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Sample records for virus 3-infected xenopus

  1. Antibody dependent enhancement of frog virus 3 infection

    Directory of Open Access Journals (Sweden)

    Penny Emily

    2010-02-01

    Full Text Available Abstract Background Viruses included in the family Iridoviridae are large, icosahedral, dsDNA viruses that are subdivided into 5 genera. Frog virus 3 (FV3 is the type species of the genus Ranavirus and the best studied iridovirus at the molecular level. Typically, antibodies directed against a virus act to neutralize the virus and limit infection. Antibody dependent enhancement occurs when viral antibodies enhance infectivity of the virus rather than neutralize it. Results Here we show that anti-FV3 serum present at the time of FV3 infection enhances infectivity of the virus in two non-immune teleost cell lines. We found that antibody dependent enhancement of FV3 was dependent on the Fc portion of anti-FV3 antibodies but not related to complement. Furthermore, the presence of anti-FV3 serum during an FV3 infection in a non-immune mammalian cell line resulted in neutralization of the virus. Our results suggest that a cell surface receptor specific to teleost cell lines is responsible for the enhancement. Conclusions This report represents the first evidence of antibody dependent enhancement in iridoviruses. The data suggests that anti-FV3 serum can either neutralize or enhance viral infection and that enhancement is related to a novel antibody dependent enhancement pathway found in teleosts that is Fc dependent.

  2. Immune roles of amphibian (Xenopus laevis) tadpole granulocytes during Frog Virus 3 ranavirus infections.

    Science.gov (United States)

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

    2017-07-01

    Infections by Frog Virus 3 (FV3) and other ranaviruses (RVs) are contributing to the amphibian declines, while the mechanisms controlling anuran tadpole susceptibility and adult frog resistance to RVs, including the roles of polymorphonuclear granulocytes (PMNs) during anti-FV3 responses, remain largely unknown. Since amphibian kidneys represent an important FV3 target, the inability of amphibian (Xenopus laevis) tadpoles to mount effective kidney inflammatory responses to FV3 is thought to contribute to their susceptibility. Here we demonstrate that a recombinant X. laevis granulocyte colony-stimulating factor (G-CSF) generates PMNs with hallmark granulocyte morphology. Tadpole pretreatment with G-CSF prior to FV3 infection reduces animal kidney FV3 loads and extends their survival. Moreover, G-CSF-derived PMNs are resistant to FV3 infection and express high levels of TNFα in response to this virus. Notably, FV3-infected tadpoles fail to recruit G-CSFR expressing granulocytes into their kidneys, suggesting that they lack an integral inflammatory effector population at this site. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Cholesterol-rich lipid rafts play a critical role in bovine parainfluenza virus type 3 (BPIV3) infection.

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    Li, Liyang; Yu, Liyun; Hou, Xilin

    2017-10-01

    Lipid rafts are specialized lipid domains enriched in cholesterol and sphingolipid, which can be utilized in the lifecycle of numerous enveloped viruses. Bovine parainfluenza virustype3 (BPIV3) entry to cell is mediated by receptor binding and membrane fusion, but how lipid rafts in host cell membrane and BPIV3 envelope affect virus infection remains unclear. In this study, we investigated the role of lipid rafts in the different stages of BPIV3 infection. The MDBK cells were treated by methyl-β-cyclodextrin (MβCD) to disrupt cellular lipid raft, and the virus infection was determined. The results showed that MβCD significantly inhibited BPIV3 infection in a dose-dependent manner, but didn't block the binding of virus to the cell membrane. Whereas, the MDBK cells treated by MβCD after virus-entry had no effects on the virus infection, to suggest that BPIV3 infection was associated with lipid rafts in cell membrane during viral entry stage. To further confirm lipid rafts in viral envelope also affected BPIV3 infection, we treated BPIV3 with MβCD to determine the virus titer. We found that disruption of the viral lipid raft caused a significant reduction of viral yield. Cholesterol reconstitution experiment showed that BPIV3 infection was successfully restored by cholesterol supplementation both in cellular membrane and viral envelope, which demonstrated that cholesterol-rich lipid rafts played a critical role in BPIV3 infection. These findings provide insights on our understanding of the mechanism of BPIV3 infection and imply that lipid raft might be a good potential therapeutic target to prevent virus infection. Copyright © 2017. Published by Elsevier Ltd.

  4. Para influenza virus 3 infection in cattle and small ruminants in Sudan

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    Intisar Kamil Saeed

    2016-09-01

    Results: Positive results were found in 29 (12.8% cattle, 31 (9.8% sheep and 11 (47.8% goat samples. All the studied areas showed positive results. Highest prevalence (66.7% was detected in the sheep and goats in Khartoum, followed by in goats in Nyala (33.3% at western Sudan. Sequence analyses of PIV3 of different regions of Sudan indicated that these were similar in sequence and length. The BLAST analysis indicated that the test sequences were closely related to the available annotated sequences at the GenBank. All these sequences matched with Bovine parainfluenza virus 3 except two those were matching with Swine parainfluenza virus 3. Conclusion: The results prove the existence of PIV3 infection in cattle, sheep and goats in the studied areas in Sudan and suggest its possible role in the respiratory infections. Genetic analysis indicate that the virus is mostly similar with bovine PIV3. [J Adv Vet Anim Res 2016; 3(3.000: 236-241

  5. Studies on the pathogenesis of a Chinese strain of bovine parainfluenza virus type 3 infection in Balb/c mice.

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    Dong, Xiu-Mei; Zhu, Yuan-Mao; Cai, Hong; Lv, Chuang; Gao, Yu-Ran; Yu, Zuo; Xue, Fei

    2012-07-06

    To date, three genotypes A, B, and C of bovine parainfluenza virus type 3 (BPIV3) have been isolated from cattle and only limited studies on the pathogenesis of the genotype A of BPIV3 infection in calves and laboratory animals have been conducted. The pathogenesis of the genotypes B and C of BPIV3 infection in calves and laboratory animals have not been reported. To alleviate the difficulties associated with sourcing suitable calves for infection studies, the establishment of BPIV3 infection model using laboratory model animals could aid in increasing the knowledge of the pathogenesis of this virus. Therefore thirty Balb/c mice were intranasally inoculated with a Chinese BPIV3 strain SD0835 which was classified as genotype C. Virus replications in mice were demonstrated by using virus isolation and titration, immunofluorescent staining, and immunohistochemistry and had occurred in the respiratory tissues as early as 24h after intranasal inoculation. The results of immunofluorescent staining and IHC implicated that the lungs and tracheas might be the major tissues in which the SD0835 infected and replicated. The histopathologic examinations revealed that alveoli septa thickening and focal cellulose pneumonia were seen in the lungs of experimentally infected mice. The aforementioned results indicated that the SD0835 of the genotype C was pathogenic to Balb/c mice and the mouse infection model could cast light on the genotype C of BPIV3 infection process and pathogenesis. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Treatment of hepatitis C virus genotype 3 infection with direct-acting antiviral agents

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    L.P. Zanaga

    Full Text Available Hepatitis C virus (HCV genotype 3 is responsible for 30.1% of chronic hepatitis C infection cases worldwide. In the era of direct-acting antivirals, these patients have become one of the most challenging to treat, due to fewer effective drug options, higher risk of developing cirrhosis and hepatocellular carcinoma and lower sustained virological response (SVR rates. Currently there are 4 recommended drugs for the treatment of HCV genotype 3: pegylated interferon (PegIFN, sofosbuvir (SOF, daclatasvir (DCV and ribavirin (RBV. Treatment with PegIFN, SOF and RBV for 12 weeks has an overall SVR rate of 83–100%, without significant differences among cirrhotic and non-cirrhotic patients. However, this therapeutic regimen has several contraindications and can cause significant adverse events, which can reduce adherence and impair SVR rates. SOF plus RBV for 24 weeks is another treatment option, with SVR rates of 82–96% among patients without cirrhosis and 62–92% among those with cirrhosis. Finally, SOF plus DCV provides 94–97% SVR rates in non-cirrhotic patients, but 59–69% in those with cirrhosis. The addition of RBV to the regimen of SOF plus DCV increases the SVR rates in cirrhotic patients above 80%, and extending treatment to 24 weeks raises SVR to 90%. The ideal duration of therapy is still under investigation. For cirrhotic patients, the optimal duration, or even the best regimen, is still uncertain. Further studies are necessary to clarify the best regimen to treat HCV genotype 3 infection.

  7. Pathogenesis of a genotype C strain of bovine parainfluenza virus type 3 infection in albino guinea pigs.

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    Shi, Hong-Fei; Zhu, Yuan-Mao; Dong, Xiu-Mei; Cai, Hong; Ma, Lei; Wang, Shu; Yan, Hao; Wang, Xue-Zhi; Xue, Fei

    2014-08-08

    Bovine parainfluenza virus type 3 (BPIV3) is one of the most important of the known viral respiratory tract agents of both young and adult cattle and widespread among cattle around the world. Up to present, three genotypes A, B and C of BPIV3 have been described on the basis of genetic and phylogenetic analysis and only limited studies on the pathogenesis of the genotype A of BPIV3 infection in calves and laboratory animals have been performed. The report about experimental infections of the genotypes B and C of BPIV3 in laboratory animals and calves was scant. Therefore, an experimental infection of guinea pigs with the Chinese BPIV3 strain SD0835 of the genotype C was performed. Sixteen guinea pigs were intranasally inoculated with the suspension of SD0835, while eight control guinea pigs were also intranasally inoculated with the same volume of supernatant from uninfected MDBK cells. The virus-inoculated guinea pigs displayed a few observable clinical signs that were related to the respiratory tract disease and two of the sixteen experimentally infected guinea pigs died at 2 and 3 days post inoculation (PI), respectively, and apparent gross pneumonic lesions were observed at necropsy. The gross pneumonic lesions in guinea pigs inoculated with SD0835 consisted of dark red, slightly depressed, irregular areas of consolidation in the lung lobes from the second to 9th day of infection at necropsy, and almost complete consolidation and atelectasis of the lung lobes were seen at 7 days PI. Histopathological changes including alveoli septa thickening and focal cellulose pneumonia were also observed in the lungs of guinea pigs experimentally infected with SD0835. Viral replication was detectable by virus isolation and titration, real-time RT-PCR and immunohistochemistry (IHC) staining in the respiratory tissues of guinea pigs as early as 24h after intranasal inoculation with SD0835. The results of virus isolation and titration showed that guinea pigs were permissive for

  8. Translation of three mouse hepatitis virus strain A59 subgenomic RNAs in Xenopus laevis oocytes

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    Horzinek, M.C.; Rottier, P.J.M.; Spaan, W.J.M.; Zeijst, B.A.M. van der

    1981-01-01

    We have purified the seven virus-specific RNAs which were previously shown to be induced in Sac(-) cells upon infection with mouse hepatitis virus strain A59 (W. J. M. Spaan, P. J. M. Rottier, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 108:424-434, 1981). The individual RNAs, prepared by

  9. Xenopus laevis

    African Journals Online (AJOL)

    Histidine and carnosine reduce muscle fatigue in. Xenopus laevis. Lesley Whitaker and G.N. Louw. Zoology Department, University of Cape Town, Rondebosch. The inclusion of either histidine or carnosine in a frog Ringer's perfusate at a concentration of 20 mmol/dm3 reduced fatigve in the gastrocnemius muscle of ...

  10. Analysis of Grape Polyamines from Grapevine leafroll associated viruses (GLRaV-2 and -3) Infected Vines

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    This is the first report comparing the free polyamine content of 'Pinot noir' grapes from vines infected with Grapevine leafroll associated viruses (GLRaV) -2 or -3, with samples taken from healthy vines. Berries were collected from three different rootstock/scion combinations, and all samples were ...

  11. Genetic Variants in the Apoptosis Gene BCL2L1 Improve Response to Interferon-Based Treatment of Hepatitis C Virus Genotype 3 Infection

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    Louise Nygaard Clausen

    2015-02-01

    Full Text Available Genetic variation upstream of the apoptosis pathway has been associated with outcome of hepatitis C virus (HCV infection. We investigated genetic polymorphisms in the intrinsic apoptosis pathway to assess their influence on sustained virological response (SVR to pegylated interferon-α and ribavirin (pegIFN/RBV treatment of HCV genotypes 1 and 3 infections. We conducted a candidate gene association study in a prospective cohort of 201 chronic HCV-infected individuals undergoing treatment with pegIFN/RBV. Differences between groups were compared in logistic regression adjusted for age, HCV viral load and interleukin 28B genotypes. Four single nucleotide polymorphisms (SNPs located in the B-cell lymphoma 2-like 1 (BCL2L1 gene were significantly associated with SVR. SVR rates were significantly higher for carriers of the beneficial rs1484994 CC genotypes. In multivariate logistic regression, the rs1484994 SNP combined CC + TC genotypes were associated with a 3.4 higher odds ratio (OR in SVR for the HCV genotype 3 (p = 0.02. The effect estimate was similar for genotype 1, but the association did not reach statistical significance. In conclusion, anti-apoptotic SNPs in the BCL2L1 gene were predictive of SVR to pegIFN/RBV treatment in HCV genotypes 1 and 3 infected individuals. These SNPs may be used in prediction of SVR, but further studies are needed.

  12. Non-travel related Hepatitis E virus genotype 3 infections in the Netherlands; A case series 2004 – 2006

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    Bosman Arnold

    2008-05-01

    Full Text Available Abstract Background Human hepatitis E virus (HEV infections are considered an emerging disease in industrialized countries. In the Netherlands, Hepatitis E virus (HEV infections have been associated with travel to high-endemic countries. Non-travel related HEV of genotype 3 has been diagnosed occasionally since 2000. A high homology of HEV from humans and pigs suggests zoonotic transmission but direct molecular and epidemiological links have yet to be established. We conducted a descriptive case series to generate hypotheses about possible risk factors for non-travel related HEV infections and to map the genetic diversity of HEV. Methods A case was defined as a person with HEV infection laboratory confirmed (positive HEV RT-PCR and/or HEV IgM after 1 January 2004, without travel to a high-endemic country three months prior to onset of illness. For virus identification 148 bp of ORF2 was sequenced and compared with HEV from humans and pigs. We interviewed cases face to face using a structured questionnaire and collected information on clinical and medical history, food preferences, animal and water contact. Results We interviewed 19 cases; 17 were male, median age 50 years (25–84 y, 12 lived in the North-East of the Netherlands and 11 had preexisting disease. Most common symptoms were dark urine (n = 16 and icterus (n = 15. Sixteen ate pork ≥ once/week and six owned dogs. Two cases had received blood transfusions in the incubation period. Seventeen cases were viremic (genotype 3 HEV, two had identical HEV sequences but no identified relation. For one case, HEV with identical sequence was identified from serum and surface water nearby his home. Conclusion The results show that the modes of transmission of genotype-3 HEV infections in the Netherlands remains to be resolved and that host susceptibility may play an important role in development of disease.

  13. Non-travel related Hepatitis E virus genotype 3 infections in the Netherlands; a case series 2004 - 2006.

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    Borgen, Katrine; Herremans, Tineke; Duizer, Erwin; Vennema, Harry; Rutjes, Saskia; Bosman, Arnold; de Roda Husman, Ana Maria; Koopmans, Marion

    2008-05-08

    Human hepatitis E virus (HEV) infections are considered an emerging disease in industrialized countries. In the Netherlands, Hepatitis E virus (HEV) infections have been associated with travel to high-endemic countries. Non-travel related HEV of genotype 3 has been diagnosed occasionally since 2000. A high homology of HEV from humans and pigs suggests zoonotic transmission but direct molecular and epidemiological links have yet to be established. We conducted a descriptive case series to generate hypotheses about possible risk factors for non-travel related HEV infections and to map the genetic diversity of HEV. A case was defined as a person with HEV infection laboratory confirmed (positive HEV RT-PCR and/or HEV IgM) after 1 January 2004, without travel to a high-endemic country three months prior to onset of illness. For virus identification 148 bp of ORF2 was sequenced and compared with HEV from humans and pigs. We interviewed cases face to face using a structured questionnaire and collected information on clinical and medical history, food preferences, animal and water contact. We interviewed 19 cases; 17 were male, median age 50 years (25-84 y), 12 lived in the North-East of the Netherlands and 11 had preexisting disease. Most common symptoms were dark urine (n = 16) and icterus (n = 15). Sixteen ate pork >/= once/week and six owned dogs. Two cases had received blood transfusions in the incubation period. Seventeen cases were viremic (genotype 3 HEV), two had identical HEV sequences but no identified relation. For one case, HEV with identical sequence was identified from serum and surface water nearby his home. The results show that the modes of transmission of genotype-3 HEV infections in the Netherlands remains to be resolved and that host susceptibility may play an important role in development of disease.

  14. Identification of candidate protein markers of Bovine Parainfluenza Virus Type 3 infection using an in vitro model.

    Science.gov (United States)

    Gray, Darren W; Welsh, Michael D; Doherty, Simon; Mooney, Mark H

    2017-05-01

    Bovine Parainfluenza Virus Type 3 (BPI3V) infections are often asymptomatic, causing respiratory tissue damage and immunosuppression, predisposing animals to severe bacterial pneumonia, the leading cause of Bovine Respiratory Disease (BRD) mortality. As with many pathogens, routine BPI3V serology does not indicate the presence of damaged respiratory tissue or active infection. In vitro proteomic marker screening using disease relevant cell models could help identify markers of infection and tissue damage that are also detectable during in vivo infections. This study utilised a proteomic approach to investigate in vitro cellular responses during BPI3V infection to enhance the current understanding of intracellular host-virus interactions and identify putative markers of in vivo infection. Through 2D gel electrophoresis proteomic analysis, BPI3V Phosphoprotein P and host T-complex Protein 1 subunit theta were found to be accumulated at the latter stages of infection within bovine fibroblasts. These proteins were subsequently detected using targeted multiple reaction monitoring (MRM) mass spectrometry in the plasma of animals challenged with BPI3V, with differential protein level profiles observed dependant on animal vaccination status. Potential mechanisms by which BPI3V overcomes host cellular immune response mechanisms allowing for replication and production of viral proteins were also revealed. Assessment of circulating protein marker levels identified through an in vitro approach as described may enable more effective diagnosis of active viral infection and diseased or damaged respiratory tissue in animals and allow for more effective utilisation of preventative therapeutic interventions prior to bacterial disease onset and significantly aid the management and control of BRD. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. All-Oral 12-Week Treatment With Daclatasvir Plus Sofosbuvir in Patients With Hepatitis C Virus Genotype 3 Infection: ALLY-3 Phase III Study

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    Nelson, David R; Cooper, James N; Lalezari, Jacob P; Lawitz, Eric; Pockros, Paul J; Gitlin, Norman; Freilich, Bradley F; Younes, Ziad H; Harlan, William; Ghalib, Reem; Oguchi, Godson; Thuluvath, Paul J; Ortiz-Lasanta, Grisell; Rabinovitz, Mordechai; Bernstein, David; Bennett, Michael; Hawkins, Trevor; Ravendhran, Natarajan; Sheikh, Aasim M; Varunok, Peter; Kowdley, Kris V; Hennicken, Delphine; McPhee, Fiona; Rana, Khurram; Hughes, Eric A

    2015-01-01

    Treatment options for patients with hepatitis C virus (HCV) genotype 3 infection are limited, with the currently approved all-oral regimens requiring 24-week treatment and the addition of ribavirin (RBV). This phase III study (ALLY-3; http://ClinicalTrials.gov: NCT02032901) evaluated the 12-week regimen of daclatasvir (DCV; pangenotypic nonstructural protein [NS]5A inhibitor) plus sofosbuvir (SOF; pangenotypic NS5B inhibitor) in patients infected with genotype 3. Patients were either treatment naïve (n = 101) or treatment experienced (n = 51) and received DCV 60 mg plus SOF 400 mg once-daily for 12 weeks. Coprimary endpoints were the proportions of treatment-naïve and treatment-experienced patients achieving a sustained virological response (SVR) at post-treatment week 12 (SVR12). SVR12 rates were 90% (91 of 101) and 86% (44 of 51) in treatment-naïve and treatment-experienced patients, respectively; no virological breakthrough was observed, and ≥99% of patients had a virological response (VR) at the end of treatment. SVR12 rates were higher in patients without cirrhosis (96%; 105 of 109) than in those with cirrhosis (63%; 20 of 32). Five of seven patients who previously failed treatment with an SOF-containing regimen and 2 of 2 who previously failed treatment with an alisporivir-containing regimen achieved SVR12. Baseline characteristics, including gender, age, HCV-RNA levels, and interleukin-28B genotype, did not impact virological outcome. DCV plus SOF was well tolerated; there were no adverse events (AEs) leading to discontinuation and only 1 serious AE on-treatment, which was unrelated to study medications. The few treatment-emergent grade 3/4 laboratory abnormalities that were observed were transient. Conclusion: A 12-week regimen of DCV plus SOF achieved SVR12 in 96% of patients with genotype 3 infection without cirrhosis and was well tolerated. Additional evaluation to optimize efficacy in genotype 3–infected patients with cirrhosis is underway

  16. Sofosbuvir plus ribavirin in treatment-naïve patients with chronic hepatitis C virus genotype 1 or 3 infection in India.

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    Shah, S R; Chowdhury, A; Mehta, R; Kapoor, D; Duseja, A; Koshy, A; Shukla, A; Sood, A; Madan, K; Sud, R; Nijhawan, S; Pawan, R; Prasad, M; Kersey, K; Jiang, D; Svarovskaia, E; Doehle, B; Kanwar, B; Subramanian, M; Acharya, S K; Sarin, S

    2017-05-01

    Until 2014, pegylated interferon plus ribavirin was the recommended standard of care for the treatment of chronic hepatitis C virus (HCV) infection in India. This open-label phase 3b study, conducted across 14 sites in India between 31 March 2014 and 30 November 2015, evaluated the efficacy and safety of sofosbuvir plus ribavirin therapy among treatment-naïve patients with chronic genotype 1 or 3 HCV infection. A total of 117 patients with genotype 1 or 3 HCV infection were randomized 1:1 to receive sofosbuvir 400 mg and weight-based ribavirin (1000 or 1200 mg) daily for 16 or 24 weeks. Among those with genotype 1 infection, the primary efficacy endpoint of sustained virologic response at 12 weeks post-treatment (SVR12) was reported in 90% (95% confidence intervals [CI], 73-98) and 96% (95% CI, 82-100) of patients following 16 and 24 weeks of treatment, respectively. For patients with genotype 3 infection, SVR12 rates were 100% (95% CI, 88-100) and 93% (95% CI, 78-99) after 16 and 24 weeks of therapy, respectively. Adverse events, most of which were mild or moderate in severity, occurred in 69% and 57% of patients receiving 16 and 24 weeks of treatment, respectively. The most common treatment-emergent adverse events were asthenia, headache and cough. Only one patient in the 24-week group discontinued treatment with sofosbuvir during this study. Overall, sofosbuvir plus ribavirin therapy achieved SVR12 rates ≥90% and was well tolerated among treatment-naïve patients with chronic genotype 1 or 3 HCV infection in India. © 2016 John Wiley & Sons Ltd.

  17. Cost-effectiveness of daclatasvir plus sofosbuvir-based regimen for treatment of hepatitis C virus genotype 3 infection in Canada.

    Science.gov (United States)

    Moshyk, A; Martel, M-J; Tahami Monfared, A A; Goeree, R

    2016-01-01

    -experienced patients. Daclatasvir plus sofosbuvir is a safe and effective option for the treatment of chronic HCV genotype 3 patients. This regimen could be considered a cost-effective option following a first-line treatment of peg-interferon/ribavirin treatment experienced patients with HCV genotype-3 infection.

  18. Ribavirin plasma concentration is a predictor of sustained virological response in patients treated for chronic hepatitis C virus genotype 2/3 infection

    DEFF Research Database (Denmark)

    Pedersen, C; Alsiö, Å; Lagging, M

    2011-01-01

    is generally recommended. The primary aim of this study was to investigate the correlation between ribavirin concentration at day 29 and therapeutic response in patients with HCV genotype 2/3 infection. A total of 382 patients were randomized to 12 or 24 weeks of treatment with pegylated interferon-alfa 2a 180...

  19. Genetic Variants in the Apoptosis Gene BCL2L1 Improve Response to Interferon-Based Treatment of Hepatitis C Virus Genotype 3 Infection

    DEFF Research Database (Denmark)

    Clausen, Louise Nygaard; Weis, Nina; Ladelund, Steen

    2015-01-01

    Genetic variation upstream of the apoptosis pathway has been associated with outcome of hepatitis C virus (HCV) infection. We investigated genetic polymorphisms in the intrinsic apoptosis pathway to assess their influence on sustained virological response (SVR) to pegylated interferon...

  20. Identification of the Best Direct-Acting Antiviral Regimen for Patients With Hepatitis C Virus Genotype 3 Infection: A Systematic Review and Network Meta-analysis

    NARCIS (Netherlands)

    Berden, F.A.C.; Aaldering, B.R.; Groenewoud, H.; Hout, J. in't; Kievit, W.; Drenth, J.P.H.

    2017-01-01

    BACKGROUND & AIMS: Direct-acting antivirals (DAAs) are effective in the treatment of chronic hepatitis C virus (HCV) infection, although results for patients infected with genotype 3 are suboptimal. There are several regimens available, however, direct comparisons have not been made and are unlikely

  1. 16, 16 Dimethyl prostaglandin E2 prevents the development of fulminant hepatitis and blocks the induction of monocyte/macrophage procoagulant activity after murine hepatitis virus strain 3 infection.

    Science.gov (United States)

    Abecassis, M; Falk, J A; Makowka, L; Dindzans, V J; Falk, R E; Levy, G A

    1987-01-01

    16, 16 Dimethyl prostaglandin E2 (dmPGE2), a known cytoprotective agent, was examined for its ability to alter the course of fulminant hepatitis in an experimental model of fulminant viral hepatitis, murine hepatitis murine hepatitis type 3 (MHV-3). Fully susceptible BALB/cJ mice, infected with 100 50% lethal doses (LD50) of MHV-3 developed histologic and biochemical evidence of fulminant hepatitis, as evidenced by massive hepatic necrosis with hypoglycemia, metabolic acidosis, and a markedly elevated serum alanine aminotransferase (ALT) (mean, 1,402 +/- 619 IU/liter). In contrast, animals treated with dmPGE2 either before or after infection (up to 48 h) demonstrated a marked reduction in both histologic and biochemical evidence of liver damage as characterized by normal blood glucose, total CO2, and ALT determinations (mean ALT, 63 +/- 40 IU/liter). Treatment of infected mice with PGF2 alpha demonstrated no cytoprotective effects. High titers of infectious virus were recovered from the livers of both dmPGE2-treated and -untreated animals throughout the course of infection. In a parallel in vitro study, dmPGE2 (10(-4)-10(-8) M) demonstrated a similar cytoprotective effect on monolayers of isolated cultured hepatocytes from fully susceptible BALB/cJ mice infected at a multiplicity of infection of 0.1, 1.0, and 10.0. In addition, splenic macrophages recovered from infected and untreated BALB/cJ mice demonstrated a marked augmentation in procoagulant activity (PCA) from a basal 10 +/- 5 mU/10(6) splenic macrophages to a maximum of 615 +/- 102 mU/10(6) splenic macrophages, whereas no increase in macrophage PCA was detected in infected animals treated with dmPGE2. These results suggest that dmPGE2, without detectably altering viral replication or infectivity in vivo, confers a marked cytoprotective effect on hepatocytes both in vivo and in vitro, and prevents the induction of macrophage PCA in vivo in fully susceptible BALB/cJ mice after murine hepatitis virus type 3

  2. Identification of the Best Direct-Acting Antiviral Regimen for Patients With Hepatitis C Virus Genotype 3 Infection: A Systematic Review and Network Meta-analysis.

    Science.gov (United States)

    Berden, Floor A C; Aaldering, Bryan R R Z; Groenewoud, Hans; IntHout, Joanna; Kievit, Wietske; Drenth, Joost P H

    2017-03-01

    Direct-acting antivirals (DAAs) are effective in the treatment of chronic hepatitis C virus (HCV) infection, although results for patients infected with genotype 3 are suboptimal. There are several regimens available, however, direct comparisons have not been made and are unlikely to occur. We aimed to identify the most effective DAA regimen for patients infected with HCV genotype 3 and to assess the role of ribavirin. We conducted a systematic search of PubMed, Embase, and Web of Science databases through March 2016. We performed a Bayesian network meta-analysis using a random-effects model to indirectly compare regimens in patients with and without cirrhosis. We calculated mean estimated sustained virologic response (SVR) with 95% credible intervals (95% CrI) per regimen and effect of ribavirin as odds ratio. We focused on current recommended regimens and regimens under evaluation by regulatory authorities. Our search identified 2167 articles; 27 studies (comprising 3415 patients) were included. Among patients without cirrhosis, the greatest rates of SVR were estimated for those receiving sofosbuvir + velpatasvir with ribavirin (99%; 95% CrI, 98%-100%) and without ribavirin (97%; 95% CrI, 95%-99%), sofosbuvir + daclatasvir + ribavirin (96%; 95% CrI, 92%-98%), and sofosbuvir + peginterferon + ribavirin (95%; 95% CrI, 91%-98%), all for 12 weeks. Among patients with cirrhosis, the highest rates of SVR were estimated for those receiving sofosbuvir + velpatasvir for 24 weeks (96%; 95% CrI, 92%-99%), sofosbuvir + daclatasvir + ribavirin for 24 weeks (94%; 95% CrI, 87%-98%), and sofosbuvir + velpatasvir + ribavirin for 12 weeks (94%; 95% CrI, 86%-98%). Ribavirin increases efficacy in patients with and without cirrhosis (odds ratio, 2.6-4.5). An indirect comparison of DAA-based treatments, using Bayesian network meta-analysis, found regimens containing sofosbuvir and velpatasvir to be the best option for patients with HCV genotype 3 infection. Our analyses

  3. Xenopus Xp54 and human RCK/p54 helicases functionally replace yeast Dhh1p in brome mosaic virus RNA replication.

    Science.gov (United States)

    Alves-Rodrigues, Isabel; Mas, Antonio; Díez, Juana

    2007-04-01

    By using a Brome mosaic virus (BMV)-Saccharomyces cerevisiae system, we previously showed that the cellular Lsm1p-7p/Pat1p/Dhh1p decapping-activator complex functions in BMV RNA translation and replication. As a first approach in investigating whether the corresponding human homologues play a similar role, we expressed human Lsm1p (hLsm1p) and RCK/p54 in yeast. Expression of RCK/p54 but not hLsm1p restored the defect in BMV RNA translation and replication observed in the dhh1Delta and lsm1Delta strains, respectively. This functional conservation, together with the common replication strategies of positive-stranded RNA viruses, suggests that RCK/p54 may also play a role in the replication of positive-stranded RNA viruses that infect humans.

  4. 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...... recombination-mediated targeted integration in Xenopus tropicalis, the usefulness, and limitation of targeted integration via the homology-independent strategy, and future directions on how to further improve targeted gene integration in Xenopus tropicalis....

  5. Amphibian (Xenopus sp.) iodothyronine deiodinase ...

    Science.gov (United States)

    The U.S. EPA-MED amphibian thyroid group is currently screening chemicals for inhibition of human iodothyronine deiodinase activity as components of the thyroid system important in human development. Amphibians are a bellwether taxonomic group to gauge toxicity of chemicals in the environment. Amphibian thyroid function is not only important in development but also metamorphosis. Xenopus sp. have been used extensively as model organisms and are well characterized genetically. We propose to screen a list of chemicals (selected from the human DIO screening results) to test for inhibition of Xenopus deiodinases. Large quantities of the enzymes will be produced using an adenovirus system. Our preliminary results show that there may be catalytic differences between human and Xenopus deiodinases. The Twin Ports Early Career Scientists is a new group formed within the Duluth-Superior scientific community. This presentation will provide a basic introduction to my research and our mission at EPA, and help to establish networking and collaboration relationships across disciplines and institutions.

  6. Simian T-cell leukemia virus (STLV) infection in wild primate populations in Cameroon: evidence for dual STLV type 1 and type 3 infection in agile mangabeys (Cercocebus agilis).

    Science.gov (United States)

    Courgnaud, Valerie; Van Dooren, Sonia; Liegeois, Florian; Pourrut, Xavier; Abela, Bernadette; Loul, Severin; Mpoudi-Ngole, Eitel; Vandamme, Annemieke; Delaporte, Eric; Peeters, Martine

    2004-05-01

    Three types of human T-cell leukemia virus (HTLV)-simian T-cell leukemia virus (STLV) (collectively called primate T-cell leukemia viruses [PTLVs]) have been characterized, with evidence for zoonotic origin from primates for HTLV type 1 (HTLV-1) and HTLV-2 in Africa. To assess human exposure to STLVs in western Central Africa, we screened for STLV infection in primates hunted in the rain forests of Cameroon. Blood was obtained from 524 animals representing 18 different species. All the animals were wild caught between 1999 and 2002; 328 animals were sampled as bush meat and 196 were pets. Overall, 59 (11.2%) of the primates had antibodies cross-reacting with HTLV-1 and/or HTLV-2 antigens; HTLV-1 infection was confirmed in 37 animals, HTLV-2 infection was confirmed in 9, dual HTLV-1 and HTLV-2 infection was confirmed in 10, and results for 3 animals were indeterminate. Prevalences of infection were significantly lower in pets than in bush meat, 1.5 versus 17.0%, respectively. Discriminatory PCRs identified STLV-1, STLV-3, and STLV-1 and STLV-3 in HTLV-1-, HTLV-2-, and HTLV-1- and HTLV-2-cross-reactive samples, respectively. We identified for the first time STLV-1 sequences in mustached monkeys (Cercopithecus cephus), talapoins (Miopithecus ogouensis), and gorillas (Gorilla gorilla) and confirmed STLV-1 infection in mandrills, African green monkeys, agile mangabeys, and crested mona and greater spot-nosed monkeys. STLV-1 long terminal repeat (LTR) and env sequences revealed that the strains belonged to different PTLV-1 subtypes. A high prevalence of PTLV infection was observed among agile mangabeys (Cercocebus agilis); 89% of bush meat was infected with STLV. Cocirculation of STLV-1 and STLV-3 and STLV-1-STLV-3 coinfections were identified among the agile mangabeys. Phylogenetic analyses of partial LTR sequences indicated that the agile mangabey STLV-3 strains were more related to the STLV-3 CTO604 strain isolated from a red-capped mangabey (Cercocebus torquatus

  7. Xenopus genomic data and browser resources.

    Science.gov (United States)

    Vize, Peter D; Zorn, Aaron M

    2017-06-15

    The two species of Xenopus most commonly used in biomedical research are the diploid Xenopus (Silurana) tropicalis and the tetraploid Xenopus laevis. The X. tropicalis genome sequence has been available since 2010 and this year the X. laevis, genome from two distinct genetic backgrounds has been published. Multiple genome assemblies available for both species and transcriptomic and epigenetic data sets are growing rapidly, all of which are available from a variety of web resources. This review describes the contents of these resources, how to locate and download genomic data, and also how to view and manipulate these data on various public genome browsers, with an emphasis on Xenbase, the Xenopus model organism database. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Xenopus oocyte electrophysiology in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans

    2009-01-01

    Deorphanization of the large group of G protein-coupled receptors (GPCRs) for which an endogenous activating ligand has not yet been identified (orphan GPCRs) has become increasingly difficult. A specialized technique that has been successfully applied to deorphanize some of these GPCRs involves...... two-electrode voltage-clamp recordings of currents through ion channels, which are activated by GPCRs heterologously expressed in Xenopus oocytes. The ion channels that couple to GPCR activation in Xenopus oocytes can be endogenous calcium-activated chloride channels (CaCCs) or heterologously...... expressed G protein-coupled inwardly rectifying potassium channels (GIRKs). We will describe a general approach for expression of GPCRs in Xenopus oocytes and characterization of these using electrophysiological recordings. We will focus on the detection of GPCR activation by recordings of currents through...

  9. Limb Regeneration in Xenopus laevis Froglet

    Directory of Open Access Journals (Sweden)

    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.

  10. Tebuconazole disrupts steroidogenesis in Xenopus laevis

    DEFF Research Database (Denmark)

    Poulsen, Rikke; Luong, Xuan; Hansen, Martin

    2015-01-01

    A 27-day controlled exposure study of adult male African clawed frogs (Xenopus laevis) was conducted to examine the mechanism by which tebuconazole may disrupt steroidogenesis. The fungicide was measured by LC-MS/MS in tank water and in target tissues (adipose, kidney, liver, and brain), and we...

  11. Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes.

    Science.gov (United States)

    Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V Bleu; Serrano, Elba E

    2011-10-01

    The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology.

  12. 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.

  13. Synthesis of bunyavirus-specific proteins in a continuous cell line (XTC-2) derived from Xenopus laevis.

    Science.gov (United States)

    Watret, G E; Pringle, C R; Elliott, R M

    1985-03-01

    The XTC-2 cell line, derived from Xenopus laevis, supported the replication of representative viruses from each of the four genera in the family Bunyaviridae. Generally, viral titres were higher in XTC-2 cells than in other susceptible cell lines, and for some viruses plaques were detected earlier in XTC-2 cells. The XTC-2 cell line permitted comparative analyses of bunyavirus-specific protein synthesis. The patterns of synthesis of viral proteins, characteristic of each of the genera, were observed with representative viruses. These studies provided biochemical characterization of two Scottish isolates, which support the inclusion of Clo Mor virus in the Nairovirus genus and St Abb's Head (M349) virus in the Uukuvirus genus.

  14. VIRUSES

    Indian Academy of Sciences (India)

    and-mouth disease in livestock was an infectious particle smaller than any bacteria. This was the first clue to the nature of viruses, genetic entities that lie somewhere in the gray area between living and non-living states.

  15. CAP1 expression is developmentally regulated in Xenopus.

    Science.gov (United States)

    KhosrowShahian, F; Hubberstey, A V; Crawford, M J

    2002-05-01

    We have cloned and characterized a Xenopus member of the cyclase associated protein (CAP) gene family. xCAP1 is expressed as a maternal transcript, but is up-regulated prior to gastrulation and subsequently localizes to head mesenchyme, lens, otic vesicle, and trunk mesoderm including the pronephros. At different stages, the gene also appears to differentiate surface from deep (sensorial) ectoderm. As in Drosophila, Xenopus CAP1 is expressed in the developing eye, specifically in the differentiating lens. However, in distinction to Drosophila, Xenopus CAP1 does not express in periodically arrayed neural bands.

  16. A Xenopus tropicalis oligonucleotide microarray works across species using RNA from Xenopus laevis.

    Science.gov (United States)

    Chalmers, Andrew D; Goldstone, Kim; Smith, James C; Gilchrist, Mike; Amaya, Enrique; Papalopulu, Nancy

    2005-03-01

    Microarrays have great potential for the study of developmental biology. As a model system Xenopus is well suited for making the most of this potential. However, Xenopus laevis has undergone a genome wide duplication meaning that most genes are represented by two paralogues. This causes a number of problems. Most importantly the presence of duplicated genes mean that a X. laevis microarray will have less or even half the coverage of a similar sized microarray from the closely related but diploid frog Xenopus tropicalis. However, to date, X. laevis is the most commonly used amphibian system for experimental embryology. Therefore, we have tested if a microarray based on sequences from X. tropicalis will work across species using RNA from X. laevis. We produced a pilot oligonucleotide microarray based on sequences from X. tropicalis. The microarray was used to identify genes whose expression levels changed during early X. tropicalis development. The same assay was then carried out using RNA from X. laevis. The cross species experiments gave similar results to those using X. tropicalis RNA. This was true at the whole microarray level and for individual genes, with most genes giving similar results using RNA from X. laevis and X. tropicalis. Furthermore, the overlap in genes identified between a X. laevis and a X. tropicalis set of experiments was only 12% less than the overlap between two sets of X. tropicalis experiments. Therefore researchers can work with X. laevis and still make use of the advantages offered by X. tropicalis microarrays.

  17. Spinal cord regeneration in Xenopus laevis.

    Science.gov (United States)

    Edwards-Faret, Gabriela; Muñoz, Rosana; Méndez-Olivos, Emilio E; Lee-Liu, Dasfne; Tapia, Victor S; Larraín, Juan

    2017-02-01

    Here we present a protocol for the husbandry of Xenopus laevis tadpoles and froglets, and procedures to study spinal cord regeneration. This includes methods to induce spinal cord injury (SCI); DNA and morpholino electroporation for genetic studies; in vivo imaging for cell analysis; a swimming test to measure functional recovery; and a convenient model for screening for new compounds that promote neural regeneration. These protocols establish X. laevis as a unique model organism for understanding spinal cord regeneration by comparing regenerative and nonregenerative stages. This protocol can be used to understand the molecular and cellular mechanisms involved in nervous system regeneration, including neural stem and progenitor cell (NSPC) proliferation and neurogenesis, extrinsic and intrinsic mechanisms involved in axon regeneration, glial response and scar formation, and trophic factors. For experienced personnel, husbandry takes 1-2 months; SCI can be achieved in 5-15 min; and swimming recovery takes 20-30 d.

  18. Neuronal, neurohormonal, and autocrine control of Xenopus melanotrope cell activity

    NARCIS (Netherlands)

    Roubos, E.W.; Scheenen, W.J.J.M.; Jenks, B.G.

    2005-01-01

    Amphibian pituitary melanotropes are used to investigate principles of neuroendocrine translation of neural input into hormonal output. Here, the steps in this translation process are outlined for the melanotrope cell of Xenopus laevis, with attention to external stimuli, neurochemical messengers,

  19. Comparison of TALEN scaffolds in Xenopus tropicalis

    Directory of Open Access Journals (Sweden)

    Keisuke Nakajima

    2013-11-01

    Transcription activator-like effector nucleases (TALENs are facile and potent tools used to modify a gene of interest for targeted gene knockout. TALENs consist of an N-terminal domain, a DNA-binding domain, and a C-terminal domain, which are derived from a transcription activator-like effector, and the non-specific nuclease domain of FokI. Using Xenopus tropicalis (X. tropicalis, we compared the toxicities and somatic mutation activities of four TALEN architectures in a side-by-side manner: a basic TALEN, a scaffold with the same truncated N- and C-terminal domains as GoldyTALEN, a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain, and a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric Sharkey nuclease domain. The strongest phenotype and targeted somatic gene mutation were induced by the injection of TALEN mRNAs containing the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain. The obligate heterodimeric TALENs exhibited reduced toxicity compared to the homodimeric TALENs, and the homodimeric GoldyTALEN-type scaffold showed both a high activity of somatic gene modification and high toxicity. The Sharkey mutation in the heterodimeric nuclease domain reduced the TALEN-mediated somatic mutagenesis.

  20. Ca2+ homeostasis regulates Xenopus oocyte maturation.

    Science.gov (United States)

    Sun, Lu; Hodeify, Rawad; Haun, Shirley; Charlesworth, Amanda; MacNicol, Angus M; Ponnappan, Subramaniam; Ponnappan, Usha; Prigent, Claude; Machaca, Khaled

    2008-04-01

    In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte's competence to undergo maturation in preparation for fertilization and embryonic development.

  1. Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins.

    Science.gov (United States)

    Murray, M T; Schiller, D L; Franke, W W

    1992-01-01

    Storage of maternal mRNAs as nontranslated ribonucleoprotein (RNP) complexes is an adaptive strategy in various vertebrate and invertebrate oocytes, for rapid translational recruitment during embryonic development. Previously, we showed that Xenopus laevis oocytes have a soluble cytoplasmic pool of mRNA-binding proteins and particles competent for messenger RNP assembly in vitro. Here we report the isolation of cDNAs for the most abundant messenger RNPs, the 54- and 56-kDa polypeptide (p54/p56) components of the approximately 6S mRNA-binding particle, from an ovarian expression library. The nucleotide sequence of p56 cDNA is almost identical to that recently reported for the putative Xenopus transcription factor FRG Y2. p54 and p56 are highly homologous and are smaller than expected by SDS/PAGE (36 kDa and 37 kDa) due to anomalous electrophoretic mobility. They lack the "RNP consensus motif" but contain four arginine-rich "basic/aromatic islands" that are similar to the RNA-binding domain of bacteriophage mRNA antiterminator proteins and of tat protein of human immunodeficiency virus. The basic/aromatic regions and a second conspicuous 100-amino acid "domain C" of p54 and p56 are conserved in the following DNA-binding proteins: human proteins dpbA, dpbB, and YB-1, rat protein EFIA, and Xenopus protein FRG Y1, all reported to bind to DNA; domain C is homologous to the major Escherichia coli cold-stress-response protein reportedly involved in translational control. Antibodies raised against a peptide of domain C have identified similar proteins in Xenopus somatic cells and in some mammalian cells and tissues. We conclude that p54 and p56 define a family of RNA-binding proteins, at least some of which may be involved in translational regulation.

  2. Chemical Screening Using Cell-FreeXenopusEgg Extract.

    Science.gov (United States)

    Broadus, Matthew R; Lee, Ethan

    2018-02-23

    Most drug screening methods use purified proteins, cultured cells, and/or small model organisms such as Xenopus , zebrafish, flies, or nematodes. These systems have proven successes in drug discovery, but they also have weaknesses. Although purified cellular components allow for identification of compounds with activity against specific targets, such systems lack the complex biological interactions present in cellular and organismal screens. In vivo systems overcome these weaknesses, but the lack of cellular permeability, efflux by cellular pumps, and/or toxicity can be major limitations. Xenopus laevis egg extract, a concentrated and biologically active cytosol, can potentially overcome these weaknesses. Drug interactions occur in a near-physiological milieu, thereby functioning in a "truer" endogenous manner than purified components. Also, Xenopus egg extract is a cell-free system that lacks intact plasma membranes that could restrict drug access to potential targets. Finally, Xenopus egg extract is readily manipulated at the protein level: Proteins are easily depleted or added to the system, an important feature for analyzing drug effects in disease states. Thus, Xenopus egg extract offers an attractive media for screening drugs that merges strengths of both in vitro and in vivo systems. © 2018 Cold Spring Harbor Laboratory Press.

  3. Early development of the thymus in Xenopus laevis

    Science.gov (United States)

    Lee, Young-Hoon; Williams, Allison; Hong, Chang-Soo; You, Youngjae; Senoo, Makoto; Saint-Jeannet, Jean-Pierre

    2012-01-01

    Background Although Xenopus laevis has been a model of choice for comparative and developmental studies of the immune system, little is known about organogenesis of the thymus, a primary lymphoid organ in vertebrates. Here we examined the expression of three transcription factors that have been functionally associated with pharyngeal gland development, gcm2, hoxa3 and foxn1, and evaluated the neural crest contribution to thymus development. Results In most species Hoxa3 is expressed in the third pharyngeal pouch endoderm where it directs thymus formation. In Xenopus, the thymus primordium is derived from the second pharyngeal pouch endoderm, which is hoxa3-negative, suggesting that a different mechanism regulates thymus formation in frogs. Unlike other species foxn1 is not detected in the epithelium of the pharyngeal pouch in Xenopus, rather, its expression is initiated as thymic epithelial cell starts to differentiate and express MHC class II molecules. Using transplantation experiments we show that while neural crest cells populate the thymus primordia, they are not required for the specification and initial development of this organ or for T cell differentiation in frogs. Conclusions These studies provide novel information on early thymus development in Xenopus, and highlight a number of features that distinguish Xenopus from other organisms. PMID:23172757

  4. Generation of Transgenic Xenopus laevis: II. Sperm Nuclei Preparation.

    Science.gov (United States)

    Ishibashi, Shoko; Kroll, Kristin L; Amaya, Enrique

    2007-09-01

    INTRODUCTIONManipulating genes specifically during later stages of amphibian embryonic development requires fine control over the time and place of expression. These protocols describe an efficient nuclear-transplantation-based method of transgenesis developed for Xenopus laevis. The approach enables stable expression of cloned gene products in Xenopus embryos. Because the transgene integrates into the genome prior to fertilization, the resulting embryos are not chimeric, eliminating the need to breed to the next generation to obtain nonmosaic transgenic animals. The procedure is based on restriction-enzyme-mediated integration (REMI) and can be divided into three parts: (I) high-speed preparation of egg extracts, (II) sperm nuclei preparation, and (III) nuclear transplantation. This protocol describes a method for the preparation of sperm nuclei from Xenopus laevis. Sperm suspensions are prepared by filtration and centrifugation, and then treated with lysolecithin to disrupt the plasma membrane of the cells. Sperm nuclei can be stored frozen in small aliquots at -80°C.

