭素珍; 曹庆珍; 刘俊奇; 施华宏

    2009-01-01

    爪蟾胚胎致畸实验(FETAX)是一种用于检测污染物发育毒性的有效方法.用CuSO4、CdCl2和三丁基锡(TBTCl)对热带爪蟾(Xenopus tropicalis)胚胎进行48 h暴露.结果表明,对照组胚胎均未出现死亡或畸形现象,Cu2+、Cd2+和TBTCl对胚胎的半致死质量浓度(LC50)分别为0.38 mg/L、4.28 mg/L和3.18 μg/L.48 h后,各暴露组胚胎出现颅面缺陷、眼睛突出、漏斗形嘴、肠道蜷曲、尾巴弯曲、水肿和色素沉着等多种畸形现象.Cu2+、Cd2+和TBTCl对胚胎的半致畸质量浓度(EC50)分别为0.16 mg/L、2.16 mg/L和2.98 μg/L,致畸指数分别为2.38、1.98和1.04.用脱碘酶抑制剂碘番酸(IOP)对非洲爪蟾胚胎进行暴露.36 h后,暴露组胚胎主要出现眼睛异常发育甚至消失等现象,这表明IOP对眼的发育有特定的毒性作用.研究表明,运用热带爪蟾胚胎检测污染物发育毒性的灵敏性并不高于非洲爪蟾(X. laevis)胚胎致畸实验,运用FETAX进行污染物发育毒性的检测并未显示出明显优势,其实验方案还有待进一步改进,但爪蟾胚胎模型在研究污染物特定的毒性机理方面具有较大潜力.鉴于爪蟾胚胎很适合进行分子操作,在现有FETAX实验的基础上增加一些分子生物学指标,将不仅能提高发育毒性效应检测的灵敏性,而且也增加了对污染物特定毒作用模式的解析功能.

  4. Asymmetric Localization of CK2α During Xenopus Oogenesis.

    Science.gov (United States)

    Imbrie, Gregory A; Wu, Hao; Seldin, David C; Dominguez, Isabel

    2012-05-05

    The establishment of the dorso-ventral axis is a fundamental process that occurs after fertilization. Dorsal axis specification in frogs starts immediately after fertilization, and depends upon activation of Wnt/β-catenin signaling. The protein kinase CK2α can modulate Wnt/β-catenin signaling and is necessary for dorsal axis specification in Xenopus laevis. Our previous experiments show that CK2α transcripts and protein are animally localized in embryos, overlapping the region where Wnt/β-catenin signaling is activated. Here we determined whether the animal localization of CK2α in the embryo is preceded by its localization in the oocyte. We found that CK2α transcripts were detected from stage I, their levels increased during oogenesis, and were animally localized as early as stage III. CK2α transcripts were translated during oogenesis and CK2α protein was localized to the animal hemisphere of stage VI oocytes. We cloned the CK2α 3'UTR and showed that the 2.8 kb CK2α transcript containing the 3'UTR was enriched during oogenesis. By injecting ectopic mRNAs, we demonstrated that both the coding and 3'UTR regions were necessary for proper CK2α transcript localization. This is the first report showing the involvement of coding and 3'UTR regions in animal transcript localization. Our findings demonstrate the pre-localization of CK2α transcript and thus, CK2α protein, in the oocyte. This may help restrict CK2α expression in preparation for dorsal axis specification.

  5. The Pesticide Malathion Disrupts "Xenopus" and Zebrafish Embryogenesis: An Investigative Laboratory Exercise in Developmental Toxicology

    Science.gov (United States)

    Chemotti, Diana C.; Davis, Sarah N.; Cook, Leslie W.; Willoughby, Ian R.; Paradise, Christopher J.; Lom, Barbara

    2006-01-01

    Malathion is an organophosphorus insecticide, which is often sprayed to control mosquitoes. When applied to aquatic habitats, malathion can also influence the embryogenesis of non-target organisms such as frogs and fish. We modified the frog embryo teratogen assay in "Xenopus" (FETAX), a standard toxicological assay, into an investigative…

  6. 甲苯喹哌对培养爪蟾胚胎肌细胞和神经细胞离子通道的作用%Effects of toquipidine on ionic channels of cultured embryonic Xenopus laevis myoblasts and neurons

    Institute of Scientific and Technical Information of China (English)

    郑平; 陈功; 史念慈; 周思平; 鲁映青; 姚明辉; 何曾佑

    1995-01-01

    目的:研究新型抗心律失常药甲苯喹哌对离子通道的作用.方法:通过膜片钳技术记录培养爪蟾胚胎肌细胞和神经细胞全细胞离子通道电流.结果:甲苯喹哌(0.1,1,10,100μmolL-1)可浓度依赖性地抑制肌细胞的钠通道,其IC50为7.2 μmol L-1(5.3-9.8 μmol L-1).甲苯喹哌(10 μmol L-1)可抑制神经细胞的高电压激活的钙通道.然而,肌细胞上的稳态外向钾电流却受到甲苯喹哌(10μmol L-1)的激活.结论:甲苯喹哌抑制钠、钙通道,但激活稳态外向钾通道.%AIM: To study the effects of toquipidine ( 1-p-methyl-phenyl- 2- ( α-piperidinoacetyl ) - 1,2, 3, 4-tetrahydroisoquinoline hydrochloride,Toq), a new anti-arrhythmic agent first synthesized in China, on ionic channels. METHODS: Ionic channel currents were recorded by whole-cell patch clamp technique in cultured embryonic Xenopus laevis myoblasts and neurons. RESULTS: Toq (0. 1, 1, 10, and 100 μmol L-1) caused a concentration-dependent inhibition of the Na+ currents with IC50 7.2μmol L-1(5.3-9.8 μmol L-1). Toq (10μmol L-1) also suppressed the high-voltageactivated Ca2+ currents in neurons. But the steady-state outward K+ currents in myoblasts were activated by Toq (10 μmol L-1).CONCLUSION: Toq blocked the Na+ andCa2+ channels and opened the steady-stateoutward K+ channels.