  5. 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

  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. Pathogenesis of a Chinese strain of bovine adenovirus type 3 infection in albino guinea pigs.

    Science.gov (United States)

    Shi, Hong-Fei; Zhu, Yuan-Mao; Yan, Hao; Ma, Lei; Wang, Xue-Zhi; Xue, Fei

    2014-12-01

    Bovine adenovirus type 3 (BAV-3) is considered one of the most important respiratory tract agents of cattle and is widespread among cattle around the world. A BAV-3 strain was isolated from a bovine nasal swab for the first time in China in 2009 and named HLJ0955. Subsequently, BAV-3 has frequently been isolated from calves with respiratory diseases in China. To date, only limited study on the pathogenesis of BAV-3 infection in cotton rats has been conducted, and the pathogenesis of BAV-3 infection in guinea pigs has not been reported. Therefore, sixteen albino guinea pigs were inoculated intranasally with HLJ0955. All of the infected guinea pigs had apparently elevated rectal temperatures (39.2 °C-39.9 °C) at 2-7 days post-inoculation (PI). Consolidation and petechial hemorrhage were also observed in guinea pigs experimentally infected with HLJ0955. Viral replication was detectable by virus isolation and titration and by immunohistochemistry in the lungs of guinea pigs as early as 24 h PI. Viral DNA was detectable in the lungs of infected guinea pigs during 11 days of observation by real-time PCR. Virus-neutralizing antibodies against BAV-3 were detectable from 11 days PI and reached a peak titer at 15 days PI. Histopathological changes mainly occurred in the lungs of infected guinea pigs and were characterized by thickening of alveolar septa, mononuclear cell infiltration, hemorrhage and alveolar epithelial necrosis. These results indicate that HLJ0955 can replicate in the lungs of guinea pigs and cause fever and gross and histological lesions. The guinea pig infection model of BAV-3 would serve as a useful system for monitoring the infection process and pathogenesis of the Chinese BAV-3 strain HLJ0955, as well as immune responses to BAV-3 vaccines.

  8. A Model of DENV-3 Infection That Recapitulates Severe Disease and Highlights the Importance of IFN-γ in Host Resistance to Infection

    Science.gov (United States)

    Valadão, Deborah F.; Cisalpino, Daniel; Dias, Ana Carolina F.; Silveira, Kátia D.; Kangussu, Lucas M.; Ávila, Thiago V.; Bonfim, Maria Rosa Q.; Bonaventura, Daniela; Silva, Tarcília A.; Sousa, Lirlândia P.; Rachid, Milene A.; Vieira, Leda Q.; Menezes, Gustavo B.; de Paula, Ana Maria; Atrasheuskaya, Alena; Ignatyev, George; Teixeira, Mauro M.; Souza, Danielle G.

    2012-01-01

    There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ−/− mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2−/− mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection. PMID:22666512

  9. Tebuconazole disrupts steroidogenesis in Xenopus laevis.

    Science.gov (United States)

    Poulsen, Rikke; Luong, Xuan; Hansen, Martin; Styrishave, Bjarne; Hayes, Tyrone

    2015-11-01

    A 27-day controlled exposure study of adult male African clawed frogs (Xenopus laevis) was conducted to examine the mechanism by which tebuconazole may disrupt steroidogenesis. The fungicide was measured by LC-MS/MS in tank water and in target tissues (adipose, kidney, liver, and brain), and we observed tissue-specific bioconcentration with BCF up to 238. Up to 10 different steroid hormones were quantified in gonads using LC-MS/MS and in plasma using GC-MS/MS and a radioimmunoassay was performed for further measurement of androgens. In order to assess whether effects increased with exposure 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 suppression dominates at higher exposure concentrations (increased androgens and decreased E2). Increased androgen levels in plasma half-way into the study and in gonads at termination may thus be explained by compensatory mechanisms, mediated through increased enzymatic expression, as prolonged exposure had no effect on circulating androgen levels. Copyright © 2015. Published by Elsevier B.V.

  10. Seeing the future: using Xenopus to understand eye regeneration.

    Science.gov (United States)

    Tseng, Ai-Sun

    2017-01-01

    Studies of Xenopus eye development have contributed considerably to the understanding of vertebrate neurogenesis, including eye field specification, cell fate determination and identification of genes critical for eye formation. This knowledge has served as a solid foundation for cellular and molecular examinations of the robust regenerative capacity of the Xenopus eye. The retina, lens, and the optic nerve are capable of regeneration after injury in both larval and adult stages. Here, we discuss the current models for studying eye regeneration in Xenopus and their potential applications for providing insights into human eye diseases. As Xenopus has many of the same tools that are available for other regeneration models, we thus highlight the distinct strengths and versatility of this organism that make it especially suited for extrapolating and testing strategies aimed at promoting regeneration and repair in eye tissues. Furthermore, we outline a promising future for the use of new techniques and approaches to address outstanding questions in understanding eye regeneration. © 2017 Wiley Periodicals, Inc.

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

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, J; Elepfandt, A

    1995-01-01

    Anesthetized clawed frogs (Xenopus laevis) were stimulated with underwater sound and the tympanic disk vibrations were studied using laser vibrometry. The tympanic disk velocities ranged from 0.01 to 0.5 mm/s (at a sound pressure of 2 Pa) in the frequency range of 0.4-4 kHz and were 20-40 dB high...

  12. 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

  13. Xenopus Dab2 is required for embryonic angiogenesis

    Directory of Open Access Journals (Sweden)

    Choi Sun-Cheol

    2006-12-01

    Full Text Available Abstract Background The molecular mechanisms governing the formation of the embryonic vascular system remain poorly understood. Here, we show that Disabled-2 (Dab2, a cytosolic adaptor protein, has a pivotal role in the blood vessel formation in Xenopus early embryogenesis. Results Xenopus Disabled-2 (XDab2 is spatially localized to the blood vessels including the intersomitic veins (ISV in early embryos. Both antisense morpholino oligonucleotide (MO-mediated knockdown and overexpression of XDab2 inhibit the formation of ISV, which arise from angiogenesis. In addition, we found that activin-like signaling is essential for this angiogenic event. Functional assays in Xenopus animal caps reveal that activin-like signals induce VEGF expression and this induction can be inhibited by XDab2 depletion. However, XDab2 MO has no effects on the induction of other target genes by activin-like signals. Furthermore, we show that the disruption of the sprouting ISV in XDab2-depleted embryos can be rescued by coexpression of VEGF. Conclusion Taking together, we suggest that XDab2 regulates the embryonic angiogenesis by mediating the VEGF induction by activin-like signaling in Xenopus early development.

  14. Xenopus laevis embryos and tadpoles as models for testing for ...

    African Journals Online (AJOL)

    Xenopus laevis embryos and tadpoles as models for testing for pollution by zinc, copper, lead and cadmium. ... Metals affected the growth of the tadpoles by reducing body length with increasing concentrations. An increase in the concentration of each metal resulted in an increase in the frequency and severity of ...

  15. Sofosbuvir based treatment of chronic hepatitis C genotype 3 infections-A Scandinavian real-life study.

    Directory of Open Access Journals (Sweden)

    Olav Dalgard

    Full Text Available Chronic hepatitis C virus (HCV genotype 3 infection with advanced liver disease has emerged as the most challenging to treat. We retrospectively assessed the treatment outcome of sofosbuvir (SOF based regimes for treatment of HCV genotype 3 infections in a real life setting in Scandinavia.Consecutive patients with chronic HCV genotype 3 infection were enrolled at 16 treatment centers in Denmark, Sweden, Norway and Finland. Patients who had received a SOF containing regimen were included. The fibrosis stage was evaluated by liver biopsy or transient liver elastography. The following treatments were given according availability and local guidelines: 1 SOF + ribavirin (RBV for 24 weeks, 2 SOF + daclatasvir (DCV +/-RBV for 12-24 weeks, 3 SOF + pegylated interferon alpha (peg-IFN-α + RBV for 12 weeks or 4 SOF/ledipasvir (LDV + RBV for 12-16 weeks. The primary endpoint was sustained virological response (SVR assessed at week 12 (SVR12 after end of treatment.We included 316 patients with a mean age of 55 years (range 24-79, 70% men, 49% treatment experienced, 58% with compensated cirrhosis and 12% with decompensated cirrhosis.In the modified intention to treat (mITT population SVR12 was achieved in 284/311 (91% patients. Among 26 treatment failures, five had non-response, 3 breakthrough and 18 relapse. Five patients were not included in the mITT population. Three patients died from reasons unrelated to treatment and two were lost to follow-up. The SVR12 rate was similar for all treatment regimens, but lower in men (p = 0.042, and in patients with decompensated liver disease (p = 0.004.We found that sofosbuvir based treatment in a real-life setting could offer SVR rates exceeding 90% in patients with HCV genotype 3 infection and advanced liver disease.

  16. 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.

  17. 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

  18. Dispatches from the DMZ: Bottle Cell Formation During Xenopus Gastrulation

    OpenAIRE

    Lee, Jen-Yi

    2011-01-01

    Bottle cell-driven blastopore lip formation externally marks the initiation of gastrulation in amphibian embryos. The blastopore groove is formed when bottle cells undergo apical constriction and transform from cuboidal to flask-shaped. Apical constriction is sufficient to cause invagination and is a highly conserved mechanism for sheet bending and folding during morphogenesis; therefore, studying apical constriction in Xenopus bottle cells could provide valuable insight into this fundamental...

  19. HDAC Activity Is Required during Xenopus Tail Regeneration

    OpenAIRE

    Ai-Sun Tseng; Kátia Carneiro; Lemire, Joan M.; Michael Levin

    2011-01-01

    The ability to fully restore damaged or lost organs is present in only a subset of animals. The Xenopus tadpole tail is a complex appendage, containing epidermis, muscle, nerves, spinal cord, and vasculature, which regenerates after amputation. Understanding the mechanisms of tail regeneration may lead to new insights to promote biomedical regeneration in non-regenerative tissues. Although chromatin remodeling is known to be critical for stem cell pluripotency, its role in complex organ regen...

  20. Induced breeding of Clarias anguillaris with Xenopus laevies ...

    African Journals Online (AJOL)

    Riped Clarias anguillaris (total body weight (TBW), 350 – 600 g; mean weight, 434.44 + 79.39 g) broodstock were treated with frog, Xenopus laevis (mean weight was 68.55±20.34 g) crude pituitary glands at three replicates of three treatment levels of 1 pituitary (T1), 2 pituitaries (T2) and 3 pituitaries (T3) per brood stock and ...

  1. Developmental role of plk4 in Xenopus laevis and Danio rerio: implications for Seckel Syndrome

    National Research Council Canada - National Science Library

    Hudson, John; Patel, Neeraj; Rapchak, Candace Elaine; Crawford, Michael

    2015-01-01

    .... Recently characterized human mutations lead to Seckel Syndrome. Riboprobe in situ hybridization revealed that plk4 is ubiquitously expressed during early stages of development of Xenopus and Danio...

  2. BMP regulates vegetal pole induction centres in early xenopus development.

    Science.gov (United States)

    Nachaliel, N; Re'Em-Kalma, Y; Eshed, O; Elias, S; Frank, D

    1998-10-01

    Bone morphogenetic protein (BMP) plays an important role in mesoderm patterning in Xenopus. The ectopic expression of BMP-4 protein hyperventralizes embryos, whereas embryos expressing a BMP-2/4 dominant-negative receptor (DNR) are hyperdorsalized. Mesoderm is initially induced in the marginal zone by cells in the underlying vegetal pole. While much is known about BMP's expression and role in patterning the marginal zone, little is known about its early role in regulating vegetal mesoderm induction centre formation. The role of BMP in regulating formation of vegetal mesoderm inducing centres during early Xenopus development was examined. Ectopic BMP-4 expression in vegetal pole cells inhibited dorsal mesoderm induction but increased ventral mesoderm induction when recombined with animal cap ectoderm in Nieuwkoop explants. 32-cell embryos injected with BMP-4 RNA in the most vegetal blastomere tier were not hyperdorsalized by LiCl treatment. The ectopic expression of Smad or Mix.1 proteins in the vegetal pole also inhibited dorsal mesoderm induction in explants and embryos. Expression of the BMP 2/4 DNR in the vegetal pole increased dorsal mesoderm induction and inhibited ventral mesoderm induction in explants and embryos. These results support a role for BMP signalling in regulating ventral vegetal and dorsal vegetal mesoderm induction centre formation during early Xenopus development.

  3. Generation of Transgenic Xenopus laevis: III. Sperm Nuclear Transplantation.

    Science.gov (United States)

    Ishibashi, Shoko; Kroll, Kristin L; Amaya, Enrique

    2007-09-01

    INTRODUCTIONManipulating genes specifically during later stages of amphibian embryonic development requires fine control over the time and place of expression. These protocols describe an efficient nuclear-transplantation-based method of transgenesis developed for Xenopus laevis. The approach enables stable expression of cloned gene products in Xenopus embryos. The procedure is based on restriction-enzyme-mediated integration (REMI) and can be divided into three parts: (I) high-speed preparation of egg extracts, (II) sperm nuclei preparation, and (III) nuclear transplantation. This protocol describes a method for the nuclear transplantation in Xenopus laevis. Permeabilized sperm nuclei are incubated briefly with linearized plasmid DNA, after which egg extract and a small amount of restriction enzyme are added. The egg extract partially decondenses the chromosomes, and the restriction enzyme stimulates recombination by creating double-strand breaks, facilitating integration of DNA into the genome. Diluted nuclei are transplanted into unfertilized eggs. Because the transgene integrates into the genome prior to fertilization, the resulting transgenic embryos are not chimeric and there is no need to breed to the next generation in order to obtain nonmosaic transgenic animals.

  4. Next generation sequencing and comparative analyses of Xenopus mitogenomes

    Directory of Open Access Journals (Sweden)

    Lloyd Rhiannon E

    2012-09-01

    Full Text Available Abstract Background Mitochondrial genomes comprise a small but critical component of the total DNA in eukaryotic organisms. They encode several key proteins for the cell’s major energy producing apparatus, the mitochondrial respiratory chain. Additonally, their nucleotide and amino acid sequences are of great utility as markers for systematics, molecular ecology and forensics. Their characterization through nucleotide sequencing is a fundamental starting point in mitogenomics. Methods to amplify complete mitochondrial genomes rapidly and efficiently from microgram quantities of tissue of single individuals are, however, not always available. Here we validate two approaches, which combine long-PCR with Roche 454 pyrosequencing technology, to obtain two complete mitochondrial genomes from individual amphibian species. Results We obtained two new xenopus frogs (Xenopus borealis and X. victorianus complete mitochondrial genome sequences by means of long-PCR followed by 454 of individual genomes (approach 1 or of multiple pooled genomes (approach 2, the mean depth of coverage per nucleotide was 9823 and 186, respectively. We also characterised and compared the new mitogenomes against their sister taxa; X. laevis and Silurana tropicalis, two of the most intensely studied amphibians. Our results demonstrate how our approaches can be used to obtain complete amphibian mitogenomes with depths of coverage that far surpass traditional primer-walking strategies, at either the same cost or less. Our results also demonstrate: that the size, gene content and order are the same among xenopus mitogenomes and that S. tropicalis form a separate clade to the other xenopus, among which X. laevis and X. victorianus were most closely related. Nucleotide and amino acid diversity was found to vary across the xenopus mitogenomes, with the greatest diversity observed in the Complex 1 gene nad4l and the least diversity observed in Complex 4 genes (cox1-3. All protein

  5. The use of Xenopus oocytes and embryos as a route towards cell ...

    Indian Academy of Sciences (India)

    2005-01-07

    Jan 7, 2005 ... When nuclei of somatic cells are transplanted to enucleated eggs of Xenopus, a complete reprogramming of nuclear function can take place. To identify mechanisms of nuclear reprogramming, somatic nuclei can be transplanted to growing meiotic oocytes of Xenopus, and stem cell genes activated without ...

  6. 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.

  7. Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes

    OpenAIRE

    Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V. Bleu; Serrano, Elba E.

    2011-01-01

    The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. ...

  8. Remobilization of Tol2 transposons in Xenopus tropicalis

    Directory of Open Access Journals (Sweden)

    Wells Dan E

    2010-01-01

    Full Text Available Abstract Background The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency. Results To test whether Tol2 transposons integrated in the Xenopus tropicalis genome are substrates for remobilization, we injected in vitro transcribed Tol2 mRNA into one-cell embryos harbouring a single copy of a Tol2 transposon. Integration site analysis of injected embryos from two founder lines showed at least one somatic remobilization event per embryo. We also demonstrate that the remobilized transposons are transmitted through the germline and re-integration can result in the generation of novel GFP expression patterns in the developing tadpole. Although the parental line contained a single Tol2 transposon, the resulting remobilized tadpoles frequently inherit multiple copies of the transposon. This is likely to be due to the Tol2 transposase acting in discrete blastomeres of the developing injected embryo during the cell cycle after DNA synthesis but prior to mitosis. Conclusions In this study, we demonstrate that single copy Tol2 transposons integrated into the Xenopus tropicalis genome are effective substrates for excision and random re-integration and that the remobilized transposons are transmitted through the germline. This is an important step in the development of 'transposon hopping' strategies for insertional mutagenesis, gene trap and enhancer trap screens in this highly tractable developmental model organism.

  9. A transgenic Xenopus laevis reporter model to study lymphangiogenesis

    Directory of Open Access Journals (Sweden)

    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.

  10. Generation of endo-siRNAs in Xenopus laevis oocytes.

    Science.gov (United States)

    Alnumeir, Sammer; Werner, Andreas

    2014-01-01

    Endogenous siRNAs (endo-siRNAs) are well documented and characterized in C. elegans and Drosophila. Endo-siRNAs can also be found in vertebrates; however, their biology is much less clear. They are thought to be produced by Dicer and to contribute to transposon silencing. Because of their generally low abundance and their similarity with miRNAs and products of physiological RNA turn-over, endo-siRNAs are difficult to investigate. Here, we report a system, oocytes from Xenopus laevis, that allows for the generation and analysis of endo-siRNAs from double-stranded RNA precursors.

  11. A dissociation factor from embryos of Xenopus laevis.

    Science.gov (United States)

    Decroly, M; Goldfinger, M

    1975-04-16

    A dissociating factor has been extracted from the ribosomal KCl wash and from the cytosol of developing embryos of Xenopus laevis. No dissociating activity could be detected in the KCl wash of ribosomes from full grown oocytes and unfertilized eggs. As in bacteria, the acitivity of the dissociation factor seems to be correlated with the rate of protein synthesis suggesting a physiological role of the dissociation factor. The possibility that the dissociation factor might be one of the components which limits the rate of protein synthesis in the oocytes is discussed.

  12. Xenopus p63 expression in early ectoderm and neurectoderm.

    Science.gov (United States)

    Lu, P; Barad, M; Vize, P D

    2001-04-01

    The tumor-suppressor protein p53 belongs to a small gene family that includes p63 and p73. While p53 and p73 regulate cell cycle progression and apoptosis, the major role of p63 appears to be in promoting ectodermal proliferation and differentiation. In this report we describe the cloning of a Xenopus orthologue of mammalian p63 that is extraordinarily conserved in sequence. The major sites of expression of Xenopus p63 mRNA are the epidermis and some neural crest and crest derivatives such as the branchial arches and tail fin. Expression is also observed in the neural plate and in the stomodeal-hypophyseal anlage. Antibodies against p63 detect a nuclear protein that is distributed in a manner similar to that of Xp63 mRNA. Both mRNA and protein are conspicuously absent from regions of the epidermal sensorial layer that are induced to form a number of (but not all) ectodermal placodes and Xp63 protein levels are particularly dynamic in the epidermis of the eye as the lens forms.

  13. PACSIN2 regulates cell adhesion during gastrulation in Xenopus laevis.

    Science.gov (United States)

    Cousin, Hélène; Desimone, Douglas W; Alfandari, Dominique

    2008-07-01

    We previously identified the adaptor protein PACSIN2 as a negative regulator of ADAM13 proteolytic function. In Xenopus embryos, PACSIN2 is ubiquitously expressed, suggesting that PACSIN2 may control other proteins during development. To investigate this possibility, we studied PACSIN2 function during Xenopus gastrulation and in XTC cells. Our results show that PACSIN2 is localized to the plasma membrane via its coiled-coil domain. We also show that increased levels of PACSIN2 in embryos inhibit gastrulation, fibronectin (FN) fibrillogenesis and the ability of ectodermal cells to spread on a FN substrate. These effects require PACSIN2 coiled-coil domain and are not due to a reduction of FN or integrin expression and/or trafficking. The expression of a Mitochondria Anchored PACSIN2 (PACSIN2-MA) sequesters wild type PACSIN2 to mitochondria, and blocks gastrulation without interfering with cell spreading or FN fibrillogenesis but perturbs both epiboly and convergence/extension. In XTC cells, the over-expression of PACSIN2 but not PACSIN2-MA prevents the localization of integrin beta1 to focal adhesions (FA) and filamin to stress fiber. PACSIN2-MA prevents filamin localization to membrane ruffles but not to stress fiber. We propose that PACSIN2 may regulate gastrulation by controlling the population of activated alpha5beta1 integrin and cytoskeleton strength during cell movement.

  14. Teratogenic effects of five anticancer drugs on Xenopus laevis embryos.

    Science.gov (United States)

    Isidori, Marina; Piscitelli, Concetta; Russo, Chiara; Smutná, Marie; Bláha, Luděk

    2016-11-01

    In recent years, the environmental presence of pharmaceuticals - including anticancer drugs - is an emerging issue. Because of the lack of appropriate critical studies about anticancer drug effects in frogs, the aim of the present study was to investigate lethal and teratogenic effects of five anticancer drugs widely used in large quantities, i.e. 5-flourouracil, capecitabine, cisplatin, etoposide, and imatinib, in the embryos of the South African clawed frog, Xenopus laevis, using FETAX - Frog Embryo Teratogenesis Assay in Xenopus. None of the studied anticancer drugs induced statistically significant mortality within the concentrations tested (0.01-50mg/L, depending on the studied compound), and no growth inhibition of embryos after a 96-h exposure was observed. Except for cisplatin, the other pharmaceuticals induced an increase of developmental malformations such as abdominal edema, axial flexure, head, eyes, gut and heart malformations with statistically significant effects observed at the highest concentrations tested (50mg/L for 5-flourouracil; 30mg/L for etoposide and 20mg/L for capecitabine and imatinib). The results indicate that anticancer drugs can affect embryogenesis mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  16. Left-Right Asymmetric Morphogenesis in the Xenopus Digestive System

    Science.gov (United States)

    Muller, Jennifer K.; Prather, D.R.; Nascone-Yoder, N. M.

    2003-01-01

    The morphogenetic mechanisms by which developing organs become left-right asymmetric entities are unknown. To investigate this issue, we compared the roles of the left and right sides of the Xenopus embryo during the development of anatomic asymmetries in the digestive system. Although both sides contribute equivalently to each of the individual digestive organs, during the initial looping of the primitive gut tube, the left side assumes concave topologies where the right side becomes convex. Of interest, the concave surfaces of the gut tube correlate with expression of the LR gene, Pitx2, and ectopic Pitx2 mRNA induces ectopic concavities in a localized manner. A morphometric comparison of the prospective concave and convex surfaces of the gut tube reveals striking disparities in their rate of elongation but no significant differences in cell proliferation. These results provide insight into the nature of symmetry-breaking morphogenetic events during left-right asymmetric organ development. ?? 2003 Wiley-Liss, Inc.

  17. Endocytosis is required for efficient apical constriction during Xenopus gastrulation.

    Science.gov (United States)

    Lee, Jen-Yi; Harland, Richard M

    2010-02-09

    Coordinated apical constriction (AC) in epithelial sheets drives tissue invagination [1, 2] and is required for diverse morphogenetic movements such as gastrulation [3], neurulation [4, 5], and organogenesis [6]. We showed previously that actomyosin contractility drives AC in Xenopus laevis bottle cells [7]; however, it remained unclear whether it does so in concert with other processes. Here we report that endocytosis-driven membrane remodeling is required for efficient AC. We found endosomes exclusively in bottle cells in the early gastrula. Disrupting endocytosis with dominant-negative dynamin or rab5 perturbed AC, with a significant decrease in constriction rate late in the process, suggesting that endocytosis operates downstream of actomyosin contractility to remove excess membrane. Additionally, disrupting endocytosis during neurulation inhibits AC in hingepoint cells, resulting in neural tube closure defects. Thus, membrane remodeling during AC could be a general mechanism to achieve efficient invagination in embryos.

  18. Deficient induction response in a Xenopus nucleocytoplasmic hybrid.

    Directory of Open Access Journals (Sweden)

    Patrick Narbonne

    2011-11-01

    Full Text Available Incompatibilities between the nucleus and the cytoplasm of sufficiently distant species result in developmental arrest of hybrid and nucleocytoplasmic hybrid (cybrid embryos. Several hypotheses have been proposed to explain their lethality, including problems in embryonic genome activation (EGA and/or nucleo-mitochondrial interactions. However, conclusive identification of the causes underlying developmental defects of cybrid embryos is still lacking. We show here that while over 80% of both Xenopus laevis and Xenopus (Silurana tropicalis same-species androgenetic haploids develop to the swimming tadpole stage, the androgenetic cybrids formed by the combination of X. laevis egg cytoplasm and X. tropicalis sperm nucleus invariably fail to gastrulate properly and never reach the swimming tadpole stage. In spite of this arrest, these cybrids show quantitatively normal EGA and energy levels at the stage where their initial gastrulation defects are manifested. The nucleocytoplasmic incompatibility between these two species instead results from a combination of factors, including a reduced emission of induction signal from the vegetal half, a decreased sensitivity of animal cells to induction signals, and differences in a key embryonic protein (Xbra concentration between the two species, together leading to inefficient induction and defective convergence-extension during gastrulation. Indeed, increased exposure to induction signals and/or Xbra signalling partially rescues the induction response in animal explants and whole cybrid embryos. Altogether, our study demonstrates that the egg cytoplasm of one species may not support the development promoted by the nucleus of another species, even if this nucleus does not interfere with the cytoplasmic/maternal functions of the egg, while the egg cytoplasm is also capable of activating the genome of that nucleus. Instead, our results provide evidence that inefficient signalling and differences in the

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

    Science.gov (United States)

    Herrgen, Leah; Akerman, Colin J

    2016-12-01

    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 increasing 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. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1328-1341, 2016. © 2016 Wiley Periodicals, Inc.

  20. FMRP regulates neurogenesis in vivo in Xenopus laevis tadpoles.

    Science.gov (United States)

    Faulkner, Regina L; Wishard, Tyler J; Thompson, Christopher K; Liu, Han-Hsuan; Cline, Hollis T

    2015-01-01

    Fragile X Syndrome (FXS) is the leading known monogenic form of autism and the most common form of inherited intellectual disability. FXS results from silencing the FMR1 gene during embryonic development, leading to loss of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein that regulates mRNA transport, stability, and translation. FXS is commonly thought of as a disease of synaptic dysfunction, however, FMRP expression is lost early in embryonic development, well before most synaptogenesis occurs. Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable. We investigated whether FMRP affects neurogenesis in Xenopus laevis tadpoles which express a homolog of FMR1. We used in vivo time-lapse imaging of neural progenitor cells and their neuronal progeny to evaluate the effect of acute loss or over-expression of FMRP on neurogenesis in the developing optic tectum. We complimented the time-lapse studies with SYTOX labeling to quantify apoptosis and CldU labeling to measure cell proliferation. Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival. They also have increased neuronal differentiation, but deficient dendritic arbor elaboration. The presence and severity of these defects was highly sensitive to FMRP levels. These data demonstrate that FMRP plays an important role in neurogenesis and suggest that endogenous FMRP levels are carefully regulated. These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS.

  1. Electroporation in the Regenerating Tail of the Xenopus Tadpole

    Science.gov (United States)

    Mochii, Makoto; Taniguchi, Yuka

    Xenopus laevis is a model system widely used to investigate embryogenesis, metamorphosis, and regeneration. The tail of the Xenopus tadpole is very useful in analyzing the molecular mechanisms underlying appendage regeneration (Slack et al., 2004; Mochii et al., 2007; Slack et al., 2008). It is transparent and suitable for whole-mount observation at the cellular level. The tail regenerates within 2 weeks of amputation. The conventional injection of blastomeres with mRNA, DNA, or antisense oligonucleotides is a powerful tool with which to study genetic mechanisms in early embryos, but it is not effective in late embryos or larvae. A transgenic approach has been used to analyze tail regeneration (Beck et al., 2003, 2006), but its success is largely dependent on the activity of the promoter used. There are limited numbers of promoters available that precisely regulate gene expression spatially and/or temporally. In vivo electroporation is an alternative method that can be used to manipulate gene expression in late embryos and larvae. The introduction of DNA or RNA into the cells of neurula and tailbud embryos has been reported (Eide et al., 2000; Sasagawa et al., 2002; Falk et al., 2007). Targeting larval tissues with in vivo electroporation also has been used to investigate neural networks, metamorphosis, and regeneration (Haas et al., 2001, 2002; Nakajima and Yaoita, 2003; Javaherian and Cline, 2005; Bestman et al., 2006; Boorse et al., 2006; Lin et al., 2007; Mochii et al., 2007). In this chapter, we report a procedure to introduce DNA into the tissues of the tadpole tail.

  2. Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

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    Desrosiers, R.R.; Romanik, E.A.; O' Connor, C.M. (Worcester Foundation for Experimental Biology, Shrewsbury, MA (USA))

    1990-12-05

    The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-(methyl-3H)methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-(methyl-3H)methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly.

  3. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below

  4. Identification of CUG-BP1/EDEN-BP target mRNAs in Xenopus tropicalis.

    OpenAIRE

    Graindorge, Antoine; Le Tonquèze, Olivier; Thuret, Raphaël; Pollet, Nicolas; Osborne, Howard Beverley; Audic, Yann

    2008-01-01

    International audience; The early development of many animals relies on the posttranscriptional regulations of maternally stored mRNAs. In particular, the translation of maternal mRNAs is tightly controlled during oocyte maturation and early mitotic cycles in Xenopus. The Embryonic Deadenylation ElemeNt (EDEN) and its associated protein EDEN-BP are known to trigger deadenylation and translational silencing to several mRNAs bearing an EDEN. This Xenopus RNA-binding protein is an ortholog of th...

  5. Identification of Xenopus Cortactin : Two Isoforms of the Transcript and Multiple Forms of the Protein(Cell Biology)

    OpenAIRE

    Atsushi, Yamashita; Takanori, KATSUBE; Naoko, Hashimoto; Kimiko, Tomita; Manabu, Takahisa; Ryu, Ueda; Shin, TOGASHI; Neurogenetics Research Project, Mitsubishi Kasei Institute of Life Sciences:(Present addresses)School of Science, Kitasato University; Neurogenetics Research Project, Mitsubishi Kasei Institute of Life Sciences(Present addresses)National Institute of Radiological Sciences; Neurogenetics Research Project, Mitsubishi Kasei Institute of Life Sciences; Neurogenetics Research Project, Mitsubishi Kasei Institute of Life Sciences(Present addresses)Laboratory of Molecular Embryology, Department of Bioscience, Kitasato University

    2001-01-01

    Cortactin was initially identified as a substrate for Src tyrosine kinase. It interacts with the filamentous actin in the cell cortex through the tandem repeats of 37-amino acid. In this report, we describe the identification of a Xenopus homolog of cortactin. The deduced amino acid sequence shares over 70% identity with human, mouse, and chicken cortactin. Northern and Western blot analyses revealed that Xenopus cortactin is widely expressed in Xenopus tissues. Analysis of the transcripts us...

  6. Prevalence of Batrachochytrium dendrobatidis in Xenopus collected in Africa (1871-2000 and in California (2001-2010.

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    Vance T Vredenburg

    Full Text Available International trade of the invasive South African clawed frog (Xenopus laevis, a subclinical carrier of the fungal pathogen Batrachochytrium dendrobatis (Bd has been proposed as a major means of introduction of Bd into naïve, susceptible amphibian populations. The historical presence of Bd in the indigenous African population of Xenopus is well documented. However, there are no reports documenting the presence of Bd in wild Xenopus populations in the US, particularly in California where introduced populations are well-established after intentional or accidental release. In this report, a survey was conducted on 178 archived specimens of 6 species of Xenopus collected in Africa from 1871-2000 and on 23 archived specimens (all wild-caught Xenopus laevis collected in California, USA between 2001 and 2010. The overall prevalence rate of Bd in the tested Xenopus was 2.8%. The earliest positive specimen was X. borealis collected in Kenya in 1934. The overall prevalence of Bd in the X. laevis collected in California was 13% with 2 positive specimens from 2001 and one positive specimen from 2003. The positive Xenopus (3/23 collected in California were collected in 2001 (2/3 and 2003 (1/3. These data document the presence of Bd-infected wild Xenopus laevis in California. The findings reported here support the prevailing hypothesis that Bd was present as a stable, endemic infection in Xenopus populations in Africa prior to their worldwide distribution likely via international live-amphibian trade.

  7. The Central Role of the Matrix Protein in Nipah Virus Assembly and Morphogenesis

    Science.gov (United States)

    2007-03-23

    virus (MeV), Sendai virus (SeV), human parainfluenza viruses (hPIV) types 1-4, Simian virus 5 (SV5), Newcastle disease virus (NDV), Mumps virus, and...Malur, and A. K. Banerjee. 2001. Polarity of human parainfluenza virus type 3 infection in polarized human lung epithelial A549 cells: role of...Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV

  8. 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.

  9. 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.

  10. Ca2+ Homeostasis Regulates Xenopus Oocyte Maturation1

    Science.gov (United States)

    Sun, Lu; Hodeify, Rawad; Haun, Shirley; Charlesworth, Amanda; MacNicol, Angus M.; Ponnappan, Subramaniam; Ponnappan, Usha; Prigent, Claude; Machaca, Khaled

    2008-01-01

    In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte’s competence to undergo maturation in preparation for fertilization and embryonic development. PMID:18094360

  11. Primordial Germ Cell Isolation from Xenopus laevis Embryos.

    Science.gov (United States)

    Butler, Amanda M; Aguero, Tristan; Newman, Karen M; King, Mary Lou

    2017-01-01

    Primordial germ cells (PGCs) are the precursors to the gametes and have the unique ability to retain full developmental potential. However, the mechanism(s) and gene-network(s) necessary for their proper specification and development are poorly understood. This is due, in part, to the challenges that must be overcome in order to identify and isolate PGCs during critical stages of development. Two distinct mechanisms have been characterized to specify the germ cell lineage in vertebrates: induction and inheritance. Regardless of mechanism, there are common developmental features shared among all vertebrates in forming the germ cell lineage. Xenopus offers several advantages for understanding the molecular mechanisms necessary to establish the germ line. Here, we provide detailed methods for isolating live PGCs at different time points: 1) just after they have segregated from the endodermal lineage, and 2) while they are migrating towards the presumptive gonad. Isolation of PGCs at these critical developmental stages will allow for the investigation of the mechanism(s) and gene-network(s) necessary for their proper specification and development.

  12. Regeneration of neural crest derivatives in the Xenopus tadpole tail

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    Slack Jonathan MW

    2007-05-01

    Full Text Available Abstract Background After amputation of the Xenopus tadpole tail, a functionally competent new tail is regenerated. It contains spinal cord, notochord and muscle, each of which has previously been shown to derive from the corresponding tissue in the stump. The regeneration of the neural crest derivatives has not previously been examined and is described in this paper. Results Labelling of the spinal cord by electroporation, or by orthotopic grafting of transgenic tissue expressing GFP, shows that no cells emigrate from the spinal cord in the course of regeneration. There is very limited regeneration of the spinal ganglia, but new neurons as well as fibre tracts do appear in the regenerated spinal cord and the regenerated tail also contains abundant peripheral innervation. The regenerated tail contains a normal density of melanophores. Cell labelling experiments show that melanophores do not arise from the spinal cord during regeneration, nor from the mesenchymal tissues of the skin, but they do arise by activation and proliferation of pre-existing melanophore precursors. If tails are prepared lacking melanophores, then the regenerates also lack them. Conclusion On regeneration there is no induction of a new neural crest similar to that seen in embryonic development. However there is some regeneration of neural crest derivatives. Abundant melanophores are regenerated from unpigmented precursors, and, although spinal ganglia are not regenerated, sufficient sensory systems are produced to enable essential functions to continue.

  13. Statistics of active transport in Xenopus melanophores cells.

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    Snezhko, A.; Barlan, K.; Aranson, I. S.; Gelfand, V. I.; Materials Science Division; Northwestern Univ.

    2010-11-01

    The transport of cell cargo, such as organelles and protein complexes in the cytoplasm, is determined by cooperative action of molecular motors stepping along polar cytoskeletal elements. Analysis of transport of individual organelles generated useful information about the properties of the motor proteins and underlying cytoskeletal elements. In this work, for the first time (to our knowledge), we study collective movement of multiple organelles using Xenopus melanophores, pigment cells that translocate several thousand of pigment granules (melanosomes), spherical organelles of a diameter of {approx} 1 {micro}m. These cells disperse melanosomes in the cytoplasm in response to high cytoplasmic cAMP, while at low cAMP melanosomes cluster at the cell center. Obtained results suggest spatial and temporal organization, characterized by strong correlations between movement of neighboring organelles, with correlation length of {approx} 4 {micro}m and pair lifetime {approx} 5 s. Furthermore, velocity statistics revealed strongly non-Gaussian velocity distribution with high velocity tails demonstrating exponential behavior suggestive of strong velocity correlations. Depolymerization of vimentin intermediate filaments using a dominant-negative vimentin mutant or actin with cytochalasin B reduced correlation of behavior of individual particles. Based on our analysis, we concluded that steric repulsion is dominant, but both intermediate filaments and actin microfilaments are involved in dynamic cross-linking organelles in the cytoplasm.

  14. 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.

  15. Calcium dependent current recordings in Xenopus laevis oocytes in microgravity

    Science.gov (United States)

    Wuest, Simon L.; Roesch, Christian; Ille, Fabian; Egli, Marcel

    2017-12-01

    Mechanical unloading by microgravity (or weightlessness) conditions triggers profound adaptation processes at the cellular and organ levels. Among other mechanisms, mechanosensitive ion channels are thought to play a key role in allowing cells to transduce mechanical forces. Previous experiments performed under microgravity have shown that gravity affects the gating properties of ion channels. Here, a method is described to record a calcium-dependent current in native Xenopus laevis oocytes under microgravity conditions during a parabolic flight. A 3-voltage-step protocol was applied to provoke a calcium-dependent current. This current increased with extracellular calcium concentration and could be reduced by applying extracellular gadolinium. The custom-made ;OoClamp; hardware was validated by comparing the results of the 3-voltage-step protocol to results obtained with a well-established two-electrode voltage clamp (TEVC). In the context of the 2nd Swiss Parabolic Flight Campaign, we tested the OoClamp and the method. The setup and experiment protocol worked well in parabolic flight. A tendency that the calcium-dependent current was smaller under microgravity than under 1 g condition could be observed. However, a conclusive statement was not possible due to the small size of the data base that could be gathered.

  16. Acoustic detection of melanosome transport in Xenopus laevis melanophores.

    Science.gov (United States)

    Frost, Rickard; Norström, Elisabeth; Bodin, Lovisa; Langhammer, Christoph; Sturve, Joachim; Wallin, Margareta; Svedhem, Sofia

    2013-04-01

    Organelle transport studies are often performed using melanophores from lower vertebrates due to the ease of inducing movements of pigment granules (melanosomes) and visualizing them by optical microscopy. Here, we present a novel methodology to monitor melanosome translocation (which is a light-sensitive process) in the dark using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. This acoustic sensing method was used to study dispersion and aggregation of melanosomes in Xenopus laevis melanophores. Reversible sensor responses, correlated to optical reflectance measurements, were obtained by alternating addition and removal of melatonin (leading to melanosome aggregation) and melanocyte-stimulating hormone (MSH) (leading to melanosome dispersion). By confocal microscopy, it was shown that a vertical redistribution of melanosomes occurred during the dispersion/aggregation processes. Furthermore, the transport process was studied in the presence of cytoskeleton-perturbing agents disrupting either actin filaments (latrunculin) or microtubules (nocodazole). Taken together, these experiments suggest that the acoustic responses mainly originate from melanosome transport along actin filaments (located close to the cell membrane), as expected based on the penetration depth of the QCM-D technique. The results clearly indicate the potential of QCM-D for studies of intracellular transport processes in melanophores. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  18. Apoptosis-Specific Protein (ASP) Identified in Apoptotic Xenopus Thymus Tumor Cells

    Science.gov (United States)

    Milner, Anne; Horton, Trudy; Ritchie, Pamela; Gascoyne, Duncan; Hewson, Tim; Hammond, Ester; Gregory, Christopher; Grand, Roger

    1998-01-01

    A novel apoptosis-specific protein (ASP) has recently been identified in the cytoplasm of apoptotic mammalian cells. This paper investigates whether ASP is found in Xenopus thymus tumor-derived lymphoid cell lines undergoing apoptosis and also in apoptotic, nontransformed splenocytes. Cultured Xenopus tumor lymphoid cells induced to undergo, apoptosis by serum deprivation or treatment with the calcium ionophore, ionomycin, displayed altered morphology typical of apoptotic cells, as judged by flow cytometric light-scatter characteristics and by fluorescence microscopy of acridine-orange-stained cells. Flow cytometry of permeabilized cells and fluorescence microscopy of acetone-fixed cytospins revealed that apoptotic Xenopus tumor cells, especially those displaying loss or condensation of DNA, displayed increased expression of epitopes recognized by a rabbit polyclonal antibody against ASP. Flow cytometry confirmed that ASP is also expressed in splenocytes induced to apoptose by culture in ionomycin or following concanavalin A stimulation. No increased expression of ASP was seen when lymphoid tumor cells or splenocytes were induced into necrosis by overdose with the antifungal agent amphotericin B. Western blotting with antibody against ASP identified the emergence of several protein bands in cell lysates from apoptotic, but not necrotic, Xenopus tumor cells. The new and simple methodology for identifying apoptotic cells described here is likely to be of value to those studying immune system development and associated programmed cell death in Xenopus. PMID:9814588

  19. interleukin-11 induces and maintains progenitors of different cell lineages during Xenopus tadpole tail regeneration.

    Science.gov (United States)

    Tsujioka, Hiroshi; Kunieda, Takekazu; Katou, Yuki; Shirahige, Katsuhiko; Fukazawa, Taro; Kubo, Takeo

    2017-09-08

    Unlike mammals, Xenopus laevis tadpoles possess high ability to regenerate their lost organs. In amphibians, the main source of regenerated tissues is lineage-restricted tissue stem cells, but the mechanisms underlying induction, maintenance and differentiation of these stem/progenitor cells in the regenerating organs are poorly understood. We previously reported that interleukin-11 (il-11) is highly expressed in the proliferating cells of regenerating Xenopus tadpole tails. Here, we show that il-11 knockdown (KD) shortens the regenerated tail length, and the phenotype is rescued by forced-il-11-expression in the KD tadpoles. Moreover, marker genes for undifferentiated notochord, muscle, and sensory neurons are downregulated in the KD tadpoles, and the forced-il-11-expression in intact tadpole tails induces expression of these marker genes. Our findings demonstrate that il-11 is necessary for organ regeneration, and suggest that IL-11 plays a key role in the induction and maintenance of undifferentiated progenitors across cell lineages during Xenopus tail regeneration. Xenopus laevis tadpoles have maintained their ability to regenerate various organs. Here, the authors show that interleukin-11 is necessary for organ regeneration, by inducing and maintaining undifferentiated progenitors across cell lineages during Xenopus tail regeneration.

  20. Xenopus laevis Kif18A is a highly processive kinesin required for meiotic spindle integrity

    Directory of Open Access Journals (Sweden)

    Martin M. Möckel

    2017-04-01

    Full Text Available The assembly and functionality of the mitotic spindle depends on the coordinated activities of microtubule-associated motor proteins of the dynein and kinesin superfamily. Our current understanding of the function of motor proteins is significantly shaped by studies using Xenopus laevis egg extract as its open structure allows complex experimental manipulations hardly feasible in other model systems. Yet, the Kinesin-8 orthologue of human Kif18A has not been described in Xenopus laevis so far. Here, we report the cloning and characterization of Xenopus laevis (Xl Kif18A. Xenopus Kif18A is expressed during oocyte maturation and its depletion from meiotic egg extract results in severe spindle defects. These defects can be rescued by wild-type Kif18A, but not Kif18A lacking motor activity or the C-terminus. Single-molecule microscopy assays revealed that Xl_Kif18A possesses high processivity, which depends on an additional C-terminal microtubule-binding site. Human tissue culture cells depleted of endogenous Kif18A display mitotic defects, which can be rescued by wild-type, but not tail-less Xl_Kif18A. Thus, Xl_Kif18A is the functional orthologue of human Kif18A whose activity is essential for the correct function of meiotic spindles in Xenopus oocytes.

  1. Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration

    Directory of Open Access Journals (Sweden)

    Love Nick R

    2011-11-01

    Full Text Available Abstract Background The molecular mechanisms governing vertebrate appendage regeneration remain poorly understood. Uncovering these mechanisms may lead to novel therapies aimed at alleviating human disfigurement and visible loss of function following injury. Here, we explore tadpole tail regeneration in Xenopus tropicalis, a diploid frog with a sequenced genome. Results We found that, like the traditionally used Xenopus laevis, the Xenopus tropicalis tadpole has the capacity to regenerate its tail following amputation, including its spinal cord, muscle, and major blood vessels. We examined gene expression using the Xenopus tropicalis Affymetrix genome array during three phases of regeneration, uncovering more than 1,000 genes that are significantly modulated during tail regeneration. Target validation, using RT-qPCR followed by gene ontology (GO analysis, revealed a dynamic regulation of genes involved in the inflammatory response, intracellular metabolism, and energy regulation. Meta-analyses of the array data and validation by RT-qPCR and in situ hybridization uncovered a subset of genes upregulated during the early and intermediate phases of regeneration that are involved in the generation of NADP/H, suggesting that these pathways may be important for proper tail regeneration. Conclusions The Xenopus tropicalis tadpole is a powerful model to elucidate the genetic mechanisms of vertebrate appendage regeneration. We have produced a novel and substantial microarray data set examining gene expression during vertebrate appendage regeneration.

  2. Global analysis of asymmetric RNA enrichment in oocytes reveals low conservation between closely related Xenopus species

    Science.gov (United States)

    Claußen, Maike; Lingner, Thomas; Pommerenke, Claudia; Opitz, Lennart; Salinas, Gabriela; Pieler, Tomas

    2015-01-01

    RNAs that localize to the vegetal cortex during Xenopus laevis oogenesis have been reported to function in germ layer patterning, axis determination, and development of the primordial germ cells. Here we report on the genome-wide, comparative analysis of differentially localizing RNAs in Xenopus laevis and Xenopus tropicalis oocytes, revealing a surprisingly weak degree of conservation in respect to the identity of animally as well as vegetally enriched transcripts in these closely related species. Heterologous RNA injections and protein binding studies indicate that the different RNA localization patterns in these two species are due to gain/loss of cis-acting localization signals rather than to differences in the RNA-localizing machinery. PMID:26337391

  3. Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

    Directory of Open Access Journals (Sweden)

    Rodrigues Alexandre

    2012-02-01

    Full Text Available Abstract Background Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations. Results Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres. Conclusions Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.

  4. Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae.

    Science.gov (United States)

    Rodrigues, Alexandre Miguel Cavaco; Christen, Bea; Martí, Mercé; Izpisúa Belmonte, Juan Carlos

    2012-02-27

    Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations. Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres. Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.

  5. Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

    Science.gov (United States)

    2012-01-01

    Background Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations. Results Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres. Conclusions Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms. PMID:22369050

  6. Histochemical identification of sialylated glycans in Xenopus laevis testis

    Science.gov (United States)

    Valbuena, Galder; Alonso, Edurne; Ubago, María Martínez; Madrid, Juan Francisco; Díaz-Flores, Lucio; Sáez, Francisco José

    2012-01-01

    Carbohydrate chains of glycoprotein and glycosphingolipids are highly diverse molecules involved in many cell functions, including cell recognition, adhesion and signalling. Sialylated glycans are of special interest because the terminal position of sialic acid (NeuAc) in glycans linked by different ways to subterminal monosaccharides has been shown to be involved in several biological processes, as occurs with gangliosides, which have been reported as being essential in spermatogenesis in mammals. Some glycan-binding proteins, the lectins, which specifically recognize glycan sequences, have been extensively used to characterize tissue and cell carbohydrates by means of cytochemical techniques. The aim of the present work was to determine the presence of NeuAc by means of histochemical techniques in the testis of Xenopus laevis, an animal model widely used in cell and molecular biology research. However, considering that some NeuAc-binding lectins are capable of binding to N-acetylglucosamine (GlcNAc), other GlcNAc-binding lectins were also assayed. The results showed that NeuAc is mainly expressed in the interstitium, and only a weak labelling in the male germ cells was observed. Most NeuAc was located in O-linked oligosaccharides, but some masked NeuAc in N-glycans were identified in primary and secondary spermatogonia and spermatocytes. By contrast, GlcNAc was widely expressed in all germ cell types. Deglycosylative pre-treatments suggest that both N- and O-glycans and/or glycolipids could be responsible for this labelling. In addition, GlcNAc in O-linked oligosaccharides has been identified in spermatogonial cells. The acrosome of spermatids was always negative. Variations of glycan expression have been found in different cell types, suggesting that glycosylation is modified during spermatogenetic development. PMID:22881213

  7. 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.

  8. Mechanisms underlying the noradrenergic modulation of longitudinal coordination during swimming in Xenopus laevis tadpoles

    DEFF Research Database (Denmark)

    Merrywest, Simon D; McDearmid, Jonathan R; Kjaerulff, Ole

    2003-01-01

    Noradrenaline (NA) is a potent modulator of locomotion in many vertebrate nervous systems. When Xenopus tadpoles swim, waves of motor neuron activity alternate across the body and propagate along it with a brief rostro-caudal delay (RC-delay) between segments. We have now investigated the mechani......Noradrenaline (NA) is a potent modulator of locomotion in many vertebrate nervous systems. When Xenopus tadpoles swim, waves of motor neuron activity alternate across the body and propagate along it with a brief rostro-caudal delay (RC-delay) between segments. We have now investigated...

  9. 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.

  10. Ebola Virus and Marburg Virus

    Science.gov (United States)

    Ebola virus and Marburg virus Overview Ebola virus and Marburg virus are related viruses that cause hemorrhagic fevers — illnesses marked by severe bleeding (hemorrhage), organ failure and, in many ...

  11. The establishment of polarized membrane traffic in Xenopus laevis embryos.

    Science.gov (United States)

    Roberts, S J; Leaf, D S; Moore, H P; Gerhart, J C

    1992-09-01

    Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin

  12. 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

  13. The organization of the histone genes in the genome of Xenopus laevis

    NARCIS (Netherlands)

    van Dongen, W.; de Laaf, L.; Zaal, R.; Moorman, A.; Destrée, O.

    1981-01-01

    We have studied the organization of the histone genes in the DNA from several individuals of Xenopus laevis. For that purpose, Southern blots of genomic DNA, that was digested with several restriction enzymes, were hybridized with radioactively labeled DNA fragments from clone X1-hi-1 (14),

  14. Twitch and Tetanic Tension during Culture of Mature Xenopus laevis Single Muscle Fibres

    NARCIS (Netherlands)

    Jaspers, R.T.; Feenstra, Hiske; Lee-de Groot, M.B.E.; Huijing, P.A.J.B.M.; van der 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

  15. Xenopus Vocalizations Are Controlled by a Sexually Differentiated Hindbrain Central Pattern Generator

    Science.gov (United States)

    Rhodes, Heather J.; Yu, Heather J.; Yamaguchi, Ayako

    2008-01-01

    Male and female African clawed frogs (Xenopus laevis) produce rhythmic, sexually distinct vocalizations as part of courtship and mating. We found that Xenopus vocal behavior is governed by a sexually dimorphic central pattern generator (CPG) and that fictive vocalizations can be elicited from an in vitro brain preparation by application of serotonin or by electrical stimulation of a premotor nucleus. Male brains produced fictive vocal patterns representing two calls commonly produced by males in vivo (advertisement and amplectant call), as well as one call pattern (release call) that is common for juvenile males and females in vivo but rare for adult males. Female brains also produced fictive release call. The production of male calls is androgen dependent in Xenopus; to test the effects of androgens on the CPG, we examined fictive calling in the brains of testosterone-treated females. Both fictive male advertisement call and release call were produced. This suggests that all Xenopus possess a sexually undifferentiated pattern generator for release call. Androgen exposure leads to a gain-of-function, allowing the production of male-specific call types without prohibiting the production of the undifferentiated call pattern. We also demonstrate that the CPG is located in the brainstem and seems to rely on the same nuclei in both males and females. Finally, we identified endogenous serotonergic inputs to both the premotor and motor nuclei in the brainstem that may regulate vocal activity in vivo. PMID:17287524

  16. 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

  17. Boundaries and functional domains in the animal/vegetal axis of Xenopus gastrula mesoderm.

    Science.gov (United States)

    Kumano, G; Ezal, C; Smith, W C

    2001-08-15

    Patterning of the Xenopus gastrula marginal zone in the axis running equatorially from the Spemann organizer-the so--called "dorsal/ventral axis"--has been extensively studied. It is now evident that patterning in the animal/vegetal axis also needs to be taken into consideration. We have shown that an animal/vegetal pattern is apparent in the marginal zone by midgastrulation in the polarized expression domains of Xenopus brachyury (Xbra) and Xenopus nodal-related factor 2 (Xnr2). In this report, we have followed cells expressing Xbra in the presumptive trunk and tail at the gastrula stage, and find that they fate to presumptive somite, but not to ventrolateral mesoderm of the tailbud embryo. From this, we speculate that the boundary between the Xbra- and Xnr2-expressing cells at gastrula corresponds to a future tissue boundary. In further experiments, we show that the level of mitogen-activated protein kinase (MAPK) activation is polarized along the animal/vegetal axis, with the Xnr2-expressing cells in the vegetal marginal zone having no detectable activated MAPK. We show that inhibition of MAPK activation in Xenopus animal caps results in the conversion of Xnr2 from a dorsal mesoderm inducer to a ventral mesoderm inducer, supporting a role for Xnr2 in induction of ventral mesoderm. Copyright 2001 Academic Press.

  18. The use of Xenopus oocytes and embryos as a route towards cell ...

    Indian Academy of Sciences (India)

    Unknown

    when this is done, substantial changes in gene expression take place in the complete absence of DNA replication. Such changes were first observed by 2D protein analysis when nuclei from amphibian or mammalian species were transplanted to Xenopus oocytes (De Robertis and Gurdon. 1977). More recently, this type of ...

  19. Absence of somatic histone H1 in oocytes and preblastula embryos of Xenopus laevis

    NARCIS (Netherlands)

    Hock, R.; Moorman, A.; Fischer, D.; Scheer, U.

    1993-01-01

    Available data on the occurrence and expression of somatic histone H1 during oogenesis and early embryogenesis of Xenopus laevis are contradictory. In particular the reported presence of a large storage pool of histone H1A in oocytes is difficult to reconcile with the high transcriptional activity

  20. The accumulation of the maternal pool of histone H1A during oogenesis in Xenopus laevis

    NARCIS (Netherlands)

    van Dongen, W. M.; Moorman, A. F.; Destrée, O. H.

    1983-01-01

    The accumulation of the maternal pool of histone H1A in Xenopus laevis oocytes was measured by the use of a semi-quantitative immunoradiographic method. This method implies the size-fractionation of total basic protein extracts from oocytes on a polyacrylamide gel, blotting of the proteins to

  1. Cloning of noggin gene from hydra and analysis of its functional conservation using Xenopus laevis embryos.

    Science.gov (United States)

    Chandramore, Kalpana; Ito, Yuzuro; Takahashi, Shuji; Asashima, Makoto; Ghaskadbi, Surendra

    2010-01-01

    Hydra, a member of phylum Cnidaria that arose early in evolution, is endowed with a defined axis, organized nervous system, and active behavior. It is a powerful model system for the elucidation of evolution of developmental mechanisms in animals. Here, we describe the identification and cloning of noggin-like gene from hydra. Noggin is a secreted protein involved at multiple stages of vertebrate embryonic development including neural induction and is known to exert its effects by inhibiting the bone morphogenetic protein (BMP)-signaling pathway. Sequence analysis revealed that hydra Noggin shows considerable similarity with its orthologs at the amino acid level. When microinjected in the early Xenopus embryos, hydra noggin mRNA induced a secondary axis in 100% of the injected embryos, demonstrating functional conservation of hydra noggin in vertebrates. This was further confirmed by the partial rescue of Xenopus embryos by hydra noggin mRNA from UV-induced ventralization. By using animal cap assay in Xenopus embryos, we demonstrate that these effects of hydra noggin in Xenopus embryos are because of inhibition of BMP signaling by Noggin. Our data indicate that BMP/Noggin antagonism predates the bilaterian divergence and is conserved during the evolution.

  2. 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…

  3. Programming Pluripotent Precursor Cells Derived from Xenopus Embryos to Generate Specific Tissues and Organs

    Directory of Open Access Journals (Sweden)

    Annette Borchers

    2010-11-01

    Full Text Available Xenopus embryos provide a rich source of pluripotent cells that can be differentiated into functional organs. Since the molecular principles of vertebrate organogenesis appear to be conserved between Xenopus and mammals, this system can provide useful guidelines for the directional manipulation of human embryonic stem cells. Pluripotent Xenopus cells can be easily isolated from the animal pole of blastula stage Xenopus embryos. These so called “animal cap” cells represent prospective ectodermal cells, but give rise to endodermal, mesodermal and neuro-ectodermal derivatives if treated with the appropriate factors. These factors include evolutionary conserved modulators of the key developmental signal transduction pathways that can be supplied either by mRNA microinjection or direct application of recombinant proteins. This relatively simple system has added to our understanding of pancreas, liver, kidney, eye and heart development. In particular, recent studies have used animal cap cells to generate ectopic eyes and hearts, setting the stage for future work aimed at programming pluripotent cells for regenerative medicine.

  4. Catecholamines are present in larval Xenopus laevis: a potential source for cardiac control.

    Science.gov (United States)

    Kloberg, Angélica Jacobsson; Fritsche, Regina

    2002-02-15

    Changes in noradrenaline (NA), adrenaline (A), and dopamine (DA) levels in the heart, kidneys, and whole body (without heart and kidneys) during embryonic development were investigated in the frog, Xenopus laevis using high-performance liquid chromatography (HPLC). In addition, the presence of cells immunoreactive to tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and/or phenylethanolamine-N-methyltransferase (PNMT) in the heart of Xenopus larvae was investigated using immunohistochemical techniques. The presence of nerve fibers was visualized using antibodies against acetylated tubulin (AcT). NA and DA concentrations in the heart were low and steady in NF 40-56, showed an increased value at NF 57, and decreased again in froglets. A trend toward higher concentrations of A was observed at NF 43-49 and NF 57. Cells immunoreactive to TH, DBH, and PNMT were found in the heart from NF 40, and the TH immunoreactive cells became more abundant in the whole heart at later stages. The presence of catecholamines in the non-innervated larval heart together with the finding of TH/DBH/PNMT immunoreactive cells suggests that catecholamines are synthesized and stored in the heart and could therefore have a paracrine role in cardiac control in Xenopus larvae. Detectable concentrations of catecholamines were also found in kidneys and whole bodies (except heart and kidneys). Therefore, catecholamine-producing cells outside the heart can be an important source of circulating catecholamines involved in adrenergic cardiac control in Xenopus larvae. Copyright 2002 Wiley‐Liss, Inc.

  5. 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

    for studying the effects of endocrine disruptors on sexual differentiation. We exposed tadpoles and metamorphs of the species Xenopus tropicalis to the synthetic estrogen ethinylestradiol and the fungicide prochloraz. Prochloraz masculinized the larynx of female frogs while ethinylestradiol had no effect...

  6. [Case of cutaneous necrosis in African clawed frogs Xenopus laevis after the topical application of eugenol].

    Science.gov (United States)

    Ross, Andréanne; Guénette, Sarah Annie; Hélie, Pierre; Vachon, Pascal

    2006-11-01

    Case of cutaneous necrosis in African Clawed frogs Xenopus laevis after the topical application of eugenol. African Clawed frogs showed necrotic cutaneous lesions after a topical application of high concentrations of eugenol, an analgesic and anesthetic agent. Microscopically, ulceration of the epidermis, a loss of mucous and serous glands as well as an infiltration of inflammatory cells were observed.

  7. The lens regenerative competency of limbal vs. central regions of mature Xenopus cornea epithelium

    Science.gov (United States)

    Hamilton, Paul W.; Henry, Jonathan J.

    2016-01-01

    The frog, Xenopus laevis, is capable of completely regenerating a lens from the cornea epithelium. Because this ability appears to be limited to the larval stages of Xenopus, virtually all the work to understand the mechanisms regulating this process has been limited to pre-metamorphic tadpoles. It has been reported that the post-metamorphic cornea is competent to regenerate under experimental conditions, despite the fact that the in vivo capacity to regenerate is lost; however, that work didn’t examine the regenerative potential of different regions of the cornea. A new model suggests that cornea-lens regeneration in Xenopus may be driven by oligopotent stem cells, and not by transdifferentiation of mature cornea cells. We investigated the regenerative potential of the limbal region in post-metamorphic cornea, where the stem cells of the cornea are thought to reside. Using EdU (5-Ethynyl-2’-deoxyuridine), we identified long-term label retaining cells in the basal cells of peripheral post-metamorphic Xenopus cornea, consistent with slow-cycling stem cells of the limbus that have been described in other vertebrates. Using this data to identify putative stem cells of the limbal region in Xenopus, we tested the regenerative competency of limbal regions and central cornea. All three regions showed a similarly high ability for the cells of the basal epithelium to express lens proteins when cultured in proximity to larval retina. Thus, the regenerative competency in post-metamorphic cornea is not restricted to stem cells of the limbal region, but also occurs in the transit amplifying cells throughout the basal layer of the cornea epithelium. PMID:27569373

  8. Daclatasvir plus peginterferon and ribavirin is noninferior to peginterferon and ribavirin alone, and reduces the duration of treatment for HCV genotype 2 or 3 infection.

    Science.gov (United States)

    Dore, Gregory J; Lawitz, Eric; Hézode, Christophe; Shafran, Stephen D; Ramji, Alnoor; Tatum, Harvey A; Taliani, Gloria; Tran, Albert; Brunetto, Maurizia R; Zaltron, Serena; Strasser, Simone I; Weis, Nina; Ghesquiere, Wayne; Lee, Samuel S; Larrey, Dominique; Pol, Stanislas; Harley, Hugh; George, Jacob; Fung, Scott K; de Lédinghen, Victor; Hagens, Peggy; McPhee, Fiona; Hernandez, Dennis; Cohen, David; Cooney, Elizabeth; Noviello, Stephanie; Hughes, Eric A

    2015-02-01

    Twenty-four weeks of treatment with peginterferon and ribavirin for chronic hepatitis C virus (HCV) genotype 2 or 3 infection produces a sustained virologic response (SVR) in 70%-80% of patients. We performed a randomized, double-blind, phase 2b study to assess whether adding daclatasvir, a nonstructural protein 5A (NS5A) inhibitor that is active against these genotypes, improves efficacy and shortens therapy. Patients with HCV genotype 2 or 3 infection (n = 151), enrolled at research centers in North America, Europe, or Australia, were assigned randomly to groups given 12 or 16 weeks of daclatasvir (60 mg once daily), or 24 weeks of placebo, each combined with peginterferon alfa-2a and ribavirin. Treatment was extended to 24 weeks for recipients of daclatasvir who did not meet the criteria for early virologic response. The primary end point was SVR at 24 weeks after treatment (SVR24). Baseline characteristics were similar among patients within each HCV genotype group. However, the 80 patients with HCV genotype 3, compared with the 71 patients with HCV genotype 2, were younger (mean age, 45 vs 53 y, respectively), and a larger proportion had cirrhosis (23% vs 1%, respectively). Among patients with HCV genotype 2 infection, an SVR24 was achieved by 83%, 83%, and 63% of those in the daclatasvir 12-week group, the daclatasvir 16-week group, or the placebo group, respectively; among patients with HCV genotype 3 infection, an SVR24 was achieved by 69%, 67%, and 59% of patients in these groups, respectively. Differences between genotypes largely were attributable to the higher frequency of post-treatment relapse among patients infected with HCV genotype 3. In both daclatasvir arms for both HCV genotypes, the lower bound of the 80% confidence interval of the difference in SVR24 rates between the daclatasvir and placebo arms was above -20%, establishing noninferiority. Safety findings were similar among groups, and were typical of those expected from peginterferon alfa and

  9. Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

    Directory of Open Access Journals (Sweden)

    Halkier Barbara A

    2006-10-01

    Full Text Available Abstract Background We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector. Results Injection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized. Conclusion Expression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified.

  10. Xp38gamma/SAPK3 promotes meiotic G(2)/M transition in Xenopus oocytes and activates Cdc25C

    DEFF Research Database (Denmark)

    Perdiguero, Eusebio; Pillaire, Marie-Jeanne; Bodart, Jean-Francois

    2003-01-01

    /SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38gamma (Xp38gamma) and show that co-expression of active MKK6 with Xp38gamma induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38gamma requires neither protein synthesis nor activation...... for the meiotic G(2)/M progression of Xenopus oocytes....

  11. Molecular cloning and characterization of the Xenopus hypoxia-inducible factor 1alpha (xHIF1alpha).

    Science.gov (United States)

    de Beaucourt, Arnaud; Coumailleau, Pascal

    2007-12-15

    We report the molecular cloning and the characterization of the Xenopus homolog of mammalian hypoxia-inducible factor 1alpha (HIF1alpha), a member of the bHLH/PAS transcription factor family. Searches in Xenopus genome sequences and phylogenetic analysis reveal the existence of HIF1alpha and HIF2alpha paralogs in the Xenopus laevis species. Sequence data analyses indicate that the organization of protein domains in Xenopus HIF1alpha (xHIF1alpha) is strongly conserved. We also show that xHIF1alpha heterodimerizes with the Xenopus Arnt1 protein (xArnt1) with the proteic complex being mediated by the HLH and PAS domains. Subcellular analysis in a Xenopus XTC cell line using chimeric GFP constructs show that over-expression of xHIF1alpha and xArnt1 allows us to detect the xHIF1alpha/xArnt1 complex in the nucleus, but only in the presence of both partners. Further analyses in XTC cell line show that over-producing xHIF1alpha and xArnt1 mediates trans-activation of the hypoxia response element (HRE) reporter. The trans-activation level can be increased in hypoxia conditions. Interestingly such trans-activation properties can be also observed when human Arnt1 is used together with the xHIF1alpha. Copyright (c) 2007 Wiley-Liss, Inc.

  12. Environmental occurrence and toxicity of bentazone on embryos of the amphibian Xenopus laevis; Ritrovamento ambientale e tossicita` del bentazone sull`embrione dell`anfibio Xenopus laevis

    Energy Technology Data Exchange (ETDEWEB)

    Bacchetta, Renato; Compagnoni, Enrico; Giarei, Cristina; Vailati, Giovanni [Milan, Univ. (Italy). Dipt. di Biologia

    1997-03-01

    In this work concentrations of bentazone at the closing section of the River Po (Pontelagoscuro, FE) are reported together with the results of toxicity test performed on embryos of the amphibian Xenopus laevis. The environmental analyses show value higher than the U E limit of 100 ng l{sup -1} for drinking water with a peak in June of 311 ng l{sup -1}. The toxicity and teratogenicity test show no significant mortality up to 10 mg l{sup -1} and developmental malformations concerning corporal axis, eyes, neural tube and coelom starting from 5 mg l{sup -1}.

  13. Tissue-specific expression of the Ets gene Xsap-1 during Xenopus laevis development.

    Science.gov (United States)

    Nentwich, O; Münchberg, F E; Frommer, G; Nordheim, A

    2001-12-01

    We report the cloning of Xenopus laevis Xsap-1 cDNA, encoding a member of the ternary complex factor subfamily of ETS transcription factors. The expression pattern of Xsap-1 was examined during Xenopus embryogenesis using whole-mount in situ hybridization. Spatial expression of Xsap-1 mRNA is first detected at the animal pole at the mid-blastula stage. During neurulation Xsap-1 is expressed in cells participating in neural tube formation, in the sensorial layer of the epidermal ectoderm, and in an anterior region of the ventral mesoderm. Later, Xsap-1 expression is observed in the eye, ear vesicle, branchial arches, heart, pronephros, in the somites, and the developing nervous system, such as fore-, mid-, and hindbrain as well as in the cranial ganglion X.

  14. Xenopus Vasa Homolog XVLG1 is Essential for Migration and Survival of Primordial Germ Cells.

    Science.gov (United States)

    Shimaoka, Kazumi; Mukumoto, Yoshiko; Tanigawa, Yoko; Komiya, Tohru

    2017-04-01

    Xenopus vasa-like gene 1 (XVLG1), a DEAD-Box Helicase 4 (DDX4) gene identified as a vertebrate vasa homologue, is required for the formation of primordial germ cells (PGCs). However, it remains to be clarified when and how XVLG1 functions in the formation of the germ cells. To gain a better understanding of the molecular mechanisms underlying XVLG1 during PGC development, we injected XVLG1 morpholino oligos into germ-plasm containing blastomeres of 32-cell stage of Xenopus embryos, and traced cell fates of the injected blastomere-derived PGCs. As a result of this procedure, migration of the PGCs was impaired and the number of PGCs derived from the blastomeres was significantly decreased. In addition, TUNEL staining in combination with in situ hybridization revealed that the loss of PGCs peaked at stage 27 was caused by apoptosis. This data strongly suggests an essential role for XVLG1 in migration and survival of the germ cells.

  15. Microvascularization and histomorphology of lateral line organs in adult Xenopus laevis.

    Science.gov (United States)

    Gerlach, Nicolas; Minnich, Bernd; Lametschwandtner, Alois

    2014-05-01

    The microvasculariaztion of the lateral line organs (LLOs) of the adult pipid frog, Xenopus laevis was studied by scanning electron microscopy of vascular corrosion casts (VCCs) and correlative light microscopy of paraplast embedded tissues sections. Scanning electron micrographs of VCCs revealed that each neuromast within the LLO rests on a distinct bowl-like capillary network (vascular bowl). One to three vascular bowls were supplied by an ascending arteriole and drained by a descending venule towards the skin deep dermal vascular network. Blood flow regulation mechanisms in form of intimal cushions were present at the origin of ascending arterioles supplying LLOs, microvenous valves were present at the confluence of deep dermal venules and veins. This together with sprouting and nonsprouting angiogenesis (intussusceptive microvascular growth) found in vascular bowls demonstrate that in adult Xenopus the capillary bed of LLO's still can be adjusted to changing energetic needs. Copyright © 2013 Wiley Periodicals, Inc.

  16. Simple and inexpensive hardware and software method to measure volume changes in Xenopus oocytes expressing aquaporins.

    Science.gov (United States)

    Dorr, Ricardo; Ozu, Marcelo; Parisi, Mario

    2007-04-15

    Water channels (aquaporins) family members have been identified in central nervous system cells. A classic method to measure membrane water permeability and its regulation is to capture and analyse images of Xenopus laevis oocytes expressing them. Laboratories dedicated to the analysis of motion images usually have powerful equipment valued in thousands of dollars. However, some scientists consider that new approaches are needed to reduce costs in scientific labs, especially in developing countries. The objective of this work is to share a very low-cost hardware and software setup based on a well-selected webcam, a hand-made adapter to a microscope and the use of free software to measure membrane water permeability in Xenopus oocytes. One of the main purposes of this setup is to maintain a high level of quality in images obtained at brief intervals (shorter than 70 ms). The presented setup helps to economize without sacrificing image analysis requirements.

  17. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

    Science.gov (United States)

    Sun, Liangliang; Bertke, Michelle M.; Champion, Matthew M.; Zhu, Guijie; Huber, Paul W.; Dovichi, Norman J.

    2014-03-01

    While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development.

  18. Elr-type proteins protect Xenopus Dead end mRNA from miR-18-mediated clearance in the soma

    OpenAIRE

    Koebernick, Katja; Loeber, Jana; Arthur, Patrick Kobina; Tarbashevich, Katsiaryna; Pieler, Tomas

    2010-01-01

    Segregation of the future germ line defines a crucial cell fate decision during animal development. In Xenopus, germ cells are specified by inheritance of vegetally localized maternal determinants, including a group of specific mRNAs. Here, we show that the vegetal localization elements (LE) of Xenopus Dead end (XDE) and of several other germ-line-specific, vegetally localized transcripts mediate germ cell-specific stabilization and somatic clearance of microinjected reporter mRNA in Xenopus ...

  19. A Matter of the Heart: The African Clawed Frog Xenopus as a Model for Studying Vertebrate Cardiogenesis and Congenital Heart Defects

    OpenAIRE

    Hempel, Annemarie; Kühl, Michael

    2016-01-01

    The African clawed frog, Xenopus, is a valuable non-mammalian model organism to investigate vertebrate heart development and to explore the underlying molecular mechanisms of human congenital heart defects (CHDs). In this review, we outline the similarities between Xenopus and mammalian cardiogenesis, and provide an overview of well-studied cardiac genes in Xenopus, which have been associated with congenital heart conditions. Additionally, we highlight advantages of modeling candidate genes d...

  20. 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...

  1. Vegetally localized Xenopus trim36 regulates cortical rotation and dorsal axis formation

    OpenAIRE

    Cuykendall, Tawny N.; Houston, Douglas W.

    2009-01-01

    Specification of the dorsoventral axis in Xenopus depends on rearrangements of the egg vegetal cortex following fertilization, concomitant with activation of Wnt/β-catenin signaling. How these processes are tied together is not clear, but RNAs localized to the vegetal cortex during oogenesis are known to be essential. Despite their importance, few vegetally localized RNAs have been examined in detail. In this study, we describe the identification of a novel localized m...

  2. Further Development and Validation of the Frog Embryo Teratogenesis Assay-Xenopus (FETAX).

    Science.gov (United States)

    1992-11-23

    high osmosis water to culture the adults with no temperature bake oven in which glassware can be apparent adverse effects on results. Only non- heated...Toxicol 14(1&2):143-160, 199 1. Laevis. Bull Environ Contain Toxicol 22:159-166, 1979. Friedman M, Rayburn JR, Bantle JA: Developmental toxicology of potato ...160. Friedman, M., Rayburn, J.R., and Bantle, J.A. 1990. Developmental toxicology of potato alkaloids in the Frog Embryo Teratogenesis Assay-Xenopus

  3. Actomyosin Contractility and Microtubules Drive Apical Constriction in Xenopus Bottle Cells

    OpenAIRE

    Lee, Jen-Yi; Harland, Richard M.

    2007-01-01

    Cell shape changes are critical for morphogenetic events such as gastrulation, neurulation, and organogenesis. However, the cell biology driving cell shape changes is poorly understood, especially in vertebrates. The beginning of Xenopus laevis gastrulation is marked by the apical constriction of bottle cells in the dorsal marginal zone, which bends the tissue and creates a crevice at the blastopore lip. We found that bottle cells contribute significantly to gastrulation, as their shape chang...

  4. Defining a large set of full-length clones from a Xenopus tropicalis EST project.

    Science.gov (United States)

    Gilchrist, Michael J; Zorn, Aaron M; Voigt, Jana; Smith, James C; Papalopulu, Nancy; Amaya, Enrique

    2004-07-15

    Amphibian embryos from the genus Xenopus are among the best species for understanding early vertebrate development and for studying basic cell biological processes. Xenopus, and in particular the diploid Xenopus tropicalis, is also ideal for functional genomics. Understanding the behavior of genes in this accessible model system will have a significant and beneficial impact on the understanding of similar genes in other vertebrate systems. Here we describe the analysis of 219,270 X. tropicalis expressed sequence tags (ESTs) from four early developmental stages. From these, we have deduced a set of unique expressed sequences comprising approximately 20,000 clusters and 16,000 singletons. Furthermore, we developed a computational method to identify clones that contain the complete coding sequence and describe the creation for the first time of a set of approximately 7000 such clones, the full-length (FL) clone set. The entire EST set is cloned in a eukaryotic expression vector and is flanked by bacteriophage promoters for in vitro transcription, allowing functional experiments to be carried out without further subcloning. We have created a publicly available database containing the FL clone set and related clustering data (http://www.gurdon.cam.ac.uk/informatics/Xenopus.html) and we make the FL clone set publicly available as a resource to accelerate the process of gene discovery and function in this model organism. The creation of the unique set of expressed sequences and the FL clone set pave the way toward a large-scale systematic analysis of gene sequence, gene expression, and gene function in this vertebrate species.

  5. Bipolarization and Poleward Flux Correlate during Xenopus Extract Spindle AssemblyV⃞

    OpenAIRE

    Mitchison, T.J.; Maddox, P.; Groen, A.; Cameron, L.; Perlman, Z.; Ohi, R.; Desai, A.; Salmon, E.D.; Kapoor, T.M.

    2004-01-01

    We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with on...

  6. 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.

    2015-01-01

    Single-wall carbon nanotubes are high aspect ratio nanomaterials that are 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. PMID:26153061

  7. Cranial Osteogenesis and Suture Morphology in Xenopus laevis: A Unique Model System for Studying Craniofacial Development

    Science.gov (United States)

    Slater, Bethany J.; Liu, Karen J.; Kwan, Matthew D.; Quarto, Natalina; Longaker, Michael T.

    2009-01-01

    Background The tremendous diversity in vertebrate skull formation illustrates the range of forms and functions generated by varying genetic programs. Understanding the molecular basis for this variety may provide us with insights into mechanisms underlying human craniofacial anomalies. In this study, we provide evidence that the anuran Xenopus laevis can be developed as a simplified model system for the study of cranial ossification and suture patterning. The head structures of Xenopus undergo dramatic remodelling during metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. Because of the extended larval period in Xenopus, the molecular basis of these alterations has not been well studied. Methodology/Principal Findings We examined late larval, metamorphosing, and post-metamorphosis froglet stages in intact and sectioned animals. Using micro-computed tomography (μCT) and tissue staining of the frontoparietal bone and surrounding cartilage, we observed that bone formation initiates from lateral ossification centers, proceeding from posterior-to-anterior. Histological analyses revealed midline abutting and posterior overlapping sutures. To determine the mechanisms underlying the large-scale cranial changes, we examined proliferation, apoptosis, and proteinase activity during remodelling of the skull roof. We found that tissue turnover during metamorphosis could be accounted for by abundant matrix metalloproteinase (MMP) activity, at least in part by MMP-1 and -13. Conclusion A better understanding of the dramatic transformation from cartilaginous head structures to bony skull during Xenopus metamorphosis may provide insights into tissue remodelling and regeneration in other systems. Our studies provide some new molecular insights into this process. PMID:19156194

  8. Caldesmon regulates actin dynamics to influence cranial neural crest migration in Xenopus

    OpenAIRE

    Nie, Shuyi; Kee, Yun; Bronner-Fraser, Marianne

    2011-01-01

    Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells t...

  9. Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

    Energy Technology Data Exchange (ETDEWEB)

    Ostrup, Olga, E-mail: osvarcova@gmail.com [Institute of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C (Denmark); Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo (Norway); Norwegian Center for Stem Cell Research, Oslo (Norway); Hyttel, Poul; Klaerke, Dan A. [Institute of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C (Denmark); Collas, Philippe, E-mail: philc@medisin.uio.no [Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo (Norway); Norwegian Center for Stem Cell Research, Oslo (Norway)

    2011-09-02

    Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.

  10. A Matter of the Heart: The African Clawed Frog Xenopus as a Model for Studying Vertebrate Cardiogenesis and Congenital Heart Defects

    Directory of Open Access Journals (Sweden)

    Annemarie Hempel

    2016-06-01

    Full Text Available The African clawed frog, Xenopus, is a valuable non-mammalian model organism to investigate vertebrate heart development and to explore the underlying molecular mechanisms of human congenital heart defects (CHDs. In this review, we outline the similarities between Xenopus and mammalian cardiogenesis, and provide an overview of well-studied cardiac genes in Xenopus, which have been associated with congenital heart conditions. Additionally, we highlight advantages of modeling candidate genes derived from genome wide association studies (GWAS in Xenopus and discuss commonly used techniques.

  11. A Matter of the Heart: The African Clawed Frog Xenopus as a Model for Studying Vertebrate Cardiogenesis and Congenital Heart Defects.

    Science.gov (United States)

    Hempel, Annemarie; Kühl, Michael

    2016-06-04

    The African clawed frog, Xenopus, is a valuable non-mammalian model organism to investigate vertebrate heart development and to explore the underlying molecular mechanisms of human congenital heart defects (CHDs). In this review, we outline the similarities between Xenopus and mammalian cardiogenesis, and provide an overview of well-studied cardiac genes in Xenopus , which have been associated with congenital heart conditions. Additionally, we highlight advantages of modeling candidate genes derived from genome wide association studies (GWAS) in Xenopus and discuss commonly used techniques.

  12. Molecular cloning of the bullfrog kisspeptin receptor GPR54 with high sensitivity to Xenopus kisspeptin.

    Science.gov (United States)

    Moon, Jung Sun; Lee, Yeo Reum; Oh, Da Young; Hwang, Jong Ik; Lee, Ju Yeon; Kim, Jae Il; Vaudry, Hubert; Kwon, Hyuk Bang; Seong, Jae Young

    2009-01-01

    Kisspeptin and its receptor, GPR54, play important roles in mammalian reproduction and cancer development. However, little is known about their function in nonmammalian species. In the present study, we have isolated the cDNA encoding the kisspeptin receptor, GPR54, from the bullfrog, Rana catesbeiana. The bullfrog GPR54 (bfGPR54) cDNA encodes a 379-amino acid heptahelical G protein-coupled receptor. bfGPR54 exhibits 45-46% amino acid identity with mammalian GPR54s and 70-74% identity with fish GPR54s. RT-PCR analysis showed that bfGPR54 mRNA is highly expressed in the forebrain, hypothalamus and pituitary. Upon stimulation by synthetic human kisspeptin-10 with Phe-amide residue at the C-terminus (h-Kiss-10F), bfGPR54 induces SRE-luc activity, a PKC-specific reporter, evidencing the PKC-linked signaling pathway of bfGPR54. Using a blast search, we found a gene encoding a kisspeptin-like peptide in Xenopus. The C-terminal decapeptide of Xenopus kisspeptin shows higher amino acid sequence identity to fish Kiss-10s than mammalian Kiss-10s. A synthetic Xenopus kisspeptin peptide (x-Kiss-12Y) showed a higher potency than mammalian Kiss-10s in the activation of bfGPR54. This study expands our understanding of the physiological roles and molecular evolution of kisspeptins and their receptors.

  13. Subcellular Localization of Class I Histone Deacetylases in the Developing Xenopus tectum.

    Science.gov (United States)

    Guo, Xia; Ruan, Hangze; Li, Xia; Qin, Liming; Tao, Yi; Qi, Xianjie; Gao, Juanmei; Gan, Lin; Duan, Shumin; Shen, Wanhua

    2015-01-01

    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 transiently localized in the mitochondria and that the subcellular distribution of class I HDACs in the Xenopus tectum is heterogeneous. Furthermore, we observed that spherical mitochondria accumulate in the cytoplasm at earlier stages, whereas elongated mitochondria are evenly distributed in the tectum at later stages. The activity of histone acetylation (H4K12) remains low in mitochondria during tectal development. Pharmacological blockades of HDACs using a broad spectrum HDAC inhibitor of Trichostatin A (TSA) or specific class I HDAC inhibitors of MS-275 and MGCD0103 decrease the number of mitochondria in the tectum at stage 34. These findings highlight a link between the subcellular distribution of class I HDACs and mitochondrial dynamics in the developing optic tectum of Xenopus laevis.

  14. Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs.

    Science.gov (United States)

    Shintomi, Keishi; Iwabuchi, Mari; Saeki, Hideaki; Ura, Kiyoe; Kishimoto, Takeo; Ohsumi, Keita

    2005-06-07

    In eukaryotic cells, genomic DNA is primarily packaged into nucleosomes through sequential ordered binding of the core and linker histone proteins. The acidic proteins termed histone chaperones are known to bind to core histones to neutralize their positive charges, thereby facilitating their proper deposition onto DNA to assemble the core of nucleosomes. For linker histones, however, little has been known about the regulatory mechanism for deposition of linker histones onto the linker DNA. Here we report that, in Xenopus eggs, the linker histone is associated with the Xenopus homologue of nucleosome assembly protein-1 (NAP-1), which is known to be a chaperone for the core histones H2A and H2B in Drosophila and mammalian cells [Ito, T., Bulger, M., Kobayashi, R. & Kadonaga, J. T. (1996) Mol. Cell Biol. 16, 3112-3124; Chang, L., Loranger, S. S., Mizzen, C., Ernst, S. G., Allis, C. D. & Annunziato, A. T. (1997) Biochemistry 36, 469-480]. We show that NAP-1 acts as the chaperone for the linker histone in both sperm chromatin remodeling into nucleosomes and linker histone binding to nucleosome core dimers. In the presence of NAP-1, the linker histone is properly deposited onto linker DNA at physiological ionic strength, without formation of nonspecific aggregates. These results strongly suggest that NAP-1 functions as a chaperone for the linker histone in Xenopus eggs.

  15. Xenopus in Space and Time: Fossils, Node Calibrations, Tip-Dating, and Paleobiogeography.

    Science.gov (United States)

    Cannatella, David

    2015-01-01

    Published data from DNA sequences, morphology of 11 extant and 15 extinct frog taxa, and stratigraphic ranges of fossils were integrated to open a window into the deep-time evolution of Xenopus. The ages and morphological characters of fossils were used as independent datasets to calibrate a chronogram. We found that DNA sequences, either alone or in combination with morphological data and fossils, tended to support a close relationship between Xenopus and Hymenochirus, although in some analyses this topology was not significantly better than the Pipa + Hymenochirus topology. Analyses that excluded DNA data found strong support for the Pipa + Hymenochirus tree. The criterion for selecting the maximum age of the calibration prior influenced the age estimates, and our age estimates of early divergences in the tree of frogs are substantially younger than those of published studies. Node-dating and tip-dating calibrations, either alone or in combination, yielded older dates for nodes than did a root calibration alone. Our estimates of divergence times indicate that overwater dispersal, rather than vicariance due to the splitting of Africa and South America, may explain the presence of Xenopus in Africa and its closest fossil relatives in South America.

  16. Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system.

    Science.gov (United States)

    Hartley, Katharine O; Nutt, Stephen L; Amaya, Enrique

    2002-02-05

    The transgenic technique in Xenopus allows one to misexpress genes in a temporally and spatially controlled manner. However, this system suffers from two experimental limitations. First, the restriction enzyme-mediated integration procedure relies on chromosomal damage, resulting in a percentage of embryos failing to develop normally. Second, every transgenic embryo has unique sites of integration and unique transgene copy number, resulting in variable transgene expression levels and variable phenotypes. For these reasons, we have adapted the Gal4-UAS method for targeted gene expression to Xenopus. This technique relies on the generation of transgenic lines that carry "activator" or "effector" constructs. Activator lines express the yeast transcription factor, Gal4, under the control of a desired promoter, whereas effector lines contain DNA-binding motifs for Gal4-(UAS) linked to the gene of interest. We show that on intercrossing of these lines, the effector gene is transcribed in the temporal and spatial manner of the activator's promoter. Furthermore, we use the Gal4-UAS system to misexpress Xvent-2, a transcriptional target of bone morphogenetic protein 4 (BMP4) signaling during early embryogenesis. Embryos inheriting both the Gal4 activator and Xvent-2 effector transgenes display a consistent microcephalic phenotype. Finally, we exploit this system to characterize the neural and mesodermal defects obtained from early misexpression of Xvent-2. These results emphasize the potential of this system for the controlled analyses of gene function in Xenopus.

  17. BDNF promotes target innervation of Xenopus mandibular trigeminal axons in vivo.

    Science.gov (United States)

    Huang, Jeffrey K; Dorey, Karel; Ishibashi, Shoko; Amaya, Enrique

    2007-05-31

    Trigeminal nerves consist of ophthalmic, maxillary, and mandibular branches that project to distinct regions of the facial epidermis. In Xenopus embryos, the mandibular branch of the trigeminal nerve extends toward and innervates the cement gland in the anterior facial epithelium. The cement gland has previously been proposed to provide a short-range chemoattractive signal to promote target innervation by mandibular trigeminal axons. Brain derived neurotrophic factor, BDNF is known to stimulate axon outgrowth and branching. The goal of this study is to determine whether BDNF functions as the proposed target recognition signal in the Xenopus cement gland. We found that the cement gland is enriched in BDNF mRNA transcripts compared to the other neurotrophins NT3 and NT4 during mandibular trigeminal nerve innervation. BDNF knockdown in Xenopus embryos or specifically in cement glands resulted in the failure of mandibular trigeminal axons to arborise or grow into the cement gland. BDNF expressed ectodermal grafts, when positioned in place of the cement gland, promoted local trigeminal axon arborisation in vivo. BDNF is necessary locally to promote end stage target innervation of trigeminal axons in vivo, suggesting that BDNF functions as a short-range signal that stimulates mandibular trigeminal axon arborisation and growth into the cement gland.

  18. Design and use of transgenic reporter strains for detecting activity of signaling pathways in Xenopus.

    Science.gov (United States)

    Tran, Hong Thi; Vleminckx, Kris

    2014-04-01

    Embryos and larvae of vertebrate species with external development are ideal subjects for investigating the dynamic spatiotemporal activity of developmental signaling pathways. The availability of efficient transgene technologies in Xenopus and zebrafish and the translucency and/or transparency of their embryos and larvae make these two species attractive for direct in vivo imaging of reporter gene expression. In this article we describe the design of efficient signaling reporters, using the Wnt/β-catenin pathway as a representative example. We define methods for validating the reporter constructs and describe how they can be used to generate stable transgenic lines in Xenopus. We provide efficient methods used in our laboratory for raising the tadpoles and froglets rapidly to sexual maturity. We further discuss how the reporter lines can be used for delineating the dynamic activity of a signaling pathway and how modulators of the pathway can be scrutinized via chemical intervention and the micro-injection of synthetic RNAs or morpholinos. The strategic outline discussed in this paper provides a template for studying other developmental signaling pathways in Xenopus. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration.

    Science.gov (United States)

    Love, Nick R; Chen, Yaoyao; Ishibashi, Shoko; Kritsiligkou, Paraskevi; Lea, Robert; Koh, Yvette; Gallop, Jennifer L; Dorey, Karel; Amaya, Enrique

    2013-02-01

    Understanding the molecular mechanisms that promote successful tissue regeneration is critical for continued advancements in regenerative medicine. Vertebrate amphibian tadpoles of the species Xenopus laevis and Xenopus tropicalis have remarkable abilities to regenerate their tails following amputation, through the coordinated activity of numerous growth factor signalling pathways, including the Wnt, Fgf, Bmp, Notch and TGF-β pathways. Little is known, however, about the events that act upstream of these signalling pathways following injury. Here, we show that Xenopus tadpole tail amputation induces a sustained production of reactive oxygen species (ROS) during tail regeneration. Lowering ROS levels, using pharmacological or genetic approaches, reduces the level of cell proliferation and impairs tail regeneration. Genetic rescue experiments restored both ROS production and the initiation of the regenerative response. Sustained increased ROS levels are required for Wnt/β-catenin signalling and the activation of one of its main downstream targets, fgf20 (ref. 7), which, in turn, is essential for proper tail regeneration. These findings demonstrate that injury-induced ROS production is an important regulator of tissue regeneration.

  20. Development of a New Decision Tree to Rapidly Screen Chemical Estrogenic Activities of Xenopus laevis.

    Science.gov (United States)

    Wang, Ting; Li, Weiying; Zheng, Xiaofeng; Lin, Zhifen; Kong, Deyang

    2014-02-01

    During the last past decades, there is an increasing number of studies about estrogenic activities of the environmental pollutants on amphibians and many determination methods have been proposed. However, these determination methods are time-consuming and expensive, and a rapid and simple method to screen and test the chemicals for estrogenic activities to amphibians is therefore imperative. Herein is proposed a new decision tree formulated not only with physicochemical parameters but also a biological parameter that was successfully used to screen estrogenic activities of the chemicals on amphibians. The biological parameter, CDOCKER interaction energy (Ebinding ) between chemicals and the target proteins was calculated based on the method of molecular docking, and it was used to revise the decision tree formulated by Hong only with physicochemical parameters for screening estrogenic activity of chemicals in rat. According to the correlation between Ebinding of rat and Xenopus laevis, a new decision tree for estrogenic activities in Xenopus laevis is finally proposed. Then it was validated by using the randomly 8 chemicals which can be frequently exposed to Xenopus laevis, and the agreement between the results from the new decision tree and the ones from experiments is generally satisfactory. Consequently, the new decision tree can be used to screen the estrogenic activities of the chemicals, and combinational use of the Ebinding and classical physicochemical parameters can greatly improves Hong's decision tree. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Role of maternal Xenopus syntabulin in germ plasm aggregation and primordial germ cell specification.

    Science.gov (United States)

    Oh, Denise; Houston, Douglas W

    2017-12-15

    The localization and organization of mitochondria- and ribonucleoprotein granule-rich germ plasm is essential for many aspects of germ cell development. In Xenopus, germ plasm is maternally inherited and is required for the specification of primordial germ cells (PGCs). Germ plasm is aggregated into larger patches during egg activation and cleavage and is ultimately translocated perinuclearly during gastrulation. Although microtubule dynamics and a kinesin (Kif4a) have been implicated in Xenopus germ plasm localization, little is known about how germ plasm distribution is regulated. Here, we identify a role for maternal Xenopus Syntabulin in the aggregation of germ plasm following fertilization. We show that depletion of sybu mRNA using antisense oligonucleotides injected into oocytes results in defects in the aggregation and perinuclear transport of germ plasm and subsequently in reduced PGC numbers. Using live imaging analysis, we also characterize a novel role for Sybu in the collection of germ plasm in vegetal cleavage furrows by surface contraction waves. Additionally, we show that a localized kinesin-like protein, Kif3b, is also required for germ plasm aggregation and that Sybu functionally interacts with Kif3b and Kif4a in germ plasm aggregation. Overall, these data suggest multiple coordinate roles for kinesins and adaptor proteins in controlling the localization and distribution of a cytoplasmic determinant in early development. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Purification and partial characterization of Xenopus laevis tenascin from the XTC cell line.