  7. Development of the retinotectal system in the direct-developing frog Eleutherodactylus coqui in comparison with other anurans

    Directory of Open Access Journals (Sweden)

    Schlosser Gerhard

    2008-06-01

    Full Text Available Abstract Background Frogs primitively have a biphasic life history with an aquatic larva (tadpole and a usually terrestrial adult. However, direct developing frogs of the genus Eleutherodactylus have lost a free living larval stage. Many larval structures never form during development of Eleutherodactylus, while limbs, spinal cord, and an adult-like cranial musculoskeletal system develop precociously. Results Here, I compare growth and differentiation of the retina and tectum and development of early axon tracts in the brain between Eleutherodactylus coqui and the biphasically developing frogs Discoglossus pictus, Physalaemus pustulosus, and Xenopus laevis using morphometry, immunohistochemical detection of proliferating cell nuclear antigen (PCNA and acetylated tubulin, biocytin tracing, and in situ hybridization for NeuroD. Findings of the present study indicate that retinotectal development was greatly altered during evolution of Eleutherodactlyus mostly due to acceleration of cell proliferation and growth in retina and tectum. However, differentiation of retina, tectum, and fiber tracts in the embryonic brain proceed along a conserved slower schedule and remain temporally coordinated with each other in E. coqui. Conclusion These findings reveal a mosaic pattern of changes in the development of the central nervous system (CNS during evolution of the direct developing genus Eleutherodactylus. Whereas differentiation events in directly interconnected parts of the CNS such as retina, tectum, and brain tracts remained coordinated presumably due to their interdependent development, they were dissociated from proliferation control and from differentiation events in other parts of the CNS such as the spinal cord. This suggests that mosaic evolutionary changes reflect the modular character of CNS development.

  8. Behavioral Repertoire of Xenopus tropicalis: Baseline Female-male Interactions during Spawning Events and Male Vocal Communication

    Science.gov (United States)

    The aquatic frog, Xenopus tropicalis, is being developed for use as a model amphibian species for inclusion in the EPA’s Endocrine Disruptor Screening Program. Current toxicity test designs do not incorporate measures of fecundity due to high variability in the responses of frog...

  9. Deiodinase activity is present in Xenopus laevis during early embryogenesis

    NARCIS (Netherlands)

    G.M. Dubois (Ghislaine Morvan); A. Sebillot (Anthony); G.G.J.M. Kuiper (George); C.H.J. Verhoelst (Carla H.); V.M. Darras (Veerle); T.J. Visser (Theo); B.A. Demeneix (Barbara)

    2006-01-01

    textabstractThyroid hormones orchestrate amphibian metamorphosis. The type 2 and type 3 deiodinases make vital contributions to this process by controlling levels of the thyroid hormones T4 and T3 available to different tissues. Because the tadpole thyroid gland is not functional until stage NF44, i

  10. Xenopus laevis embryos: biochemical evaluations in simulated microgravity condition

    Directory of Open Access Journals (Sweden)

    B. Berra

    2009-01-01

    Full Text Available Exposure to space-flight environment, notably microgravity and radiations, can induce changes in living systems. Life in space increases the amount of stress hormones, insulin resistance, altered musculoskeletal system structure and function, inflammation and mitochondrial function with increased oxidative stress (Biolo et al., 2003; Zhang et al., 2007; Shatten et al., 2001.

  11. Thyroxine Induced Resorption of Xenopus Laevis Tail Tissue in Vitro.

    Science.gov (United States)

    Scadding, Steven R.

    1984-01-01

    A simple method of studying thyroxine-induced resorption of tadpole tails in vitro is described. This procedure demonstrates that resorption is dependent on thyroxine and requires protein synthesis. It introduces students to the use of tissue culture methods. (Author)

  12. Histological development of the gonad in juvenile Xenopus laevis

    Science.gov (United States)

    As directed by the Food Quality Protection Act, the US Environmental Protection Agency is developing a screening program for endocrine disrupting compounds. The Larval Amphibian Growth and Development Assay (LAGDA) is a tier II test intended to identify and characterize the adver...

  13. Histological development of the gonad in juvenile Xenopus laevis

    Science.gov (United States)

    As directed by the Food Quality Protection Act, the US Environmental Protection Agency is developing a screening program for endocrine disrupting compounds. The Larval Amphibian Growth and Development Assay (LAGDA) is a tier II test intended to identify and characterize the adver...

  14. Fibre-selective recording from the peripheral nerves of frogs using a multi-electrode cuff

    Science.gov (United States)

    Schuettler, Martin; Donaldson, Nick; Seetohul, Vipin; Taylor, John

    2013-06-01

    Objective. We investigate the ability of the method of velocity selective recording (VSR) to determine the fibre types that contribute to a compound action potential (CAP) propagating along a peripheral nerve. Real-time identification of the active fibre types by determining the direction of action potential propagation (afferent or efferent) and velocity might allow future neural prostheses to make better use of biological sensor signals and provide a new and simple tool for use in fundamental neuroscience. Approach. Fibre activity was recorded from explanted Xenopus Laevis frog sciatic nerve using a single multi-electrode cuff that records whole nerve activity with 11 equidistant ring-shaped electrodes. The recorded signals were amplified, delayed against each other with variable delay times, added and band-pass filtered. Finally, the resulting amplitudes were measured. Main Result. Our experiments showed that electrically evoked frog CAP was dominated by two fibre populations, propagating at around 20 and 40 m/s, respectively. The velocity selectivity, i.e. the ability of the system to discriminate between individual populations was increased by applying band-pass filtering. The method extracted an entire velocity spectrum from a 10 ms CAP recording sample in real time. Significance. Unlike the techniques introduced in the 1970s and subsequently, VSR requires only a single nerve cuff and does not require averaging to provide velocity spectral information. This makes it potentially suitable for the generation of highly-selective real-time control-signals for future neural prostheses. In our study, electrically evoked CAPs were analysed and it remains to be proven whether the method can reliably classify physiological nerve traffic. The work presented here was carried out at the laboratories of the Implanted Devices Group, Department of Medical Physics and Bioengineering, University College London, UK.