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    Riou, J F; Alfandari, D; Eppe, M; Tacchetti, C; Chiquet, M; Boucaut, J C; Thiery, J P; Levi, G

    1991-02-25

    We report here the purification of tenascin, an extracellular matrix molecule involved in the control of morphogenesis, from the conditioned medium of the Xenopus XTC cell line. Tenascin was purified by affinity chromatography on a column of the monoclonal antibody mAb TnM1; the molecule eluted from this column has a relative molecular mass of 210 kDa after reduction. Electrophoretic analysis under non-reducing conditions shows that the purified components are oligomeric disulfide-linked complexes which barely enter a 4% polyacrylamide gel. Upon rotary shadowing these molecules appear to possess a central globular domain to which pairs or triplets of arms are attached. Polyclonal antibodies have been raised against purified Xenopus tenascin. They recognise specifically the antigen on Western blots of XTC conditioned medium and adult brain, by immunofluorescence, these antibodies reveal large amounts of tenascin in the secretory vesicles as well as in the extracellular matrix of XTC cells. In the Xenopus tadpole, they stain the developing cartilage, the basal lamina of skin epidermis, myotendinous ligaments and restricted regions of the central nervous system.

  3. Subcellular localization of class I histone deacetylases in the developing Xenopus tectum

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    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 transiently localized in the mitochondria and that the subcellular distribution of class I HDACs in the Xenopus tectum is heterogeneous. Furthermore, we observed that spherical mitochondria accumulate in the cytoplasm at earlier stages, whereas elongated mitochondria are evenly distributed in the tectum at later stages. The activity of histone acetylation (H4K12 remains low in mitochondria during tectal development. Pharmacological blockades of HDACs using a broad spectrum HDAC inhibitor of Trichostatin A (TSA or specific class I HDAC inhibitors of MS-275 and MGCD0103 decrease the number of mitochondria in the tectum at stage 34. These findings highlight a link between the subcellular distribution of class I HDACs and mitochondrial dynamics in the developing optic tectum of Xenopus laevis.

  4. 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

  5. Subcellular distribution of the Xenopus p58/lamin B receptor in oocytes and eggs.

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    Gajewski, A; Krohne, G

    1999-08-01

    The p58/lamin B receptor of vertebrates is localized in the inner nuclear membrane. Antibodies raised against the bacterially expressed amino-terminal half of Xenopus p58 (Xp58) revealed that in Xenopus oocytes the vast majority of this membrane protein is localized in cytoplasmic membranes. Only very small amounts of p58 not detectable by immunofluorescence microscopy were contained in the oocyte nuclear envelope. In contrast, nuclear membranes of 2-cell stage embryos were successfully stained with p58 antibodies, nuclei reconstituted in vitro in Xenopus egg extracts contained p58, and the nucleoplasmic domain of Xp58 could be specifically bound to sperm chromatin in vitro. One major difference between oocytes and early embryonic cells is that no chromatin is associated with the oocyte inner nuclear membrane whereas the complement of lamins is identical in both cell types. To gain insight into the properties of oocyte p58 we microinjected isolated nuclei of cultured rat cells into the cytoplasm of Xenopus oocytes. The oocyte p58 was detectable by immunofluorescence microscopy within 16-20 hours in the nuclear membrane of rat nuclei. Our data indicate that the peripheral chromatin but not lamins are required for the retention of p58 in the inner nuclear membrane. Sucrose step gradient centrifugation of total oocyte membranes revealed that the oocyte p58 was predominantly recovered in membrane fractions that did not contain lamins whereas membrane associated lamins and p58 of unfertilized eggs were found in the same fractions. By electron microscopical immunolocalizations one major population of meiotic p58 vesicles was identified that contained exclusively p58 and a second minor population (ca. 11% of p58 vesicles) contained in addition to p58 membrane bound B-type lamins. Egg vesicles containing pore membrane proteins were predominantly recovered in gradient fractions that did not contain p58 and B-type lamins. Our data indicate that the targeting of p58 to

  6. PLD1 regulates Xenopus convergent extension movements by mediating Frizzled7 endocytosis for Wnt/PCP signal activation.

    Science.gov (United States)

    Lee, Hyeyoon; Lee, Seung Joon; Kim, Gun-Hwa; Yeo, Inchul; Han, Jin-Kwan

    2016-03-01

    Phospholipase D (PLD) is involved in the regulation of receptor-associated signaling, cell movement, cell adhesion and endocytosis. However, its physiological role in vertebrate development remains poorly understood. In this study, we show that PLD1 is required for the convergent extension (CE) movements during Xenopus gastrulation by activating Wnt/PCP signaling. Xenopus PLD1 protein is specifically enriched in the dorsal region of Xenopus gastrula embryo and loss or gain-of-function of PLD1 induce defects in gastrulation and CE movements. These defective phenotypes are due to impaired regulation of Wnt/PCP signaling pathway. Biochemical and imaging analysis using Xenopus tissues reveal that PLD1 is required for Fz7 receptor endocytosis upon Wnt11 stimulation. Moreover, we show that Fz7 endocytosis depends on dynamin and regulation of GAP activity of dynamin by PLD1 via its PX domain is crucial for this process. Taken together, our results suggest that PLD1 acts as a new positive mediator of Wnt/PCP signaling by promoting Wnt11-induced Fz7 endocytosis for precise regulation of Xenopus CE movements. Copyright © 2016 Elsevier Inc. All rights reserved.

  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

    2011-05-01

    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.

  8. Characterization of Xenopus tissue inhibitor of metalloproteinases-2: a role in regulating matrix metalloproteinase activity during development.

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    Liezhen Fu

    Full Text Available BACKGROUND: Frog metamorphosis is totally dependent on thyroid hormone (T3 and mimics the postembryonic period around birth in mammals. It is an excellent model to study the molecular basis of postembryonic development in vertebrate. We and others have shown that many, if not all, matrix metalloproteinases (MMPs, which cleave proteins of the extracellular matrix as well as other substrates, are induced by T3 and important for metamorphosis. MMP activity can be inhibited by tissue inhibitors of metalloproteinase (TIMPs. There are 4 TIMPs in vertebrates and their roles in postembryonic development are poorly studied. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the TIMP2 genes in Xenopus laevis and the highly related species Xenopus tropicalis and discovered that TIMP2 is a single copy gene in Xenopus tropicalis as in mammals but is duplicated in Xenopus laevis. Furthermore, the TIMP2 locus in Xenopus tropicalis genome is different from that in human, suggesting an evolutionary reorganization of the locus. More importantly, we found that the duplicated TIMP2 genes were similarly regulated in the developing limb, remodeling intestine, resorbing tail during metamorphosis. Unexpectedly, like its MMP target genes, the TIMP2 genes were upregulated by T3 during both natural and T3-induced metamorphosis. CONCLUSIONS/SIGNIFICANCE: Our results indicate that TIMP2 is highly conserved among vertebrates and that the TIMP2 locus underwent a chromosomal reorganization during evolution. Furthermore, the unexpected upregulation of TIMP2 genes during metamorphosis suggests that proper balance of MMP activity is important for metamorphosis.

  9. ECHO virus

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/001340.htm ECHO virus To use the sharing features on this page, please enable JavaScript. Enteric cytopathic human orphan (ECHO) viruses are a group of viruses that can lead ...

  10. Role of TAK1 and TAB1 in BMP signaling in early Xenopus development.

    Science.gov (United States)

    Shibuya, H; Iwata, H; Masuyama, N; Gotoh, Y; Yamaguchi, K; Irie, K; Matsumoto, K; Nishida, E; Ueno, N

    1998-01-01

    Transforming growth factor-beta (TGF-beta) superfamily members elicit signals through stimulation of serine/threonine kinase receptors. Recent studies of this signaling pathway have identified two types of novel mediating molecules, the Smads and TGF-beta activated kinase 1 (TAK1). Smads were shown to mimic the effects of bone morphogenetic protein (BMP), activin and TGF-beta. TAK1 and TAB1 were identified as a MAPKKK and its activator, respectively, which might be involved in the up-regulation of TGF-beta superfamily-induced gene expression, but their biological role is poorly understood. Here, we have examined the role of TAK1 and TAB1 in the dorsoventral patterning of early Xenopus embryos. Ectopic expression of Xenopus TAK1 (xTAK1) in early embryos induced cell death. Interestingly, however, concomitant overexpression of bcl-2 with the activated form of xTAK1 or both xTAK1 and xTAB1 in dorsal blastomeres not only rescued the cells but also caused the ventralization of the embryos. In addition, a kinase-negative form of xTAK1 (xTAK1KN) which is known to inhibit endogenous signaling could partially rescue phenotypes generated by the expression of a constitutively active BMP-2/4 type IA receptor (BMPR-IA). Moreover, xTAK1KN could block the expression of ventral mesoderm marker genes induced by Smad1 or 5. These results thus suggest that xTAK1 and xTAB1 function in the BMP signal transduction pathway in Xenopus embryos in a cooperative manner. PMID:9463380

  11. A distinct mechanism of vascular lumen formation in Xenopus requires EGFL7.

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    Marta S Charpentier

    Full Text Available During vertebrate blood vessel development, lumen formation is the critical process by which cords of endothelial cells transition into functional tubular vessels. Here, we use Xenopus embryos to explore the cellular and molecular mechanisms underlying lumen formation of the dorsal aorta and the posterior cardinal veins, the primary major vessels that arise via vasculogenesis within the first 48 hours of life. We demonstrate that endothelial cells are initially found in close association with one another through the formation of tight junctions expressing ZO-1. The emergence of vascular lumens is characterized by elongation of endothelial cell shape, reorganization of junctions away from the cord center to the periphery of the vessel, and onset of Claudin-5 expression within tight junctions. Furthermore, unlike most vertebrate vessels that exhibit specialized apical and basal domains, we show that early Xenopus vessels are not polarized. Moreover, we demonstrate that in embryos depleted of the extracellular matrix factor Epidermal Growth Factor-Like Domain 7 (EGFL7, an evolutionarily conserved factor associated with vertebrate vessel development, vascular lumens fail to form. While Claudin-5 localizes to endothelial tight junctions of EGFL7-depleted embryos in a timely manner, endothelial cells of the aorta and veins fail to undergo appropriate cell shape changes or clear junctions from the cell-cell contact. Taken together, we demonstrate for the first time the mechanisms by which lumens are generated within the major vessels in Xenopus and implicate EGFL7 in modulating cell shape and cell-cell junctions to drive proper lumen morphogenesis.

  12. The protocadherin PAPC establishes segmental boundaries during somitogenesis in xenopus embryos.

    Science.gov (United States)

    Kim, S H; Jen, W C; De Robertis, E M; Kintner, C

    2000-07-13

    One prominent example of segmentation in vertebrate embryos is the subdivision of the paraxial mesoderm into repeating, metameric structures called somites. During this process, cells in the presomitic mesoderm (PSM) are first patterned into segments leading secondarily to differences required for somite morphogenesis such as the formation of segmental boundaries. Recent studies have shown that a segmental pattern is generated in the PSM of Xenopus embryos by genes encoding a Mesp-like bHLH protein called Thylacine 1 and components of the Notch signaling pathway. These genes establish a repeating pattern of gene expression that subdivides cells in the PSM into anterior and posterior half segments, but how this pattern of gene expression leads to segmental boundaries is unknown. Recently, a member of the protocadherin family of cell adhesion molecules, called PAPC, has been shown to be expressed in the PSM of Xenopus embryos in a half segment pattern, suggesting that it could play a role in restricting cell mixing at the anterior segmental boundary. Here, we examine the expression and function of PAPC during segmentation of the paraxial mesoderm in Xenopus embryos. We show that Thylacine 1 and the Notch pathway establish segment identity one segment prior to the segmental expression of PAPC. Altering segmental identity in embryos by perturbing the activity of Thylacine 1 and the Notch pathway, or by treatment with a protein synthesis inhibitor, cycloheximide, leads to the predicted changes in the segmental expression of PAPC. By disrupting PAPC function in embryos using a putative dominant-negative or an activated form of PAPC, we show that segmental PAPC activity is required for proper somite formation as well as for maintaining segmental gene expression within the PSM. Segmental expression of PAPC is established in the PSM as a downstream consequence of segmental patterning by Thylacine 1 and the Notch pathway. We propose that PAPC is part of the mechanism that

  13. Growth-arrest-specific protein 2 inhibits cell division in Xenopus embryos.

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    Tong Zhang

    Full Text Available Growth-arrest-specific 2 gene was originally identified in murine fibroblasts under growth arrest conditions. Furthermore, serum stimulation of quiescent, non-dividing cells leads to the down-regulation of gas2 and results in re-entry into the cell cycle. Cytoskeleton rearrangements are critical for cell cycle progression and cell division and the Gas2 protein has been shown to co-localize with actin and microtubules in interphase mammalian cells. Despite these findings, direct evidence supporting a role for Gas2 in the mechanism of cell division has not been reported.To determine whether the Gas2 protein plays a role in cell division, we over-expressed the full-length Gas2 protein and Gas2 truncations containing either the actin-binding CH domain or the tubulin-binding Gas2 domain in Xenopus laevis embryos. We found that both the full-length Gas2 protein and the Gas2 domain, but not the CH domain, inhibited cell division and resulted in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that Gas2 function is mediated by microtubule binding. Gas2 co-localized with microtubules at the cell cortex of Gas2-injected Xenopus embryos using cryo-confocal microscopy and co-sedimented with microtubules in cytoskeleton co-sedimentation assays. To investigate the mechanism of Gas2-induced cell division arrest, we showed, using a wound-induced contractile array assay, that Gas2 stabilized microtubules. Finally, electron microscopy studies demonstrated that Gas2 bundled microtubules into higher-order structures.Our experiments show that Gas2 inhibits cell division in Xenopus embryos. We propose that Gas2 function is mediated by binding and bundling microtubules, leading to cell division arrest.

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

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    Tatiana Tatum Parker

    Full Text Available 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. Keywords: Cigarette butt leachate, Xenopus laevis, Development

  15. RNA sequencing reveals a diverse and dynamic repertoire of the Xenopus tropicalis transcriptome over development.

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    Tan, Meng How; Au, Kin Fai; Yablonovitch, Arielle L; Wills, Andrea E; Chuang, Jason; Baker, Julie C; Wong, Wing Hung; Li, Jin Billy

    2013-01-01

    The Xenopus embryo has provided key insights into fate specification, the cell cycle, and other fundamental developmental and cellular processes, yet a comprehensive understanding of its transcriptome is lacking. Here, we used paired end RNA sequencing (RNA-seq) to explore the transcriptome of Xenopus tropicalis in 23 distinct developmental stages. We determined expression levels of all genes annotated in RefSeq and Ensembl and showed for the first time on a genome-wide scale that, despite a general state of transcriptional silence in the earliest stages of development, approximately 150 genes are transcribed prior to the midblastula transition. In addition, our splicing analysis uncovered more than 10,000 novel splice junctions at each stage and revealed that many known genes have additional unannotated isoforms. Furthermore, we used Cufflinks to reconstruct transcripts from our RNA-seq data and found that ∼13.5% of the final contigs are derived from novel transcribed regions, both within introns and in intergenic regions. We then developed a filtering pipeline to separate protein-coding transcripts from noncoding RNAs and identified a confident set of 6686 noncoding transcripts in 3859 genomic loci. Since the current reference genome, XenTro3, consists of hundreds of scaffolds instead of full chromosomes, we also performed de novo reconstruction of the transcriptome using Trinity and uncovered hundreds of transcripts that are missing from the genome. Collectively, our data will not only aid in completing the assembly of the Xenopus tropicalis genome but will also serve as a valuable resource for gene discovery and for unraveling the fundamental mechanisms of vertebrate embryogenesis.

  16. 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.

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

    Science.gov (United States)

    Dzementsei, Aliaksandr; Schneider, David; Janshoff, Andreas; Pieler, Tomas

    2013-12-15

    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.

  18. Stable magnetic field gradient levitation of Xenopus laevis: toward low-gravity simulation.

    Science.gov (United States)

    Valles, J M; Lin, K; Denegre, J M; Mowry, K L

    1997-08-01

    We have levitated, for the first time, living biological specimens, embryos of the frog Xenopus laevis, using a large inhomogeneous magnetic field. The magnetic field/field gradient product required for levitation was 1430 kG2/cm, consistent with the embryo's susceptibility being dominated by the diamagnetism of water and protein. We show that unlike any other earth-based technique, magnetic field gradient levitation of embryos reduces the body forces and gravity-induced stresses on them. We discuss the use of large inhomogeneous magnetic fields as a probe for gravitationally sensitive phenomena in biological specimens.

  19. Single blastomere expression profiling of Xenopus laevis embryos of 8 to 32-cells reveals developmental asymmetry

    OpenAIRE

    Flachsova, Monika; Sindelka, Radek; Kubista, Mikael

    2013-01-01

    We have measured the expression of 41 maternal mRNAs in individual blastomeres collected from the 8 to 32-cell Xenopus laevis embryos to determine when and how asymmetry in the body plan is introduced. We demonstrate that the asymmetry along the animal-vegetal axis in the oocyte is transferred to the daughter cells during early cell divisions. All studied mRNAs are distributed evenly among the set of animal as well as vegetal blastomeres. We find no asymmetry in mRNA levels that might be ascr...

  20. Stability and movement of mRNAs and their encoded proteins in Xenopus oocytes

    OpenAIRE

    1985-01-01

    The stability and movement of several polyadenylated (poly A+) and nonpolyadenylated (poly A-) mRNAs in Xenopus oocytes have been examined. At least 50% of the poly A+ mRNA molecules (9S rabbit globin mRNA, chicken ovalbumin, and lysozyme) were stable in oocytes over a 48- h period, irrespective of the amount injected. About 50% of injected poly A- reovirus mRNAs was degraded within the first 24 h of injection, irrespective of the amount injected, although no further degradation was observed ...

  1. Relocation of mitochondria to the prospective dorsal marginal zone during Xenopus embryogenesis

    Science.gov (United States)

    Yost, H. J.; Phillips, C. R.; Boore, J. L.; Bertman, J.; Whalon, B.; Danilchik, M. V.

    1995-01-01

    Dorsal-ventral axis formation in Xenopus laevis begins with a cytoplasmic rotation during the first cell cycle and culminates in a series of cell interactions and movements during gastrulation and neurulation that lead to the formation of dorsal-anterior structures. Evidence reported here indicates that mitochondria are differentially redistributed along the prospective dorsal-ventral axis as a consequence of the cortical-cytoplasmic rotation during the first cell cycle. This finding reinvigorates a possibility that has been considered for many years: asymmetries in cytoplasmic components and metabolic activities contribute to the development of morphological asymmetries.

  2. The biological effects of XTC-MIF: quantitative comparison with Xenopus bFGF.

    Science.gov (United States)

    Green, J B; Howes, G; Symes, K; Cooke, J; Smith, J C

    1990-01-01

    Mesoderm in Xenopus and other amphibian embryos is induced by signals from the vegetal hemisphere acting on equatorial or animal hemisphere cells. These signals are diffusible and two classes of candidate signal molecule have been identified: the fibroblast growth factor (FGF) and transforming growth factor beta (TGF-beta) types. In this paper, we compare the effects of cloned Xenopus basic FGF (XbFGF) and electophoretically homogeneous XTC-MIF (a TGF-beta-like factor obtained from a Xenopus cell line) on animal pole explants. We find that they have a similar minimum active concentration (0.1-0.2 ng ml-1) but that, nonetheless, XTC-MIF is at least 40 times more active in inducing muscle. In general, we find that the two factors cause inductions of significantly different characters in terms of tissue type, morphology, gene expression and timing. At low concentrations (0.1-1.0 ng ml-1) both factors induce the differentiation of 'mesenchyme' and 'mesothelium' as well as blood-like cells. These latter cells do not, however, react with an antibody to Xenopus globin. This raised the possibility that the identification of red blood cells in other studies on mesoderm induction might have been mistaken, but combinations of animal pole regions with ventral vegetal pole regions confirmed that genuine erythrocytes are formed. The identity of the blood-like cells formed in response to the inducing factors remains unknown. At higher concentrations XTC-MIF induces neural tissue, notochord, pronephros and substantial and often segmented muscle. By contrast, XbFGF only induces significant amounts of muscle above 24 ng ml-1 and even then this is much less than that induced by XTC-MIF. For both factors an exposure of less than 30 min is effective. Competence of animal pole cells to respond to XbFGF is completely lost by the beginning of gastrulation (stage 10) while competence to XTC-MIF is detectable until somewhat later (stage 11). Since animal pole tissue is known to be able to

  3. SOX7 and SOX18 are essential for cardiogenesis in Xenopus1

    OpenAIRE

    Zhang, Chi; Basta, Tamara; Klymkowsky, Michael W.

    2005-01-01

    Early in vertebrate development, endodermal signals act on mesoderm to induce cardiogenesis. The F-type SOXs SOX7 and SOX18β are expressed in the cardiogenic region of the early Xenopus embryo. Injection of RNAs encoding SOX7 or SOX18β, but not the related F-type SOX, SOX17, leads to the nodal-dependent expression of markers of cardiogenesis in animal cap explants. Injection of morpholinos directed against either SOX7 or SOX18 mRNAs lead to a partial inhibition of cardiogenesis in vivo, while...

  4. sox4 And sox11 Function during Xenopus laevis Eye Development

    OpenAIRE

    Wiebke Cizelsky; Annemarie Hempel; Marlen Metzig; Si Tao; Thomas Hollemann; Michael Kühl; Kühl, Susanne J.

    2013-01-01

    SoxC genes are involved in many developmental processes such as cardiac, lymphoid, and bone development. The SoxC gene family is represented by Sox4, Sox11, and Sox12. Loss of either Sox4 or Sox11 function is lethal during mouse embryogenesis. Here, we demonstrate that sox4 and sox11 are strongly expressed in the developing eye, heart as well as brain in Xenopus laevis. Morpholino oligonucleotide mediated knock-down approaches in anterior neural tissue revealed that interference with either S...

  5. Feedback from luminosity horizontal cells mediates depolarizing responses of chromaticity horizontal cells in the Xenopus retina.

    OpenAIRE

    Witkovsky, P; Gabriel, R; Krizaj, D; Akopian, A.

    1995-01-01

    It has been proposed that the depolarizing responses of chromaticity horizontal cells (C-HCs) to red light depend on a feedback signal from luminosity horizontal cells (L-HCs) to short-wavelength-sensitive cones in the retinas of lower vertebrates. In this regard we studied the C-HCs of the Xenopus retina. C-HCs and L-HCs were identified by physiological criteria and then injected with neurobiotin. The retina then was incubated with peanut agglutinin, which stains red-but not blue-sensitive c...

  6. Demonstration of thymus-independent immune system in Xenopus laevis. Response to polyvinylpyrrolidone.

    Science.gov (United States)

    Tochinai, S

    1976-01-01

    Larvae of Xenopus laevis were thymectomized at stage 45 (4 days old), and raised beyond metamorphosis. Thymectomized toads, when injected with polyvinylpyrrolidone (PVP), produced the same titres of antibodies as non-thymectomized animals, providing strong evidence that the thymus-independent immune response is present in this anuran. Antibodies were of exclusively high molecular weight, heat-stable, and 2-mercaptoethanol sensitive. After immunization, lymphoid accumulations were evident in the splenic white pulp of both thymectomized and non-thymectomized toads, but in the red pulp the accumulations were prominent only in non-thymectomized toads. Images Figure 3 PMID:1027717

  7. Visualisation of cerebrospinal fluid flow patterns in albino Xenopus larvae in vivo

    Science.gov (United States)

    2012-01-01

    Background It has long been known that cerebrospinal fluid (CSF), its composition and flow, play an important part in normal brain development, and ependymal cell ciliary beating as a possible driver of CSF flow has previously been studied in mammalian fetuses in vitro. Lower vertebrate animals are potential models for analysis of CSF flow during development because they are oviparous. Albino Xenopus laevis larvae are nearly transparent and have a straight, translucent brain that facilitates the observation of fluid flow within the ventricles. The aim of these experiments was to study CSF flow and circulation in vivo in the developing brain of living embryos, larvae and tadpoles of Xenopus laevis using a microinjection technique. Methods The development of Xenopus larval brain ventricles and the patterns of CSF flow were visualised after injection of quantum dot nanocrystals and polystyrene beads (3.1 or 5.8 μm in diameter) into the fourth cerebral ventricle at embryonic/larval stages 30-53. Results The fluorescent nanocrystals showed the normal development of the cerebral ventricles from embryonic/larval stages 38 to 53. The polystyrene beads injected into stage 47-49 larvae revealed three CSF flow patterns, left-handed, right-handed and non-biased, in movement of the beads into the third ventricle from the cerebral aqueduct (aqueduct of Sylvius). In the lateral ventricles, anterior to the third ventricle, CSF flow moved anteriorly along the outer wall of the ventricle to the inner wall and then posteriorly, creating a semicircle. In the cerebral aqueduct, connecting the third and fourth cerebral ventricles, CSF flow moved rostrally in the dorsal region and caudally in the ventral region. Also in the fourth ventricle, clear dorso-ventral differences in fluid flow pattern were observed. Conclusions This is the first visualisation of the orchestrated CSF flow pattern in developing vertebrates using a live animal imaging approach. CSF flow in Xenopus albino larvae

  8. Visualizing and Analyzing Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts and TIRF Microscopy.

    Science.gov (United States)

    King, Matthew; Petry, Sabine

    2016-01-01

    Mitotic and meiotic spindles consist primarily of microtubules, which originate from centrosomes and within the vicinity of chromatin. Indirect evidence suggested that microtubules also originate throughout the spindle, but the high microtubule density within the spindle precludes the direct observation of this phenomenon. By using meiotic Xenopus laevis egg extract and employing total internal reflection (TIRF) microscopy, microtubule nucleation from preexisting microtubules could be demonstrated and analyzed. Branching microtubule nucleation is an ideal mechanism to assemble and maintain a mitotic spindle, because microtubule numbers are amplified while preserving their polarity. Here, we describe the assays that made these findings possible and the experiments that helped identify the key molecular players involved.

  9. 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

  10. Cost-effectiveness analysis of therapeutic options for chronic hepatitis C genotype 3 infected patients.

    Science.gov (United States)

    Gimeno-Ballester, Vicente; Mar, Javier; O'Leary, Aisling; Adams, Róisín; San Miguel, Ramón

    2017-01-01

    This study provides a cost-effectiveness analysis of therapeutic strategies for chronic hepatitis C genotype 3 infected patients in Spain. A Markov model was designed to simulate the progression in a cohort of patients aged 50 years over a lifetime horizon. Sofosbuvir (SOF) plus peginterferon and ribavirin for 12 weeks was a cost-effective option when compared to standard of care (SoC) in the treatment of both 'moderate fibrosis' and 'cirrhotic' patients. Incremental cost-effectiveness ratios were €35,276/QALY and €18,374/QALY respectively. ICERs for SOF plus daclatasvir (DCV) regimens versus SoC were over the threshold limit considered, at €56,178/QALY and €77,378/QALY for 'moderate fibrosis' and 'cirrhotic' patients respectively. Addition of SOF to IFN-based regimens for genotype 3 was cost-effective for both 'moderate fibrosis' and 'cirrhotic' patients. IFN-free options including SOF and DCV association required price reductions lower than the list prices to be considered cost-effective.

  11. 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.

  12. Two Piwi proteins, Xiwi and Xili, are expressed in the Xenopus female germline.

    Science.gov (United States)

    Wilczynska, Anna; Minshall, Nicola; Armisen, Javier; Miska, Eric A; Standart, Nancy

    2009-02-01

    The Argonaute superfamily is a large family of RNA-binding proteins involved in gene regulation mediated by small noncoding RNA and characterized by the presence of PAZ and PIWI domains. The family consists of two branches, the Ago and the Piwi clade. Piwi proteins bind to 21-30-nucleotide-long Piwi-interacting RNAs (piRNAs), which map primarily to transposons and repeated sequence elements. Piwi/piRNAs are important regulators of gametogenesis and have been proposed to play roles in transposon silencing, DNA methylation, transcriptional silencing, and/or post-transcriptional control of translation and RNA stability. Most reports to date have concentrated on the Piwi family members in the male germline. We have identified four Piwi proteins in Xenopus and demonstrate that two, namely, Xiwi1b and Xili, are expressed in the oocyte and early embryo. Xiwi1 and Xili are predominantly found in small, separate complexes, and we do not detect significant interaction of Piwi proteins with the cap-binding complex. Putative nuclear localization and export signals were identified in Xiwi1 and Xili, supporting our observation that Xiwi1, but not Xili, is a nucleo-cytoplasmic protein. Furthermore, by immunoprecipitation of small RNAs, we establish Xiwi1 as a bona fide Piwi protein. These results suggest that the Piwi/piRNA pathway is active in translationally repressed oocytes. This is a significant finding as the Xenopus model provides an excellent tool to study post-transcriptional mechanisms.

  13. Xenopus embryos lacking specific isoforms of the corepressor SMRT develop abnormal heads.

    Science.gov (United States)

    Malartre, Marianne; Short, Stephen; Sharpe, Colin

    2006-04-15

    The corepressor SMRT acts on a range of transcription factors, including the retinoid and thyroid hormone nuclear receptors. The carboxy-terminal region of SMRT contains CoRNR box motifs that mediate these interactions. We have shown, in Xenopus, that SMRT can exist as isoforms containing either two or three CoRNR boxes depending on the alternative splicing of exon 37b. The number of SMRT transcript isoforms expressed increases during development until all sixteen possible isoforms are identified in the swimming tadpole. To eliminate specific SMRT isoforms, we have developed a process that uses an antisense morpholino oligonucleotide in Xenopus to dictate the outcome of alternative splicing at a defined exon and used this to inhibit the formation of transcripts containing exon 37b. These embryos are therefore limited to the expression of SMRT isoforms that contain two rather than three CoRNR boxes. Analysis of responsive genes in these embryos shows that targets of thyroid hormone, but not retinoid signaling are affected by the elimination of exon 37b. Morpholino-injected embryos have swimming abnormalities and develop altered head morphology, an expanded olfactory epithelium and disorganized peripheral axons. These experiments indicate a critical role for the alternative splicing of SMRT in development.

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

    Science.gov (United States)

    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.

  15. Ca-sensitive sodium absorption in the colon of Xenopus laevis.

    Science.gov (United States)

    Krattenmacher, R; Voigt, R; Clauss, W

    1990-01-01

    Transepithelial electrogenic Na transport (INa) was investigated in the colon of the frog Xenopus laevis with electrophysiological methods in vitro. The short circuit current (Isc) of the voltage-clamped tissue was 24.2 +/- 1.8 microA.cm-2 (n = 10). About 60% of this current was generated by electrogenic Na transport. Removal of Ca2+ from the mucosal Ringer solution stimulated INa by about 120%. INa was not blockable by amiloride (0.1 mmol.l-1), a specific Na-channel blocker in epithelia, but a fully and reversible inhibition was achieved by mucosal application of 1 mmol.l-1 lanthanum (La3+). No Na-self-inhibition was found, because INa increased linearly with the mucosal Na concentration. A stimulation of INa by antidiuretic hormones was not possible. The analysis of fluctuations in the short circuit current (noise analysis) indicated that Na ions pass the apical cell membrane via a Ca-sensitive ion channel. The results clearly demonstrate that in the colon of Xenopus laevis Na ions are absorbed through Ca-sensitive apical ion channels. They differ considerably in their properties and regulation from the amiloride-sensitive Na channel which is "typically" found in the colon of vertebrates.

  16. Spatially restricted translation of the xCR1 mRNA in Xenopus embryos.

    Science.gov (United States)

    Zhang, Yan; Forinash, Kara D; McGivern, Jered; Fritz, Brian; Dorey, Karel; Sheets, Michael D

    2009-07-01

    The xCR1 protein is a maternal determinant and cofactor for nodal signaling in vertebrate embryos. The xCR1 protein accumulates specifically in the animal cells of Xenopus embryos, but maternal xCR1 mRNA is distributed equally throughout all embryonic cells. Here, we show that vegetal cell-specific translational repression of xCR1 mRNA contributes to this spatially restricted accumulation of the xCR1 protein in Xenopus embryos. xCR1 mRNA was associated with polyribosomes in animal cells but not vegetal cells. A 351-nucleotide region of xCR1 mRNA's 3' untranslated region was sufficient to confer a spatially restricted pattern of translation to a luciferase reporter mRNA by repressing translation in vegetal cells. Repression depended upon the mRNA's 5' cap but not its 3' poly(A) tail. Furthermore, the region of xCR1 mRNA sufficient to confer vegetal cell-specific repression contained both Pumilio binding elements (PBEs) and binding sites for the CUG-BP1 protein. The PBEs and the CUG-BP1 sites were necessary but not sufficient for translation repression. Our studies of xCR1 mRNA document the first example of spatially regulated translation in controlling the asymmetric distribution of a maternal determinant in vertebrates.

  17. EphA7 modulates apical constriction of hindbrain neuroepithelium during neurulation in Xenopus.

    Science.gov (United States)

    Wang, Xiaolei; Sun, Jian; Li, Chaocui; Mao, Bingyu

    2016-10-28

    Eph receptor tyrosine kinases (RTKs) and their ephrin ligands play multiple roles in the developing nervous system, including cell segregation, axon guidance and synaptic plasticity. Here we report the expression and function of EphA7 in Xenopus hindbrain development. EphA7 is specifically expressed in the hindbrain throughout neurulation in Xenopus embryos. Knockdown of EphA7 by specific morpholino oligonucleotide (MO) disrupted cranial neural tube closure and disturbed apical constriction of hindbrain neuroepithelial cells, indicating weakened cell surface tension. In neural plate explants, EphA7 knockdown inhibited apical filamentous actin (F-actin) accumulation. We further showed that EphA7 is involved in the phosphorylation and activation of focal adhesion kinase (FAK) in vivo and in vitro, a key regulator of actin assembly. Our findings reveal that EphA7 functions as a critical regulator of apical constriction of hindbrain neuroepithelial cells. Copyright © 2016. Published by Elsevier Inc.

  18. Generation of Transgenic Xenopus laevis: I. High-Speed Preparation of Egg Extracts.

    Science.gov (United States)

    Ishibashi, Shoko; Kroll, Kristin L; Amaya, Enrique

    2007-09-01

    INTRODUCTIONManipulating genes specifically during later stages of amphibian embryonic development requires fine control over the time and place of expression. These protocols describe an efficient nuclear-transplantation-based method of transgenesis developed for Xenopus laevis. The approach enables stable expression of cloned gene products in Xenopus embryos. Because the transgene integrates into the genome prior to fertilization, the resulting embryos are not chimeric, eliminating the need to breed to the next generation to obtain nonmosaic transgenic animals. The procedure is based on restriction-enzyme-mediated integration (REMI) and can be divided into three parts: (I) high-speed preparation of egg extracts, (II) sperm nuclei preparation, and (III) nuclear transplantation. This protocol describes the method for the high-speed preparation of egg extracts. Briefly, a crude, cytostatic factor (CSF)-arrested egg extract (i.e., cytoplasm arrested in meiotic metaphase) is prepared. These extracts are driven into the interphase stage of the cell cycle by addition of calcium, and high-speed centrifugation is performed to obtain a purer cytoplasmic fraction. This fraction promotes swelling of sperm nuclei, but does not promote DNA replication. By adding the egg extract to the reaction, the sperm chromatin partially decondenses, facilitating integration of plasmid DNA into the genome.

  19. Novel gene expression domains reveal early patterning of the Xenopus endoderm.

    Science.gov (United States)

    Costa, Ricardo M B; Mason, Julia; Lee, Monica; Amaya, Enrique; Zorn, Aaron M

    2003-08-01

    The endoderm gives rise the respiratory and digestive tract epithelia as well as associated organs such as the liver, lungs and pancreas. Investigations examining the molecular basis of embryonic endodermal patterning and organogenesis have been hampered by the lack of regionally expressed molecular markers in the early endoderm. By differentially screening an arrayed cDNA library, combined with an in situ hybridization screen we identified 13 new genes regionally expressed in the early tailbud endoderm of the Xenopus embryo. The putative proteins encoded by these cDNAs include a cell surface transporter, secreted proteins, a protease, a protease inhibitor, an RNA-binding protein, a phosphatase inhibitor and several enzymes. We find that the expression of these genes falls into one of three re-occurring domains in the tailbud embryo; (1). a ventral midgut, (2). posterior to the midgut and (3). in the dorsal endoderm beneath the notochord. Several of these genes are also regionally expressed at gastrula and neurula stages and appear to mark territories that were previously only predicted by the endoderm fate map. This indicates that there is significant positional identity in the early endoderm long before stages 28-32 when regional specification of the endoderm is thought to occur. These new genes provide valuable tools for studying endodermal patterning and organogenesis in Xenopus.

  20. 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. © 2016 Wiley Periodicals, Inc.

  1. Teratogenic effects of organic extracts from the Pearl River sediments on Xenopus laevis embryos.

    Science.gov (United States)

    Zhang, Cong; Liu, Xinhui; Wu, Dan; Liu, Guannan; Tao, Li; Fu, Wenjun; Hou, Jing

    2014-01-01

    Toxicity of organic extracts from the Pearl River sediments was investigated with Xenopus laevis embryos. The effects of sediment organic extracts on the mortality, body length and malformation of X. laevis embryos were tested by the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). The 96-h LC₅₀ values for X. laevis embryos ranged from 62 to 137 g/L (g extracted sediment per L), and the toxicity effect on body length of larvae was not significant under 20 g/L. However, the teratogenic effects produced by sediment organic extracts were diverse, including edema, hypopigmentation, cardiac and ocular malformations, abdomen recurved and curved spine. The percentage of malformations increased with increasing sediment organic extracts, and even reached almost 100% at 10 and 20 g/L in Guangzhou district. A gradient of pollution in the Pearl River sediments was discerned from the teratogenic toxicity. Guangzhou district showed higher teratogenic toxicity compared with Panyu and Nansha districts as a possible consequence of high levels of PAHs, PCBs, OCPs and NP in the sediments. The teratogenic effects of organic extracts from the Pearl River sediments were successfully assessed which indicated the feasibility of teratogenic potential studies of sediments using X. laevis embryos. Copyright © 2013. Published by Elsevier B.V.

  2. 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.

  3. Cadherin transfection of Xenopus XTC cells downregulates expression of substrate adhesion molecules.

    Science.gov (United States)

    Finnemann, S; Kühl, M; Otto, G; Wedlich, D

    1995-09-01

    Cadherins are discussed not in terms of their adhesive function but rather as morphoregulatory proteins. Changes in gene expression following cadherin transfection of cells in culture or by overexpression in embryos have, until now, not been reported. We established a protocol for stable transfection of Xenopus XTC cells and generated cells bearing high levels of membrane-integrated mouse uvomorulin (E-cadherin) or Xenopus XB-cadherin. These cell lines showed drastically impaired substrate adhesion on fibronectin and laminin. In immunoblot and radioimmunoprecipitation experiments, we found that fibronectin and alpha 3/beta 1 integrin are downregulated. The reduced amounts of proteins result from a decrease of the respective mRNAs as proven by RNase protection assays. Coprecipitations revealed that transfected cadherin molecules are complexed with alpha-catenin and beta-catenin at plasma membranes. However, the alpha-catenin present in the XB-cadherin complex differs immunologically from that found in the uvomorulin complex. When a truncated form of XB-cadherin lacking 38 of the most C-terminal amino acids was expressed in XTC cells, complex formation with endogenous catenins was abolished. In these transfectants, substrate adhesion was not affected. These results prove that complex formation of transfected cadherins in XTC cells with endogenous beta-catenin correlates with altered synthesis of certain substrate adhesion molecules.

  4. Autoinduction of thyroid hormone receptor during metamorphosis is reproduced in Xenopus XTC-2 cells.

    Science.gov (United States)

    Machuca, I; Tata, J R

    1992-09-01

    To determine if the autoinduction of thyroid hormone receptor (TR) alpha and beta mRNAs during metamorphosis in Xenopus tadpoles can be reproduced in cultured cells, we have screened four Xenopus cell lines (XTC-2, XL-177, XL2 and Kr) for receptor transcripts and their response to thyroid hormone. Exposure of XTC-2 cells to 10(-9) M triiodothyronine (T3) for 24 h upregulated TR alpha and beta mRNAs by 2-4- and 10-40-fold, respectively. In view of the marked similarity of the differential distribution of the two transcripts and their upregulation by T3 to the pattern of autoinduction seen in whole tadpoles, the process was studied in greater detail in XTC-2 cells. The time-course of autoinduction of TR alpha and beta mRNAs in these cells also resembled that in vivo, the two transcripts being significantly induced by 3-6 h after T3. Dose-response to T3, and the relative responses to its active and inactive analogs, confirmed that the process of autoinduction was initiated by thyroid hormone receptor with the same functional characteristics as that found in all amphibian and mammalian tissues. Experiments performed with cycloheximide suggested that intermediary protein(s) were involved in autoinduction, so that TR genes cannot be considered as 'immediate early' genes for this process. The possible advantages of studying thyroid hormone action in metamorphosis in XTC-2 cells are briefly discussed.

  5. Activin-like factor from a Xenopus laevis cell line responsible for mesoderm induction.

    Science.gov (United States)

    van den Eijnden-Van Raaij, A J; van Zoelent, E J; van Nimmen, K; Koster, C H; Snoek, G T; Durston, A J; Huylebroeck, D

    1990-06-21

    Induction of mesoderm during early amphibian embryogenesis can be mimicked in vitro by adding growth factors, including heparin-binding and type-beta transforming growth factors (TGF-beta), to isolated ectoderm explants from Xenopus laevis embryos. Although the mesoderm-inducing factor (MIF) from X. laevis XTC cells (XTC-MIF) has properties similar to TGF-beta, this factor is still unidentified. Recently, we obtained a number of homogeneous cell lines from the heterogeneous XTC population, which differ in their MIF production. Only one, XTC-GTX-11, produced MIF, although it was similar to the rest of the clones in its production of known growth factors, including TGF-beta activity. This observation, together with the identification of activin A as a potent MIF led us to study the parallel activities of MIF and activin. Here we report an analysis of activin-like activity from XTC cells and some of the XTC clones, including XTC-GTX-11. There is a clear consistent correlation between MIF activity and presence of activin activity, indicating that XTC-MIF is the Xenopus homologue of mammalian activin.

  6. Phylogenetic and expression analysis of amphibian Xenopus Toll-like receptors.

    Science.gov (United States)

    Ishii, Akihiro; Kawasaki, Miyuki; Matsumoto, Misako; Tochinai, Shin; Seya, Tsukasa

    2007-04-01

    An anuran amphibian, South African clawed frog (Xenopus laevis), is used to study the immune system, as it possesses a set of acquired immune system represented by T and B lymphocytes and the immunoglobulins. The acquired immune system is impaired throughout the larva and the metamorphosis stage in the amphibians. On the other hand, the role of innate immune system in the tadpole remains unclear. Recently, insect Toll protein homologues, namely, Toll-like receptors (TLRs), have been identified as sensors recognizing microbe-pattern molecules in vertebrates. Whole-genome analysis of Xenopus tropicalis supported the existence of the tlr genes in the frog. In this study, we annotated 20 frog tlr gene nucleotide sequences from the latest genome assembly version 4.1 on the basis of homology and identified cDNAs of the predicted frog TLR proteins. Phylogenetic analysis showed that the repertoire of the frog TLRs consisted of both fish- and mammalian-type TLRs. We showed that the frog TLRs are constitutively expressed in the tadpole as well as in the adult frog. Our results suggest that tadpoles are protected from microbes by the innate system that includes TLRs, despite impaired acquired immune system in tadpoles. This is the first report on the properties of TLRs in the most primitive terrestrial animals like amphibia.

  7. Effects of barium, lanthanum and gadolinium on endogenous chloride and potassium currents in Xenopus oocytes.

    Science.gov (United States)

    Tokimasa, T; North, R A

    1996-01-01

    1. The effects of multivalent cations on membrane currents recorded from Xenopus oocytes were studied. 2. The hyperpolarization-activated chloride current was reversibly blocked by lanthanum; half-maximal block occurred at a concentration of 8 microM. Zinc, cadmium, cobalt and nickel were less potent than lanthanum, and gadolinium, manganese, barium and strontium had no effect at a concentration of 100 microM. 3. The calcium-activated chloride current was blocked by gadolinium (50 microM), and lanthanum, cadmium, cobalt, nickel and manganese were equally effective. The actions of gadolinium and lanthanum were almost irreversible, while partial (30-80%) recovery was observed with the other cations. Zinc (100 microM) had no effect. 4. In lanthanum (100 microM), membrane depolarizations from -70 mV activated an outward potassium current that was partially blocked by barium (0.1-2 mM). The barium-sensitive current was confined to potentials less negative than -70 mV. The current consisted of a time-independent as well as a time-dependent component, the latter of which had voltage dependence similar to the M-current. 5. It is proposed that lanthanum, gadolinium and barium can usefully separate these endogenous membrane currents in Xenopus oocytes. PMID:8930835

  8. Involvement of Neptune in induction of the hatching gland and neural crest in the Xenopus embryo.

    Science.gov (United States)

    Kurauchi, Takayuki; Izutsu, Yumi; Maéno, Mitsugu

    2010-01-01

    Neptune, a Krüppel-like transcription factor, is expressed in various regions of the developing Xenopus embryo and it has multiple functions in the process of development in various organs. In situ hybridization analysis showed that Neptune is expressed in the boundary region between neural and non-neural tissues at the neurula stage, but little is known about the function of Neptune in this region. Here, we examined the expression and function of Neptune in the neural plate border (NPB) in the Xenopus embryo. Depletion of Neptune protein in developing embryos by using antisense MO caused loss of the hatching gland and otic vesicle as well as malformation of neural crest-derived cranial cartilages and melanocytes. Neptune MO also suppressed the expression of hatching gland and neural crest markers such as he, snail2, sox9 and msx1 at the neurula stage. Subsequent experiments showed that Neptune is necessary and sufficient for the differentiation of hatching gland cells and that it is located downstream of pax3 in the signal regulating the differentiation of these cells. Thus, Neptune is a new member of hatching gland specifier and plays a physiological role in determination and specification of multiple lineages derived from the NPB region.

  9. Eugenol Anesthesia in African Clawed Frogs (Xenopus laevis) of Different Body Weights

    Science.gov (United States)

    Goulet, Félix; Hélie, Pierre; Vachon, Pascal

    2010-01-01

    The objective of this prospective study was to determine the duration of anesthesia in Xenopus laevis frogs of different body weights relative to exposure time in a eugenol (350 µL/L) bath. Two groups of 5 female frogs each weighing 7.5 ± 2.1 g (small frogs) or 29.2 ± 7.4 g (medium frogs) were used. The acetic acid test (AAT), withdrawal reflex, righting reflex, heart rate, and blood oxygen saturation were used to evaluate CNS depression after eugenol bath administration. No responses to the AAT, withdrawal reflex, and righting reflex were seen for 1 h (small frogs) or 0.5 h (medium frogs) after immersion in a eugenol bath for 5 or 10 min, respectively. Oxygen saturation was not affected by anesthesia, but heart rate was depressed for as long as 1 h in both groups of frogs. Surgical anesthesia evaluated by using skin and abdominal incisions revealed that small frogs were anesthetized for a maximum of 15 min compared with 30 min in medium frogs. Frogs showed no ill effects 24 h after eugenol bath administration. These results suggest that body weight is an important parameter to consider when using a eugenol bath for anesthesia of Xenopus frogs. PMID:20819393

  10. Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts.

    Science.gov (United States)

    Bermudez, Jessica G; Chen, Hui; Einstein, Lily C; Good, Matthew C

    2017-01-01

    Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery. © 2017 Wiley Periodicals, Inc.