  15. Triclosan exposure alters postembryonic development in a Pacific tree frog (Pseudacris regilla) Amphibian Metamorphosis Assay (TREEMA)

    Energy Technology Data Exchange (ETDEWEB)

    Marlatt, Vicki L. [Nautilus Environmental, 8864 Commerce Court, Burnaby, B.C. V5A 4N7 (Canada); Veldhoen, Nik [Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055 Stn CSC, Victoria, B.C. V8W 3P6 (Canada); Lo, Bonnie P. [Nautilus Environmental, 8864 Commerce Court, Burnaby, B.C. V5A 4N7 (Canada); Bakker, Dannika; Rehaume, Vicki; Vallee, Kurtis [Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055 Stn CSC, Victoria, B.C. V8W 3P6 (Canada); Haberl, Maxine; Shang, Dayue; Aggelen, Graham C. van; Skirrow, Rachel C. [Pacific and Yukon Laboratory for Environmental Testing, Emergencies Operational Analytical Laboratories and Research Support Division, Environment Canada, 2645 Dollarton Highway, North Vancouver, B.C. V7H 1B1 (Canada); Elphick, James R. [Nautilus Environmental, 8864 Commerce Court, Burnaby, B.C. V5A 4N7 (Canada); Helbing, Caren C., E-mail: chelbing@uvic.ca [Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055 Stn CSC, Victoria, B.C. V8W 3P6 (Canada)

    2013-01-15

    The Amphibian Metamorphosis Assay (AMA), developed for Xenopus laevis, is designed to identify chemicals that disrupt thyroid hormone (TH)-mediated biological processes. We adapted the AMA for use on an ecologically-relevant North American species, the Pacific tree frog (Pseudacris regilla), and applied molecular endpoints to evaluate the effects of the antibacterial agent, triclosan (TCS). Premetamorphic (Gosner stage 26-28) tadpoles were immersed for 21 days in solvent control, 1.5 {mu}g/L thyroxine (T{sub 4}), 0.3, 3 and 30 {mu}g/L (nominal) TCS, or combined T{sub 4}/TCS treatments. Exposure effects were scored by morphometric (developmental stage, wet weight, and body, snout-vent and hindlimb lengths) and molecular (mRNA abundance using quantitative real time polymerase chain reaction) criteria. T{sub 4} treatment alone accelerated development concomitant with altered levels of TH receptors {alpha} and {beta}, proliferating cell nuclear antigen, and gelatinase B mRNAs in the brain and tail. We observed TCS-induced perturbations in all of the molecular and morphological endpoints indicating that TCS exposure disrupts coordination of postembryonic tadpole development. Clear alterations in molecular endpoints were evident at day 2 whereas the earliest morphological effects appeared at day 4 and were most evident at day 21. Although TCS alone (3 and 30 {mu}g/L) was protective against tadpole mortality, this protection was lost in the presence of T{sub 4}. The Pacific tree frog is the most sensitive species examined to date displaying disruption of TH-mediated development by a common antimicrobial agent.

  16. Diseases of frogs and toads

    Science.gov (United States)

    Green, D.E.; Converse, K.A.; Majumdar, S.K.; Huffman, J.E.; Brenner, F.J.; Panah, A.I.

    2005-01-01

    This chapter presents information on infectious diseases of free-living frogs and toads that have completed metamorphosis. The diseases discussed in this chapter pertain principally to sub-adult and adult frogs and toads that are at least 60-90 days removed from completion of metamorphosis. The main emphasis of this chapter is the diseases found in amphibians of Canada and the United States. Diseases of recent metamorphs, larvae and amphibian eggs are presented in the chapters Diseases of Amphibian Eggs and Embryos and Diseases of Tadpoles. The smallest disease agents (viruses and bacteria) are presented first, followed by fungi, protozoa, helminths and ectoparasites. Diseases presented in this chapter are Ranaviral (iridovirus) infection Lucke frog herpesvirus (kidney cancer) Frog erythrocytic virus West Nile virus Red-leg disease (bacterial septicemia) Salmonellosis Chytrid fungal infection Basidiobolus fungi Dermosporidiosis Ichthyophoniasis Dermocystidium & Dermomycoides Myxozoa Ribeiroia flukes and Amphibian malformations Clinostomum metacercaria Aspects of each disease are presented to assist the biologist with recognition of diseases in the field. Hence, the major emphases for identification of diseases are the epizootiological aspects (host species, life stage, casualty numbers, etc) and gross findings ('lesions'). Descriptions of the microscopical, ultrastructural and cultural characteristics of each infectious agent were considered beyond the scope of this text. Detailed cultural and microscopical features of these disease agents are available in other reviews (Taylor et al., 2001; Green, 2001). Some diseases, while common in captive and zoo amphibians, are exceptionally rare in free-living frogs and toads, and therefore are omitted from this review. Among the diseases not presented are infections by chlamydia and mycobacteria, which occur principally in captive colonies of African clawed frogs (Xenopus, Hymenochirus, et al.) and northern leopard frogs

  17. Teratogenic effect and genotoxicity of acetochlor on embryos and tadpoles of West-African clawed frog (Xenopus tropicalis)%乙草胺对热带爪蟾的早期致畸效应及遗传毒性研究

    Institute of Scientific and Technical Information of China (English)

    李贤宾; 李少南; 袁丙强; 朱国念

    2011-01-01

    为了评价乙草胺对热带爪蟾Xenopus tropicalis早期发育阶段的影响,采用爪蟾胚胎致畸试验(FETAX)测定了乙草胺对爪蟾胚胎发育的致畸效应.结果表明,乙草胺对热带爪蟾胚胎的致畸中浓度(TC50)为有效成分1.20 mg/L,且能引起爪蟾腹部水肿、面部畸形、脊柱弯曲、尾巴弯曲、头部畸形等多种胚胎畸形.乙草胺的致畸指数(TI)为1.88,具有潜在的致畸风险.爪蟾蝌蚪在亚致死浓度的乙草胺中暴露4 d后,微核和单细胞凝胶电泳试验结果显示,乙草胺可导致蝌蚪血细胞的微核率显著升高,其尾部DNA含量和Olive尾矩两个指标也均显示细胞DNA受到损伤,且随着药剂浓度的增加,对DNA的损伤程度增强.%In order to assess the developmental toxicity and genotoxicity of acetochlor on the early development of Xenopus tropicalis, the teratogenicity on embryos exposed to acetochlor was investigated by modified frog embryo teratogenesis assay-Xenopus (FETAX). The teratogenic median concentration (TC50) value of acetochlor was 1.20 mg (a. i)/L. Acetochlor could induce a variety of malformations on X. tropicalis embryos, such as facial abnormality, pericardial edema, notochord and tail flexures,cephalic dysplasia and gut malformation. The calculated teratogenic index (TI) value was 1.88, suggesting that acetochlor had a teratogenic potential. Furthermore, the DNA damage was observed on the erythrocytes of tadpoles 4 days after exposed to sub-lethal concentrations of acetochlor by micronucleus test and alkaline single-cell gel electrophoresis. Compared to the control group, the results showed that the micronucleus frequencies were increased significantly, and the tail DNA and Olive tail moment of Comet picture had significant difference after treatment of acetochlor, which indicated that the DNA of erythrocytes was damaged.

  18. Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts.

    Science.gov (United States)

    Wangh, L J; DeGrace, D; Sanchez, J A; Gold, A; Yeghiazarians, Y; Wiedemann, K; Daniels, S

    1995-06-01

    Rapid genome replication is one of the hallmarks of the frog embryonic cell cycle. We report here that complete reactivation of quiescent somatic cell nuclei in Xenopus egg extracts depends on prior restructuring of the nuclear substrate and prior preparation of cytoplasmic extract with the highest capacity to initiate and sustain DNA synthesis. Nuclei from mature erythrocytes swell, replicate their DNA efficiently, and enter mitosis in frozen/thawed extracts prepared from activated Xenopus eggs, provided the nuclei are first treated with trypsin, heparin, and an extract prepared from unactivated, meiotically arrested, eggs. Optimal replicating extracts are prepared from large batches of unfertilized eggs that are synchronously activated into the cell cycle for 28 minutes (at 20 degrees C). Because the Xenopus cell cycle progresses so rapidly, extracts prepared just a few minutes before or after this time have substantially lower DNA synthetic capacities. At the optimal time and temperature, eggs have just reached the G1/S boundary of the first cell cycle. This fact was revealed by injecting and replicating an SV40 plasmid in intact unfertilized eggs as described previously. We estimate that under optimal conditions approximately 6.14 x 10(9) base pairs of DNA/per nucleus are synthesized in 30-40 minutes, a rate that rivals that observed in the zygotic nucleus. The findings reported here are one step in our long term effort to develop a new in vitro/in vivo approach to nuclear transplantation. Nuclear transplantation in amphibian embryos has been used to establish that the genomes of many types of differentiated somatic cells are pluripotent. But very few such nuclei have ever developed into advanced tadpoles or adult frogs, probably because somatic nuclei injected directly into activated eggs fail to reactivate quickly enough to avoid being damaged during first mitosis. We have already shown that unfertilized eggs can be injected prior to activation of the first

  19. Evolution of Courtship Songs in Xenopus : Vocal Pattern Generation and Sound Production.

    Science.gov (United States)

    Leininger, Elizabeth C; Kelley, Darcy B

    2015-01-01

    The extant species of African clawed frogs (Xenopus and Silurana) provide an opportunity to link the evolution of vocal characters to changes in the responsible cellular and molecular mechanisms. In this review, we integrate several robust lines of research: evolutionary trajectories of Xenopus vocalizations, cellular and circuit-level mechanisms of vocalization in selected Xenopus model species, and Xenopus evolutionary history and speciation mechanisms. Integrating recent findings allows us to generate and test specific hypotheses about the evolution of Xenopus vocal circuits. We propose that reduced vocal sex differences in some Xenopus species result from species-specific losses of sexually differentiated neural and neuromuscular features. Modification of sex-hormone-regulated developmental mechanisms is a strong candidate mechanism for reduced vocal sex differences.

  20. A simple method of transgenesis using I-SceI meganuclease in Xenopus.

    Science.gov (United States)

    Ishibashi, Shoko; Love, Nick R; Amaya, Enrique

    2012-01-01

    Here we present a protocol for generating transgenic embryos in Xenopus using I-SceI meganuclease. This method relies on integration of DNA constructs, containing one or two I-SceI meganuclease sites. It is a simpler method than the REMI method of transgenesis, and it is ideally suited for generating transgenic lines in Xenopus laevis and Xenopus tropicalis. In addition to it being simpler than the REMI method, this protocol also results in single copy integration events rather than tandem concatemers. Although the protocol we describe is for X. tropicalis, the method can also be used to generate transgenic lines in X. laevis. We also describe a convenient method for designing and generating complex constructs for transgenesis, named pTransgenesis, based on the Multisite Gateway(®) cloning, which include I-SceI sites and Tol2 elements to facilitate genome integration.

  1. Conservation Biology of Xenopus Longipes

    Science.gov (United States)

    Quock, R.; Blackburn, D. C.; Ghose, S.

    2014-12-01

    For the past 9 months, we have been studying the presence of disease and genetic variation in the Cameroonian species Xenopus longipes, found only in a lake on Mount Oku. During research trips to this lake (Lake Oku) over the past decade, mortalities of this species have been observed, and in addition there may be evidence of declines in other frog species in these mountains. It is well understood that in many parts of the world, amphibians are currently declining due to disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), and possibly also by the iridovirus ranavirus. A previous study suggested that ranavirus could be found in Lake Oku, and also that Bd may be present. Using 25 X. longipes liver samples collected during the summer of 2013 and 10 samples collected during the summer of 2011, we screened for Ranavirus through PCR amplification and sequencing, and screened for Bd in our 25 samples from 2013 through quantitative PCR. We also PCR amplified and sequenced 1950bp of the X. longipes 16S gene to look for genetic variation. We did not find ranavirus present on these frogs, and we found low prevalence (4%) of Bd. Through our analysis of 16S data, we found low genetic variation among the X. longipes, with a maximum divergence of 0.37% observed between any two individuals. Time is of the essence and it is crucial that the causes of these die offs be identified. While there have been observed mortalities of X. longipes since 2006, and this species remains on the Critically Endangered List, the cause of these mortalities is still unknown. If and when a cause can be identified, it would be monumental for this species' population and can hopefully be used to preserve and save these frogs.