  11. Fishing on chips: up-and-coming technological advances in analysis of zebrafish and Xenopus embryos.

    Science.gov (United States)

    Zhu, Feng; Skommer, Joanna; Huang, Yushi; Akagi, Jin; Adams, Dany; Levin, Michael; Hall, Chris J; Crosier, Philip S; Wlodkowic, Donald

    2014-11-01

    Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

  12. Ectopic eyes outside the head in Xenopus tadpoles provide sensory data for light-mediated learning.

    Science.gov (United States)

    Blackiston, Douglas J; Levin, Michael

    2013-03-15

    A major roadblock in the biomedical treatment of human sensory disorders, including blindness, has been an incomplete understanding of the nervous system and its ability to adapt to changes in sensory modality. Likewise, fundamental insight into the evolvability of complex functional anatomies requires understanding brain plasticity and the interaction between the nervous system and body architecture. While advances have been made in the generation of artificial and biological replacement components, the brain's ability to interpret sensory information arising from ectopic locations is not well understood. We report the use of eye primordia grafts to create ectopic eyes along the body axis of Xenopus tadpoles. These eyes are morphologically identical to native eyes and can be induced at caudal locations. Cell labeling studies reveal that eyes created in the tail send projections to the stomach and trunk. To assess function we performed light-mediated learning assays using an automated machine vision and environmental control system. The results demonstrate that ectopic eyes in the tail of Xenopus tadpoles could confer vision to the host. Thus ectopic visual organs were functional even when present at posterior locations. These data and protocols demonstrate the ability of vertebrate brains to interpret sensory input from ectopic structures and incorporate them into adaptive behavioral programs. This tractable new model for understanding the robust plasticity of the central nervous system has significant implications for regenerative medicine and sensory augmentation technology.

  13. Xenopus egg extract to study regulation of genome-wide and locus-specific DNA replication.

    Science.gov (United States)

    Raspelli, Erica; Falbo, Lucia; Costanzo, Vincenzo

    2017-01-01

    Faithful DNA replication, coupled with accurate repair of DNA damage, is essential to maintain genome stability and relies on different DNA metabolism genes. Many of these genes are involved in the assembly of replication origins, in the coordination of DNA repair to protect replication forks progression in the presence of DNA damage and in the replication of repetitive chromatin regions. Some DNA metabolism genes are essential in higher eukaryotes, suggesting the existence of specialized mechanisms of repair and replication in organisms with complex genomes. The impact on cell survival of many of these genes has so far precluded in depth molecular analysis of their function. The cell-free Xenopus laevis egg extract represents an ideal system to overcome survival issues and to facilitate the biochemical study of replication-associated functions of essential proteins in vertebrate organisms. Here, we will discuss how Xenopus egg extracts have been used to study cellular and molecular processes, such as DNA replication and DNA repair. In particular, we will focus on innovative imaging and proteomic-based experimental approaches to characterize the molecular function of a number of essential DNA metabolism factors involved in the duplication of complex vertebrate genomes. © 2017 Wiley Periodicals, Inc.

  14. Coexistence of Y, W, and Z sex chromosomes in Xenopus tropicalis

    Science.gov (United States)

    Roco, Álvaro S.; Olmstead, Allen W.; Degitz, Sigmund J.; Amano, Tosikazu; Zimmerman, Lyle B.; Bullejos, Mónica

    2015-01-01

    Homomorphic sex chromosomes and rapid turnover of sex-determining genes can complicate establishing the sex chromosome system operating in a given species. This difficulty exists in Xenopus tropicalis, an anuran quickly becoming a relevant model for genetic, genomic, biochemical, and ecotoxicological research. Despite the recent interest attracted by this species, little is known about its sex chromosome system. Direct evidence that females are the heterogametic sex, as in the related species Xenopus laevis, has yet to be presented. Furthermore, X. laevis’ sex-determining gene, DM-W, does not exist in X. tropicalis, and the sex chromosomes in the two species are not homologous. Here we identify X. tropicalis’ sex chromosome system by integrating data from (i) breeding sex-reversed individuals, (ii) gynogenesis, (iii) triploids, and (iv) crosses among several strains. Our results indicate that at least three different types of sex chromosomes exist: Y, W, and Z, observed in YZ, YW, and ZZ males and in ZW and WW females. Because some combinations of parental sex chromosomes produce unisex offspring and other distorted sex ratios, understanding the sex-determination systems in X. tropicalis is critical for developing this flexible animal model for genetics and ecotoxicology. PMID:26216983

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

    Science.gov (United States)

    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.

  16. Serotonin signaling is required for Wnt-dependent GRP specification and leftward flow in Xenopus.

    Science.gov (United States)

    Beyer, Tina; Danilchik, Michael; Thumberger, Thomas; Vick, Philipp; Tisler, Matthias; Schneider, Isabelle; Bogusch, Susanne; Andre, Philipp; Ulmer, Bärbel; Walentek, Peter; Niesler, Beate; Blum, Martin; Schweickert, Axel

    2012-01-10

    In vertebrates, most inner organs are asymmetrically arranged with respect to the main body axis [1]. Symmetry breakage in fish, amphibian, and mammalian embryos depends on cilia-driven leftward flow of extracellular fluid during neurulation [2-5]. Flow induces the asymmetric nodal cascade that governs asymmetric organ morphogenesis and placement [1, 6, 7]. In the frog Xenopus, an alternative laterality-generating mechanism involving asymmetric localization of serotonin at the 32-cell stage has been proposed [8]. However, no functional linkage between this early localization and flow at neurula stage has emerged. Here, we report that serotonin signaling is required for specification of the superficial mesoderm (SM), which gives rise to the ciliated gastrocoel roof plate (GRP) where flow occurs [5, 9]. Flow and asymmetry were lost in embryos in which serotonin signaling was downregulated. Serotonin, which we found uniformly distributed along the main body axes in the early embryo, was required for Wnt signaling, which provides the instructive signal to specify the GRP. Importantly, serotonin was required for Wnt-induced double-axis formation as well. Our data confirm flow as primary mechanism of symmetry breakage and suggest a general role of serotonin as competence factor for Wnt signaling during axis formation in Xenopus. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. High-Throughput Sequencing of MicroRNAs in Adenovirus Type 3 Infected Human Laryngeal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yuhua Qi

    2010-01-01

    Full Text Available Adenovirus infection can cause various illnesses depending on the infecting serotype, such as gastroenteritis, conjunctivitis, cystitis, and rash illness, but the infection mechanism is still unknown. MicroRNAs (miRNA have been reported to play essential roles in cell proliferation, cell differentiation, and pathogenesis of human diseases including viral infections. We analyzed the miRNA expression profiles from adenovirus type 3 (AD3 infected Human laryngeal epithelial (Hep2 cells using a SOLiD deep sequencing. 492 precursor miRNAs were identified in the AD3 infected Hep2 cells, and 540 precursor miRNAs were identified in the control. A total of 44 miRNAs demonstrated high expression and 36 miRNAs showed lower expression in the AD3 infected cells than control. The biogenesis of miRNAs has been analyzed, and some of the SOLiD results were confirmed by Quantitative PCR analysis. The present studies may provide a useful clue for the biological function research into AD3 infection.

  18. 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.

    1975-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  20. Skeletal callus formation is a nerve‐independent regenerative response to limb amputation in mice and Xenopus

    Science.gov (United States)

    Miura, Shinichirou; Takahashi, Yumiko; Satoh, Akira

    2015-01-01

    Abstract To clarify the mechanism of limb regeneration that differs between mammals (non‐regenerative) and amphibians (regenerative), responses to limb amputation and the accessory limb inducible surgery (accessory limb model, ALM) were compared between mice and Xenopus, focusing on the events leading to blastema formation. In both animals, cartilaginous calluses were formed around the cut edge of bones after limb amputation. They not only are morphologically similar but show other similarities, such as growth driven by undifferentiated cell proliferation and macrophage‐dependent and nerve‐independent induction. It appears that amputation callus formation is a common nerve‐independent regenerative response in mice and Xenopus. In contrast, the ALM revealed that the wound epithelium (WE) in Xenopus was innervated by many regenerating axons when a severed nerve ending was placed underneath it, whereas only a few axons were found within the WE in mice. Since nerves are involved in induction of the regeneration‐permissive WE in amphibians, whether or not nerves can interact with the WE might be one of the key processes separating successful nerve‐dependent blastema formation in Xenopus and failure in mice. PMID:27499875

  1. Proteomic analysis of fibroblastema formation in regenerating hind limbs of Xenopus laevis froglets and comparison to axolotl

    Science.gov (United States)

    2014-01-01

    Background To gain insight into what differences might restrict the capacity for limb regeneration in Xenopus froglets, we used High Performance Liquid Chromatography (HPLC)/double mass spectrometry to characterize protein expression during fibroblastema formation in the amputated froglet hindlimb, and compared the results to those obtained previously for blastema formation in the axolotl limb. Results Comparison of the Xenopus fibroblastema and axolotl blastema revealed several similarities and significant differences in proteomic profiles. The most significant similarity was the strong parallel down regulation of muscle proteins and enzymes involved in carbohydrate metabolism. Regenerating Xenopus limbs differed significantly from axolotl regenerating limbs in several ways: deficiency in the inositol phosphate/diacylglycerol signaling pathway, down regulation of Wnt signaling, up regulation of extracellular matrix (ECM) proteins and proteins involved in chondrocyte differentiation, lack of expression of a key cell cycle protein, ecotropic viral integration site 5 (EVI5), that blocks mitosis in the axolotl, and the expression of several patterning proteins not seen in the axolotl that may dorsalize the fibroblastema. Conclusions We have characterized global protein expression during fibroblastema formation after amputation of the Xenopus froglet hindlimb and identified several differences that lead to signaling deficiency, failure to retard mitosis, premature chondrocyte differentiation, and failure of dorsoventral axial asymmetry. These differences point to possible interventions to improve blastema formation and pattern formation in the froglet limb. PMID:25063185

  2. Isolation and characterization of TGF-beta 2 and TGF-beta 5 from medium conditioned by Xenopus XTC cells.

    Science.gov (United States)

    Roberts, A B; Rosa, F; Roche, N S; Coligan, J E; Garfield, M; Rebbert, M L; Kondaiah, P; Danielpour, D; Kehrl, J H; Wahl, S M

    1990-01-01

    TGF-beta 2 and -beta 5 have been purified from medium conditioned by Xenopus cultured cells (XTC) and identified based on their N-terminal amino acid sequence analysis and biological activity. When applied in high concentrations, Xenopus TGF-beta 2, like porcine TGF-beta 2, induces expression of mesodermal markers from cultured Xenopus ectodermal explants, whereas TGF-beta 5 is inactive in this assay. However, the TGF-beta 's could be separated from the major mesoderm-inducing activity present in XTC medium. Xenopus TGF-beta 2 and -beta 5 are approximately equivalent to TGF-beta 1 in their abilities to inhibit the growth of mink lung CCL-64 cells, induce anchorage-independent growth of rat NRK cells, inhibit the proliferation and antibody secretion of human B-lymphocytes, and stimulate chemotaxis of human monocytes. These data establish the functional activity of TGF-beta 5 and suggest that more complex multicellular systems, in contrast to most isolated cells, discriminate between the different TGF-beta s.

  3. 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…

  4. Toxicity of CuO nanoparticles and Cu ions to tight epithelial cells from Xenopus laevis (A6)

    DEFF Research Database (Denmark)

    Thit, Amalie; Selck, Henriette; Bjerregaard, Henning F.

    2013-01-01

    Nanoparticles (NPs) have unique chemical and physical properties caused by their small size (1–100 nm) and high surface to volume ratio. This means that the NPs are potentially more toxic than their bulk counterparts. In the present study a cultured epithelial cell line from Xenopus laevis (A6...

  5. A chemical and pharmacological study on the role of catecholamines in the dispersion reaction of Xenopus laevis

    NARCIS (Netherlands)

    Brouwer, E.

    Chemical analyses have been made of dopamine in the skin of black backgroundapted Xenopus laevis treated with α-methyl-p-tyrosine (α-MPT), an inhibitor of tyrosine hydroxylase, and without such treatment. Based on the assumption that dopamine is involved in the dispersion reaction induced by MSH,

  6. Calcium influx factor (CIF) as a diffusible messenger for the activation of capacitative calcium entry in Xenopus oocytes.

    Science.gov (United States)

    Kim, H Y; Hanley, M R

    1999-06-30

    Acid extracts of thapsigargin-treated Xenopus oocytes revealed Ca2(+)-dependent Cl- currents by microinjection into Xenopus oocytes. These currents were detected in highly purified fractions by carrying out a sequence of purification steps including gel filtration chromatography and high performance thin layer chromatography. The nature of the membrane currents evoked by the highly purified fractions were carried by chloride ions as blockade by the selective chloride channel blocker 1 mM niflumic acid. Injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) eradicated the current activities, indicating that the current responses are completely Ca2(+)-dependent. Moreover, the currents were sensitive to the removal of extracellular calcium, indicating the dependence on calcium entry through plasma membrane calcium entry channels. These results elucidate that the highly purified fractions aquired by thapsigargin-stimulated oocytes is an authentic calcium influx factor (CIF). Thus, the detection of increased CIF production from thapsigargin treatment in Xenopus oocytes would give strong support for the existence of CIF as a diffusible messenger for the activation of capacitative calcium entry pathways in Xenopus oocytes.

  7. 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.

  8. Chikungunya virus

    Science.gov (United States)

    Chikungunya virus infection; Chikungunya ... Where Chikungunya is Found Before 2013, the virus was found in Africa, Asia, Europe, and the Indian and Pacific oceans. In late 2013, outbreaks occurred for the first time in the ...

  9. Zika Virus

    Science.gov (United States)

    ... through blood transfusions. There have been outbreaks of Zika virus in the United States, Africa, Southeast Asia, the ... not travel to areas where there is a Zika virus outbreak. If you do decide to travel, first ...

  10. Chikungunya Virus

    Science.gov (United States)

    ... Gaines, PhD, MPH, MA, CHES Differentiating Chikungunya From Dengue: A Clinical Challenge For Travelers CDC Travelers' Health Chikungunya Virus Home Prevention Transmission Symptoms & Treatment Geographic Distribution Chikungunya virus in the United States ...

  11. Zika Virus

    Science.gov (United States)

    ... Funding CDC Activities For Healthcare Providers Clinical Evaluation & Disease Sexual Transmission HIV Infection & Zika Virus Testing for Zika Test Specimens – At Time of Birth Diagnostic Tests Understanding Zika Virus Test Results ...

  12. Desynchronizing Embryonic Cell Division Waves Reveals the Robustness of Xenopus laevis Development.

    Science.gov (United States)

    Anderson, Graham A; Gelens, Lendert; Baker, Julie C; Ferrell, James E

    2017-10-03

    The early Xenopus laevis embryo is replete with dynamic spatial waves. One such wave, the cell division wave, emerges from the collective cell division timing of first tens and later hundreds of cells throughout the embryo. Here, we show that cell division waves do not propagate between neighboring cells and do not rely on cell-to-cell coupling to maintain their division timing. Instead, intrinsic variation in division period autonomously and gradually builds these striking patterns of cell division. Disrupting this pattern of division by placing embryos in a temperature gradient resulted in highly asynchronous entry to the midblastula transition and misexpression of the mesodermal marker Xbra. Remarkably, this gene expression defect is corrected during involution, resulting in delayed yet normal Xbra expression and viable embryos. This implies the existence of a previously unknown mechanism for normalizing mesodermal gene expression during involution. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Polyunsaturated fatty acids are potent openers of human M-channels expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Liin, Sara I; Karlsson, Urban; Bentzen, Bo Hjorth

    2016-01-01

    the threshold current to evoke action potentials in dorsal root ganglion neurons. The polyunsaturated fatty acids docosahexaenoic acid, α-linolenic acid, and eicosapentaenoic acid facilitated opening of the human M-channel, comprised of the heteromeric human KV 7.2/3 channel expressed in Xenopus oocytes......, by shifting the conductance-versus-voltage curve towards more negative voltages (by -7.4 to -11.3 mV by 70 μM). Uncharged docosahexaenoic acid methyl ester and monounsaturated oleic acid did not facilitate opening of the human KV 7.2/3 channel. CONCLUSIONS: These findings suggest that circulating...... polyunsaturated fatty acids, with a minimum requirement of multiple double bonds and a charged carboxyl group, dampen excitability by opening neuronal M-channels. Collectively, our data bring light to the molecular targets of polyunsaturated fatty acids and thus a possible mechanism by which polyunsaturated fatty...

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

    Science.gov (United States)

    Lu, Ping; Ren, Min; Zhai, Zhonghe

    2002-12-01

    The cell-free extracts from animal Xenopus laevis egg could induce chromatin decondensation and pronuclear formation from demembranated plant (Orychophragmus violaceu) 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.

  15. Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts.

    Directory of Open Access Journals (Sweden)

    David Halpin

    2011-12-01

    Full Text Available During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 µm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1, the Guanine Nucleotide Exchange Factor (GEF for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.

  16. Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

    DEFF Research Database (Denmark)

    Østrup, Olga; Hyttel, Poul; Klærke, Dan Arne

    2011-01-01

    complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract......Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression....... This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling...

  17. First parasitological study of the African clawed frog (Xenopus laevis, Amphibia in Chile

    Directory of Open Access Journals (Sweden)

    Cristóbal Castillo

    Full Text Available Abstract Introduced species can arrive into new territories with parasites; however, these species are expected to face lower parasite richness than in their original regions. Both introduced hosts and parasites can affect native fauna. Since their release into the wild in Chile following laboratory use, Xenopus laevis Daudin, 1802 has widely spread throughout central Chile. The only pathogen described on the host is the fungus Batrachochytrium dendrobatidis Longcore, Pessier, Nichols, 1999; thus, this is the first parasitological study of this species in Chile. In 10 localities in central Chile, 179 specimens of X. laevis were captured and examined for parasites in the gastrointestinal tube, cavities, lungs, liver, and skin. Only nine specimens of the genus Contracaecum Railliet, Henry, 1912 were found in six specimens of X. laevis from a private dam in La Patagua. It is likely that these parasites originated from species of native birds. This is the first record of Contracaecum sp. in Chilean amphibians.

  18. Pilot morpholino screen in Xenopus tropicalis identifies a novel gene involved in head development.

    Science.gov (United States)

    Kenwrick, Sue; Amaya, Enrique; Papalopulu, Nancy

    2004-02-01

    The diploid frog X. tropicalis has recently been adopted as a model genetic system, but loss-of-function screens in Xenopus have not yet been performed. We have undertaken a pilot functional knockdown screen in X. tropicalis for genes involved in nervous system development by injecting antisense morpholino (MO) oligos directed against X. tropicalis mRNAs. Twenty-six genes with primary expression in the nervous system were selected as targets based on an expression screen previously conducted in X. laevis. Reproducible phenotypes were observed for six and for four of these, a second MO gave a similar result. One of these genes encodes a novel protein with previously unknown function. Knocking down this gene, designated pinhead, results in severe microcephaly, whereas, overexpression results in macrocephaly. Together with the early embryonic expression in the anterior neural plate, these data indicate that pinhead is a novel gene involved in controlling head development. Copyright 2003 Wiley-Liss, Inc.

  19. Maintenance of motor neuron progenitors in Xenopus requires a novel localized cyclin.

    Science.gov (United States)

    Chen, Jun-An; Chu, Sin-Tak; Amaya, Enrique

    2007-03-01

    The ventral spinal cord contains a pool of motor neuron progenitors (pMNs), which sequentially generate motor neurons and oligodendrocytes in the embryo. The mechanisms responsible for the maintenance of pMNs are not clearly understood. We have identified a novel cyclin, cyclin Dx (ccndx), which is specifically expressed in pMNs in Xenopus. Here, we show that inhibition of ccndx causes paralysis in embryos. Furthermore, we show that maintenance of pMNs requires ccndx function. In addition, inhibition of ccndx results in the specific loss of differentiated motor neurons. However, the expression of interneuron or sensory neuron markers is unaffected in these embryos, suggesting that the role of ccndx is specifically to maintain pMNs. Thus, we have identified, for the first time, a tissue-specific cell-cycle regulator that is essential for the maintenance of a pool of neural progenitors in the vertebrate spinal cord.

  20. Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes.

    Science.gov (United States)

    Jarecki, Brian W; Makino, Shin-ichi; Beebe, Emily T; Fox, Brian G; Chanda, Baron

    2013-01-01

    Voltage-gated ion channels are a class of membrane proteins that temporally orchestrate the ion flux critical for chemical and electrical signaling in excitable cells. Current methods to investigate the function of these channels rely on heterologous expression in living systems or reconstitution into artificial membranes; however these approaches have inherent drawbacks which limit potential biophysical applications. Here, we describe a new integrated approach combining cell-free translation of membrane proteins and in vivo expression using Xenopus laevis oocytes. In this method, proteoliposomes containing Shaker potassium channels are synthesized in vitro and injected into the oocytes, yielding functional preparations as shown by electrophysiological and fluorescence measurements within few hours. This strategy for studying eukaryotic ion channels is contrasted with existing, laborious procedures that require membrane protein extraction and reconstitution into synthetic lipid systems.

  1. Neurotransmitter signaling pathways required for normal development in Xenopus laevis embryos: a pharmacological survey screen.

    Science.gov (United States)

    Sullivan, Kelly G; Levin, Michael

    2016-10-01

    Neurotransmitters are not only involved in brain function but are also important signaling molecules for many diverse cell types. Neurotransmitters are widely conserved, from evolutionarily ancient organisms lacking nervous systems through man. Here, results are reported from a loss- and gain-of-function survey, using pharmacological modulators of several neurotransmitter pathways to examine possible roles for these pathways in normal embryogenesis. Applying reagents targeting the glutamatergic, adrenergic and dopaminergic pathways to embryos of Xenopus laevis from gastrulation to organogenesis stages, we observed and quantified numerous malformations, including craniofacial defects, hyperpigmentation, muscle mispatterning and miscoiling of the gut. These data implicate several key neurotransmitters in new embryonic patterning roles, reveal novel earlier stages for processes involved in eye development, suggest new targets for subsequent molecular-genetic investigation, and highlight the necessity for in-depth toxicology studies of psychoactive compounds to which human embryos might be exposed during pregnancy. © 2016 Anatomical Society.

  2. Uptake of /sup 35/S sulphate by Xenopus cartilage. The influence of growth hormone and prolactin

    Energy Technology Data Exchange (ETDEWEB)

    Ishii, Takehisa; Kikuyama, Sakae (Waseda Univ., Tokyo (Japan))

    1984-08-01

    Hypohysectomized juvenile Xenopus were injected with growth hormone (GH) or prolactin (PRL) of either ovine or bullfrog origin. The growth promoting activity of these hormones was measured by monitoring the uptake of /sup 35/S sulphate by the xiphisternal cartilage in vitro. Analysis of the labelled cartilage revealed that the acid mucopolysaccharide fraction contained about 60-80 % of the label most of which was incorporated into chondroitin sulphates. All of the hormones tested enhaced the /sup 35/S sulphate uptake dose-dependently. Among them bullfrog GH was most effective, then followed ovine GH and ovine PRL. Bullfrog PRL was far less effective than other three. The sensitive assay for frog GH developed in the present experiment may be applicable to the assay for somatomedin-like activity and contribute to the analysis of the mode of action of GH in amphibians.

  3. Movements of the large intestine in the anuran larvae, Xenopus laevis.

    Science.gov (United States)

    Naitoh, T; Miura, A; Akiyoshi, H; Wassersug, R J

    1990-01-01

    1. The contractile behavior of the large intestine of Xenopus laevis tadpoles was studied. 2. The large intestine is divided into a colon and rectum, and shows three types of movements: rhythmic ascending (antiperistaltic) waves of contraction originating at the anal end of the large bowel, rhythmic longitudinal contractions in the rectum and colon, and irregular contractions. The first two patterns occur in the large bowel in situ and thus appear mature. The last one occurred only in older preparations, and thus appeared pathological. 3. Antiperistaltic waves of contractions and longitudinal contractions are generated independent of each other, suggesting that circular muscles and longitudinal muscles contract separately. 4. Acetylcholine, adrenaline and noradrenaline augment motility. 5. The premetamorphic motility of the large bowel is similar to that seen in adult frogs. Comparable motility was not observed elsewhere in the larval alimentary tract. The large intestine appears to be the first portion of the anuran alimentary tract to acquire the adult physiological and morphological profile.

  4. 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 nociception induced by an abdominal laparotomy and consequently cannot be used to evaluate analgesics in X. leavis frogs. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  5. Pre-meiotic transformation of germplasm-related structures during male gamete differentiation in Xenopus laevis.

    Science.gov (United States)

    Reunov, Arkadiy A; Reunova, Yulia A

    2016-02-01

    To highlight the ultrastructural features of transformation occurring with germplasm-related structures (GPRS), the spermatogenic cells of Xenopus laevis were studied by transmission electron microscopy and quantitative analysis. It was determined that in spermatogonia and spermatocytes, the compact germinal granules underwent fragmentation into particles comparable with inter-mitochondrial cement (IMC). Fragments of IMC agglutinated some cell mitochondria and resulted in the creation of mitochondrial clusters. Clustered mitochondria responded with loss of their membranes that occurred by the twisting of membranous protrusions around themselves until multi-layered membranes were formed. The mitochondrial affinity of multi-layered membranes was proven by an immunopositive test for mitochondrial dihydrolipoamide acetyltransferase. As a consequence of mitochondrial membrane twisting, the naked mitochondrial cores appeared and presumably underwent dispersion, which is the terminal stage of GPRS transformation. As no GPRS were observed in spermatids and sperm, it was assumed that these structures are functionally assigned to early stages of meiotic differentiation.

  6. Bioconcentration and Metabolism of Pyriproxyfen in Tadpoles of African Clawed Frogs, Xenopus laevis.

    Science.gov (United States)

    Ose, Keiko; Miyamoto, Mitsugu; Fujisawa, Takuo; Katagi, Toshiyuki

    2017-11-22

    Bioconcentration and metabolism of pyriproxyfen uniformly labeled with 14C at the phenoxyphenyl ring were studied using tadpoles of African clawed frog, Xenopus laevis, exposed to water at the nominal concentrations of 3 and 300 ppb for 22 days under the flow-through conditions, with a following 3 day depuration phase. Neither meaningful mortality nor abnormal behavior was observed in control and exposure groups throughout the study. After the rapid uptake to tadpoles, pyriproxyfen was extensively metabolized and excreted, and as a result, steady-state bioconcentration factors and depuration half-lives ranged from 550 to 610 and from 0.34 to 0.54 days, respectively. The metabolites were mostly distributed in the liver or gastrointestinal tract. The major metabolic reactions were hydroxylation at the 4' position of the phenoxyphenyl group and cleavage of the ether linkage, followed by sulfate conjugation.

  7. The Xenopus Emx genes identify presumptive dorsal telencephalon and are induced by head organizer signals.

    Science.gov (United States)

    Pannese, M; Lupo, G; Kablar, B; Boncinelli, E; Barsacchi, G; Vignali, R

    1998-04-01

    We have isolated and studied the expression pattern of Xemx1 and Xemx2 genes in Xenopus laevis. Xemx genes are the homologues of mouse Emx genes, related to Drosophila empty spiracles. They are expressed in selected regions of the developing brain, particularly in the telencephalon, and, outside the brain, in the otic vesicles, olfactory placodes, visceral arches and the developing excretory system. We also report on experiments concerning the tissue and molecular signals responsible for their activation in competent ectoderm. Xemx genes are activated in ectoderm conjugated with head organizer tissue, but not with tail organizer tissue. Furthermore, they are not activated in animal cap either by noggin or by Xnr3, thus suggesting that a different inducer or the integration of several signals may be responsible for their activation.

  8. 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

    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....... 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...

  9. Efficient Preparation of High-Complexity ChIP-Seq Profiles from Early Xenopus Embryos.

    Science.gov (United States)

    Gentsch, George E; Smith, James C

    2017-01-01

    Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) has become a powerful tool to acquire a precise and genome-wide snapshot of many chromatin features in vivo. These chromatin profiles are obtained by immunoprecipitation of cross-linked chromatin fragments to enrich the feature of interest. Sequencing and aligning the underlying DNA sequences to the genome make it possible to virtually reconstruct the global distribution of most chromatin features. We present here recent improvements to the ChIP-seq protocol by means of Xenopus embryos to prepare high-complexity DNA libraries from small amounts of biological material. This approach allows researchers to explore the landscape of chromatin regulators and states in early vertebrate embryos or in any biological entity with small numbers of cells.

  10. Involvement of slingshot in the Rho-mediated dephosphorylation of ADF/cofilin during Xenopus cleavage.

    Science.gov (United States)

    Tanaka, Kenji; Okubo, Yoshiko; Abe, Hiroshi

    2005-09-01

    ADF/cofilin is a key regulator for actin dynamics during cytokinesis. Its activity is suppressed by phosphorylation and reactivated by dephosphorylation. Little is known, however, about regulatory mechanisms of ADF/cofilin function during formation of contractile ring actin filaments. Using Xenopus cycling extracts, we found that ADF/cofilin was dephosphorylated at prophase and telophase. In addition, constitutively active Rho GTPase induced dephosphorylation of ADF/cofilin in the egg extracts. This dephosphorylation was inhibited by Na(3)VO (4) but not by other conventional phosphatase-inhibitors. We cloned a Xenopus homologue of Slingshot phosphatase (XSSH), originally identified in Drosophila and human as an ADF/cofilin phosphatase, and raised antibody specific for the catalytic domain of XSSH. This inhibitory antibody significantly suppressed the Rho-induced dephosphorylation of ADF/cofilin in extracts, suggesting that the dephosphorylation at telophase is dependent on XSSH. XSSH bound to actin filaments with a dissociation constant of 0.4 microM, and the ADF/cofilin phosphatase activity was increased in the presence of F-actin. When latrunculin A, a G-actin-sequestering drug, was added to extracts, both Rho-induced actin polymerization and dephosphorylation of ADF/cofilin were markedly inhibited. Jasplakinolide, an actin-stabilizing drug, alone induced actin polymerization in the extracts and lead to dephosphorylation of ADF/cofilin. These results suggest that Rho-induced dephosphorylation of ADF/cofilin is dependent on the XSSH activation that is caused by increase in the amount of F-actin induced by Rho signaling. XSSH colocalized with both actin filaments and ADF/cofilin in the actin patches formed on the surface of the early cleavage furrow. Injection of inhibitory antibody blocked cleavage of blastomeres. Thus, XSSH may reorganize actin filaments through dephosphorylation and reactivation of ADF/cofilin at early stage of contractile ring formation.

  11. Mechanical roles of apical constriction, cell elongation, and cell migration during neural tube formation in Xenopus.

    Science.gov (United States)

    Inoue, Yasuhiro; Suzuki, Makoto; Watanabe, Tadashi; Yasue, Naoko; Tateo, Itsuki; Adachi, Taiji; Ueno, Naoto

    2016-12-01

    Neural tube closure is an important and necessary process during the development of the central nervous system. The formation of the neural tube structure from a flat sheet of neural epithelium requires several cell morphogenetic events and tissue dynamics to account for the mechanics of tissue deformation. Cell elongation changes cuboidal cells into columnar cells, and apical constriction then causes them to adopt apically narrow, wedge-like shapes. In addition, the neural plate in Xenopus is stratified, and the non-neural cells in the deep layer (deep cells) pull the overlying superficial cells, eventually bringing the two layers of cells to the midline. Thus, neural tube closure appears to be a complex event in which these three physical events are considered to play key mechanical roles. To test whether these three physical events are mechanically sufficient to drive neural tube formation, we employed a three-dimensional vertex model and used it to simulate the process of neural tube closure. The results suggest that apical constriction cued the bending of the neural plate by pursing the circumference of the apical surface of the neural cells. Neural cell elongation in concert with apical constriction further narrowed the apical surface of the cells and drove the rapid folding of the neural plate, but was insufficient for complete neural tube closure. Migration of the deep cells provided the additional tissue deformation necessary for closure. To validate the model, apical constriction and cell elongation were inhibited in Xenopus laevis embryos. The resulting cell and tissue shapes resembled the corresponding simulation results.

  12. 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

  13. Vestibular lesion-induced developmental plasticity in spinal locomotor networks during Xenopus laevis metamorphosis.

    Directory of Open Access Journals (Sweden)

    Anna Beyeler

    Full Text Available During frog metamorphosis, the vestibular sensory system remains unchanged, while spinal motor networks undergo a massive restructuring associated with the transition from the larval to adult biomechanical system. We investigated in Xenopus laevis the impact of a pre- (tadpole stage or post-metamorphosis (juvenile stage unilateral labyrinthectomy (UL on young adult swimming performance and underlying spinal locomotor circuitry. The acute disruptive effects on locomotion were similar in both tadpoles and juvenile frogs. However, animals that had metamorphosed with a preceding UL expressed restored swimming behavior at the juvenile stage, whereas animals lesioned after metamorphosis never recovered. Whilst kinematic and electrophysiological analyses of the propulsive system showed no significant differences in either juvenile group, a 3D biomechanical simulation suggested that an asymmetry in the dynamic control of posture during swimming could account for the behavioral restoration observed in animals that had been labyrinthectomized before metamorphosis. This hypothesis was subsequently supported by in vivo electromyography during free swimming and in vitro recordings from isolated brainstem/spinal cord preparations. Specifically, animals lesioned prior to metamorphosis at the larval stage exhibited an asymmetrical propulsion/posture coupling as a post-metamorphic young adult. This developmental alteration was accompanied by an ipsilesional decrease in propriospinal coordination that is normally established in strict left-right symmetry during metamorphosis in order to synchronize dorsal trunk muscle contractions with bilateral hindlimb extensions in the swimming adult. Our data thus suggest that a disequilibrium in descending vestibulospinal information during Xenopus metamorphosis leads to an altered assembly of adult spinal locomotor circuitry. This in turn enables an adaptive compensation for the dynamic postural asymmetry induced by the

  14. Molecular asymmetry in the 8-cell stage Xenopus tropicalis embryo described by single blastomere transcript sequencing.

    Science.gov (United States)

    De Domenico, Elena; Owens, Nick D L; Grant, Ian M; Gomes-Faria, Rosa; Gilchrist, Michael J

    2015-12-15

    Correct development of the vertebrate body plan requires the early definition of two asymmetric, perpendicular axes. The first axis is established during oocyte maturation, and the second is established by symmetry breaking shortly after fertilization. The physical processes generating the second asymmetric, or dorsal-ventral, axis are well understood, but the specific molecular determinants, presumed to be maternal gene products, are poorly characterized. Whilst enrichment of maternal mRNAs at the animal and vegetal poles in both the oocyte and the early embryo has been studied, little is known about the distribution of maternal mRNAs along either the dorsal-ventral or left-right axes during the early cleavage stages. Here we report an unbiased analysis of the distribution of maternal mRNA on all axes of the Xenopus tropicalis 8-cell stage embryo, based on sequencing of single blastomeres whose positions within the embryo are known. Analysis of pooled data from complete sets of blastomeres from four embryos has identified 908 mRNAs enriched in either the animal or vegetal blastomeres, of which 793 are not previously reported as enriched. In contrast, we find no evidence for asymmetric distribution along either the dorsal-ventral or left-right axes. We confirm that animal pole enrichment is on average distinctly lower than vegetal pole enrichment, and that considerable variation is found between reported enrichment levels in different studies. We use publicly available data to show that there is a significant association between genes with human disease annotation and enrichment at the animal pole. Mutations in the human ortholog of the most animally enriched novel gene, Slc35d1, are causative for Schneckenbecken dysplasia, and we show that a similar phenotype is produced by depletion of the orthologous protein in Xenopus embryos. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Stage-specific histone modification profiles reveal global transitions in the Xenopus embryonic epigenome.

    Directory of Open Access Journals (Sweden)

    Tobias D Schneider

    Full Text Available Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development.

  16. Exploring nervous system transcriptomes during embryogenesis and metamorphosis in Xenopus tropicalis using EST analysis

    Directory of Open Access Journals (Sweden)

    Wegnez Maurice

    2007-05-01

    Full Text Available Abstract Background The western African clawed frog Xenopus tropicalis is an anuran amphibian species now used as model in vertebrate comparative genomics. It provides the same advantages as Xenopus laevis but is diploid and has a smaller genome of 1.7 Gbp. Therefore X. tropicalis is more amenable to systematic transcriptome surveys. We initiated a large-scale partial cDNA sequencing project to provide a functional genomics resource on genes expressed in the nervous system during early embryogenesis and metamorphosis in X. tropicalis. Results A gene index was defined and analysed after the collection of over 48,785 high quality sequences. These partial cDNA sequences were obtained from an embryonic head and retina library (30,272 sequences and from a metamorphic brain and spinal cord library (27,602 sequences. These ESTs are estimated to represent 9,693 transcripts derived from an estimated 6,000 genes. Comparison of these cDNA sequences with protein databases indicates that 46% contain their start codon. Further annotation included Gene Ontology functional classification, InterPro domain analysis, alternative splicing and non-coding RNA identification. Gene expression profiles were derived from EST counts and used to define transcripts specific to metamorphic stages of development. Moreover, these ESTs allowed identification of a set of 225 polymorphic microsatellites that can be used as genetic markers. Conclusion These cDNA sequences permit in silico cloning of numerous genes and will facilitate studies aimed at deciphering the roles of cognate genes expressed in the nervous system during neural development and metamorphosis. The genomic resources developed to study X. tropicalis biology will accelerate exploration of amphibian physiology and genetics. In particular, the model will facilitate analysis of key questions related to anuran embryogenesis and metamorphosis and its associated regulatory processes.

  17. Regeneration of Xenopus laevis spinal cord requires Sox2/3 expressing cells

    Science.gov (United States)

    Muñoz, Rosana; Edwards-Faret, Gabriela; Moreno, Mauricio; Zuñiga, Nikole; Cline, Hollis; Larraín, Juan

    2016-01-01

    Spinal cord regeneration is very inefficient in humans, causing paraplegia and quadriplegia. Studying model organisms that can regenerate the spinal cord in response to injury could be useful for understanding the cellular and molecular mechanisms that explain why this process fails in humans. Here, we use Xenopus laevis as a model organism to study spinal cord repair. Histological and functional analyses showed that larvae at pre-metamorphic stages restore anatomical continuity of the spinal cord and recover swimming after complete spinal cord transection. These regenerative capabilities decrease with onset of metamorphosis. The ability to study regenerative and non-regenerative stages in Xenopus laevis makes it a unique model system to study regeneration. We studied the response of Sox2/3 expressing cells to spinal cord injury and their function in the regenerative process. We found that cells expressing Sox2 and/or Sox3 are present in the ventricular zone of regenerative animals and decrease in non-regenerative froglets. Bromodeoxyuridine (BrdU) experiments and in vivo time-lapse imaging studies using green fluorescent protein (GFP) expression driven by the Sox3 promoter showed a rapid, transient and massive proliferation of Sox2/3+ cells in response to injury in the regenerative stages. The in vivo imaging also demonstrated that Sox2/3+ neural progenitor cells generate neurons in response to injury. In contrast, these cells showed a delayed and very limited response in non-regenerative froglets. Sox2 knockdown and overexpression of a dominant negative form of Sox2 disrupts locomotor and anatomical-histological recovery. We also found that neurogenesis markers increase in response to injury in regenerative but not in non-regenerative animals. We conclude that Sox2 is necessary for spinal cord regeneration and suggest a model whereby spinal cord injury activates proliferation of Sox2/3 expressing cells and their differentiation into neurons, a mechanism that is

  18. Anaesthetic sensitivity of fMLP-induced cell signalling in Xenopus oocytes.

    Science.gov (United States)

    Wittmann, Sigrid; Fröhlich, Dieter; Mietens, Andrea; Daniels, Stephen

    2006-01-01

    FMLP stimulation of Xenopus oocytes expressing fMLP receptors leads to a concentration-dependent biphasic inward current. To identify the evolution of these currents we have examined the effects of blocking various cell signalling pathways. In addition we have analysed the effects of three intravenous anaesthetics on these fMLP-induced currents. Xenopus oocytes were microinjected with cRNA encoding the fMLP receptor and fMLP-stimulated (100 nM) currents measured, using two-electrode voltage-clamp (-70 mV), before and after injection of heparin (120 ng ml-1), wortmannin (1 microM), U73122 (5 microM) or buffer. Concentration-response curves were established for the action on fMLP-stimulated currents of thiopentone (5-500 microM), methohexitone (0.2-200 microM) and propofol (0.5-500 microM). Heparin significantly enhanced the fast current (pPLC/PKC pathway because it is reduced by the PLC inhibitor U73122, (b) the PI3K- and PLD-mediated pathways are not involved because wortmannin had no effect and (c) activation of the two conductance channels must be different because U73122 reduced the slow but not the fast current. Since both currents are decreased by all three anaesthetics, their inhibition might be mediated through an action at the agonist/receptor, although, since the slow current is consistently more sensitive than the fast, there may be additionally an action on cell signalling.

  19. Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus.

    Science.gov (United States)

    Reisoli, Elisa; De Lucchini, Stefania; Nardi, Irma; Ori, Michela

    2010-09-01

    Serotonin (5-HT) is a neuromodulator that plays many different roles in adult and embryonic life. Among the 5-HT receptors, 5-HT2B is one of the key mediators of 5-HT functions during development. We used Xenopus laevis as a model system to further investigate the role of 5-HT2B in embryogenesis, focusing on craniofacial development. By means of gene gain- and loss-of-function approaches and tissue transplantation assays, we demonstrated that 5-HT2B modulates, in a cell-autonomous manner, postmigratory skeletogenic cranial neural crest cell (NCC) behavior without altering early steps of cranial NCC development and migration. 5-HT2B overexpression induced the formation of an ectopic visceral skeletal element and altered the dorsoventral patterning of the branchial arches. Loss-of-function experiments revealed that 5-HT2B signaling is necessary for jaw joint formation and for shaping the mandibular arch skeletal elements. In particular, 5-HT2B signaling is required to define and sustain the Xbap expression necessary for jaw joint formation. To shed light on the molecular identity of the transduction pathway acting downstream of 5-HT2B, we analyzed the function of phospholipase C beta 3 (PLC) in Xenopus development and showed that PLC is the effector of 5-HT2B during craniofacial development. Our results unveiled an unsuspected role of 5-HT2B in craniofacial development and contribute to our understanding of the interactive network of patterning signals that is involved in the development and evolution of the vertebrate mandibular arch.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Caging, but not air deprivation, slows tadpole growth and development in the amphibian Xenopus laevis.

    Science.gov (United States)

    Rose, Christopher S

    2014-08-01

    Xenopus laevis tadpoles raised in submerged cages in normoxic water develop more slowly than tadpoles raised with access to air. This study distinguishes between the effects of being caged and being deprived access to air on development and growth. Tadpoles were raised in high and low density control tanks and in cages in the same tank that were either completely submerged or with the top exposed to air. Experiments were repeated with the cages in different positions relative to the air stones and with and without the water flow from air stones supplemented with a pump. Whereas caging tadpoles has a large effect on their development and growth, additionally depriving them of air has a small effect and this effect can be removed by optimizing water flow through the cage. The effect of caging, though significant in this study, is small compared to the variation in growth and developmental rates that is commonly encountered within and among controls in lab studies. Caging effects can also be diminished by optimizing rearing conditions and/or having exceptionally vigorous tadpoles. The effects of air deprivation and caging thus pose less of a problem for experimenting on air-deprived (AD) and air-restored Xenopus tadpoles than their inherent variability in growth and developmental rates and their susceptibility to growth and developmental arrest. Further, the effect of air deprivation in this air-breathing amphibian does not pose a conflict with evolutionary hypotheses for lung loss involving lengthening of the larval period and delay in the onset of air breathing. © 2014 Wiley Periodicals, Inc.

  3. HDAC1 regulates the proliferation of radial glial cells in the developing Xenopus tectum.

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    Yi Tao

    Full Text Available In the developing central nervous system (CNS, progenitor cells differentiate into progeny to form functional neural circuits. Radial glial cells (RGs are a transient progenitor cell type that is present during neurogenesis. It is thought that a combination of neural trophic factors, neurotransmitters and electrical activity regulates the proliferation and differentiation of RGs. However, it is less clear how epigenetic modulation changes RG proliferation. We sought to explore the effect of histone deacetylase (HDAC activity on the proliferation of RGs in the visual optic tectum of Xenopus laevis. We found that the number of BrdU-labeled precursor cells along the ventricular layer of the tectum decrease developmentally from stage 46 to stage 49. The co-labeling of BrdU-positive cells with brain lipid-binding protein (BLBP, a radial glia marker, showed that the majority of BrdU-labeled cells along the tectal midline are RGs. BLBP-positive cells are also developmentally decreased with the maturation of the brain. Furthermore, HDAC1 expression is developmentally down-regulated in tectal cells, especially in the ventricular layer of the tectum. Pharmacological blockade of HDACs using Trichostatin A (TSA or Valproic acid (VPA decreased the number of BrdU-positive, BLBP-positive and co-labeling cells. Specific knockdown of HDAC1 by a morpholino (HDAC1-MO decreased the number of BrdU- and BLBP-labeled cells and increased the acetylation level of histone H4 at lysine 12 (H4K12. The visual deprivation-induced increase in BrdU- and BLBP-positive cells was blocked by HDAC1 knockdown at stage 49 tadpoles. These data demonstrate that HDAC1 regulates radial glia cell proliferation in the developing optical tectum of Xenopus laevis.

  4. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos.

    Science.gov (United States)

    Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Takai, Jun-Ichi; Yoshida, Junki; Mishina, Takamichi; Fuchimukai, Kota; Ogasawara, Tsukasa; Kariya, Taro; Tashiro, Kosuke; Igarashi, Kazuei

    2010-02-01

    We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

  6. 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).

  7. Elr-type proteins protect Xenopus Dead end mRNA from miR-18-mediated clearance in the soma.

    Science.gov (United States)

    Koebernick, Katja; Loeber, Jana; Arthur, Patrick Kobina; Tarbashevich, Katsiaryna; Pieler, Tomas

    2010-09-14

    Segregation of the future germ line defines a crucial cell fate decision during animal development. In Xenopus, germ cells are specified by inheritance of vegetally localized maternal determinants, including a group of specific mRNAs. Here, we show that the vegetal localization elements (LE) of Xenopus Dead end (XDE) and of several other germ-line-specific, vegetally localized transcripts mediate germ cell-specific stabilization and somatic clearance of microinjected reporter mRNA in Xenopus embryos. The part of XDE-LE critical for somatic RNA clearance exhibits homology to zebrafish nanos1 and appears to be targeted by Xenopus miR-18 for somatic mRNA clearance. Xenopus Elr-type proteins of the vegetal localization complex can alleviate somatic RNA clearance of microinjected XDE-LE and endogenous XDE mRNA. ElrB1 synergizes with Xenopus Dead end protein in the stabilization of XDE-LE mRNA. Taken together, our findings unveil a functional link of vegetal mRNA localization and the protection of germ-line mRNAs from somatic clearance.

  8. HTLV-3 infection and AIDS: risk of spread by heterosexual contact.

    Science.gov (United States)

    Craske, J

    1986-02-01

    This article reviews current research evidence on the natural history, epidemiology, and clinical features of acquired immunodeficiency disease (AIDS) and presents guidelines for controlling the sexual transmission of human lymphotropic virus type III (HTLV-III) infection. The rapid spread of HTLV-III infection through homosexual communities in the US and Europe and its association with promiscuity initially obscured the fact that heterosexual transmission is also a significant risk factor for infection. Public health workers and epidemiologists are examining which sexual practices are most associated with the transmission of HTLV-III infection. Case-control studies in homosexuals have suggested that promiscuity, passive anal intercourse, and other sexual practices associated with rectal trauma and bleeding correlate with infection. Similar studies involving heterosexuals have not been conducted. However, the following guidelines have been proposed for couples where 1 partner has been found to be positive for HTLV-III antibodies: 1) sexual partners should be confined to established relationships; 2) anal intercourse should be avoided, even if the male uses a condom; 3) no oral contact with semen should occur; 4) if vaginal intercourse is practiced, the use of condom is essential; and 5) the only practices that are free from risk of infection are mutual masturbation and hand caresses. Since a high proportion of children of women with HTLV-III develop severe immunodeficiency, it is undesirable for women who are HTLV-III antibody positive to become pregnant. Furthermore, there is evidence that women who are HTLV-III antibody positive are more likely to develop AIDS if they become pregnant. A reliable method of permanent or reversible contraception is recommended for these women. Finally, men who are antibody positive should not donate sperm to a sperm bank.