  2. Application of algal growth inhibition test and frog embryo teratogenicity test to sewage samples; Sorui zoshoku sogai shiken oyobi kaeru haisai kikeisei shiken no kisoteki kento to gesui shiryo eno tekiyo

    Energy Technology Data Exchange (ETDEWEB)

    Matsubara, M.; Harada, A.; Tanaka, H. [Public Works Research Institute, Tsukuba (Japan)

    1997-11-10

    As sewerage has spread in Japan, evaluation of the effects of sewage and treated sewage on aquatic organisms has been becoming important. In this paper, applicability of two bioassays to sewage samples was discussed. One is an algal growth inhibition test using the green alga, Selenastrum capricornutum, proposed by Environment Canada that is conducted in a microplate for simplicity of handling. Because sewage samples include high concentration of nutrients, the test medium was modified here. The other is teratogenicity test with embryos of the frog, Xenopus laevis proposed by ASTM. Application of both the assays to sewage samples has been little reported. Therefore, we applied these bioassays to sewage, treated sewage and its receiving water at three sewage treatment plants (STPs). MICROTOX acute toxicity test was also conducted for comparison with the above bioassays. As a result from all the bioassays, the following findings were observed: (1) significant reduction of the toxicity of sewage during biological treatment in all the STPs, (2) no significant toxicity of disinfection with ozone and low-chlorine-dose, (3) high toxicity due to disinfection with high-chlorine-dose from MICROTOX test, (4) toxicity due to high-chlorine-dose from algal test even after dechlorination, (5) no significant toxicity of high-chlorine-dose from frog embryo test. Considering the above results, these bioassays are useful for evaluating the toxicity of sewage samples to each organism. 14 refs., 8 figs., 4 tabs.

  3. Dermaseptins and Magainins: Antimicrobial Peptides from Frogs' Skin—New Sources for a Promising Spermicides Microbicides—A Mini Review

    Directory of Open Access Journals (Sweden)

    Amira Zairi

    2009-01-01

    Full Text Available Sexually transmitted infections (STIs and human immunodeficiency virus (HIV, the causative agents of acquired immunodeficiency syndrome (AIDS, are two great concerns in the reproductive health of women. Thus, the challenge is to find products with a double activity, on the one hand having antimicrobial/antiviral properties with a role in the reduction of STI, and on the other hand having spermicidal action to be used as a contraceptive. In the absence of an effective microbicide along with the disadvantages of the most commonly used spermicidal contraceptive worldwide, nonoxynol-9, new emphasis has been focused on the development of more potential intravaginal microbicidal agents. Topical microbicides spermicides would ideally provide a female-controlled method of self-protection against HIV as well as preventing pregnancies. Nonoxynol-9, the only recommended microbicide spermicide, damages cervicovaginal epithelium because of its membrane-disruptive properties. Clearly, there is an urgent need to identify new compounds with dual potential microbicidal properties; antimicrobial peptides should be candidates for such investigations. Dermaseptins and magainins are two classes of cationic, amphipathic α-helical peptides that have been identified in the skin extracts of frogs Phyllomedusa sauvagei and Xenopus laevis. Regarding their contraceptive activities and their effect against various STI-causing pathogens, we believe that these two peptides are appropriate candidates in the evaluation of newer and safer microbicides spermicides in the future.

  4. Action of the pyrethroid insecticide cypermethrin on rat brain IIa sodium channels expressed in xenopus oocytes.

    Science.gov (United States)

    Smith, T J; Soderlund, D M

    1998-12-01

    Pyrethroid insecticides bind to a unique site on voltage-dependent sodium channels and prolong sodium currents, leading to repetitive bursts of action potentials or use-dependent nerve block. To further characterize the site and mode of action of pyrethroids on sodium channels, we injected synthetic mRNA encoding the rat brain IIa sodium channel alpha subunit, either alone or in combination with synthetic mRNA encoding the rat sodium channel beta1 subunit, into oocytes of the frog Xenopus laevis and assessed the actions of the pyrethroid insecticide [1R,cis,alphaS]-cypermethrin on expressed sodium currents by two-electrode voltage clamp. In oocytes expressing only the rat brain IIa alpha subunit, cypermethrin produced a slowly-decaying sodium tail current following a depolarizing pulse. In parallel experiments using oocytes expressing the rat brain IIa alpha subunit in combination with the rat beta1 subunit, cypermethrin produced qualitatively similar tail currents following a depolarizing pulse and also induced a sustained component of the sodium current measured during a step depolarization of the oocyte membrane. The voltage dependence of activation and steady-state inactivation of the cypermethrin-dependent sustained current were identical to those of the peak transient sodium current measured in the absence of cypermethrin. Concentration-response curves obtained using normalized tail current amplitude as an index of the extent of sodium channel modification by cypermethrin revealed that coexpression of the rat brain IIa alpha subunit with the rat beta1 subunit increased the apparent affinity of the sodium channel binding site for cypermethrin by more than 20-fold. These results confirm that the pyrethroid binding site is intrinsic to the sodium channel alpha subunit and demonstrate that coexpression of the rat brain IIa alpha subunit with the rat beta1 subunit alters the apparent affinity of this site for pyrethroids.

  5. Fantastic Frogs!

    Science.gov (United States)

    Scott, Kym

    2002-01-01

    Number rhymes can be used in many exciting and different ways to support the early learning goals for mathematics. The rhyme "five little speckled frogs" provides the theme for this display, which was set up in Lewisham's professional development center. It provides a range of ideas which would help develop young children's mathematical learning…

  6. Fantastic Frogs!

    Science.gov (United States)

    Scott, Kym

    2002-01-01

    Number rhymes can be used in many exciting and different ways to support the early learning goals for mathematics. The rhyme "five little speckled frogs" provides the theme for this display, which was set up in Lewisham's professional development center. It provides a range of ideas which would help develop young children's mathematical learning…

  7. Characterization of the Xenopus homolog of an immediate early gene associated with cell activation:sequence analysis and regulation of its expression by thyroid hormone during amphibian metamorphosis

    Institute of Scientific and Technical Information of China (English)

    VIVIACTLIANG; TIFFANYSEDGWICK; 等

    1997-01-01

    The complex transformation of a tadpole to a frog during amphibian development is under the control of thyroid hormone (T3).T3 is known to regulate gene transcription through its nuclear receptors.We have previously isolated many genes which are up-regulated by T3 in the intestine of Xenopus laevis tadpoles.We have now cloned a full-length cDNA for one such gene (IU12).Sequence analysis shows that the IU12 cDNA encodes a plasma membrane protein with 12 transmembrane domains and homologous to a mammalian gene associated with cell activation and organ development.Similarly,we have found that IU12 is activated during intestinal remodeling when both cell death and proliferation take place.Furthermore,IU12 is an early T3-response gene and its expression in the intestine during T3-induced metamorphosis mimics that during normal development.These results argue for a role of IU 12 in the signal transduction pathways leading to intestinal metamorphosis.

  8. The need of MMP-2 on the sperm surface for Xenopus fertilization: its role in a fast electrical block to polyspermy.

    Science.gov (United States)

    Iwao, Yasuhiro; Shiga, Keiko; Shiroshita, Ayumi; Yoshikawa, Tomoyasu; Sakiie, Maho; Ueno, Tomoyo; Ueno, Shuichi; Ijiri, Takashi W; Sato, Ken-ichi

    2014-11-01

    Monospermic fertilization in the frog, Xenopus laevis, is ensured by a fast-rising, positive fertilization potential to prevent polyspermy on the fertilized egg, followed by a slow block with the formation of a fertilization envelope over the egg surface. In this paper, we found that not only the enzymatic activity of sperm matrix metalloproteinase-2 (MMP-2) was necessary for a sperm to bind and/or pass through the extracellular coat of vitelline envelope, but also the hemopexin (HPX) domain of MMP-2 on the sperm surface was involved in binding and membrane fusion between the sperm and eggs. A peptide with a partial amino acid sequence of the HPX domain caused egg activation accompanied by an increase in [Ca(2+)]i in a voltage-dependent manner, similar to that in fertilization. The membrane microdomain (MD) of unfertilized eggs bound the HPX peptide, and this was inhibited by ganglioside GM1 distributed in the MD. The treatment of sperm with GM1 or anti-MMP-2 HPX antibody allows the sperm to fertilize an egg clamped at 0 mV, which untreated sperm cannot achieve. We propose a model accounting for the mechanism of voltage-dependent fertilization based on an interaction between the positively charged HPX domain in the sperm membrane and negatively-charged GM1 in the egg plasma membrane. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  9. I-SceI meganuclease-mediated transgenesis in Xenopus.

    Science.gov (United States)

    Pan, Fong Cheng; Chen, Yonglong; Loeber, Jana; Henningfeld, Kristine; Pieler, Tomas

    2006-01-01

    Several experimental approaches have been described to generate transgenic frogs. Here, we report on the application of a novel method in Xenopus, making use of I-SceI meganuclease. The characteristic feature of this endonuclease is that it has an extended recognition site of 18 bp, which is expected to exist only once in 7 x 10(10) bp of random DNA sequences. Various reporter constructs flanked by two I-SceI recognition sites were injected together with the I-SceI meganuclease into one-cell stage Xenopus embryos. We observed an overall transgenesis frequency of 10% or more under optimized condition. The injected genes were integrated into the genome and transmitted to F1 offspring. Southern blot analysis showed that between one and eight copies of the transgene were integrated. Meganuclease-aided transgenesis, thus, provides a simple and highly efficient tool for transgenesis in Xenopus.

  10. Xenopus: An Emerging Model for Studying Congenital Heart Disease

    Science.gov (United States)

    Kaltenbrun, Erin; Tandon, Panna; Amin, Nirav M.; Waldron, Lauren; Showell, Chris; Conlon, Frank L.

    2011-01-01

    Congenital heart defects affect nearly 1% of all newborns and are a significant cause of infant death. Clinical studies have identified a number of congenital heart syndromes associated with mutations in genes that are involved in the complex process of cardiogenesis. The African clawed frog, Xenopus, has been instrumental in studies of vertebrate heart development and provides a valuable tool to investigate the molecular mechanisms underlying human congenital heart diseases. In this review, we discuss the methodologies that make Xenopus an ideal model system to investigate heart development and disease. We also outline congenital heart conditions linked to cardiac genes that have been well-studied in Xenopus and describe some emerging technologies that will further aid in the study of these complex syndromes. PMID:21538812

  11. Xenopus pancreas development.

    Science.gov (United States)

    Pearl, Esther J; Bilogan, Cassandra K; Mukhi, Sandeep; Brown, Donald D; Horb, Marko E

    2009-06-01

    Understanding how the pancreas develops is vital to finding new treatments for a range of pancreatic diseases, including diabetes and pancreatic cancer. Xenopus is a relatively new model organism for the elucidation of pancreas development, and has already made contributions to the field. Recent studies have shown benefits of using Xenopus for understanding both early patterning and lineage specification aspects of pancreas organogenesis. This review focuses specifically on Xenopus pancreas development, and covers events from the end of gastrulation, when regional specification of the endoderm is occurring, right through metamorphosis, when the mature pancreas is fully formed. We have attempted to cover pancreas development in Xenopus comprehensively enough to assist newcomers to the field and also to enable those studying pancreas development in other model organisms to better place the results from Xenopus research into the context of the field in general and their studies specifically. Developmental Dynamics 238:1271-1286, 2009. (c) 2009 Wiley-Liss, Inc.

  12. Observation of nuclei reassembled from demembranated Xenopus sperm nuclei and analysis of their lamina components

    Institute of Scientific and Technical Information of China (English)

    QUJIAN; CHUANMAOZHANG; 等

    1994-01-01

    A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei.Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear memblane,nuclear pore complexes,and decondensed chromatin etc.Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei.Western-blotting results showed that lamin LII was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only egg derived lamin LIII.