  9. Cloning and expression of a novel zinc finger gene, Fez, transcribed in the forebrain of Xenopus and mouse embryos.

    Science.gov (United States)

    Matsuo-Takasaki, M; Lim, J H; Beanan, M J; Sato, S M; Sargent, T D

    2000-05-01

    We have identified and cloned a novel zinc finger gene, Fez (forebrain embryonic zinc-finger), as a potential downstream determinant of anterior neural plate formation in Xenopus. Fez was isolated as one of several neural-specific genes that was induced by the neuralizing factor, noggin (Smith and Harland, 1992. Cell 70, 829-840), in uncommitted ectoderm. Fez has an open reading frame comprising 466 amino acids, and contains six C(2)H(2) type zinc finger domains, which are highly conserved among Drosophila, zebrafish, mouse, and human. In Xenopus, the expression of Fez begins at stage 12 in the rostral end of the neural plate, and by stage 45, it is localized to several telencephalic regions, including the olfactory bulbs, nervus terminalis, and ventricular zone. The mouse homologue of Fez is similarly expressed in the mouse forebrain by embryonic day 11.

  10. Nuclear reconstitution of plant (Orychophragmus violaceus) demembranated sperm in cell-free extracts from animal (Xenopus laevis) eggs.

    Science.gov (United States)

    Lu, P; Ren, M; Zhai, Z H

    2001-11-01

    Cell-free extracts from animal Xenopus laevis egg could induce chromatin decondensation and pronuclear formation from demembranated plant (Orychophragmus violaceus) sperm. When incubated with Xenopus egg extracts, the demembranated sperm began to swell and then gradually decondensed. The assembly of the nuclear envelope in the reconstituted nuclei was visualized by means of electron microscopy and fluorescence microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nuclei, with a double membrane, was similar to that of nuclei after fertilization. The electron micrograph of the whole-mount prepared nuclear matrix--lamina showed the reconstituted nucleus to be filled with a dense network. (C)2001 Elsevier Science (USA).

  11. EphrinB2 affects apical constriction in Xenopus embryos and is regulated by ADAM10 and flotillin-1

    Science.gov (United States)

    Ji, Yon Ju; Hwang, Yoo-Seok; Mood, Kathleen; Cho, Hee-Jun; Lee, Hyun-Shik; Winterbottom, Emily; Cousin, Hélène; Daar, Ira O.

    2014-03-01

    The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development. Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis. Such a decrease in ephrinB2 protein levels is observed on the loss of flotillin-1 scaffold protein, a newly identified ephrinB2-binding partner. This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10. These findings indicate that flotillin-1 regulates ephrinB2 protein levels through ADAM10, and is required for appropriate neural tube morphogenesis in the Xenopus embryo.

  12. Mef2d acts upstream of muscle identity genes and couples lateral myogenesis to dermomyotome formation in Xenopus laevis.

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    Bruno Della Gaspera

    Full Text Available Xenopus myotome is formed by a first medial and lateral myogenesis directly arising from the presomitic mesoderm followed by a second myogenic wave emanating from the dermomyotome. Here, by a series of gain and loss of function experiments, we showed that Mef2d, a member of the Mef2 family of MADS-box transcription factors, appeared as an upstream regulator of lateral myogenesis, and as an inducer of dermomyotome formation at the beginning of neurulation. In the lateral presomitic cells, we showed that Mef2d transactivates Myod expression which is necessary for lateral myogenesis. In the most lateral cells of the presomitic mesoderm, we showed that Mef2d and Paraxis (Tcf15, a member of the Twist family of transcription factors, were co-localized and activate directly the expression of Meox2, which acts upstream of Pax3 expression during dermomyotome formation. Cell tracing experiments confirm that the most lateral Meox2 expressing cells of the presomitic mesoderm correspond to the dermomyotome progenitors since they give rise to the most dorsal cells of the somitic mesoderm. Thus, Xenopus Mef2d couples lateral myogenesis to dermomyotome formation before somite segmentation. These results together with our previous works reveal striking similarities between dermomyotome and tendon formation in Xenopus: both develop in association with myogenic cells and both involve a gene transactivation pathway where one member of the Mef2 family, Mef2d or Mef2c, cooperates with a bHLH protein of the Twist family, Paraxis or Scx (Scleraxis respectively. We propose that these shared characteristics in Xenopus laevis reflect the existence of a vertebrate ancestral mechanism which has coupled the development of the myogenic cells to the formation of associated tissues during somite compartmentalization.

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

    OpenAIRE

    ROMO MARTY, XIMENA CAROLINA; 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.

  14. 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

    Transient receptor potential vanilloid subtype 1 (TRPV1) channels are essential nociceptive integrators in primary afferent neurons. These nonselective cation channels are inhibited by local anesthetic compounds through an undefined mechanism. Here, we show that lidocaine inhibits TRPV1 channels...... 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...

  15. Insights on the evolution of prolyl 3-hydroxylation sites from comparative analysis of chicken and Xenopus fibrillar collagens.

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    David M Hudson

    2011-05-01

    Full Text Available Recessive mutations that prevent 3-hydroxyproline formation in type I collagen have been shown to cause forms of osteogenesis imperfecta. In mammals, all A-clade collagen chains with a GPP sequence at the A1 site (P986, except α1(III, have 3Hyp at residue P986. Available avian, amphibian and reptilian type III collagen sequences from the genomic database (Ensembl all differ in sequence motif from mammals at the A1 site. This suggests a potential evolutionary distinction in prolyl 3-hydroxylation between mammals and earlier vertebrates. Using peptide mass spectrometry, we confirmed that this 3Hyp site is fully occupied in α1(III from an amphibian, Xenopus laevis, as it is in chicken. A thorough characterization of all predicted 3Hyp sites in collagen types I, II, III and V from chicken and xenopus revealed further differences in the pattern of occupancy of the A3 site (P707. In mammals only α2(I and α2(V chains had any 3Hyp at the A3 site, whereas in chicken all α-chains except α1(III had A3 at least partially 3-hydroxylated. The A3 site was also partially 3-hydroxylated in xenopus α1(I. Minor differences in covalent cross-linking between chicken, xenopus and mammal type I and III collagens were also found as a potential index of evolving functional differences. The function of 3Hyp is still unknown but observed differences in site occupancy during vertebrate evolution are likely to give important clues.

  16. The repetitive portion of the Xenopus IgH Mu switch region mediates orientation-dependent class switch recombination.

    Science.gov (United States)

    Zhang, Zheng Z; Pannunzio, Nicholas R; Lu, Zhengfei; Hsu, Ellen; Yu, Kefei; Lieber, Michael R

    2015-10-01

    Vertebrates developed immunoglobulin heavy chain (IgH) class switch recombination (CSR) to express different IgH constant regions. Most double-strand breaks for Ig CSR occur within the repetitive portion of the switch regions located upstream of each set of constant domain exons for the Igγ, Igα or Igϵ heavy chain. Unlike mammalian switch regions, Xenopus switch regions do not have a high G-density on the non-template DNA strand. In previous studies, when Xenopus Sμ DNA was moved to the genome of mice, it is able to support substantial CSR when it is used to replace the murine Sγ1 region. Here, we tested both the 2kb repetitive portion and the 4.6 kb full-length portions of the Xenopus Sμ in both their natural (forward) orientation relative to the constant domain exons, as well as the opposite (reverse) orientation. Consistent with previous work, we find that the 4.6 kb full-length Sμ mediates similar levels of CSR in both the forward and reverse orientations. Whereas, the forward orientation of the 2kb portion can restore the majority of the CSR level of the 4.6 kb full-length Sμ, the reverse orientation poorly supports R-looping and no CSR. The forward orientation of the 2kb repetitive portion has more GG dinucleotides on the non-template strand than the reverse orientation. The correlation of R-loop formation with CSR efficiency, as demonstrated in the 2kb repetitive fragment of the Xenopus switch region, confirms a role played by R-looping in CSR that appears to be conserved through evolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

  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. Copyright

  18. Xenopus Sprouty2 inhibits FGF-mediated gastrulation movements but does not affect mesoderm induction and patterning

    Science.gov (United States)

    Nutt, Stephen L.; Dingwell, Kevin S.; Holt, Christine E.; Amaya, Enrique

    2001-01-01

    Signal transduction through the FGF receptor is essential for the specification of the vertebrate body plan. Blocking the FGF pathway in early Xenopus embryos inhibits mesoderm induction and results in truncation of the anterior–posterior axis. The Drosophila gene sprouty encodes an antagonist of FGF signaling, which is transcriptionally induced by the pathway, but whose molecular functions are poorly characterized. We have cloned Xenopus sprouty2 and show that it is expressed in a similar pattern to known FGFs and is dependent on the FGF/Ras/MAPK pathway for its expression. Overexpression of Xsprouty2 in both embryos and explant assays results in the inhibition of the cell movements of convergent extension. Although blocking FGF/Ras/MAPK signaling leads to an inhibition of mesodermal gene expression, these markers are unaffected by Xsprouty2, indicating that mesoderm induction and patterning occurs normally in these embryos. Finally, using Xenopus oocytes we show that Xsprouty2 is an intracellular antagonist of FGF-dependent calcium signaling. These results provide evidence for at least two distinct FGF-dependent signal transduction pathways: a Sprouty-insensitive Ras/MAPK pathway required for the transcription of most mesodermal genes, and a Sprouty-sensitive pathway required for coordination of cellular morphogenesis. PMID:11331610

  19. Identification of novel genes affecting mesoderm formation and morphogenesis through an enhanced large scale functional screen in Xenopus.

    Science.gov (United States)

    Chen, Jun-An; Voigt, Jana; Gilchrist, Mike; Papalopulu, Nancy; Amaya, Enrique

    2005-03-01

    The formation of mesoderm is an important developmental process of vertebrate embryos, which can be broken down into several steps; mesoderm induction, patterning, morphogenesis and differentiation. Although mesoderm formation in Xenopus has been intensively studied, much remains to be learned about the molecular events responsible for each of these steps. Furthermore, the interplay between mesoderm induction, patterning and morphogenesis remains obscure. Here, we describe an enhanced functional screen in Xenopus designed for large-scale identification of genes controlling mesoderm formation. In order to improve the efficiency of the screen, we used a Xenopus tropicalis unique set of cDNAs, highly enriched in full-length clones. The screening strategy incorporates two mesodermal markers, Xbra and Xmyf-5, to assay for cell fate specification and patterning, respectively. In addition we looked for phenotypes that would suggest effects in morphogenesis, such as gastrulation defects and shortened anterior-posterior axis. Out of 1728 full-length clones we isolated 82 for their ability to alter the phenotype of tadpoles and/or the expression of Xbra and Xmyf-5. Many of the clones gave rise to similar misexpression phenotypes (synphenotypes) and many of the genes within each synphenotype group appeared to be involved in similar pathways. We determined the expression pattern of the 82 genes and found that most of the genes were regionalized and expressed in mesoderm. We expect that many of the genes identified in this screen will be important in mesoderm formation.

  20. 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.

  1. Glider and Vision: two new families of miniature inverted-repeat transposable elements in Xenopus laevis genome.

    Science.gov (United States)

    Lepetit, D; Pasquet, S; Olive, M; Thézé, N; Thiébaud, P

    2000-01-01

    We have characterised from Xenopus laevis two new short interspersed repetitive elements, we have named Glider and Vision, that belong to the family of miniature inverted-repeat transposable elements (MITEs). Glider was first characterised in an intronic region of the alpha-tropomyosin (alpha-TM) gene and database search has revealed the presence of this element in 10 other Xenopus laevis genes. Glider elements are about 150 bp long and for some of them, their terminal inverted repeats are flanked by potential target-site duplications. Evidence for the mobility of Glider element has been provided by the presence/absence of one element at corresponding location in duplicated alpha-TM genes. Vision element has been identified in the promoter region of the cyclin dependant kinase 2 gene (cdk2) where it is boxed in a Glider element. Vision is 284bp long and is framed by 14-bp terminal inverted repeats that are flanked by 7-bp direct repeats. We have estimated that there are about 20,000 and 300 copies of Glider and Vision respectively scattered throughout the Xenopus laevis genome. Every MITEs elements but two described in our study are found either in 5' or in 3' regulatory regions of genes suggesting a potential role in gene regulation.

  2. Phylogeny, Functional Annotation, and Protein Interaction Network Analyses of the Xenopus tropicalis Basic Helix-Loop-Helix Transcription Factors

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    Wuyi Liu

    2013-01-01

    Full Text Available The previous survey identified 70 basic helix-loop-helix (bHLH proteins, but it was proved to be incomplete, and the functional information and regulatory networks of frog bHLH transcription factors were not fully known. Therefore, we conducted an updated genome-wide survey in the Xenopus tropicalis genome project databases and identified 105 bHLH sequences. Among the retrieved 105 sequences, phylogenetic analyses revealed that 103 bHLH proteins belonged to 43 families or subfamilies with 46, 26, 11, 3, 15, and 4 members in the corresponding supergroups. Next, gene ontology (GO enrichment analyses showed 65 significant GO annotations of biological processes and molecular functions and KEGG pathways counted in frequency. To explore the functional pathways, regulatory gene networks, and/or related gene groups coding for Xenopus tropicalis bHLH proteins, the identified bHLH genes were put into the databases KOBAS and STRING to get the signaling information of pathways and protein interaction networks according to available public databases and known protein interactions. From the genome annotation and pathway analysis using KOBAS, we identified 16 pathways in the Xenopus tropicalis genome. From the STRING interaction analysis, 68 hub proteins were identified, and many hub proteins created a tight network or a functional module within the protein families.

  3. CHLORELLA VIRUSES

    Science.gov (United States)

    Yamada, Takashi; Onimatsu, Hideki; Van Etten, James L.

    2007-01-01

    Chlorella viruses or chloroviruses are large, icosahedral, plaque‐forming, double‐stranded‐DNA—containing viruses that replicate in certain strains of the unicellular green alga Chlorella. DNA sequence analysis of the 330‐kbp genome of Paramecium bursaria chlorella virus 1 (PBCV‐1), the prototype of this virus family (Phycodnaviridae), predict ∼366 protein‐encoding genes and 11 tRNA genes. The predicted gene products of ∼50% of these genes resemble proteins of known function, including many that are completely unexpected for a virus. In addition, the chlorella viruses have several features and encode many gene products that distinguish them from most viruses. These products include: (1) multiple DNA methyltransferases and DNA site‐specific endonucleases, (2) the enzymes required to glycosylate their proteins and synthesize polysaccharides such as hyaluronan and chitin, (3) a virus‐encoded K+ channel (called Kcv) located in the internal membrane of the virions, (4) a SET domain containing protein (referred to as vSET) that dimethylates Lys27 in histone 3, and (5) PBCV‐1 has three types of introns; a self‐splicing intron, a spliceosomal processed intron, and a small tRNA intron. Accumulating evidence indicates that the chlorella viruses have a very long evolutionary history. This review mainly deals with research on the virion structure, genome rearrangements, gene expression, cell wall degradation, polysaccharide synthesis, and evolution of PBCV‐1 as well as other related viruses. PMID:16877063

  4. Virus Crystallography

    Science.gov (United States)

    Fry, Elizabeth; Logan, Derek; Stuart, David

    Crystallography provides a means of visualizing intact virus particles as well as their isolated constituent proteins and enzymes (1-3) at near-atomic resolution, and is thus an extraordinarily powerful tool in the pursuit of a fuller understanding of the functioning of these simple biological systems. We have already expanded our knowledge of virus evolution, assembly, antigenic variation, and host-cell interactions; further studies will no doubt reveal much more. Although the rewards are enormous, an intact virus structure determination is not a trivial undertaking and entails a significant scaling up in terms of time and resources through all stages of data collection and processing compared to a traditional protein crystallographic structure determination. It is the methodology required for such studies that will be the focus of this chapter. The computational requirements were satisfied in the late 1970s, and when combined with the introduction of phase improvement techniques utilizing the virus symmetry (4,5), the application of crystallography to these massive macromolecular assemblies became feasible. This led to the determination of the first virus structure (the small RNA plant virus, tomato bushy stunt virus), by Harrison and coworkers in 1978 (6). The structures of two other plant viruses followed rapidly (7,8). In the 1980s, a major focus of attention was a family of animal RNA viruses; the Picornaviridae.

  5. 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

  6. Protein 4.1 and its interaction with other cytoskeletal proteins in Xenopus laevis oogenesis.

    Science.gov (United States)

    Carotenuto, Rosa; Petrucci, Tamara C; Correas, Isabel; Vaccaro, Maria C; De Marco, Nadia; Dale, Brian; Wilding, Martin

    2009-06-01

    In human red blood cells, protein 4.1 (4.1R) is an 80-kDa polypeptide that stabilizes the spectrin-actin network and anchors it to the plasma membrane. In non-erythroid cells there is a great variety of 4.1R isoforms, mainly generated by alternative pre-mRNA splicing, which localize at various intracellular sites, including the nucleus. We studied protein 4.1R distribution in relation to beta-spectrin, actin and cytokeratin during Xenopus oogenesis. Immunoprecipitation experiments indicate that at least two isoforms of protein 4.1R are present in Xenopus laevis oocytes: a 56-kDa form in the cytoplasm and a 37-kDa form in the germinal vesicle (GV). Antibodies to beta-spectrin reveal two bands of 239 and 100 kDa in the cytoplasm. Coimmunoprecipitation experiments indicate that both the 37- and 56-kDa isoforms of protein 4.1R associate with the 100-kDa isoform of beta-spectrin. Moreover, the 56-kDa form coimmunoprecipitates with a cytokeratin of the same molecular weight. Confocal immunolocalization shows that protein 4.1R distribution is in the peripheral cytoplasm, in the mitochondrial cloud (MC) and in the GV of previtellogenic oocytes. In the cytoplasm of vitellogenic oocytes, a loose network of fibers stained by the anti-protein 4.1R antibody spreads across the cytoplasm. beta-Spectrin has a similar distribution. Protein 4.1R was found to colocalize with actin in the cortex of oocytes in the form of fluorescent dots. Double immunolocalization of protein 4.1R and cytokeratin depicts two separate networks that overlap throughout the whole cytoplasm. Protein 4.1R filaments partially colocalize with cytokeratin in both the animal and vegetal hemispheres. We hypothesize that protein 4.1R could function as a linker protein between cytokeratin and the actin-based cytoskeleton.

  7. N1-Src Kinase Is Required for Primary Neurogenesis in Xenopus tropicalis.

    Science.gov (United States)

    Lewis, Philip A; Bradley, Isobel C; Pizzey, Alastair R; Isaacs, Harry V; Evans, Gareth J O

    2017-08-30

    The presence of the neuronal-specific N1-Src splice variant of the C-Src tyrosine kinase is conserved through vertebrate evolution, suggesting an important role in complex nervous systems. Alternative splicing involving an N1-Src-specific microexon leads to a 5 or 6 aa insertion into the SH3 domain of Src. A prevailing model suggests that N1-Src regulates neuronal differentiation via cytoskeletal dynamics in the growth cone. Here we investigated the role of n1-src in the early development of the amphibian Xenopus tropicalis, and found that n1-src expression is regulated in embryogenesis, with highest levels detected during the phases of primary and secondary neurogenesis. In situ hybridization analysis, using locked nucleic acid oligo probes complementary to the n1-src microexon, indicates that n1-src expression is highly enriched in the open neural plate during neurula stages and in the neural tissue of adult frogs. Given the n1-src expression pattern, we investigated a possible role for n1-src in neurogenesis. Using splice site-specific antisense morpholino oligos, we inhibited n1-src splicing, while preserving c-src expression. Differentiation of neurons in the primary nervous system is reduced in n1-src-knockdown embryos, accompanied by a severely impaired touch response in later development. These data reveal an essential role for n1-src in amphibian neural development and suggest that alternative splicing of C-Src in the developing vertebrate nervous system evolved to regulate neurogenesis.SIGNIFICANCE STATEMENT The Src family of nonreceptor tyrosine kinases acts in signaling pathways that regulate cell migration, cell adhesion, and proliferation. Srcs are also enriched in the brain, where they play key roles in neuronal development and neurotransmission. Vertebrates have evolved a neuron-specific splice variant of C-Src, N1-Src, which differs from C-Src by just 5 or 6 aa. N1-Src is poorly understood and its high similarity to C-Src has made it difficult to

  8. Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes.

    Science.gov (United States)

    Nakhoul, N L; Davis, B A; Romero, M F; Boron, W F

    1998-02-01

    It is generally accepted that gases such as CO2 cross cell membranes by dissolving in the membrane lipid. No role for channels or pores in gas transport has ever been demonstrated. Here we ask whether expression of the water channel aquaporin-1 (AQP1) enhances the CO2 permeability of Xenopus oocytes. We expressed AQP1 in Xenopus oocytes by injecting AQP1 cRNA, and we assessed CO2 permeability by using microelectrodes to monitor the changes in intracellular pH (pHi) produced by adding 1.5% CO2/10 mM HCO3- to (or removing it from) the extracellular solution. Oocytes normally have an undetectably low level of carbonic anhydrase (CA), which eliminates the CO2 hydration reaction as a rate-limiting step. We found that expressing AQP1 (vs. injecting water) had no measurable effect on the rate of CO2-induced pHi changes in such low-CA oocytes: adding CO2 caused pHi to fall at a mean initial rate of 11.3 x 10(-4) pH units/s in control oocytes and 13.3 x 10(-4) pH units/s in oocytes expressing AQP1. When we injected oocytes with water, and a few days later with CA, the CO2-induced pHi changes in these water/CA oocytes were more than fourfold faster than in water-injected oocytes (acidification rate, 53 x 10(-4) pH units/s). Ethoxzolamide (ETX; 10 microM), a membrane-permeant CA inhibitor, greatly slowed the pHi changes (16.5 x 10(-4) pH units/s). When we injected oocytes with AQP1 cRNA and then CA, the CO2-induced pHi changes in these AQP1/CA oocytes were approximately 40% faster than in the water/CA oocytes (75 x 10(-4) pH units/s), and ETX reduced the rates substantially (14.7 x 10(-4) pH units/s). Thus, in the presence of CA, AQP1 expression significantly increases the CO2 permeability of oocyte membranes. Possible explanations include 1) AQP1 expression alters the lipid composition of the cell membrane, 2) AQP1 expression causes overexpression of a native gas channel, and/or 3) AQP1 acts as a channel through which CO2 can permeate. Even if AQP1 should mediate a CO2 flux

  9. Regeneration of Xenopus laevis spinal cord requires Sox2/3 expressing cells.

    Science.gov (United States)

    Muñoz, Rosana; Edwards-Faret, Gabriela; Moreno, Mauricio; Zuñiga, Nikole; Cline, Hollis; Larraín, Juan

    2015-12-15

    Spinal cord regeneration is very inefficient in humans, causing paraplegia and quadriplegia. Studying model organisms that can regenerate the spinal cord in response to injury could be useful for understanding the cellular and molecular mechanisms that explain why this process fails in humans. Here, we use Xenopus laevis as a model organism to study spinal cord repair. Histological and functional analyses showed that larvae at pre-metamorphic stages restore anatomical continuity of the spinal cord and recover swimming after complete spinal cord transection. These regenerative capabilities decrease with onset of metamorphosis. The ability to study regenerative and non-regenerative stages in Xenopus laevis makes it a unique model system to study regeneration. We studied the response of Sox2(/)3 expressing cells to spinal cord injury and their function in the regenerative process. We found that cells expressing Sox2 and/or Sox3 are present in the ventricular zone of regenerative animals and decrease in non-regenerative froglets. Bromodeoxyuridine (BrdU) experiments and in vivo time-lapse imaging studies using green fluorescent protein (GFP) expression driven by the Sox3 promoter showed a rapid, transient and massive proliferation of Sox2(/)3(+) cells in response to injury in the regenerative stages. The in vivo imaging also demonstrated that Sox2(/)3(+) neural progenitor cells generate neurons in response to injury. In contrast, these cells showed a delayed and very limited response in non-regenerative froglets. Sox2 knockdown and overexpression of a dominant negative form of Sox2 disrupts locomotor and anatomical-histological recovery. We also found that neurogenesis markers increase in response to injury in regenerative but not in non-regenerative animals. We conclude that Sox2 is necessary for spinal cord regeneration and suggest a model whereby spinal cord injury activates proliferation of Sox2/3 expressing cells and their differentiation into neurons, a mechanism

  10. Effects of 17α-trenbolone and melengestrol acetate on Xenopus laevis growth, development, and survival.

    Science.gov (United States)

    Finch, Bryson E; Blackwell, Brett R; Faust, Derek R; Wooten, Kimberly J; Maul, Jonathan D; Cox, Stephen B; Smith, Philip N

    2013-02-01

    The synthetic growth-promoting hormones trenbolone and melengestrol acetate have been detected in the environment near beef cattle feedlots and are reportedly transported via wind-borne particulate matter. Therefore, movement of synthetic hormones from beef cattle feedlots to water bodies via particulate matter is possible. Our objective was to evaluate potential effects of 17α-trenbolone (17α-TB), melengestrol acetate (MGA), and combinations of both on growth, development, and survival of Xenopus laevis larvae. On post-hatch day 2 (stage 33/34), X. laevis larvae were exposed to three nominal concentrations of 17α-TB (10, 100, and 500 ng/L), MGA (1, 10, and 100 ng/L), a combination of both (1/10, 10/100, and 100/500 ng/L MGA/17α-TB), frog embryo teratogenesis assay-Xenopus medium, or a solvent control. Significant increases in all X. laevis growth metrics were observed among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB and 10 ng/L MGA + 100 ng/L 17α-TB treatments. Stage of development was increased among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB treatment group and significantly decreased among those in the 500 ng/L 17α-TB treatment. Total body mass and snout-vent length of X. laevis larvae were significantly reduced in the 100 ng/L MGA and 100 ng/L MGA + 500 ng/L 17α-TB treatment groups. Larvae exposed to 500 ng/L 17α-TB had decreased total body mass, snout-vent length, and total length. In general, growth measurements decreased with increasing concentration of MGA, 17α-TB, or a combination of both. Survival among all treatments was not significantly different from controls. Amphibians exposed to MGA and 17α-TB in the environment may experience alterations in growth and development.

  11. A glyphosate micro-emulsion formulation displays teratogenicity in Xenopus laevis.

    Science.gov (United States)

    Bonfanti, Patrizia; Saibene, M; Bacchetta, R; Mantecca, P; Colombo, A

    2018-02-01

    Glyphosate is the active ingredient in broad-spectrum herbicide formulations used in agriculture, domestic area and aquatic weed control worldwide. Its market is growing steadily concurrently with the cultivation of glyphosate-tolerant transgenic crops and emergence of weeds less sensitive to glyphosate. Ephemeral and lentic waters near to agricultural lands, representing favorite habitats for amphibian reproduction and early life-stage development, may thus be contaminated by glyphosate based herbicides (GBHs) residues. Previous studies on larval anuran species highlighted increased mortality and growth effects after exposure to different GBHs in comparison to glyphosate itself, mainly because of the surfactants such as polyethoxylated tallow amine present in the formulations. Nevertheless, these conclusions are not completely fulfilled when the early development, characterized by primary organogenesis events, is considered. In this study, we compare the embryotoxicity of Roundup ® Power 2.0, a new GBH formulation currently authorized in Italy, with that of technical grade glyphosate using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Our results evidenced that glyphosate was not embryolethal and only at the highest concentration (50 mg a.e./L) caused edemas. Conversely, Roundup ® Power 2.0 exhibited a 96 h LC50 of 24.78 mg a.e./L and a 96 h EC50 of 7.8 mg a.e./L. A Teratogenic Index of 3.4 was derived, pointing out the high teratogenic potential of the Roundup ® Power 2.0. Specific concentration-dependent abnormal phenotypes, such as craniofacial alterations, microphthalmia, narrow eyes and forebrain regionalization defects were evidenced by gross malformation screening and histopathological analysis. These phenotypes are coherent with those evidenced in Xenopus laevis embryos injected with glyphosate, allowing us to hypothesize that the teratogenicity observed for Roundup ® Power 2.0 may be related to the improved efficacy in delivering

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

    Directory of Open Access Journals (Sweden)

    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

  13. CHANDIPURA VIRUS

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. CHANDIPURA VIRUS. First isolated from a village called Chandipura near Nagpur in 1965 in India. Belongs to rhabdoviridae family. Used as a Model System to study RNA virus multiplication in the infected cell at molecular level. Notes:

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

    Science.gov (United States)

    Shrestha, Bindesh; Sripadi, Prabhakar; Reschke, Brent R; Henderson, Holly D; Powell, Matthew J; Moody, Sally A; Vertes, Akos

    2014-01-01

    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.

  15. Dynamic properties of calcium-activated chloride currents in Xenopus laevis oocytes.

    Science.gov (United States)

    M De la Fuente, Ildefonso; Malaina, Iker; Pérez-Samartín, Alberto; Boyano, María Dolores; Pérez-Yarza, Gorka; Bringas, Carlos; Villarroel, Álvaro; Fedetz, María; Arellano, Rogelio; Cortes, Jesus M; Martínez, Luis

    2017-02-13

    Chloride is the most abundant permeable anion in the cell, and numerous studies in the last two decades highlight the great importance and broad physiological role of chloride currents mediated anion transport. They participate in a multiplicity of key processes, as for instance, the regulation of electrical excitability, apoptosis, cell cycle, epithelial secretion and neuronal excitability. In addition, dysfunction of Cl(-) channels is involved in a variety of human diseases such as epilepsy, osteoporosis and different cancer types. Historically, chloride channels have been of less interest than the cation channels. In fact, there seems to be practically no quantitative studies of the dynamics of chloride currents. Here, for the first time, we have quantitatively studied experimental calcium-activated chloride fluxes belonging to Xenopus laevis oocytes, and the main results show that the experimental Cl(-) currents present an informational structure characterized by highly organized data sequences, long-term memory properties and inherent "crossover" dynamics in which persistent correlations arise at short time intervals, while anti-persistent behaviors become dominant in long time intervals. Our work sheds some light on the understanding of the informational properties of ion currents, a key element to elucidate the physiological functional coupling with the integrative dynamics of metabolic processes.

  16. Tbx6, Thylacine1, and E47 synergistically activate bowline expression in Xenopus somitogenesis.

    Science.gov (United States)

    Hitachi, Keisuke; Kondow, Akiko; Danno, Hiroki; Inui, Masafumi; Uchiyama, Hideho; Asashima, Makoto

    2008-01-15

    T-box factor, Tbx6, is a prerequisite for somite segmentation in vertebrates. We recently identified a negative regulator of Tbx6, Bowline, which represses the expression of genes involved in somite segmentation by suppressing the transcriptional activity of Tbx6. According to this function, bowline gene expression is restricted to the most anterior presomitic mesoderm where the somite segmentation program terminates, although it remains unclear how bowline expression is activated. To address this, we investigated the cis-regulatory region of bowline. Measuring luciferase activity driven by the bowline promoter, we found that Tbx6, Thylacine1, and E47 synergistically activate bowline expression in vitro. We also found that Tbx6, Thylacine1, and E47 are spatiotemporally sufficient to induce bowline expression in Xenopus somitogenesis. Our findings indicated that besides being a negative regulator of Tbx6, bowline itself is also regulated by Tbx6, suggesting the negative feedback loop of Tbx6-Bowline in the termination step of somite segmentation.

  17. Discovering novel phenotypes with automatically inferred dynamic models: a partial melanocyte conversion in Xenopus

    Science.gov (United States)

    Lobo, Daniel; Lobikin, Maria; Levin, Michael

    2017-01-01

    Progress in regenerative medicine requires reverse-engineering cellular control networks to infer perturbations with desired systems-level outcomes. Such dynamic models allow phenotypic predictions for novel perturbations to be rapidly assessed in silico. Here, we analyzed a Xenopus model of conversion of melanocytes to a metastatic-like phenotype only previously observed in an all-or-none manner. Prior in vivo genetic and pharmacological experiments showed that individual animals either fully convert or remain normal, at some characteristic frequency after a given perturbation. We developed a Machine Learning method which inferred a model explaining this complex, stochastic all-or-none dataset. We then used this model to ask how a new phenotype could be generated: animals in which only some of the melanocytes converted. Systematically performing in silico perturbations, the model predicted that a combination of altanserin (5HTR2 inhibitor), reserpine (VMAT inhibitor), and VP16-XlCreb1 (constitutively active CREB) would break the all-or-none concordance. Remarkably, applying the predicted combination of three reagents in vivo revealed precisely the expected novel outcome, resulting in partial conversion of melanocytes within individuals. This work demonstrates the capability of automated analysis of dynamic models of signaling networks to discover novel phenotypes and predictively identify specific manipulations that can reach them.

  18. Species-specific voltage-gating properties of connexin-45 junctions expressed in Xenopus oocytes.

    Science.gov (United States)

    Barrio, L C; Capel, J; Jarillo, J A; Castro, C; Revilla, A

    1997-08-01

    Gap junctions composed of connexin-45 (Cx45) homologs from four species, zebrafish, chicken, mouse, and human, were expressed in pairs of Xenopus oocytes. The macroscopic conductance (gj) of all Cx45 junctions was modulated by transjunctional voltage (Vj) and by the inside-outside voltage (Vm), and the modulation was species specific. Although their gating characteristics varied in voltage sensitivity and kinetics, the four Cx45 junctions shared 1) maximum conductance at Vj = 0 and symmetrical gj reduction in response to positive and negative Vj of low amplitude, with little residual conductance; and 2) gj increases in response to simultaneous depolarization of the paired cells. The formation of hybrid channels, comprising Cx45 hemichannels from different species, allowed us to infer that two separate gates exist, one in each hemichannel, and that each Cx45 hemichannel is closed by the negativity of Vj on its cytoplasmic side. Interestingly, the Vm dependence of hybrid channels also suggests the presence of two gates in series, one Vm gate in each hemichannel. Thus the Vj and Vm dependence provides evidence that two independent voltage gates in each Cx45 hemichannel exist, reacting through specific voltage sensors and operating by different mechanisms, properties that have evolved divergently among species.

  19. Folate receptor 1 is necessary for neural plate cell apical constriction during Xenopus neural tube formation.

    Science.gov (United States)

    Balashova, Olga A; Visina, Olesya; Borodinsky, Laura N

    2017-04-15

    Folate supplementation prevents up to 70% of neural tube defects (NTDs), which result from a failure of neural tube closure during embryogenesis. The elucidation of the mechanisms underlying folate action has been challenging. This study introduces Xenopus laevis as a model to determine the cellular and molecular mechanisms involved in folate action during neural tube formation. We show that knockdown of folate receptor 1 (Folr1; also known as FRα) impairs neural tube formation and leads to NTDs. Folr1 knockdown in neural plate cells only is necessary and sufficient to induce NTDs. Folr1-deficient neural plate cells fail to constrict, resulting in widening of the neural plate midline and defective neural tube closure. Pharmacological inhibition of folate action by methotrexate during neurulation induces NTDs by inhibiting folate interaction with its uptake systems. Our findings support a model in which the folate receptor interacts with cell adhesion molecules, thus regulating the apical cell membrane remodeling and cytoskeletal dynamics necessary for neural plate folding. Further studies in this organism could unveil novel cellular and molecular events mediated by folate and lead to new ways of preventing NTDs. © 2017. Published by The Company of Biologists Ltd.

  20. Multiple inhibitory actions of lidocaine on Torpedo nicotinic acetylcholine receptors transplanted to Xenopus oocytes.

    Science.gov (United States)

    Alberola-Die, Armando; Martinez-Pinna, Juan; González-Ros, José Manuel; Ivorra, Isabel; Morales, Andrés

    2011-06-01

    Lidocaine is a local anaesthetic that blocks sodium channels, but also inhibits several ligand-gated ion-channels. The aim of this work was to unravel the mechanisms by which lidocaine blocks Torpedo nicotinic receptors transplanted to Xenopus oocytes. Acetylcholine-elicited currents were reversibly blocked by lidocaine, in a concentration dependent manner. At doses lower than the IC(50) , lidocaine blocked nicotinic receptors only at negative potentials, indicating an open-channel blockade; the binding site within the channel was at about 30% of the way through the electrical field across the membrane. In the presence of higher lidocaine doses, nicotinic receptors were blocked both at positive and negative potentials, acetylcholine dose-response curve shifted to the right and lidocaine pre-application, before its co-application with acetylcholine, enhanced the current inhibition, indicating all together that lidocaine also blocked resting receptors; besides, it increased the current decay rate. When lidocaine, at low doses, was co-applied with 2-(triethylammonio)-N-(2,6-dimethylphenyl) acetamide bromide, edrophonium or 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, which are quaternary-ammonium molecules that also blocked nicotinic receptors, there was an additive inhibitory effect, indicating that these molecules bound to different sites within the channel pore. These results prove that lidocaine blocks nicotinic receptors by several independent mechanisms and evidence the diverse and complex modulation of this receptor by structurally related molecules. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  1. Extinction of an introduced warm-climate alien species, Xenopus laevis, by extreme weather events.

    Science.gov (United States)

    Tinsley, Richard C; Stott, Lucy C; Viney, Mark E; Mable, Barbara K; Tinsley, Matthew C

    Invasive, non-native species represent a major threat to biodiversity worldwide. The African amphibian Xenopus laevis is widely regarded as an invasive species and a threat to local faunas. Populations originating at the Western Cape, South Africa, have been introduced on four continents, mostly in areas with a similar Mediterranean climate. Some introduced populations are also established in cooler environments where persistence for many decades suggests a capacity for long-term adaptation. In these cases, recent climate warming might enhance invasion ability, favouring range expansion, population growth and negative effects on native faunas. In the cool temperate UK, populations have been established for about 50 years in Wales and for an unknown period, probably >20 years, in England (Lincolnshire). Our field studies over 30 and 10 years, respectively, show that in favourable conditions there may be good recruitment, fast individual growth rates and large body size; maximum longevity exceeds 23 years. Nevertheless, areas of distribution remained limited, with numbers extreme cold and drought (December 2010 was the coldest in 120 years and the third driest in 100 years). The extinction of X. laevis in these areas indicates that even relatively long-established alien species remain vulnerable to rare extreme weather conditions.

  2. Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes.

    Science.gov (United States)

    Labokha, Aksana A; Gradmann, Sabine; Frey, Steffen; Hülsmann, Bastian B; Urlaub, Henning; Baldus, Marc; Görlich, Dirk

    2013-01-23

    Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules 30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98-derived hydrogel. It fully blocks entry of GFP-sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β-type NTRs at its surface. O-GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid-state NMR spectroscopy revealed that the O-GlcNAc-modified Nup98 gel lacks amyloid-like β-structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC-like permeability.

  3. Nuclear protein synthesis in animal and vegetal hemispheres of Xenopus oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Feldherr, C.M.; Hodges, P. (Univ. of Florida, Gainesville (USA)); Paine, P.L. (Michigan Cancer Foundation, Detroit (USA))

    1988-12-01

    Experiments were conducted to determine if nuclear proteins are preferentially synthesized in the vicinity of the nucleus, a factor which could facilitate nucleocytoplasmic exchange. Using Xenopus oocytes, animal and vegetal hemispheres were separated by bisecting the cells in paraffin oil. It was initially established that protein synthesis is not affected by the bisecting procedure. To determine if nuclear protein synthesis is restricted to the animal hemisphere (which contains the nucleus), vegetal halves and enucleated animal halves were injected with ({sup 3}H)leucine and incubated in oil for 90 min. The labeled cell halves were then fused with unlabeled, nucleated animal hemispheres that had been previously injected with puromycin in amounts sufficient to prevent further protein synthesis. Thus, labeled polypeptides which subsequently entered the nuclei were synthesized before fusion. Three hours after fusion, the nuclei were isolated, run on two-dimensional gels, and fluorographed. Approximately 200 labeled nuclear polypeptides were compared, and only 2 were synthesized in significantly different amounts in the animal and vegetal hemispheres. The results indicate that nuclear protein synthesis is not restricted to the cytoplasm adjacent to the nucleus.

  4. Vegetally localized Xenopus trim36 regulates cortical rotation and dorsal axis formation.

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    Cuykendall, Tawny N; Houston, Douglas W

    2009-09-01

    Specification of the dorsoventral axis in Xenopus depends on rearrangements of the egg vegetal cortex following fertilization, concomitant with activation of Wnt/beta-catenin signaling. How these processes are tied together is not clear, but RNAs localized to the vegetal cortex during oogenesis are known to be essential. Despite their importance, few vegetally localized RNAs have been examined in detail. In this study, we describe the identification of a novel localized mRNA, trim36, and characterize its function through maternal loss-of-function experiments. We find that trim36 is expressed in the germ plasm and encodes a ubiquitin ligase of the Tripartite motif-containing (Trim) family. Depletion of maternal trim36 using antisense oligonucleotides results in ventralized embryos and reduced organizer gene expression. We show that injection of wnt11 mRNA rescues this effect, suggesting that Trim36 functions upstream of Wnt/beta-catenin activation. We further find that vegetal microtubule polymerization and cortical rotation are disrupted in trim36-depleted embryos, in a manner dependent on Trim36 ubiquitin ligase activity. Additionally, these embryos can be rescued by tipping the eggs 90 degrees relative to the animal-vegetal axis. Taken together, our results suggest a role for Trim36 in controlling the stability of proteins regulating microtubule polymerization during cortical rotation, and subsequently axis formation.

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

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    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.

  6. Actomyosin contractility and microtubules drive apical constriction in Xenopus bottle cells.

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    Lee, Jen-Yi; Harland, Richard M

    2007-11-01

    Cell shape changes are critical for morphogenetic events such as gastrulation, neurulation, and organogenesis. However, the cell biology driving cell shape changes is poorly understood, especially in vertebrates. The beginning of Xenopus laevis gastrulation is marked by the apical constriction of bottle cells in the dorsal marginal zone, which bends the tissue and creates a crevice at the blastopore lip. We found that bottle cells contribute significantly to gastrulation, as their shape change can generate the force required for initial blastopore formation. As actin and myosin are often implicated in contraction, we examined their localization and function in bottle cells. F-actin and activated myosin accumulate apically in bottle cells, and actin and myosin inhibitors either prevent or severely perturb bottle cell formation, showing that actomyosin contractility is required for apical constriction. Microtubules were localized in apicobasally directed arrays in bottle cells, emanating from the apical surface. Surprisingly, apical constriction was inhibited in the presence of nocodazole but not taxol, suggesting that intact, but not dynamic, microtubules are required for apical constriction. Our results indicate that actomyosin contractility is required for bottle cell morphogenesis and further suggest a novel and unpredicted role for microtubules during apical constriction.

  7. Rapamycin treatment causes developmental delay, pigmentation defects, and gastrointestinal malformation on Xenopus embryogenesis

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    Moriyama, Yuki [Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529 (Japan); Ohata, Yoshihisa [Department of Education (Sciences), Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529 (Japan); Mori, Shoko [Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529 (Japan); Matsukawa, Shinya [Department of Education (Sciences), Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529 (Japan); Michiue, Tatsuo [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Asashima, Makoto [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Baien, Tsukuba, Ibaraki 305-8562 (Japan); Kuroda, Hiroki, E-mail: ehkurod@ipc.shizuoka.ac.jp [Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529 (Japan); Department of Education (Sciences), Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529 (Japan)

    2011-01-28

    Research highlights: {yields} Does famous anti-aging drug rapamycin work from the beginning of life? The answer is yes. {yields} This study shows that developmental speed of frog embryo was dose-dependently decreased by rapamycin treatment. {yields} In additions, morphogenetic effects such as less pigmentations and gut malformation are occurred by rapamycin. -- Abstract: Rapamycin is a drug working as an inhibitor of the TOR (target of rapamycin) signaling pathway and influences various life phenomena such as cell growth, proliferation, and life span extension in eukaryote. However, the extent to which rapamycin controls early developmental events of amphibians remains to be understood. Here we report an examination of rapamycin effects during Xenopus early development, followed by a confirmation of suppression of TOR downstream kinase S6K by rapamycin treatment. First, we found that developmental speed was declined in dose-dependent manner of rapamycin. Second, black pigment spots located at dorsal and lateral skin in tadpoles were reduced by rapamycin treatment. Moreover, in tadpole stages severe gastrointestinal malformations were observed in rapamycin-treated embryos. Taken together with these results, we conclude that treatment of the drug rapamycin causes enormous influences on early developmental period.

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

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    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.

  9. Tail structure is formed when blastocoel roof contacts blastocoel floor in Xenopus laevis.

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    Nishihara, Akiha; Hashimoto, Chikara

    2014-04-01

    The tail organizer has been assessed by such transplantation methods as the Einsteck procedure. However, we found that simple wounding of blastocoel roof (BCR) made it possible to form secondary tails without any transplantation in Xenopus laevis. We revealed that the ectopic expression of Xbra was blocked by inhibiting the contact between BCR and blastocoel floor (BCF), and wounding per se seemed to be not directly related to the secondary tail formation. Therefore, the secondary tail might be induced by the contact between BCR and BCF due to the leak of blastocoel fluid from the wound. This secondary tail was similar to the original tail in the expression pattern of tail genes, and in the fact that the inhibition of fibroblast growth factor signaling prevented the secondary tail induction. Our results imply that the secondary tail formation reflects the developmental processes of the original tail, indicating that simple wounding of BCR is useful for the analysis of tail formation in normal development. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

  10. Reconstitution of the Golgi apparatus after microinjection of rat liver Golgi fragments into Xenopus oocytes

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    Paiement, J.; Jolicoeur, M.; Fazel, A.; Bergeron, J.J.

    1989-04-01

    We have studied the reconstitution of the Golgi apparatus in vivo using an heterologous membrane transplant system. Endogenous glycopeptides of rat hepatic Golgi fragments were radiolabeled in vitro with (3H)sialic acid using detergent-free conditions. The Golgi fragments consisting of dispersed vesicles and tubules with intraluminal lipoprotein-like particles were then microinjected into Xenopus oocytes and their fate studied by light (LM) and electron microscope (EM) radioautography. 3 h after microinjection, radiolabel was observed by LM radioautography over yolk platelet-free cytoplasmic regions near the injection site. EM radioautography revealed label over Golgi stacked saccules containing the hepatic marker of intraluminal lipoprotein-like particles. At 14 h after injection, LM radioautographs revealed label in the superficial cortex of the oocytes between the yolk platelets and at the oocyte surface. EM radioautography identified the labeled structures as the stacked saccules of the Golgi apparatus, the oocyte cortical granules, and the plasmalemma, indicating that a proportion of microinjected material was transferred to the surface via the secretion pathway of the oocyte. The efficiency of transport was low, however, as biochemical studies failed to show extensive secretion of radiolabel into the extracellular medium by 14 h with approximately half the microinjected radiolabeled constituents degraded. Vinblastine (50 microM) administered to oocytes led to the formation of tubulin paracrystals. Although microinjected Golgi fragments were able to effect the formation of stacked saccules in vinblastine-treated oocytes, negligible transfer of heterologous material to the oocyte surface could be detected by radioautography.

  11. 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.

  12. Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Schmalzing, G.; Eckard, P.; Kroener, S.P.; Passow, H. (Max-Planck-Institut fuer Biophysik, Frankfurt (Germany, F.R.))

    1990-01-01

    During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by (gamma-32P)ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from (3H)ouabain bound to the cell surface before maturation could be phosphorylated with inorganic (32P)phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.