  13. Nonsense suppression in eukaryotes: the use of the Xenopus oocyte as an in vivo assay system.

    Science.gov (United States)

    Bienz, M; Kubli, E; Kohli, J; de Henau, S; Grosjean, H

    1980-11-25

    Amber, ochre, and opal nonsense suppressor tRNAs isolated from yeast were injected into Xenopus laevis oocytes together with purified mRNAs (globin mRNA from rabbit, tobacco mosaic virus-RNA). Yeast opal suppressor tRNA is able to read the UGA stop codon of the rabbit beta-globin mRNA, thus producing a readthrough protein. A large readthrough product is also obtained upon coinjection of yeast amber or ochre suppressor tRNA with TMV-RNA. The amount of readthrough product is dependent on the amount of injected suppressor tRNA. The suppression of the terminator codon of TMV-RNA is not susceptible to Mg++ concentration or polyamine addition. Therefore, the Xenopus laevis oocyte provides a simple, sensitive, and well buffered in vivo screening system for all three types of eukaryotic nonsense suppressor tRNAs.

  14. Spatial and temporal regulation of collagenases—3,—4,and stromelysin —3 implicates distinct functions in apoptosis and tissue remodeling during frog metamorphosis

    Institute of Scientific and Technical Information of China (English)

    DAMJANOVSKISASHKO; ATSUKOISHIZUYAOKA; 等

    1999-01-01

    Matrix metalloproteinases (MMPs) are a family of extracellular proteases capable of degrading various proteinaceous components of the extracellular matrix(ECM).They have been implicated to play important roles in a number of developmental and pathological processes,such as tumor metastasis and inflammation.Relatively few studies have been carried out to investigate the function of MMPs during postembryonic organ-development.Using Xenopus laevis development as a model system,we demonstrate here that three MMPs,stromelysin-3(ST3),collagenases-3(Col3),and Col4,have distinct spatial and temporal expression profiles during metamorphosis as the tadpole transforms into a frog.In situ hybridizations reveal a tight,but distinct,association of individual MMPs with tissue remodeling in the tail and intestine during metamorphosis.In particular,ST3 expression is strongly correlated with apoptosis in both organs as demonstrated by analyses of serial sections with in situ hybridization for ST3 mRNA and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling)for apoptosis,respectively.On the other hand,Col3 and Col4 are present in regions where extensive connective tissue remodeling take place.These results indicate that ST3 is likely to play a role in ECM-remodeling that facilitate apoptotic tissue remodeling or resorption while Col3 and Col4 appear to participate in connective tissue degradation during development.

  15. Genetic diversity of MHC class I loci in six non-model frogs is shaped by positive selection and gene duplication

    Science.gov (United States)

    Kiemnec-Tyburczy, K M; Richmond, J Q; Savage, A E; Lips, K R; Zamudio, K R

    2012-01-01

    Comparative studies of major histocompatibility complex (MHC) genes across vertebrate species can reveal the evolutionary processes that shape the structure and function of immune regulatory proteins. In this study, we characterized MHC class I sequences from six frog species representing three anuran families (Hylidae, Centrolenidae and Ranidae). Using cDNA from our focal species, we amplified a total of 79 unique sequences spanning exons 2–4 that encode the extracellular domains of the functional alpha chain protein. We compared intra- and interspecific nucleotide and amino-acid divergence, tested for recombination, and identified codon sites under selection by estimating the rate of non-synonymous to synonymous substitutions with multiple codon-based maximum likelihood methods. We determined that positive (diversifying) selection was acting on specific amino-acid sites located within the domains that bind pathogen-derived peptides. We also found significant signals of recombination across the physical distance of the genes. Finally, we determined that all the six species expressed two or three putative classical class I loci, in contrast to the single locus condition of Xenopus laevis. Our results suggest that MHC evolution in anurans is a dynamic process and that variation in numbers of loci and genetic diversity can exist among taxa. Thus, the accumulation of genetic data for more species will be useful in further characterizing the relative importance of processes such as selection, recombination and gene duplication in shaping MHC loci among amphibian lineages. PMID:22549517

  16. Real-time automated measurement of Xenopus leavis tadpole behavior and behavioral response following triphenyltin exposure using the multispecies freshwater biomonitor (MFB)

    NARCIS (Netherlands)

    Schriks, M.; Hoorn, van M.K.; Faassen, E.J.; Dam, van J.W.; Murk, A.J.

    2006-01-01

    The present study examines whether behavior of Xenopus laevis tadpoles, when measured with the multispecies freshwater biomonitor (MFB), can be a sensitive and practical parameter for quantification of behavioral effects induced by toxic compounds. The MFB system is capable of automated simultaneous

  17. Heat-shock mediated overexpression of HNF1β mutations has differential effects on gene expression in the Xenopus pronephric kidney.

    Directory of Open Access Journals (Sweden)

    Kathrin Sauert

    Full Text Available The transcription factor HNF1B, encoded by the TCF2 gene, plays an important role in the organogenesis of vertebrates. In humans, heterozygous mutations of HNF1B are associated with several diseases, such as pancreatic β-cell dysfunction leading to maturity-onset diabetes of the young (MODY5, defective kidney development, disturbed liver function, pancreas atrophy, and malformations of the genital tract. The African claw frog Xenopus laevis is an excellent model to study the processes involved in embryogenesis and organogenesis, as it can be manipulated easily with a series of methods. In the present study, we overexpressed HNF1β mutants in the developing Xenopus embryo to assess their roles during organogenesis, particularly in the developing pronephric kidney. Towards this goal, we developed a heat-shock inducible binary Cre/loxP system with activator and effector strains. Heat-shock activation of the mutant HNF1B variants P328L329del and A263insGG resulted in malformations of various organs and the affected larvae developed large edemas. Defects in the pronephros were primarily confined to malformed proximal tubules. Furthermore, the expression of the proximal tubule marker genes tmem27 and slc3a1, both involved in amino acid transport, was affected. Both P328L329del and A263insGG downregulated expression of slc3a1. In addition, P328L329del reduced tmem27 expression while A263insGG overexpression decreased expression of the chloride channel clcnk and the transcription factor pax2. Overexpression of two mutant HNF1B derivatives resulted in distinct phenotypes reflected by either a reduction or an enlargement of pronephros size. The expression of selected pronephric marker genes was differentially affected upon overexpression of HNF1B mutations. Based on our findings, we postulate that HNF1B mutations influence gene regulation upon overexpression in specific and distinct manners. Furthermore, our study demonstrates that the newly established

  18. Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs).

    Science.gov (United States)

    Lei, Yong; Guo, Xiaogang; Liu, Yun; Cao, Yang; Deng, Yi; Chen, Xiongfeng; Cheng, Christopher H K; Dawid, Igor B; Chen, Yonglong; Zhao, Hui

    2012-10-23

    Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F(1) frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus.

  19. Differential regulation of two period genes in the Xenopus eye.

    Science.gov (United States)

    Zhuang, M; Wang, Y; Steenhard, B M; Besharse, J C

    2000-10-20

    The recent identification and analysis of mammalian homologues of the well characterized Drosophila circadian clock gene, Period (Per), has led to the idea that key features of vertebrate circadian rhythmicity are conserved at the molecular level. The Xenopus laevis retina contains a circadian clock mechanism that can be studied in vitro. To study the rhythmic expression of Per in the Xenopus retina, we used a degenerate RT-PCR strategy to obtain cDNA clones covering the entire 1427 amino acid coding region of a Xenopus homologue of Per2 and a partial cDNA sequence for a Xenopus homologue of Per1. Northern blot analysis shows that xPer1 and xPer2 transcripts are expressed most abundantly in the eye and the brain. However, rhythmic expression of xPer2 transcripts in the retina and retinal pigment epithelium (RPE) is light dependent and occurs only under 12 h light/12 h dark (LD) conditions, not in constant dark (DD). In contrast, xPer1 mRNA accumulation is rhythmic under both LD and DD conditions. Light dependent regulation of xPer2 mRNA and circadian regulation of xPer1 mRNA in the Xenopus retina differs from that in Drosophila and mammals. Light dependence of xPer2 mRNA levels and the offset phase relationship of the xPer2 rhythm to that for xPer1 suggests a role for xPer2 in circadian entrainment.

  20. Frog eat frog: exploring variables influencing anurophagy

    Directory of Open Access Journals (Sweden)

    G. John Measey

    2015-08-01

    Full Text Available Background. Frogs are generalist predators of a wide range of typically small prey items. But descriptions of dietary items regularly include other anurans, such that frogs are considered to be among the most important of anuran predators. However, the only existing hypothesis for the inclusion of anurans in the diet of post-metamorphic frogs postulates that it happens more often in bigger frogs. Moreover, this hypothesis has yet to be tested.Methods. We reviewed the literature on frog diet in order to test the size hypothesis and determine whether there are other putative explanations for anurans in the diet of post-metamorphic frogs. In addition to size, we recorded the habitat, the number of other sympatric anuran species, and whether or not the population was invasive. We controlled for taxonomic bias by including the superfamily in our analysis.Results. Around one fifth of the 355 records included anurans as dietary items of populations studied, suggesting that frogs eating anurans is not unusual. Our data showed a clear taxonomic bias with ranids and pipids having a higher proportion of anuran prey than other superfamilies. Accounting for this taxonomic bias, we found that size in addition to being invasive, local anuran diversity, and habitat produced a model that best fitted our data. Large invasive frogs that live in forests with high anuran diversity are most likely to have a higher proportion of anurans in their diet.Conclusions. We confirm the validity of the size hypothesis for anurophagy, but show that there are additional significant variables. The circumstances under which frogs eat frogs are likely to be complex, but our data may help to alert conservationists to the possible dangers of invading frogs entering areas with threatened anuran species.

  1. Embryogenesis and laboratory maintenance of the foam-nesting túngara frogs, genus Engystomops (= Physalaemus).

    Science.gov (United States)

    Romero-Carvajal, Andrés; Sáenz-Ponce, Natalia; Venegas-Ferrín, Michael; Almeida-Reinoso, Diego; Lee, Chanjae; Bond, Jennifer; Ryan, Michael J; Wallingford, John B; Del Pino, Eugenia M

    2009-06-01

    The vast majority of embryological research on amphibians focuses on just a single genus of frogs, Xenopus. To attain a more comprehensive understanding of amphibian development, experimentation on non-model frogs will be essential. Here, we report on the early development, rearing, and embryological analysis of túngara frogs (genus Engystomops, also called Physalaemus). The frogs Engystomops pustulosus, Engystomops coloradorum, and Engystomops randi construct floating foam-nests with small eggs. We define a table of 23 stages for the developmental period in the foam-nest. Embryos were immunostained against Lim1, neural, and somite-specific proteins and the expression pattern of RetinoBlastoma Binding Protein 6 (RBBP6) was analyzed by in situ hybridization. Due to their brief life-cycle, frogs belonging to the genus Engystomops are attractive for comparative and genetic studies of development. Developmental Dynamics 238:1444-1454, 2009. (c) 2009 Wiley-Liss, Inc.

  2. Complete Genome Sequence of the Frog Pathogen Mycobacterium ulcerans Ecovar Liflandii

    NARCIS (Netherlands)

    Tobias, Nicholas J.; Doig, Kenneth D.; Medema, Marnix H.; Chen, Honglei; Haring, Volker; Moore, Robert; Seemann, Torsten; Stinear, Timothy P.

    2013-01-01

    In 2004, a previously undiscovered mycobacterium resembling Mycobacterium ulcerans (the agent of Buruli ulcer) was reported in an outbreak of a lethal mycobacteriosis in a laboratory colony of the African clawed frog Xenopus tropicalis. This mycobacterium makes mycolactone and is one of several stra

  3. Subcellular localization of class I histone deacetylases in the developing Xenopus tectum

    Directory of Open Access Journals (Sweden)

    Xia eGuo

    2016-01-01

    Full Text Available Histone deacetylases (HDACs are thought to localize in the nucleus to regulate gene transcription and play pivotal roles in neurogenesis, apoptosis and plasticity. However, the subcellular distribution of class I HDACs in the developing brain remains unclear. Here, we show that HDAC1 and HDAC2 are located in both the mitochondria and the nucleus in the Xenopus laevis stage 34 tectum and are mainly restricted to the nucleus following further brain development. HDAC3 is widely present in the mitochondria, nucleus and cytoplasm during early tectal development and is mainly distributed in the nucleus in stage 45 tectum. In contrast, HDAC8 is broadly located in the mitochondria, nucleus and cytoplasm during tectal development. These data demonstrate that HDAC1, HDAC2 and HDAC3 are