  13. NF2/Merlin is required for the axial pattern formation in the Xenopus laevis embryo.

    Science.gov (United States)

    Zhu, Xuechen; Min, Zheying; Tan, Renbo; Tao, Qinghua

    2015-11-01

    The NF2 gene product Merlin is a FERM-domain protein possessing a broad tumor-suppressing function. NF2/Merlin has been implicated in regulating multiple signaling pathways critical for cell growth and survival. However, it remains unknown whether NF2/Merlin regulates Wnt/β-catenin signaling during vertebrate embryogenesis. Here we demonstrate that NF2/Merlin is required for body pattern formation in the Xenopus laevis embryo. Depletion of the maternal NF2/Merlin enhances organizer gene expression dependent on the presence of β-catenin, and causes dorsanteriorized development; Morpholino antisense oligo-mediated knockdown of the zygotic NF2/Merlin shifts posterior genes anteriorwards and reduces the anterior development. We further demonstrate that targeted depletion of NF2 in the presumptive dorsal tissues increases the levels of nuclear β-catenin in the neural epithelial cells. Biochemical analyses reveal that NF2 depletion promotes the production of active β-catenin and concurrently decreases the level of N-terminally phosphorylated β-catenin under the stimulation of the endogenous Wnt signaling. Our findings suggest that NF2/Merlin negatively regulates the Wnt/β-catenin signaling activity during the pattern formation in early X. laevis embryos. Copyright © 2015. Published by Elsevier Ireland Ltd.

  14. Metabolic cost of osmoregulation in a hypertonic environment in the invasive African clawed frog Xenopus laevis

    Science.gov (United States)

    Peña-Villalobos, Isaac; Narváez, Cristóbal

    2016-01-01

    ABSTRACT Studies of aquatic invertebrates reveal that salinity affects feeding and growth rates, reproduction, survival, and diversity. Little is known, however, about how salinity impacts the energy budget of vertebrates and amphibians in particular. The few studies focused on this topic in vertebrates suggest that the ingestion of salts and the resulting osmoregulatory activity is energetically expensive. We analyzed the effect of saline acclimation on standard metabolic rates (SMR) and the activities of metabolic enzymes of internal organs and osmoregulatory variables (plasma osmolality and urea plasma level) in females of Xenopus laevis by means of acclimating individuals to an isosmotic (235 mOsm NaCl; ISO group) and hyper-osmotic (340 mOsm NaCl; HYP group) environment for 40 days. After acclimation, we found that total and mass-specific SMR was approximately 80% higher in the HYP group than those found in the ISO group. These changes were accompanied by higher citrate synthase activities in liver and heart in the HYP group than in the ISO group. Furthermore, we found a significant and positive correlation between metabolic rates and plasma urea, and citrate synthase activity in liver and heart. These results support the notion that the cost of osmoregulation is probably common in most animal species and suggest the existence of a functional association between metabolic rates and the adjustments in osmoregulatory physiology, such as blood distribution and urea synthesis. PMID:27334694

  15. Metabolic cost of osmoregulation in a hypertonic environment in the invasive African clawed frog Xenopus laevis

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    Isaac Peña-Villalobos

    2016-07-01

    Full Text Available Studies of aquatic invertebrates reveal that salinity affects feeding and growth rates, reproduction, survival, and diversity. Little is known, however, about how salinity impacts the energy budget of vertebrates and amphibians in particular. The few studies focused on this topic in vertebrates suggest that the ingestion of salts and the resulting osmoregulatory activity is energetically expensive. We analyzed the effect of saline acclimation on standard metabolic rates (SMR and the activities of metabolic enzymes of internal organs and osmoregulatory variables (plasma osmolality and urea plasma level in females of Xenopus laevis by means of acclimating individuals to an isosmotic (235 mOsm NaCl; ISO group and hyper-osmotic (340 mOsm NaCl; HYP group environment for 40 days. After acclimation, we found that total and mass-specific SMR was approximately 80% higher in the HYP group than those found in the ISO group. These changes were accompanied by higher citrate synthase activities in liver and heart in the HYP group than in the ISO group. Furthermore, we found a significant and positive correlation between metabolic rates and plasma urea, and citrate synthase activity in liver and heart. These results support the notion that the cost of osmoregulation is probably common in most animal species and suggest the existence of a functional association between metabolic rates and the adjustments in osmoregulatory physiology, such as blood distribution and urea synthesis.

  16. Evidence for an RNA polymerization activity in axolotl and Xenopus egg extracts.

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    Hélène Pelczar

    2010-12-01

    Full Text Available We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3'dAMP, but sensitive to cordycepin 5'-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii the RNA polymerization is not a 3' end labelling and that iv the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp.

  17. Structural and functional characteristics of xenavidin, the first frog avidin from Xenopus tropicalis

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    Airenne Tomi T

    2009-09-01

    Full Text Available Abstract Background Avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of avidins is not fully understood. Here we structurally and functionally characterise a novel avidin named xenavidin, which is to our knowledge the first reported avidin from a frog. Results Xenavidin was identified from an EST sequence database for Xenopus tropicalis and produced in insect cells using a baculovirus expression system. The recombinant xenavidin was found to be homotetrameric based on gel filtration analysis. Biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenavidin - it binds biotin with high affinity though less tightly than do chicken avidin and bacterial streptavidin. X-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. The location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenavidin. Conclusion The novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. This information may facilitate the discovery of novel tools for biotechnology.

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

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    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 (poogenesis 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Upstream stimulatory factors, USF1 and USF2 are differentially expressed during Xenopus embryonic development.

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    Fujimi, Takahiko J; Aruga, Jun

    2008-07-01

    Upstream stimulatory factors (USF) 1 and 2 are members of the basic helix-loop-helix leucine zipper transcription factor family. They are considered to play critical roles in cell-cycle regulation and chromatin remodeling. Their gene expression patterns are considered ubiquitous but have not been fully investigated in terms of embryogenesis. We examined the expression of the genes encoding USF1 and USF2 in Xenopus laevis during embryonic development. Expression of both genes was first detected as maternal transcripts and was observed continuously throughout development. However, in situ hybridization analysis revealed that the two genes were expressed differentially. In the late blastula, both genes were expressed in the blastocoel roof and marginal zone. At the gastrula stage, USF2 was strongly expressed in the sensorial layer of the ectoderm and in the mesoderm, whereas USF1 expression was hardly detectable. From the neurula stage onward, expression of both genes was markedly enhanced in the neural tissues, neural crest, eye and otic vesicle. However, spatial expression of the genes within the neural tube differed in that the strongest USF1 signals were observed in the lateral region of the basal plate and the strongest USF2 ones in the dorsal region of the neural tube. Expression of the two genes occurred in different mesoderm derivatives at the tailbud stage (USF1, somite; USF2, pronephros and lateral plate mesoderm of the tail region). USF1 was expressed in the notochord of the early neurula, but was lost at the stage.

  20. Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development.

    Science.gov (United States)

    Kaeser, Gwendolyn E; Rabe, Brian A; Saha, Margaret S

    2011-04-01

    Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABA(A) and metabotropic GABA(B) receptors. During the development of the nervous system, GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation, and activity-dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABA(A) and GABA(B) receptors during early development of Xenopus laevis. Using in situ hybridization and qRT-PCR, GABA(A) α2 was detected as a maternal mRNA. All other α-subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern, suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. Copyright © 2011 Wiley-Liss, Inc.

  1. Xenopus as a model system for studying pancreatic development and diabetes.

    Science.gov (United States)

    Kofent, Julia; Spagnoli, Francesca M

    2016-03-01

    Diabetes is a chronic disease caused by the loss or dysfunction of the insulin-producing β-cells in the pancreas. To date, much of our knowledge about β-cells in humans comes from studying rare monogenic forms of diabetes. Importantly, the majority of mutations so far associated to monogenic diabetes are in genes that exert a regulatory role in pancreatic development and/or β-cell function. Thus, the identification and study of novel mutations open an unprecedented window into human pancreatic development. In this review, we summarize major advances in the genetic dissection of different types of monogenic diabetes and the insights gained from a developmental perspective. We highlight future challenges to bridge the gap between the fast accumulation of genetic data through next-generation sequencing and the need of functional insights into disease mechanisms. Lastly, we discuss the relevance and advantages of studying candidate gene variants in vivo using the Xenopus as model system. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. The repertoire of G-protein-coupled receptors in Xenopus tropicalis

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    Hu Yinghe

    2009-06-01

    Full Text Available Abstract Background The G-protein-coupled receptor (GPCR superfamily represents the largest protein family in the human genome. These proteins have a variety of physiological functions that give them well recognized roles in clinical medicine. In Xenopus tropicalis, a widely used animal model for physiology research, the repertoire of GPCRs may help link the GPCR evolutionary history in vertebrates from teleost fish to mammals. Results We have identified 1452 GPCRs in the X. tropicalis genome. Phylogenetic analyses classified these receptors into the following seven families: Glutamate, Rhodopsin, Adhesion, Frizzled, Secretin, Taste 2 and Vomeronasal 1. Nearly 70% of X. tropicalis GPCRs are represented by the following three types of receptors thought to receive chemosensory information from the outside world: olfactory, vomeronasal 1 and vomeronasal 2 receptors. Conclusion X. tropicalis shares a more similar repertoire of GPCRs with mammals than it does with fish. An examination of the three major groups of receptors related to olfactory/pheromone detection shows that in X. tropicalis, these groups have undergone lineage specific expansion. A comparison of GPCRs in X. tropicalis, teleost fish and mammals reveals the GPCR evolutionary history in vertebrates.

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

    Science.gov (United States)

    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. The Effect of Plasma Exposure on Tail Regeneration of Tadpoles Xenopus Laevis

    Science.gov (United States)

    June, Joyce; Rivie, Adonis; Ezuduemoih, Raphael; Menon, Jaishri; Martus, Kevin

    2014-03-01

    Wound healing requires a balanced combination of nutrients and growth factors for healing and tissue regeneration. The effect of plasma exposure on tail regeneration of tadpoles, Xenopus laevis is investigated. The exposure of the wound to the helium plasma immediately followed the amputation of 40% of the tail. Amputation of the tail initiates regeneration of spinal cord, muscle, notochord, skin and connective tissues. By 24 h, the wound was covered by wound epithelium and blastema was formed by day 5. There was increased angiogenesis in plasma exposed tail regenerate compared to the control following 5 d post amputation. Observed was an increase in NO production in the regenerate of plasma exposed tadpoles was derived from increased activity of nNOS and iNOS. Western blot analysis for vascular endothelial growth factor showed stronger bands for the protein in amputated tadpoles of both the groups. Analysis of the composition and characteristics of the plasma using optical emission spectroscopy indicates excited state species consisting of N2, N2+,and OH is present in the plasma. This study was supported, in part, by the NSF Grant 1040108.

  5. Spinal cord regeneration in Xenopus tadpoles proceeds through activation of Sox2-positive cells

    Science.gov (United States)

    2012-01-01

    Background In contrast to mammals, amphibians, such as adult urodeles (for example, newts) and anuran larvae (for example, Xenopus) can regenerate their spinal cord after injury. However, the cellular and molecular mechanisms involved in this process are still poorly understood. Results Here, we report that tail amputation results in a global increase of Sox2 levels and proliferation of Sox2+ cells. Overexpression of a dominant negative form of Sox2 diminished proliferation of spinal cord resident cells affecting tail regeneration after amputation, suggesting that spinal cord regeneration is crucial for the whole process. After spinal cord transection, Sox2+ cells are found in the ablation gap forming aggregates. Furthermore, Sox2 levels correlated with regenerative capabilities during metamorphosis, observing a decrease in Sox2 levels at non-regenerative stages. Conclusions Sox2+ cells contribute to the regeneration of spinal cord after tail amputation and transection. Sox2 levels decreases during metamorphosis concomitantly with the lost of regenerative capabilities. Our results lead to a working hypothesis in which spinal cord damage activates proliferation and/or migration of Sox2+ cells, thus allowing regeneration of the spinal cord after tail amputation or reconstitution of the ependymal epithelium after spinal cord transection. PMID:22537391

  6. Xenopus Nanos1 is required to prevent endoderm gene expression and apoptosis in primordial germ cells

    Science.gov (United States)

    Lai, Fangfang; Singh, Amar; King, Mary Lou

    2012-01-01

    Nanos is expressed in multipotent cells, stem cells and primordial germ cells (PGCs) of organisms as diverse as jellyfish and humans. It functions together with Pumilio to translationally repress targeted mRNAs. Here we show by loss-of-function experiments that Xenopus Nanos1 is required to preserve PGC fate. Morpholino knockdown of maternal Nanos1 resulted in a striking decrease in PGCs and a loss of germ cells from the gonads. Lineage tracing and TUNEL staining reveal that Nanos1-deficient PGCs fail to migrate out of the endoderm. They appear to undergo apoptosis rather than convert to normal endoderm. Whereas normal PGCs do not become transcriptionally active until neurula, Nanos1-depleted PGCs prematurely exhibit a hyperphosphorylated RNA polymerase II C-terminal domain at the midblastula transition. Furthermore, they inappropriately express somatic genes characteristic of endoderm regulated by maternal VegT, including Xsox17α, Bix4, Mixer, GATA4 and Edd. We further demonstrate that Pumilio specifically binds VegT RNA in vitro and represses, along with Nanos1, VegT translation within PGCs. Repressed VegT RNA in wild-type PGCs is significantly less stable than VegT in Nanos1-depleted PGCs. Our data indicate that maternal VegT RNA is an authentic target of Nanos1/Pumilio translational repression. We propose that Nanos1 functions to translationally repress RNAs that normally specify endoderm and promote apoptosis, thus preserving the germline. PMID:22399685

  7. Delayed fertilization of anuran amphibian (Xenopus) eggs leads to reduced numbers of primordial germ cells

    Science.gov (United States)

    Wakahara, M.; Neff, A. W.; Malacinski, G. M.

    1984-01-01

    Several media were tested for the extent to which they promoted high fertilization efficiencies in ovulated, stripped Xenopus eggs. One medium was selected for maintaining eggs in a 'delayed fertilization' (DelF) condition. DelF eggs displayed several unusual characteristics, including shift of the center of gravity, prominent sperm entrance site, and occasional polyspermy. The frequency of normal pattern formation varied according to the length of time eggs were maintained in the DelF condition. Various developmental abnormalities were observed during gastrulation, neurulation, and organogenesis. Most abnormalities appeared, however, to be related to morphogenesis of the endoderm. Primordial germ cell (PGC) development was examined in DelF eggs which displayed normal external morphological features at the swimming tadpole stage. PGC counts were usually normal in short-duration (eg, 5 hr) DelF eggs, but frequently substantially reduced or completely diminished in longer-duration (eg, 25h) tadpoles. Six spawnings were compared and shown to exhibit considerable variability in fertility, morphogenesis, and PGC development. Yolk platelet shifts and developmental parameters were examined in two additional spawnings. The subcortical cytoplasm in which the germ plasm is normally localized appeared to be disrupted in longer duration DelF eggs. That observation may account for low PGC counts in DelF tadpoles.

  8. A GTPase controls cell-substrate adhesion in Xenopus XTC fibroblasts.

    Science.gov (United States)

    Symons, M H; Mitchison, T J

    1992-09-01

    Cell-substrate adhesion is crucial at various stages of development and for the maintenance of normal tissues. Little is known about the regulation of these adhesive interactions. To investigate the role of GTPases in the control of cell morphology and cell-substrate adhesion we have injected guanine nucleotide analogs into Xenopus XTC fibroblasts. Injection of GTP gamma S inhibited ruffling and increased spreading, suggesting an increase in adhesion. To further investigate this, we made use of GRGDSP, a peptide which inhibits binding of integrins to vitronectin and fibronectin. XTC fibroblasts injected with non-hydrolyzable analogs of GTP took much more time to round up than mock-injected cells in response to treatment with GRGDSP, while GDP beta S-injected cells rounded up in less time than controls. Injection with GTP gamma S did not inhibit cell rounding induced by trypsin however, showing that cell contractility is not significantly affected by the activation of GTPases. These data provide evidence for the existence of a GTPase which can control cell-substrate adhesion from the cytoplasm. Treatment of XTC fibroblasts with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate reduced cell spreading and accelerated cell rounding in response to GRGDSP, which is essentially opposite to the effect exerted by non-hydrolyzable GTP analogs. These results suggest the existence of at least two distinct pathways controlling cell-substrate adhesion in XTC fibroblasts, one depending on a GTPase and another one involving protein kinase C.

  9. 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.

  10. Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis

    Directory of Open Access Journals (Sweden)

    Yergeau Donald A

    2011-11-01

    Full Text Available Abstract Background The Sleeping Beauty (SB transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. Results Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. Conclusions In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.

  11. Lateral Line Scene Analysis in the Purely Aquatic Frog Xenopus laevis Daudin (Pipidae).

    Science.gov (United States)

    Elepfandt, Andreas; Lebrecht, Silke; Schroedter, Kirsten; Brudermanns, Britta; Hillig, Renate; Schuberth, Claire; Fliess, Alexander

    2016-01-01

    The ability to locate and discriminate water surface waves that impinge simultaneously from multiple directions was studied in the clawed frog, Xenopus laevis. Monofrequency waves of 5-30 Hz were presented from point sources at a distance of 10 cm from the frog, unless stated otherwise, and the animal's response turn towards the wave origin examined. Two-choice conditioning with two simultaneous frontal waves at a 90-degree inter-wave angle revealed discrimination thresholds lower than 1 Hz for 10- to 20-Hz source wave frequencies. Smaller inter-wave angles resulted in larger thresholds, and no discrimination was found below 40°. If a third wave was added from behind, the frequency discrimination of the two frontal waves deteriorated, with 18 Hz being discriminated from waves differing by at least 2.75 Hz. Subjects also discriminated between two simultaneous waves of equal frequency presented from differing distances. At a distance of 10 cm, the discrimination threshold was 0.95 cm. Thus, X. laevis is capable of discriminating source distances in an overlap on the basis of wave curvatures. The detection of source directions among four, six or eight waves of equal frequency and distance was investigated by measuring the angular distribution of the response turns. Turns were significantly more closely oriented towards sources than to intermediate directions. The orientation accuracy did not degrade with the number of waves. © 2016 S. Karger AG, Basel.

  12. Neptune is involved in posterior axis and tail formation in Xenopus embryogenesis.

    Science.gov (United States)

    Takeda, Masatoshi; Kurauchi, Takayuki; Yamazaki, Takeshi; Izutsu, Yumi; Maéno, Mitsugu

    2005-09-01

    In order to elucidate the molecular mechanisms underlying the posterior axis and tail formation in embryogenesis, the function of Neptune, a zinc-finger transcription factor, in Xenopus laevis embryos was investigated. Injection of neptune mRNA into the animal pole area of embryos resulted in the formation of an additional tail structure that included a neural tube and muscle tissue. This activity required FGF signaling since coinjection of a dominant-negative FGF receptor RNA (XFD) completely blocked the formation of a tail structure. A loss-of-function experiment using a fusion construct of neptune and Drosophila engrailed (en-neptune) RNA showed that endogenous Neptune is necessary for formation of the posterior trunk and tail. Furthermore, activity of Neptune was necessary for the endogenous expression of brachyury and fgf-8 at the late gastrula stage. These findings demonstrate a novel function of Neptune in the process of anterior-posterior axis formation through the FGF and brachyury signaling cascades. An experiment using a combination explant with ventral and dorsal marginal tissues showed that cooperation of these two distinct tissues is important for the tail formation and that expression of Neptune in prospective ventral cells may be involved in the activation of the process of tail formation. Copyright 2005 Wiley-Liss, Inc.

  13. 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.

  14. NH3 and NH4+ permeability in aquaporin-expressing Xenopus oocytes

    DEFF Research Database (Denmark)

    Holm, Lars M.; Jahn, Thomas Paul; Møller, Anders Laurell Blom

    2005-01-01

    We have shown recently, in a yeast expression system, that some aquaporins are permeable to ammonia. In the present study, we expressed the mammalian aquaporins AQP8, AQQP9, AQP3, AQP1 and a plant aquaporin TIP2;1 in Xenopus oocytes to study the transport of ammonia (NH3) and ammonium (NH4+) under...... opencircuit and voltage-clamped conditions. TIP2;1 was tested as the wild-type and in a mutated version (tip2;1) in which the water permeability is intact. When AQP8-, AQP9-, AQP3- and TIP2;1-expressing oocytes were placed in a well-stirred bathing medium of low buffer capacity, NH3 permeability was evident...... from the acidification of the bathing medium; the effects observed with AQP1 and tip2;1 did not exceed that of native oocytes. AQP8, AQP9, AQP3, and TIP2;1 were permeable to larger amides, while AQP1 was not. Under voltage-clamp conditions, given sufficient NH3, AQP8, AQP9, AQP3, and TIP2;1 supported...

  15. Caldesmon regulates actin dynamics to influence cranial neural crest migration in Xenopus.

    Science.gov (United States)

    Nie, Shuyi; Kee, Yun; Bronner-Fraser, Marianne

    2011-09-01

    Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells to migrate a significantly shorter distance, prevents their segregation into distinct migratory streams, and later results in severe defects in cartilage formation. Demonstrating specificity, these effects are rescued by adding back exogenous CaD. Interestingly, CaD proteins with mutations in the Ca(2+)-calmodulin-binding sites or ErK/Cdk1 phosphorylation sites fail to rescue the knockdown phenotypes, whereas mutation of the PAK phosphorylation site is able to rescue them. Analysis of neural crest explants reveals that CaD is required for the dynamic arrangements of actin and, thus, for cell shape changes and process formation. Taken together, these results suggest that the actin-modulating activity of CaD may underlie its critical function and is regulated by distinct signaling pathways during normal neural crest migration.

  16. 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.

  17. Overland movement in African clawed frogs (Xenopus laevis): empirical dispersal data from within their native range.

    Science.gov (United States)

    De Villiers, F André; Measey, John

    2017-01-01

    Dispersal forms are an important component of the ecology of many animals, and reach particular importance for predicting ranges of invasive species. African clawed frogs (Xenopus laevis) move overland between water bodies, but all empirical studies are from invasive populations with none from their native southern Africa. Here we report on incidents of overland movement found through a capture-recapture study carried out over a three year period in Overstrand, South Africa. The maximum distance moved was 2.4 km with most of the 91 animals, representing 5% of the population, moving ∼150 m. We found no differences in distances moved by males and females, despite the former being smaller. Fewer males moved overland, but this was no different from the sex bias found in the population. In laboratory performance trials, we found that males outperformed females, in both distance moved and time to exhaustion, when corrected for size. Overland movement occurred throughout the year, but reached peaks in spring and early summer when temporary water bodies were drying. Despite permanent impoundments being located within the study area, we found no evidence for migrations of animals between temporary and permanent water bodies. Our study provides the first dispersal kernel for X. laevis and suggests that it is similar to many non-pipid anurans with respect to dispersal.

  18. Defining synphenotype groups in Xenopus tropicalis by use of antisense morpholino oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Amer Ahmed Rana

    2006-11-01

    Full Text Available To identify novel genes involved in early development, and as proof-of-principle of a large-scale reverse genetics approach in a vertebrate embryo, we have carried out an antisense morpholino oligonucleotide (MO screen in Xenopus tropicalis, in the course of which we have targeted 202 genes expressed during gastrula stages. MOs were designed to complement sequence between -80 and +25 bases of the initiating AUG codons of the target mRNAs, and the specificities of many were tested by (i designing different non-overlapping MOs directed against the same mRNA, (ii injecting MOs differing in five bases, and (iii performing "rescue" experiments. About 65% of the MOs caused X. tropicalis embryos to develop abnormally (59% of those targeted against novel genes, and we have divided the genes into "synphenotype groups," members of which cause similar loss-of-function phenotypes and that may function in the same developmental pathways. Analysis of the expression patterns of the 202 genes indicates that members of a synphenotype group are not necessarily members of the same synexpression group. This screen provides new insights into early vertebrate development and paves the way for a more comprehensive MO-based analysis of gene function in X. tropicalis.

  19. The mucosubstance coating the pneumonocytes in the lungs of Xenopus laevis and Lacerta viridis.

    Science.gov (United States)

    Meban, C

    1975-01-01

    The layer of mucosubstance that is associated with the free surface membranes of the pneumonocytes in the lungs of the toad Xenopus laevis and the lizard Lacerta viridis was demonstrated by electron microscopy using iron oxide stain. The form and staining reactions of the mucosubstance layer were similar in both animals. In electron micrographs the mucosubstance was represented by a band of densely stained material (25-50 nm thick) which coated the entire free surface of the pneumonocytes. It appeared to be firmly attached to the outer leaflet of the superficial plasma membrane. Short lengths of osmiophilic membranes, presumed to be fragments of pulmonary surfactant, were often observed lying free in the air spaces but they did not show any affinity for iron stain. Incubation of lung sections in a solution of neuraminidase produced a marked decrease in the intensity of the surface staining; no change was detected after incubation in trypsin, papain, hyaluronidase, N-acetyl cysteine, or phosphate buffer. It is, therefore, concluded that the pneumonocyte surface coat consists mainly of a sialomucin.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Augmin promotes meiotic spindle formation and bipolarity in Xenopus egg extracts.

    Science.gov (United States)

    Petry, Sabine; Pugieux, Céline; Nédélec, François J; Vale, Ronald D

    2011-08-30

    Female meiotic spindles in many organisms form in the absence of centrosomes, the organelle typically associated with microtubule (MT) nucleation. Previous studies have proposed that these meiotic spindles arise from RanGTP-mediated MT nucleation in the vicinity of chromatin; however, whether this process is sufficient for spindle formation is unknown. Here, we investigated whether a recently proposed spindle-based MT nucleation pathway that involves augmin, an 8-subunit protein complex, also contributes to spindle morphogenesis. We used an assay system in which hundreds of meiotic spindles can be observed forming around chromatin-coated beads after introduction of Xenopus egg extracts. Spindles forming in augmin-depleted extracts showed reduced rates of MT formation and were predominantly multipolar, revealing a function of augmin in stabilizing the bipolar shape of the acentrosomal meiotic spindle. Our studies also have uncovered an apparent augmin-independent MT nucleation process from acentrosomal poles, which becomes increasingly active over time and appears to partially rescue the spindle defects that arise from augmin depletion. Our studies reveal that spatially and temporally distinct MT generation pathways from chromatin, spindle MTs, and acentrosomal poles all contribute to robust bipolar spindle formation in meiotic extracts.

  2. Structure of the SANT domain from the Xenopus chromatin remodeling factor ISWI

    Energy Technology Data Exchange (ETDEWEB)

    Horton, John R.; Elgar, Stuart J.; Khan, Seema I.; Zhang, Xing; Wade, Paul A.; Cheng, Xiaodong (Emory-MED)

    2008-09-17

    The SANT (Swi3, Ada2, N-Cor, and TFIIIB) module was first described as a putative DNA-binding domain with strong similarity to the helix-turn-helix DNA binding domain of Myb-related proteins. The X-ray structure of the C-terminal one third portion of the ATPase ISWI of Drosophila melangoaster, containing both SANT and SLIDE (SANT-Like ISWI Domain), confirmed the overall helix-turn-helix structural architecture of SANT as well as SLIDE. However, the DNA-contacting residues in Myb are not conserved in SANT and the structurally corresponding residues in the ISWI SANT domain are acidic, and therefore incompatible with DNA interaction. Recent studies suggested that SANT domains might be a histone-tail-binding module, including the DNA binding SANT domain of c-Myb. Here they present the X-ray structure of Xenopus laevis ISWI SANT domain, derived from limited proteolysis of a C-terminal fragment of ISWI protein.

  3. 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.

  4. Targeting kidney CLC-K channels: pharmacological profile in a human cell line versus Xenopus oocytes.

    Science.gov (United States)

    Imbrici, Paola; Liantonio, Antonella; Gradogna, Antonella; Pusch, Michael; Camerino, Diana Conte

    2014-10-01

    CLC-K chloride channels play a crucial role in kidney physiology and genetic mutations, affecting their function are responsible for severe renal salt loss in humans. Thus, compounds that selectively bind to CLC-Ka and/or CLC-Kb channels and modulate their activity may have a significant therapeutic potential. Here, we compare the biophysical and pharmacological behaviors of human CLC-K channels expressed either in HEK293 cells or in Xenopus oocytes and we show that CLC-K channel properties are greatly influenced by the biochemical environment surrounding the channels. Indeed, in HEK293 cells the potentiating effect of niflumic acid (NFA) on CLC-Ka/barttin and CLC-Kb/barttin channels seems to be absent while the blocking efficacy of niflumic acid and benzofuran derivatives observed in oocytes is preserved. The NFA block does not seem to involve the accessory subunit barttin on CLC-K1 channels. In addition, the sensitivity of CLC-Ks to external Ca(2+) is reduced in HEK293 cells. Based on our findings, we propose that mammalian cell lines are a suitable expression system for the pharmacological profiling of CLC-Ks. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. The effect of food properties on grasping and manipulation in the aquatic frog, Xenopus laevis.

    Science.gov (United States)

    Anzeraey, Aude; Aumont, Madeleine; Decamps, Thierry; Herrel, Anthony; Pouydebat, Emmanuelle

    2017-10-05

    The ability to grasp an object is fundamental from an evolutionary perspective. Involved in many daily activities, grasping has been extensively studied in primates and other mammals. Yet, other groups of tetrapods, including anurans, have also evolved significant forelimb prehensile capacities that are often thought to have originated in an arboreal context. However, grasping is also observed in aquatic species. Yet, how aquatic frogs use their forelimbs to capture and manipulate prey remains largely unknown. The aim of this study is to explore how the grasping and manipulation of food items in aquatic frogs is impacted by food properties such as size and mobility. To do so we use the aquatic frog Xenopus laevis and quantified the use of the hands and fingers while processing mobile and stationary prey of different sizes (small, intermediate, and large prey). Our results show that X. laevis is able to individualize the digits and that the mobility and the length of the prey significantly influence the kind of grasping pattern used. Grasping abilities are thus not specific to terrestrial, nor arboreal species. These results illustrate how prey properties impact grasping and manipulation strategies in an aquatic frog and shed further light on the ecological contexts that may have given rise to the origin of grasping in frogs. © 2017. Published by The Company of Biologists Ltd.

  6. Hspa9 is required for pronephros specification and formation in Xenopus laevis.

    Science.gov (United States)

    Gassié, Lionel; Lombard, Aude; Moraldi, Tiphanie; Bibonne, Anne; Leclerc, Catherine; Moreau, Marc; Marlier, Arnaud; Gilbert, Thierry

    2015-12-01

    Development of the pronephros in Xenopus laevis is largely dependent on retinoic acid signaling at the time of kidney field specification with the simultaneous occurrence of a necessary calcium signaling. At the crossroads of these two signaling pathways, we studied the role of Hspa9 (heat shock 70 kDa protein 9) encoding a mitochondrial chaperone in pronephros development. We first showed that Hspa9 is highly expressed in the pronephros territory and elongating nephric duct. We then observed that upon reduced retinoic acid signaling hspa9 expression was reduced as pax8 and pax2. Overexpression of hspa9 enlarged the pax8 positive pronephros territory, leading to a larger pronephric tubule. Loss of function of hspa9 in the kidney field using morpholino approach severely reduced pax8 expression and pronephros formation. Phenotypic rescue was achieved by co-injection of the full-length murine Hspa9 mRNA. However, no rescue was observed when Hspa9 mRNA lacking the mitochondrial-targeting sequence was injected, as this truncated form is able to interfere with pronephros formation when injected solely. Hspa9 is an important mediator for pronephros development through modulation of pax8. Mitochondrial functions of hspa9 are likely to be involved in specification of pronephric cell fate. © 2015 Wiley Periodicals, Inc.

  7. Signaling and transcriptional regulation in neural crest specification and migration: lessons from xenopus embryos.

    Science.gov (United States)

    Pegoraro, Caterina; Monsoro-Burq, Anne H

    2013-01-01

    The neural crest is a population of highly migratory and multipotent cells, which arises from the border of the neural plate in vertebrate embryos. In the last few years, the molecular actors of neural crest early development have been intensively studied, notably by using the frog embryo, as a prime model for the analysis of the earliest embryonic inductions. In addition, tremendous progress has been made in understanding the molecular and cellular basis of Xenopus cranial neural crest migration, by combining in vitro and in vivo analysis. In this review, we examine how the action of previously known neural crest-inducing signals [bone morphogenetic protein (BMP), wingless-int (Wnt), fibroblast growth factor (FGF)] is controlled by newly discovered modulators during early neural plate border patterning and neural crest specification. This regulation controls the induction of key transcription factors that cooperate to pattern the premigratory neural crest progenitors. These data are discussed in the perspective of the gene regulatory network that controls neural and neural crest patterning. We then address recent findings on noncanonical Wnt signaling regulation, cell polarization, and collective cell migration which highlight how cranial neural crest cells populate their target tissue, the branchial arches, in vivo. More than ever, the neural crest stands as a powerful and attractive model to decipher complex vertebrate regulatory circuits in vivo. Copyright © 2012 Wiley Periodicals, Inc.

  8. 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.

  9. 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.

  10. Features of vestibuloocular reflex modulations induced by altered gravitational forces in tadpoles ( Xenopus laevis)

    Science.gov (United States)

    Sebastian, C.; Horn, E.

    2001-01-01

    In Xenopus laevis tadpoles, we studied the static vestibuloocular reflex (rVOR) in relation to modifications of the gravitational environment to find basic mechanisms of how altered gravitational forces (AGF) affect this reflex. Animals were exposed to microgravity during space flight or hypergravity (3g) for 4 to 12 days. Basic observations were that (1) the development of the rVOR is significantly affected by altered gravitational conditions, (2) the duration of 1g-readaptation depends on the strength of the test stimulus, (3) μg induces malformations of the body which are related to the rVOR depression. Future studies are based on the hypotheses (1) that the vestibular nuclei play a key roll in the adaptation to AGF conditions, (2) that the stimulus transducing systems in the sense organ are affected by AGF conditions, and (3) that fertilized eggs will be converted to normal adults guided by physiological and morphological set points representing the genetic programs. Developmental retardation or acceleration, or otherwise occurring deviations from standard development during embryonic and postembryonic life will activate genes that direct the developmental processes towards normality.

  11. Thyroid disruption by technical decabromodiphenyl ether (DE-83R) at low concentrations in Xenopus laevis.

    Science.gov (United States)

    Qin, Xiaofei; Xia, Xijuan; Yang, Zhongzhi; Yan, Shishuai; Zhao, Yaxian; Wei, Rongguo; Li, Yan; Tian, Mi; Zhao, Xingru; Qin, Zhanfen; Xu, Xiaobai

    2010-01-01

    Decabromodiphenyl ether (decaBDE), as a flame retardant, is widely produced and used. To study the thyroid disruption by technical decaBDE at low concentrations, Xenopus laevis tadpoles were exposed to technical decaBDE mixture DE-83R (1-1000 ng/L) in water from stage 46/47 (free swimming larvae, system of Nieuwkoop and Faber) to stage 62. DE-83R at concentration of 1000 ng/L significantly delayed the time to metamorphosis (presented by forelimb emergence, FLE). Histological examination showed that DE-83R at all tested concentrations caused histological alterations - multilayer follicular epithelial cell and markedly increased follicle size accompanied by partial colloid depletion and increase in the peripheral colloid vacuolation, in thyroid glands. All tested concentrations of DE-83R also induced a down-regulation of thyroid receptor mRNA expression. These results demonstrated that technical decaBDE disrupted the thyroid system in X. laevis tadpoles. Analysis of polybrominated diphenyl ethers (PBDEs) (sum of 39 congeners) in X. laevis indicated that mean concentrations of total PBDEs in X. laevis exposed to 1, 10, 100, 1000 ng/L were 11.0, 128.1, 412.1, 1400.2 ng/g wet weight, respectively. Considering that PBDEs burden ofX. laevis tadpoles was close to PBDEs levels in amphibians as reported in previous studies, our study has raised new concerns for thyroid disruption in amphibians of technical decaBDE at environmentally relevant concentrations.

  12. Development of the Flight Hardware for the Experiment XENOPUS on the Kubik BIO4-Mission

    Science.gov (United States)

    Horn, Eberhard R.; Böser, Sybille; Franz, Markus; Gabriel, Martin; Hiesgen, Norbert; Kübler, Ulrich; Porciani, Massimiliano; Schwarzwälder, Achim; Zolesi, Valfredo

    2011-02-01

    The needs of developing aquatic animals depend on their age. For example, amphibian tadpole stages require regular food supply while embryos use their yolk as food source. Thus, life support systems have to be adapted to the different ages; an efficient control for water cleanness and steady food supply is mandatory for a safe flight in microgravity. A list of biological and technical requirements prompted the concept of the Dornier-Mini-System and the design for the Astrium SUPPLY Unit. These life support systems are connected with the Astrium miniaquarium that was used several times for the transport of small aquatic animals in space. Scientific experience from this concept was considered by Kayser Italia to design and develop a space suitable hardware. Its functionality was successfully demonstrated by the experiment XENOPUS that flew on the Soyuz TMA13/TMA12 mission in 2008. From 36 launched tadpoles, 35 returned back to Earth after the 12 days lasting space flight in physiologically stable conditions.

  13. TLR3 is required for survival following Coxsackievirus B3 infection by driving T lymphocyte activation and polarization: The role of dendritic cells

    National Research Council Canada - National Science Library

    Renata Sesti-Costa; Marcela Cristina Santiago Françozo; Grace Kelly Silva; José Luiz Proenca-Modena; João Santana Silva

    2017-01-01

    .... In the current study, we found that TLR3 expression in dendritic cells plays a role in their activation upon CVB3 infection in vitro, as TLR3-deficient dendritic cells up-regulate CD80 and CD86...

  14. Computer viruses

    Science.gov (United States)

    Denning, Peter J.

    1988-01-01

    The worm, Trojan horse, bacterium, and virus are destructive programs that attack information stored in a computer's memory. Virus programs, which propagate by incorporating copies of themselves into other programs, are a growing menace in the late-1980s world of unprotected, networked workstations and personal computers. Limited immunity is offered by memory protection hardware, digitally authenticated object programs,and antibody programs that kill specific viruses. Additional immunity can be gained from the practice of digital hygiene, primarily the refusal to use software from untrusted sources. Full immunity requires attention in a social dimension, the accountability of programmers.

  15. Extraterrestrial Viruses?

    OpenAIRE

    Jurado Hernández, Daniel José

    2017-01-01

    Fundamentals of Life - Origin and Fundamentals of Living Things. Evaluation rubric to evaluate the debate and presentation about the point of view regarding the possibility of viruses from the outer space.

  16. Zika Virus

    OpenAIRE

    Musso, Didier; Gubler, Duane J.

    2016-01-01

    Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) in the genus Flavivirus and the family Flaviviridae. ZIKV was first isolated from a nonhuman primate in 1947 and from mosquitoes in 1948 in Africa, and ZIKV infections in humans were sporadic for half a century before emerging in the Pacific and the Americas. ZIKV is usually transmitted by the bite of infected mosquitoes. The clinical presentation of Zika fever is nonspecific and can be misdiagnosed as other infectious diseases, especi...

  17. Maternal Wnt/β-catenin signaling coactivates transcription through NF-κB binding sites during Xenopus axis formation.

    Directory of Open Access Journals (Sweden)

    Neil J Armstrong

    Full Text Available Maternal Wnt/β-Catenin signaling establishes a program of dorsal-specific gene expression required for axial patterning in Xenopus. We previously reported that a subset of dorsally expressed genes depends not only on Wnt/β-Catenin stimulation, but also on a MyD88-dependent Toll-like receptor/IL1-receptor (TLR/IL1-R signaling pathway. Here we show that these two signal transduction cascades converge in the nucleus to coactivate gene transcription in blastulae through a direct interaction between β-Catenin and NF-κB proteins. A transdominant inhibitor of NF-κB, ΔNIκBα, phenocopies loss of MyD88 protein function, implicating Rel/NF-κB proteins as selective activators of dorsal-specific gene expression. Sensitive axis formation assays in the embryo demonstrate that dorsalization by Wnt/β-Catenin requires NF-κB protein activity, and vice versa. Xenopus nodal-related 3 (Xnr3 is one of the genes with dual β-Catenin/NF-κB input, and a proximal NF-κB consensus site contributes to the regional activity of its promoter. We demonstrate in vitro binding of Xenopus β-Catenin to several XRel proteins. This interaction is observed in vivo upon Wnt-stimulation. Finally, we show that a synthetic luciferase reporter gene responds to both endogenous and exogenous β-Catenin levels in an NF-κB motif dependent manner. These results suggest that β-Catenin acts as a transcriptional co-activator of NF-κB-dependent transcription in frog primary embryonic cells.

  18. Maternal Wnt/β-Catenin Signaling Coactivates Transcription through NF-κB Binding Sites during Xenopus Axis Formation

    Science.gov (United States)

    Armstrong, Neil J.; Fagotto, François; Prothmann, Christian; Rupp, Ralph A. W.

    2012-01-01

    Maternal Wnt/β-Catenin signaling establishes a program of dorsal-specific gene expression required for axial patterning in Xenopus. We previously reported that a subset of dorsally expressed genes depends not only on Wnt/β-Catenin stimulation, but also on a MyD88-dependent Toll-like receptor/IL1-receptor (TLR/IL1-R) signaling pathway. Here we show that these two signal transduction cascades converge in the nucleus to coactivate gene transcription in blastulae through a direct interaction between β-Catenin and NF-κB proteins. A transdominant inhibitor of NF-κB, ΔNIκBα, phenocopies loss of MyD88 protein function, implicating Rel/NF-κB proteins as selective activators of dorsal-specific gene expression. Sensitive axis formation assays in the embryo demonstrate that dorsalization by Wnt/β-Catenin requires NF-κB protein activity, and vice versa. Xenopus nodal-related 3 (Xnr3) is one of the genes with dual β-Catenin/NF-κB input, and a proximal NF-κB consensus site contributes to the regional activity of its promoter. We demonstrate in vitro binding of Xenopus β-Catenin to several XRel proteins. This interaction is observed in vivo upon Wnt-stimulation. Finally, we show that a synthetic luciferase reporter gene responds to both endogenous and exogenous β-Catenin levels in an NF-κB motif dependent manner. These results suggest that β-Catenin acts as a transcriptional co-activator of NF-κB-dependent transcription in frog primary embryonic cells. PMID:22590521

  19. Planar cell polarity enables posterior localization of nodal cilia and left-right axis determination during mouse and Xenopus embryogenesis.

    Directory of Open Access Journals (Sweden)

    Dragana Antic

    2010-02-01

    Full Text Available Left-right asymmetry in vertebrates is initiated in an early embryonic structure called the ventral node in human and mouse, and the gastrocoel roof plate (GRP in the frog. Within these structures, each epithelial cell bears a single motile cilium, and the concerted beating of these cilia produces a leftward fluid flow that is required to initiate left-right asymmetric gene expression. The leftward fluid flow is thought to result from the posterior tilt of the cilia, which protrude from near the posterior portion of each cell's apical surface. The cells, therefore, display a morphological planar polarization. Planar cell polarity (PCP is manifested as the coordinated, polarized orientation of cells within epithelial sheets, or as directional cell migration and intercalation during convergent extension. A set of evolutionarily conserved proteins regulates PCP. Here, we provide evidence that vertebrate PCP proteins regulate planar polarity in the mouse ventral node and in the Xenopus gastrocoel roof plate. Asymmetric anterior localization of VANGL1 and PRICKLE2 (PK2 in mouse ventral node cells indicates that these cells are planar polarized by a conserved molecular mechanism. A weakly penetrant Vangl1 mutant phenotype suggests that compromised Vangl1 function may be associated with left-right laterality defects. Stronger functional evidence comes from the Xenopus GRP, where we show that perturbation of VANGL2 protein function disrupts the posterior localization of motile cilia that is required for leftward fluid flow, and causes aberrant expression of the left side-specific gene Nodal. The observation of anterior-posterior PCP in the mouse and in Xenopus embryonic organizers reflects a strong evolutionary conservation of this mechanism that is important for body plan determination.

  20. Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

    Science.gov (United States)

    Orsini, Francesco; Santacroce, Massimo; Cremona, Andrea; Gosvami, Nitya N; Lascialfari, Alessandro; Hoogenboom, Bart W

    2014-11-01

    Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.

  1. Ascl1 phospho-status regulates neuronal differentiation in a Xenopus developmental model of neuroblastoma

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    Luke A. Wylie

    2015-05-01

    Full Text Available Neuroblastoma (NB, although rare, accounts for 15% of all paediatric cancer mortality. Unusual among cancers, NBs lack a consistent set of gene mutations and, excluding large-scale chromosomal rearrangements, the genome seems to be largely intact. Indeed, many interesting features of NB suggest that it has little in common with adult solid tumours but instead has characteristics of a developmental disorder. NB arises overwhelmingly in infants under 2 years of age during a specific window of development and, histologically, NB bears striking similarity to undifferentiated neuroblasts of the sympathetic nervous system, its likely cells of origin. Hence, NB could be considered a disease of development arising when neuroblasts of the sympathetic nervous system fail to undergo proper differentiation, but instead are maintained precociously as progenitors with the potential for acquiring further mutations eventually resulting in tumour formation. To explore this possibility, we require a robust and flexible developmental model to investigate the differentiation of NB's presumptive cell of origin. Here, we use Xenopus frog embryos to characterise the differentiation of anteroventral noradrenergic (AVNA cells, cells derived from the neural crest. We find that these cells share many characteristics with their mammalian developmental counterparts, and also with NB cells. We find that the transcriptional regulator Ascl1 is expressed transiently in normal AVNA cell differentiation but its expression is aberrantly maintained in NB cells, where it is largely phosphorylated on multiple sites. We show that Ascl1's ability to induce differentiation of AVNA cells is inhibited by its multi-site phosphorylation at serine-proline motifs, whereas overexpression of cyclin-dependent kinases (CDKs and MYCN inhibit wild-type Ascl1-driven AVNA differentiation, but not differentiation driven by a phospho-mutant form of Ascl1. This suggests that the maintenance of ASCL1

  2. Genome-wide transcriptional response of Silurana (Xenopus tropicalis to infection with the deadly chytrid fungus.

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    Erica Bree Rosenblum

    Full Text Available Emerging infectious diseases are of great concern for both wildlife and humans. Several highly virulent fungal pathogens have recently been discovered in natural populations, highlighting the need for a better understanding of fungal-vertebrate host-pathogen interactions. Because most fungal pathogens are not fatal in the absence of other predisposing conditions, host-pathogen dynamics for deadly fungal pathogens are of particular interest. The chytrid fungus Batrachochytrium dendrobatidis (hereafter Bd infects hundreds of species of frogs in the wild. It is found worldwide and is a significant contributor to the current global amphibian decline. However, the mechanism by which Bd causes death in amphibians, and the response of the host to Bd infection, remain largely unknown. Here we use whole-genome microarrays to monitor the transcriptional responses to Bd infection in the model frog species, Silurana (Xenopus tropicalis, which is susceptible to chytridiomycosis. To elucidate the immune response to Bd and evaluate the physiological effects of chytridiomycosis, we measured gene expression changes in several tissues (liver, skin, spleen following exposure to Bd. We detected a strong transcriptional response for genes involved in physiological processes that can help explain some clinical symptoms of chytridiomycosis at the organismal level. However, we detected surprisingly little evidence of an immune response to Bd exposure, suggesting that this susceptible species may not be mounting efficient innate and adaptive immune responses against Bd. The weak immune response may be partially explained by the thermal conditions of the experiment, which were optimal for Bd growth. However, many immune genes exhibited decreased expression in Bd-exposed frogs compared to control frogs, suggesting a more complex effect of Bd on the immune system than simple temperature-mediated immune suppression. This study generates important baseline data for ongoing

  3. A role for biliverdin IXα in dorsal axis development of Xenopus laevis embryos

    Science.gov (United States)

    Falchuk, Kenneth H.; Contin, Jennifer M.; Dziedzic, T. Scott; Feng, Zhongling; French, Thayer C.; Heffron, Gregory J.; Montorzi, Marcelo

    2002-01-01

    The determinants of Xenopus laevis embryos that act before their first cell division are mandatory for the formation of mRNas required to establish the dorsal axis. Although their chemical identities are unknown, a number of their properties have long been recognized. One of the determinants is present in the cytoplasm and is sensitive to UV light. Thus, exposing stage 1 embryos to either standard 254-nm or, as shown here, to 366-nm UV light during the 0.3–0.4 time fraction of their first cycle inactivates the cytoplasmic determinant. As a consequence, both types of irradiated embryos fail to express dorsal markers, e.g., goosecoid and chordin, without affecting formation of ventral markers, e.g., Vent-1. The developmental outcome is dorsal axis-deficient morphology. We report here that biliverdin IXα, a normal constituent of cytoplasmic yolk platelets, is photo-transformed by irradiation with either 254- or 366-nm UV light and that the transformation triggers the dorsal axis deficiency. When the 254- or 366-nm UV-irradiated embryos, fated to dorsal axis deficiency, are incubated solely with μM amounts of biliverdin, they recover and form the axis. In contrast, incubation with either in vitro photo-transformed biliverdin or biliverdin IXα dimethyl ester does not induce recovery. The results define an approach to produce dorsal axis-deficient embryos by photo-transforming its biliverdin by irradiation with 366-nm UV light and identify an unsuspected role for biliverdin IXα in X. laevis embryogenesis. PMID:11782548

  4. hmmr mediates anterior neural tube closure and morphogenesis in the frog Xenopus.

    Science.gov (United States)

    Prager, Angela; Hagenlocher, Cathrin; Ott, Tim; Schambony, Alexandra; Feistel, Kerstin

    2017-10-01

    Development of the central nervous system requires orchestration of morphogenetic processes which drive elevation and apposition of the neural folds and their fusion into a neural tube. The newly formed tube gives rise to the brain in anterior regions and continues to develop into the spinal cord posteriorly. Conspicuous differences between the anterior and posterior neural tube become visible already during neural tube closure (NTC). Planar cell polarity (PCP)-mediated convergent extension (CE) movements are restricted to the posterior neural plate, i.e. hindbrain and spinal cord, where they propagate neural fold apposition. The lack of CE in the anterior neural plate correlates with a much slower mode of neural fold apposition anteriorly. The morphogenetic processes driving anterior NTC have not been addressed in detail. Here, we report a novel role for the breast cancer susceptibility gene and microtubule (MT) binding protein Hmmr (Hyaluronan-mediated motility receptor, RHAMM) in anterior neurulation and forebrain development in Xenopus laevis. Loss of hmmr function resulted in a lack of telencephalic hemisphere separation, arising from defective roof plate formation, which in turn was caused by impaired neural tissue narrowing. hmmr regulated polarization of neural cells, a function which was dependent on the MT binding domains. hmmr cooperated with the core PCP component vangl2 in regulating cell polarity and neural morphogenesis. Disrupted cell polarization and elongation in hmmr and vangl2 morphants prevented radial intercalation (RI), a cell behavior essential for neural morphogenesis. Our results pinpoint a novel role of hmmr in anterior neural development and support the notion that RI is a major driving force for anterior neurulation and forebrain morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. 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.

  6. Transgenic analysis of signaling pathways required for Xenopus tadpole spinal cord and muscle regeneration.

    Science.gov (United States)

    Lin, Gufa; Chen, Ying; Slack, Jonathan M W

    2012-10-01

    The Xenopus tadpole has the capacity fully to regenerate its tail after amputation. Previously, we have established that this regeneration process requires the operation of several signaling pathways including the bone morphogenic protein, Wnt, and Fgf pathways. Here, we have addressed the signaling requirements for spinal cord and muscle regeneration in a tissue-specific manner. Two methods were used namely grafts of transgenic spinal cord to a wild type host, and the use of the Tet-on conditional transgenic system to express inhibitors in the individual tissues. For the grafting experiments, the tail was amputated through the graft, which contained a temperature inducible inhibitor of the Wnt-β-catenin pathway. For the Tet-on experiments, treatment with doxycycline was used to induce cell autonomous inhibitors of the Wnt-β-catenin or the Fgf pathway in either spinal cord or muscle. The results show that both spinal cord and muscle regeneration depend on both the Wnt-β-catenin and the Fgf pathways. This experimental design also enables us to observe the effect of inhibition of regeneration of one tissue on the regeneration of the others. Regardless of the method of inhibition, we find that reduction of spinal cord regeneration reduces regeneration of other parts of the tail, including the myotomal muscles. In contrast, reduction of muscle regeneration has no effect on the regeneration of the spinal cord. In common with other regeneration systems, this indicates that soluble factors from the spinal cord are needed to promote the regeneration of the other tissues in the tail. Copyright © 2012 Wiley Periodicals, Inc.

  7. Application of frog embryo teratogenesis assay-Xenopus to ecological risk assessment.

    Science.gov (United States)

    Hoke, Robert A; Ankley, Gerald T

    2005-10-01

    An expert workshop recently was convened to consider the frog embryo teratogenesis assay-Xenopus (FETAX) as a screening method for identifying the potential developmental toxicity of single chemicals and chemical mixtures. One recommendation from the workshop was that, in order to determine the utility of FETAX for ecological risk assessments, additional consideration of how the assay is conducted is necessary. In addition, a comparative evaluation would be useful of FETAX endpoints (i.e., survival, malformations, growth) versus each other, endpoints from aquatic toxicity tests using more commonly tested species of cladocerans and fish, and tests with other amphibian species. This review provides an evaluation and critique of the current FETAX protocol from two perspectives: Practical considerations relative to conducting the test and sensitivity of the assay (and associated endpoints) compared to tests with other species. Several aspects of the current standard protocol, including test temperature, diet, loading rates, and chemical exposure options, need to be modified to ensure that the assay is robust technically. Evaluation of FETAX data from the open literature indicates that growth is the most sensitive endpoint in the assay, followed by malformations and then survival; unfortunately, the growth endpoint often is not considered or reported in the assay. Comparison of FETAX data with acute toxicity data from tests with other amphibians or traditional aquatic test species indicates FETAX is relatively insensitive. This suggests that environmental risk assessments using acute hazard data from tests with traditional aquatic test species usually would be more protective of native amphibian species than risk assessments that use hazard data from FETAX.

  8. The inhibitory effect of the antipsychotic drug haloperidol on HERG potassium channels expressed in Xenopus oocytes

    Science.gov (United States)

    Suessbrich, H; Schönherr, R; Heinemann, S H; Attali, B; Lang, F; Busch, A E

    1997-01-01

    The antipsychotic drug haloperidol can induce a marked QT prolongation and polymorphic ventricular arrhythmias. In this study, we expressed several cloned cardiac K+ channels, including the human ether-a-go-go related gene (HERG) channels, in Xenopus oocytes and tested them for their haloperidol sensitivity.Haloperidol had only little effects on the delayed rectifier channels Kv1.1, Kv1.2, Kv1.5 and IsK, the A-type channel Kv1.4 and the inward rectifier channel Kir2.1 (inhibition <6% at 3 μM haloperidol).In contrast, haloperidol blocked HERG channels potently with an IC50 value of approximately 1 μM. Reduced haloperidol, the primary metabolite of haloperidol, produced a block with an IC50 value of 2.6 μM.Haloperidol block was use- and voltage-dependent, suggesting that it binds preferentially to either open or inactivated HERG channels. As haloperidol increased the degree and rate of HERG inactivation, binding to inactivated HERG channels is suggested.The channel mutant HERG S631A has been shown to exhibit greatly reduced C-type inactivation which occurs only at potentials greater than 0 mV. Haloperidol block of HERG S631A at 0 mV was four fold weaker than for HERG wild-type channels. Haloperidol affinity for HERG S631A was increased four fold at +40 mV compared to 0 mV.In summary, the data suggest that HERG channel blockade is involved in the arrhythmogenic side effects of haloperidol. The mechanism of haloperidol block involves binding to inactivated HERG channels. PMID:9138706

  9. Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance

    Science.gov (United States)

    Boulin, T; Fauvin, A; Charvet, CL; Cortet, J; Cabaret, J; Bessereau, J-L; Neveu, C

    2011-01-01

    BACKGROUND AND PURPOSE The cholinergic agonist levamisole is widely used to treat parasitic nematode infestations. This anthelmintic drug paralyses worms by activating a class of levamisole-sensitive acetylcholine receptors (L-AChRs) expressed in nematode muscle cells. However, levamisole efficacy has been compromised by the emergence of drug-resistant parasites, especially in gastrointestinal nematodes such as Haemonchus contortus. We report here the first functional reconstitution and pharmacological characterization of H. contortus L-AChRs in a heterologous expression system. EXPERIMENTAL APPROACH In the free-living nematode Caenorhabditis elegans, five AChR subunit and three ancillary protein genes are necessary in vivo and in vitro to synthesize L-AChRs. We have cloned the H. contortus orthologues of these genes and expressed them in Xenopus oocytes. We reconstituted two types of H. contortus L-AChRs with distinct pharmacologies by combining different receptor subunits. KEY RESULTS The Hco-ACR-8 subunit plays a pivotal role in selective sensitivity to levamisole. As observed with C. elegans L-AChRs, expression of H. contortus receptors requires the ancillary proteins Hco-RIC-3, Hco-UNC-50 and Hco-UNC-74. Using this experimental system, we demonstrated that a truncated Hco-UNC-63 L-AChR subunit, which was specifically detected in a levamisole-resistant H. contortus isolate, but not in levamisole-sensitive strains, hampers the normal function of L-AChRs, when co-expressed with its full-length counterpart. CONCLUSIONS AND IMPLICATIONS We provide the first functional evidence for a putative molecular mechanism involved in levamisole resistance in any parasitic nematode. This expression system will provide a means to analyse molecular polymorphisms associated with drug resistance at the electrophysiological level. PMID:21486278

  10. Sex chromosome differentiation and the W- and Z-specific loci in Xenopus laevis.

    Science.gov (United States)

    Mawaribuchi, Shuuji; Takahashi, Shuji; Wada, Mikako; Uno, Yoshinobu; Matsuda, Yoichi; Kondo, Mariko; Fukui, Akimasa; Takamatsu, Nobuhiko; Taira, Masanori; Ito, Michihiko

    2017-06-15

    Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W- and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278kb W-specific and 83kb Z-specific sequences on chromosome 2Lq32-33, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X. laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Hypotonic regulation of mouse epithelial sodium channel in Xenopus laevis oocytes.

    Science.gov (United States)

    Galizia, Luciano; Marino, Gabriela I; Ojea, Alejandro; Kotsias, Basilio A

    2013-12-01

    The regulation of the epithelial Na⁺ channel (ENaC) during cell swelling is relevant in cellular processes in which cell volume changes occur, i.e., migration, proliferation and cell absorption. Its sensitivity to hypotonically induced swelling was investigated in the Xenopus oocyte expression system with the injection of the three subunits of mouse ENaC. We used voltage-clamp techniques to study the amiloride-sensitive Na⁺ currents (INa(amil)) and video microscopic methodologies to assess oocyte volume changes. Under conditions of mild swelling (25 % reduced hypotonicity) inward current amplitude decreased rapidly over 1.5 min. In contrast, there was no change in current amplitude of H₂O-injected oocytes to the osmotic insult. INa(amil) kinetics analysis revealed a decrease in the slower inactivation time constant during the hypotonic stimuli. Currents from ENaC-injected oocytes were not sensitive to external Cl⁻ reduction. Neither short- nor long-term cytochalasin D treatment affected the observed response. Oocytes expressing a DEG mutant β-ENaC subunit (β-S518K) with an open probability of 1 had reduced INa(amil) hypotonic response compared to oocytes injected with wild-type ENaC subunits. Finally, during the hypotonic response ENaC-injected oocytes did not show a cell volume difference compared with water-injected oocytes. On this basis we suggest that hypotonicity-dependent ENaC inhibition is principally mediated through an effect on open probability of channels in the membrane.

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

    Directory of Open Access Journals (Sweden)

    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. Williams Syndrome Transcription Factor is critical for neural crest cell function in Xenopus laevis.

    Science.gov (United States)

    Barnett, Chris; Yazgan, Oya; Kuo, Hui-Ching; Malakar, Sreepurna; Thomas, Trevor; Fitzgerald, Amanda; Harbour, William; Henry, Jonathan J; Krebs, Jocelyn E

    2012-01-01

    Williams Syndrome Transcription Factor (WSTF) is one of ∼25 haplodeficient genes in patients with the complex developmental disorder Williams Syndrome (WS). WS results in visual/spatial processing defects, cognitive impairment, unique behavioral phenotypes, characteristic "elfin" facial features, low muscle tone and heart defects. WSTF exists in several chromatin remodeling complexes and has roles in transcription, replication, and repair. Chromatin remodeling is essential during embryogenesis, but WSTF's role in vertebrate development is poorly characterized. To investigate the developmental role of WSTF, we knocked down WSTF in Xenopus laevis embryos using a morpholino that targets WSTF mRNA. BMP4 shows markedly increased and spatially aberrant expression in WSTF-deficient embryos, while SHH, MRF4, PAX2, EPHA4 and SOX2 expression are severely reduced, coupled with defects in a number of developing embryonic structures and organs. WSTF-deficient embryos display defects in anterior neural development. Induction of the neural crest, measured by expression of the neural crest-specific genes SNAIL and SLUG, is unaffected by WSTF depletion. However, at subsequent stages WSTF knockdown results in a severe defect in neural crest migration and/or maintenance. Consistent with a maintenance defect, WSTF knockdowns display a specific pattern of increased apoptosis at the tailbud stage in regions corresponding to the path of cranial neural crest migration. Our work is the first to describe a role for WSTF in proper neural crest function, and suggests that neural crest defects resulting from WSTF haploinsufficiency may be a major contributor to the pathoembryology of WS. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  14. The morphology and attachment of Protopolystoma xenopodis (Monogenea: Polystomatidae infecting the African clawed frog Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Theunissen Maxine

    2014-01-01

    Full Text Available The African clawed frog Xenopus laevis (Anura: Pipidae is host to more than 25 parasite genera encompassing most of the parasitic invertebrate groups. Protopolystoma xenopodis Price, 1943 (Monogenea: Polystomatidae is one of two monogeneans infecting X. laevis. This study focussed on the external morphology of different developmental stages using scanning electron microscopy, histology and light microscopy. Eggs are released continuously and are washed out when the frog urinates. After successful development, an active swimming oncomiracidium leaves the egg capsule and locates a potential post-metamorphic clawed frog. The oncomiracidium migrates to the kidney where it attaches and starts to feed on blood. The parasite then migrates to the urinary bladder where it reaches maturity. Eggs are fusiform, about 300 μm long, with a smooth surface and are operculated. Oncomiracidia are elongated and cylindrical in shape, with an oval posterior cup-shaped haptor that bears a total of 20 sclerites; 16 marginal hooklets used for attachment to the kidney of the host and two pairs of hamulus primordia. Cilia from the 64 ciliated cells enable the oncomiracidium to swim for up to 24 h when the cilia subsequently curl up, become non-functional and are shed from the body. The tegument between the ciliated cells bears a series of sensory papillae. The body of the mature parasite is elongated and pyriform and possesses an opisthaptor armed with three pairs of suckers and two pairs of falciform hooks to ensure a firm grip on the flexible internal surface of the urinary bladder.

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

    Science.gov (United States)

    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.

  16. 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.

  17. Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes.

    Science.gov (United States)

    Mertl, Manuela; Daniel, Hannelore; Kottra, Gabor

    2008-11-01

    The adaptation of the capacity of the intestinal peptide transporter PEPT1 to varying substrate concentrations may be important with respect to its role in providing bulk quantities of amino acids for growth, development, and other nutritional needs. In the present study, we describe a novel phenomenon of the regulation of PEPT1 in the Xenopus oocyte system. Using electrophysiological and immunofluorescence methods, we demonstrate that a prolonged substrate exposure of rabbit PEPT1 (rPEPT1) caused a retrieval of transporters from the membrane. Capacitance as a measure of membrane surface area was increased in parallel with the increase in rPEPT1-mediated transport currents with a slope of approximately 5% of basal surface per 100 nA. Exposure of oocytes to the model peptide Gly-l-Gln for 2 h resulted in a decrease in maximal transport currents with no change of membrane capacitance. However, exposure to substrate for 5 h decreased transport currents but also, in parallel, surface area by endocytotic removal of transporter proteins from the surface. The reduction of the surface expression of rPEPT1 was confirmed by presteady-state current measurements and immunofluorescent labeling of rPEPT1. A similar simultaneous decrease of current and surface area was also observed when endocytosis was stimulated by the activation of PKC. Cytochalasin D inhibited all changes evoked by either dipeptide or PKC stimulation, whereas the PKC-selective inhibitor bisindolylmaleimide only affected PKC-stimulated endocytotic processes but not substrate-dependent retrieval of rPEPT1. Coexpression experiments with human Na(+)-glucose transporter 1 (hSGLT1) revealed that substrate exposure selectively affected PEPT1 but not the activity of hSGLT1.

  18. Dehydration triggers differential microRNA expression in Xenopus laevis brain.

    Science.gov (United States)

    Luu, Bryan E; Storey, Kenneth B

    2015-11-15

    African clawed frogs, Xenopus laevis, although primarily aquatic, have a high tolerance for dehydration, being capable of withstanding the loss of up to 32-35% of total water body water. Recent studies have shown that microRNAs play a role in the response to dehydration by the liver, kidney and ventral skin of X. laevis. MicroRNAs act by modulating the expression of mRNA transcripts, thereby affecting diverse biochemical pathways. In this study, 43 microRNAs were assessed in frog brains comparing control and dehydrated (31.2±0.83% of total body water lost) conditions. MicroRNAs of interest were measured using a modified protocol which employs polyadenylation of microRNAs prior to reverse transcription and qPCR. Twelve microRNAs that showed a significant decrease in expression (to 41-77% of control levels) in brains from dehydrated frogs (xla-miR-15a, -150, -181a, -191, -211, -218, -219b, -30c, -30e, -31, -34a, and -34b) were identified. Genomic analysis showed that the sequences of these dehydration-responsive microRNAs were highly conserved as compared with the comparable microRNAs of mice (91-100%). Suppression of these microRNAs implies that translation of the mRNA transcripts under their control could be enhanced in response to dehydration. Bioinformatic analysis using the DIANA miRPath program (v.2.0) predicted the top two KEGG pathways that these microRNAs collectively regulate: 1. Axon guidance, and 2. Long-term potentiation. Previous studies indicated that suppression of these microRNAs promotes neuroprotective pathways by increasing the expression of brain-derived neurotrophic factor and activating anti-apoptotic pathways. This suggests that similar actions may be triggered in X. laevis brains as a protective response to dehydration. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  19. A comparative survey of the frequency and distribution of polymorphism in the genome of Xenopus tropicalis.

    Directory of Open Access Journals (Sweden)

    Chris Showell

    Full Text Available Naturally occurring DNA sequence variation within a species underlies evolutionary adaptation and can give rise to phenotypic changes that provide novel insight into biological questions. This variation exists in laboratory populations just as in wild populations and, in addition to being a source of useful alleles for genetic studies, can impact efforts to identify induced mutations in sequence-based genetic screens. The Western clawed frog Xenopus tropicalis (X. tropicalis has been adopted as a model system for studying the genetic control of embryonic development and a variety of other areas of research. Its diploid genome has been extensively sequenced and efforts are underway to isolate mutants by phenotype- and genotype-based approaches. Here, we describe a study of genetic polymorphism in laboratory strains of X. tropicalis. Polymorphism was detected in the coding and non-coding regions of developmental genes distributed widely across the genome. Laboratory strains exhibit unexpectedly high frequencies of genetic polymorphism, with alleles carrying a variety of synonymous and non-synonymous codon substitutions and nucleotide insertions/deletions. Inter-strain comparisons of polymorphism uncover a high proportion of shared alleles between Nigerian and Ivory Coast strains, in spite of their distinct geographical origins. These observations will likely influence the design of future sequence-based mutation screens, particularly those using DNA mismatch-based detection methods which can be disrupted by the presence of naturally occurring sequence variants. The existence of a significant reservoir of alleles also suggests that existing laboratory stocks may be a useful source of novel alleles for mapping and functional studies.

  20. Geminin is required for zygotic gene expression at the Xenopus mid-blastula transition.

    Directory of Open Access Journals (Sweden)

    Sarah L Kerns

    Full Text Available In many organisms early development is under control of the maternal genome and zygotic gene expression is delayed until the mid-blastula transition (MBT. As zygotic transcription initiates, cell cycle checkpoints become activated and the tempo of cell division slows. The mechanisms that activate zygotic transcription at the MBT are incompletely understood, but they are of interest because they may resemble mechanisms that cause stem cells to stop dividing and terminally differentiate. The unstable regulatory protein Geminin is thought to coordinate cell division with cell differentiation. Geminin is a bi-functional protein. It prevents a second round of DNA replication during S and G2 phase by binding and inhibiting the essential replication factor Cdt1. Geminin also binds and inhibits a number of transcription factors and chromatin remodeling proteins and is thought to keep dividing cells in an undifferentiated state. We previously found that the cells of Geminin-deficient Xenopus embryos arrest in G2 phase just after the MBT then disintegrate at the onset of gastrulation. Here we report that they also fail to express most zygotic genes. The gene expression defect is cell-autonomous and is reproduced by over-expressing Cdt1 or by incubating the embryos in hydroxyurea. Geminin deficient and hydroxyurea-treated blastomeres accumulate DNA damage in the form of double stranded breaks. Bypassing the Chk1 pathway overcomes the cell cycle arrest caused by Geminin depletion but does not restore zygotic gene expression. In fact, bypassing the Chk1 pathway by itself induces double stranded breaks and abolishes zygotic transcription. We did not find evidence that Geminin has a replication-independent effect on transcription. We conclude that Geminin is required to maintain genome integrity during the rapid cleavage divisions, and that DNA damage disrupts zygotic gene transcription at the MBT, probably through activation of DNA damage checkpoint pathways.

  1. Mechanics of Fluid-Filled Interstitial Gaps. II. Gap Characteristics in Xenopus Embryonic Ectoderm.

    Science.gov (United States)

    Barua, Debanjan; Parent, Serge E; Winklbauer, Rudolf

    2017-08-22

    The ectoderm of the Xenopus embryo is permeated by a network of channels that appear in histological sections as interstitial gaps. We characterized this interstitial space by measuring gap sizes, angles formed between adjacent cells, and curvatures of cell surfaces at gaps. From these parameters, and from surface-tension values measured previously, we estimated the values of critical mechanical variables that determine gap sizes and shapes in the ectoderm, using a general model of interstitial gap mechanics. We concluded that gaps of 1-4 μm side length can be formed by the insertion of extracellular matrix fluid at three-cell junctions such that cell adhesion is locally disrupted and a tension difference between cell-cell contacts and the free cell surface at gaps of 0.003 mJ/m2 is generated. Furthermore, a cell hydrostatic pressure of 16.8 ± 1.7 Pa and an interstitial pressure of 3.9 ± 3.6 Pa, relative to the central blastocoel cavity of the embryo, was found to be consistent with the observed gap size and shape distribution. Reduction of cell adhesion by the knockdown of C-cadherin increased gap volume while leaving intracellular and interstitial pressures essentially unchanged. In both normal and adhesion-reduced ectoderm, cortical tension of the free cell surfaces at gaps does not return to the high values characteristic of the free surface of the whole tissue. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  2. Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vg1.

    OpenAIRE

    Dale, L; Matthews, G.; Colman, A

    1993-01-01

    Vg1 is a maternal mRNA localized to the vegetal hemisphere of Xenopus embryos during blastula stages, a region responsible for the induction of mesoderm in the adjacent marginal zone. Its homology to the transforming growth factor-beta family, which includes several proteins with mesoderm-inducing activity, suggests a role for Vg1 as an endogenous mesoderm-inducing factor. However, expression of Vg1 protein in the animal hemisphere, following injection of synthetic mRNA, has no effect on deve...

  3. Cell colony formation induced by Xenopus egg extract as a marker for improvement of cloned blastocyst formation in pig

    DEFF Research Database (Denmark)

    Liu, Ying; Østrup, Olga; Li, Juan

    2011-01-01

    Treatment with cytoplasmic extracts from Xenopus laevis eggs represents a potential tool for universal cellular reprogramming. However, the biochemical activity and quality of the extract vary from batch to batch. This study aimed to evaluate three different extract batches prepared by the same...... colonies in treated cells was counted on Day 7 after extract treatment and significant variability was detected between different batches of extract. Similarly, when using cells from colonies at Days 7 to 8 after treatment for handmade cloning, increased blastocyst formation rates were observed after...

  4. Change in desensitization of cat muscle acetylcholine receptor caused by coexpression of Torpedo acetylcholine receptor subunits in Xenopus oocytes.

    OpenAIRE

    Sumikawa, K; Miledi, R

    1989-01-01

    Cat muscle acetylcholine receptors (AcChoR) expressed in Xenopus oocytes desensitized more slowly than Torpedo electric organ AcChoRs, also expressed in oocytes. To examine the bases for the different degrees of desensitization, cat-Torpedo AcChoR hybrids were formed by injecting oocytes with cat denervated muscle mRNA mixed with a large excess of cloned Torpedo AcChoR subunit mRNAs. Hybrid AcChoRs formed by coinjection of cat muscle mRNA with the Torpedo beta or delta subunit mRNAs desensiti...

  5. Developmental expression of the protein product of Vg1, a localized maternal mRNA in the frog Xenopus laevis.

    OpenAIRE

    Dale, L; Matthews, G; Tabe, L; Colman, A

    1989-01-01

    Vg1 is a maternal mRNA localized in the vegetal cortex of Xenopus laevis oocytes, that encodes a protein homologous to the mammalian growth factor TGF-beta. Using a polyclonal antibody to a T7-Vg1 fusion protein, we have identified the native protein. We find that a single protein of Mr 40 kd is immunoprecipitated following in vitro translation of oocyte poly(A)+ RNA, whilst two proteins of Mr 45 and 43.5 kd are immunoprecipitated from oocyte and embryo extracts. Synthesis of at least the 40 ...

  6. Glycine antagonist action of 1-aminocyclobutane-1-carboxylate (ACBC) in Xenopus oocytes injected with rat brain mRNA.

    Science.gov (United States)

    Watson, G B; Bolanowski, M A; Baganoff, M P; Deppeler, C L; Lanthorn, T H

    1989-08-22

    ACBC has been reported to have the binding profile of an antagonist at the glycine site of the NMDA receptor. In Xenopus oocytes injected with rat brain mRNA, we have confirmed the antagonist action of ACBC on NMDA responses. ACBC and HA-966, a known glycine antagonist, blocked NMDA responses in a non-competitive manner and blocked the potentiation of NMDA responses by glycine in a competitive manner. We conclude that ACBC blocks NMDA responses via a competitive interaction at the glycine modulatory site.

  7. Newcastle Disease Virus (PDQ)

    Science.gov (United States)

    ... to Ask about Your Treatment Research Newcastle Disease Virus (PDQ®)–Patient Version Overview Go to Health Professional ... Question 8 ). Questions and Answers About Newcastle Disease Virus What is Newcastle disease virus? Newcastle disease virus ( ...

  8. Powassan (POW) Virus Basics

    Science.gov (United States)

    ... Professionals Related Topics For International Travelers Powassan (POW) Virus Basics Download this fact sheet formatted for print: ... POW) Virus Fact Sheet (PDF) What is Powassan virus? Powassan (POW) virus is a flavivirus that is ...

  9. Computer Viruses. Technology Update.

    Science.gov (United States)

    Ponder, Tim, Comp.; Ropog, Marty, Comp.; Keating, Joseph, Comp.

    This document provides general information on computer viruses, how to help protect a computer network from them, measures to take if a computer becomes infected. Highlights include the origins of computer viruses; virus contraction; a description of some common virus types (File Virus, Boot Sector/Partition Table Viruses, Trojan Horses, and…

  10. Viruses Avian influenza, bovine herpes, bovine viral diarrhea virus ...

    Indian Academy of Sciences (India)

    ... human cytomegalovirus, herpes simplex virus, human immunodeficiency virus I, influenza, lymphocytic choriomeningitis virus, measles, papilloma, rabies, respiratory syncitial virus, simian immunodeficiency virus, simian virus 40. Bacteria Borrelia burgdorferi (Lyme disease), Moraxella bovis, Mycobacterium tuberculosis, ...

  11. Computer viruses

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, F.B.

    1986-01-01

    This thesis investigates a recently discovered vulnerability in computer systems which opens the possibility that a single individual with an average user's knowledge could cause widespread damage to information residing in computer networks. This vulnerability is due to a transitive integrity corrupting mechanism called a computer virus which causes corrupted information to spread from program to program. Experiments have shown that a virus can spread at an alarmingly rapid rate from user to user, from system to system, and from network to network, even when the best-availability security techniques are properly used. Formal definitions of self-replication, evolution, viruses, and protection mechanisms are used to prove that any system that allows sharing, general functionality, and transitivity of information flow cannot completely prevent viral attack. Computational aspects of viruses are examined, and several undecidable problems are shown. It is demonstrated that a virus may evolve so as to generate any computable sequence. Protection mechanisms are explored, and the design of computer networks that prevent both illicit modification and dissemination of information are given. Administration and protection of information networks based on partial orderings are examined, and probably correct automated administrative assistance is introduced.

  12. Iron is a substrate of the Plasmodium falciparum chloroquine resistance transporter PfCRT in Xenopus oocytes.

    Science.gov (United States)

    Bakouh, Naziha; Bellanca, Sebastiano; Nyboer, Britta; Moliner Cubel, Sonia; Karim, Zoubida; Sanchez, Cecilia P; Stein, Wilfred D; Planelles, Gabrielle; Lanzer, Michael

    2017-09-29

    The chloroquine resistance transporter of the human malaria parasite Plasmodium falciparum, PfCRT, is an important determinant of resistance to several quinoline and quinoline-like antimalarial drugs. PfCRT also plays an essential role in the physiology of the parasite during development inside erythrocytes. However, the function of this transporter besides its role in drug resistance is still unclear. Using electrophysiological and flux experiments conducted on PfCRT-expressing Xenopus laevis oocytes, we show here that both wild-type PfCRT and a PfCRT variant associated with chloroquine resistance transport both ferrous and ferric iron, albeit with different kinetics. In particular, we found that the ability to transport ferrous iron is reduced by the specific polymorphisms acquired by the PfCRT variant as a result of chloroquine selection. We further show that iron and chloroquine transport via PfCRT is electrogenic. If these findings in the Xenopus model extend to P. falciparum in vivo, our data suggest that PfCRT might play a role in iron homeostasis, which is essential for the parasite's development in erythrocytes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Inhibitory effects of tramadol on nicotinic acetylcholine receptors in adrenal chromaffin cells and in Xenopus oocytes expressing α7 receptors

    Science.gov (United States)

    Shiraishi, Munehiro; Minami, Kouichiro; Uezono, Yasuhito; Yanagihara, Nobuyuki; Shigematsu, Akio; Shibuya, Izumi

    2002-01-01

    Tramadol has been used clinically as an analgesic; however, the mechanism of its analgesic effects is still unknown.We used bovine adrenal chromaffin cells to investigate effects of tramadol on catecholamine secretion, nicotine-induced cytosolic Ca2+ concentration ([Ca2+]i) increases and membrane current changes. We also investigated effects of tramadol on α7 nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes.Tramadol concentration-dependently suppressed carbachol-induced catecholamine secretion to 60% and 27% of the control at the concentration of 10 and 100 μM, respectively, whereas it had little effect on veratridine- or high K+-induced catecholamine secretion.Tramadol also suppressed nicotine-induced ([Ca2+]i) increases in a concentration-dependent manner. Tramadol inhibited nicotine-induced inward currents, and the inhibition was unaffected by the opioid receptor antagonist naloxone.Tramadol inhibited nicotinic currents carried by α7 receptors expressed in Xenopus oocytes.Tramadol inhibited both α-bungarotoxin-sensitive and -insensitive nicotinic currents in bovine adrenal chromaffin cells.In conclusion, tramadol inhibits catecholamine secretion partly by inhibiting nicotinic AChR functions in a naloxone-insensitive manner and α7 receptors are one of those inhibited by tramadol. PMID:12010769

  14. RNA localization in Xenopus oocytes uses a core group of trans-acting factors irrespective of destination

    Science.gov (United States)

    Snedden, Donald D.; Bertke, Michelle M.; Vernon, Dominic; Huber, Paul W.

    2013-01-01

    The 3′ untranslated region of mRNA encoding PHAX, a phosphoprotein required for nuclear export of U-type snRNAs, contains cis-acting sequence motifs E2 and VM1 that are required for localization of RNAs to the vegetal hemisphere of Xenopus oocytes. However, we have found that PHAX mRNA is transported to the opposite, animal, hemisphere. A set of proteins that cross-link to the localization elements of vegetally localized RNAs are also cross-linked to PHAX and An1 mRNAs, demonstrating that the composition of RNP complexes that form on these localization elements is highly conserved irrespective of the final destination of the RNA. The ability of RNAs to bind this core group of proteins is correlated with localization activity. Staufen1, which binds to Vg1 and VegT mRNAs, is not associated with RNAs localized to the animal hemisphere and may determine, at least in part, the direction of RNA movement in Xenopus oocytes. PMID:23645708

  15. Enabled (Xena) regulates neural plate morphogenesis, apical constriction, and cellular adhesion required for neural tube closure in Xenopus.

    Science.gov (United States)

    Roffers-Agarwal, Julaine; Xanthos, Jennifer B; Kragtorp, Katherine A; Miller, Jeffrey R

    2008-02-15

    Regulation of cellular adhesion and cytoskeletal dynamics is essential for neurulation, though it remains unclear how these two processes are coordinated. Members of the Ena/VASP family of proteins are localized to sites of cellular adhesion and actin dynamics and lack of two family members, Mena and VASP, in mice results in failure of neural tube closure. The precise mechanism by which Ena/VASP proteins regulate this process, however, is not understood. In this report, we show that Xenopus Ena (Xena) is localized to apical adhesive junctions of neuroepithelial cells during neurulation and that Xena knockdown disrupts cell behaviors integral to neural tube closure. Changes in the shape of the neural plate as well as apical constriction within the neural plate are perturbed in Xena knockdown embryos. Additionally, we demonstrate that Xena is essential for cell-cell adhesion. These results demonstrate that Xena plays an integral role in coordinating the regulation of cytoskeletal dynamics and cellular adhesion during neurulation in Xenopus.

  16. Metabolomic approach for identifying and visualizing molecular tissue markers in tadpoles of Xenopus tropicalis by mass spectrometry imaging

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    Naoko Goto-Inoue

    2016-09-01

    Full Text Available In developmental and cell biology it is crucial to evaluate the dynamic profiles of metabolites. An emerging frog model system using Xenopus tropicalis, whose genome sequence and inbred strains are available, is now ready for metabolomics investigation in amphibians. In this study we applied matrix-assisted laser desorption/ionization (MALDI-mass spectrometry imaging (MSI analysis to identify and visualize metabolomic molecular markers in tadpoles of Xenopus tropicalis. We detected tissue-specific peaks and visualized their distribution in tissues, and distinguished 19 tissues and their specific peaks. We identified, for the first time, some of their molecular localizations via tandem mass spectrometric analysis: hydrocortisone in artery, L-DOPA in rhombencephalon, taurine in eye, corticosterone in gill, heme in heart, inosine monophosphate and carnosine in muscle, dopamine in nerves, and phosphatidylethanolamine (16:0/20:4 in pharynx. This is the first MALDI-MSI study of X. tropicalis tadpoles, as in small tadpoles it is hard to distinguish and dissect the various organs. Furthermore, until now there has been no data about the metabolomic profile of each organ. Our results suggest that MALDI-MSI is potentially a powerful tool for examining the dynamics of metabolomics in metamorphosis as well as conformational changes due to metabolic changes.

  17. A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.

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    Siwei Zhang

    Full Text Available Embryonic development requires exquisite regulation of several essential processes, such as patterning of tissues and organs, cell fate decisions, and morphogenesis. Intriguingly, these diverse processes are controlled by only a handful of signalling pathways, and mis-regulation in one or more of these pathways may result in a variety of congenital defects and diseases. Consequently, investigating how these signalling pathways are regulated at the molecular level is essential to understanding the mechanisms underlying vertebrate embryogenesis, as well as developing treatments for human diseases. Here, we designed and performed a large-scale gain-of-function screen in Xenopus embryos aimed at identifying new regulators of MAPK/Erk, PI3K/Akt, BMP, and TGF-β/Nodal signalling pathways. Our gain-of-function screen is based on the identification of gene products that alter the phosphorylation state of key signalling molecules, which report the activation state of the pathways. In total, we have identified 20 new molecules that regulate the activity of one or more signalling pathways during early Xenopus development. This is the first time that such a functional screen has been performed, and the findings pave the way toward a more comprehensive understanding of the molecular mechanisms regulating the activity of important signalling pathways under normal and pathological conditions.

  18. Nitric oxide donor s-nitroso-n-acetyl penicillamine (SNAP) alters meiotic spindle morphogenesis in Xenopus oocytes.

    Science.gov (United States)

    Gelaude, Armance; Marin, Matthieu; Cailliau, Katia; Jeseta, Michal; Lescuyer-Rousseau, Arlette; Vandame, Pauline; Nevoral, Jan; Sedmikova, Marketa; Martoriati, Alain; Bodart, Jean-François

    2015-11-01

    Nitric Oxide (NO) has been involved in both intra- and extra-cellular signaling pathways in a wide range of organisms, and can be detected in some reproductive tissues. Based upon previous results reporting that NO-donor SNAP (s-nitroso-n-acetyl penicillamine) promoted the release from the metaphase II-anaphase II block in amphibian eggs, the aim of the present study was to assess the influence of SNAP on the activation of the molecular mechanisms triggering meiotic resumption of Xenopus oocytes, analogous to G2/M transition of the cell cycle. A high concentration of SNAP (2.5 mM) was found to inhibit the appearance of the white spot (meiotic resumption) and promoted alteration of spindle morphogenesis leading to atypical structures lacking bipolarity and correct chromosomes equatorial alignment. The medium acidification (pH = 4) promoted by SNAP specifically impacted the white spot occurrence. However, even when pH was restored to 7.4 in SNAP medium, observed spindles remained atypical (microtubule disorganization), suggesting SNAP impacted spindle assembly regardless of the pH. n-Acetyl-d,l-penicillamine disulfide, a degradation product of SNAP with the same molecular characteristics, albeit without release of NO, yielded spindle assemblies typical of metaphase II suggesting the specificity of NO action on meiotic spindle morphogenesis in Xenopus oocytes. © 2015 Wiley Periodicals, Inc.

  19. Development of a new approach for targeted gene editing in primordial germ cells using TALENs in Xenopus

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    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.

  20. Ancient T-Independence of Mucosal IgX/A: Gut Microbiota Unaffected by Larval Thymectomy in Xenopus laevis

    Science.gov (United States)

    Mashoof, Sara; Goodroe, Anna; Du, Christina C.; Eubanks, Jeannine O.; Jacobs, Natalie; Steiner, Jörg M.; Tizard, Ian; Suchodolski, Jan S.; Criscitiello, Michael F.

    2012-01-01

    Many studies address the influence of the gut microbiome on the immune system, but few dissect the effect of T cells on gut microbiota and mucosal responses. We have employed larval thymectomy in Xenopus to study the gut microbiota with and without the influence of T lymphocytes. Pyrosequencing of 16S rRNA genes was used to assess the relative abundance of bacterial groups present in the stomach, small and large intestine. Clostridiaceae was the most abundant family throughout the gut, while Bacteroidaceae, Enterobacteriaceae and Flavobacteriaceae also were well represented. Unifrac analysis revealed no differences in microbiota distribution between thymectomized and unoperated frogs. This is consistent with immunization data showing that levels of the mucosal immunoglobulin IgX are not altered significantly by thymectomy. This study in Xenopus represents the oldest organisms that exhibit class switch to a mucosal isotype and is relevant to mammalian immunology, as IgA appears to have evolved from IgX based upon phylogeny, genomic synteny, and function. PMID:22929561

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

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    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.

  2. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Mutant analysis of Cdt1's function in suppressing nascent strand elongation during DNA replication in Xenopus egg extracts.

    Science.gov (United States)

    Nakazaki, Yuta; Tsuyama, Takashi; Azuma, Yutaro; Takahashi, Mikiko; Tada, Shusuke

    2017-09-02

    The initiation of DNA replication is strictly regulated by multiple mechanisms to ensure precise duplication of chromosomes. In higher eukaryotes, activity of the Cdt1 protein is temporally regulated during the cell cycle, and deregulation of Cdt1 induces DNA re-replication. In previous studies, we showed that excess Cdt1 inhibits DNA replication by suppressing progression of replication forks in Xenopus egg extracts. Here, we investigated the functional regions of Cdt1 that are required for the inhibition of DNA replication. We constructed a series of N-terminally or C-terminally deleted mutants of Cdt1 and examined their inhibitory effects on DNA replication in Xenopus egg extracts. Our results showed that the region spanning amino acids (a. a.) 255-620 is required for efficient inhibition of DNA replication, and that, within this region, a. a. 255-289 have a critical role in inhibition. Moreover, one of the Cdt1 mutants, Cdt1 R285A, was compromised with respect to the licensing activity but still inhibited DNA replication. This result suggests that Cdt1 has an unforeseen function in the negative regulation of DNA replication, and that this function is located within a molecular region that is distinct from those required for the licensing activity. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. 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.

  5. DIF-1, an anti-tumor substance found in Dictyostelium discoideum, inhibits progesterone-induced oocyte maturation in Xenopus laevis.

    Science.gov (United States)

    Kubohara, Yuzuru; Hanaoka, Yoichi; Akaishi, Emi; Kobayashi, Hisae; Maeda, Mineko; Hosaka, Kohei

    2003-01-24

    Differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) is a putative morphogen that induces stalk-cell formation in the cellular slime mold Dictyostelium discoideum. DIF-1 has previously been shown to suppress cell growth in mammalian cells. In this study, we examined the effects of DIF-1 on the progesterone-induced germinal vesicle breakdown in Xenopus laevis, which is thought to be mediated by a decrease in intracellular cAMP and the subsequent activation of mitogen-activated protein kinase (MAPK) and maturation-promoting factor, a complex of cdc2 and cyclin B, which regulates germinal vesicle breakdown. DIF-1 at 10-40 microM inhibited progesterone-induced germinal vesicle breakdown in de-folliculated oocytes in a dose-dependent manner. Progesterone-induced cdc2 activation, MAPK activation, and c-Mos accumulation were inhibited by DIF-1. Furthermore, DIF-1 was found to inhibit the progesterone-induced cAMP decrease in the oocytes. These results indicate that DIF-1 inhibits progesterone-induced germinal vesicle breakdown possibly by blocking the progesterone-induced decrease in [cAMP](i) and the subsequent events in Xenopus oocytes.

  6. Hendra virus.

    Science.gov (United States)

    Middleton, Deborah

    2014-12-01

    Hendra virus infection of horses occurred sporadically between 1994 and 2010 as a result of spill-over from the viral reservoir in Australian mainland flying-foxes, and occasional onward transmission to people also followed from exposure to affected horses. An unprecedented number of outbreaks were recorded in 2011 leading to heightened community concern. Release of an inactivated subunit vaccine for horses against Hendra virus represents the first commercially available product that is focused on mitigating the impact of a Biosafety Level 4 pathogen. Through preventing the development of acute Hendra virus disease in horses, vaccine use is also expected to reduce the risk of transmission of infection to people. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

  7. Marburg virus.

    Science.gov (United States)

    Dowdle, W R

    1976-01-01

    Marburg virus disease, which produced 20 per cent mortality when it first occured during 1967 in Germany and Yugoslavia, recently appeared again in South Africa. The source of the first outbreak was monkeys shipped from Africa; the origin of the second episode is unclear. Because distribution of the virus in nature is unknown, its threat to man cannot be readily determined. Differential laboratory diagnoses of hemorrhagic fevers should be encouraged in order to learn more about the epidemiology of these diseases and to better assess the risks which their etiologic agents may pose for attending medical personnel.

  8. Identification of metalloprotease/disintegrins in Xenopus laevis testis with a potential role in fertilization.

    Science.gov (United States)

    Shilling, F M; Krätzschmar, J; Cai, H; Weskamp, G; Gayko, U; Leibow, J; Myles, D G; Nuccitelli, R; Blobel, C P

    1997-06-15

    Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein-receptor interactions are necessary for fertilization to occur.

  9. High-avidity, low-affinity multivalent interactions and the block to polyspermy in Xenopus laevis.

    Science.gov (United States)

    Arranz-Plaza, Esther; Tracy, Alex S; Siriwardena, Aloysius; Pierce, J Michael; Boons, Geert-Jan

    2002-11-06

    The interaction of the lectin XL35 with the jelly coat protein (JCP) surrounding oocytes in Xenopus laevis is essential for the block to polyspermy. The molecular details of this event are poorly understood, and the present study has been undertaken with a view to delineating the mechanism of formation of the fertilization envelope. A range of JCP-derived oligosaccharides were synthesized, and all were installed with an artificial aminopropyl arm. This arm allowed the preparation of monovalent derivatives by acetylation of the amino group or the synthesis of polyvalent compounds by attachment to an activated polyacrylamide polymer. A number of analytical techniques, including enzyme-linked lectin assays and surface plasmon resonance, have been developed and utilized to study the interactions of the mono- and polyvalent compounds with XL35. The results reveal that the lectin XL35 has remarkably broad specificity for galactose-containing saccharides and the affinities are only slightly modulated by secondary features, such as anomeric configuration of the terminal sugar or the identity and linkage pattern of branching sugars. Broad specificity was also observed when the saccharides were presented in a polyvalent fashion. The glycopolymers displayed 10-20-fold increases in valency-corrected affinities compared to the corresponding monovalent counterparts. Although the synthetic polymers are not as potent as the JCP, the kinetics of their interactions mirror closely those of the native ligand, and in each case extremely long-lived interactions were observed. The results of this study indicate that, in X. laevis, the true biological function of multivalency is not to create an extremely tightly binding complex between XL35 and its natural ligand but, instead, to create a very stable protective layer that will not dissociate and is yet flexible enough to encapsulate the developing embryo. It is postulated that, even if these partners are unable to attain true equilibrium

  10. Hypergravity susceptibility of ventral root activity during fictive swimming in tadpoles (Xenopus laevis).

    Science.gov (United States)

    Böser, S; Horn, E R

    2006-05-01

    1. Fictive swimming is an experimental model to study early motor development. As vestibular activity also affects the development of spinal motor projections, the present study focused on the question whether in Xenopus laevis tadpoles, the rhythmic activity of spinal ventral roots (VR) during fictive swimming revealed age-dependent modifications after hypergravity exposure. In addition, developmental characteristics for various features of fictive swimming between stages 37/38 and 47 were determined. Parameters of interest were duration of fictive swimming episodes, burst duration, burst frequency (i.e., cycle length), and rostrocaudal delay. 2. Ventral root recordings were performed between developmental stage 37/38, which is directly after hatching and stage 47 when the hind limb buds appear. The location of recording electrodes extended from myotome 4 to 17. 3. Hypergravity exposure by 3 g-centrifugation lasted 9 to 11 days. It started when embryos had just terminated gastrulation (stage 11/19-group), when first rhythmical activity in the ventral roots appeared (stage 24/27-group), and immediately after hatching (stage 37/41-group). Ventral root recordings were taken for 8 days after termination of 3 g-exposure. 4. Between stage 37/38 (hatching) and stage 47 (hind limb bud stage) burst duration, cycle length and rostrocaudal delay recorded between the 10th and 14th postotic myotome increased while episode duration decreased significantly. In tadpoles between stage 37 and 43, the rostrocaudal delay in the proximal tail part was as long as in older tadpoles while in caudal tail parts, it was shorter. During this period of development, there was also an age-dependent progression of burst extension in the proximal tail area that could not be observed between the 10th and 14th myotome. 6. After termination of the 3 g-exposure, the mean burst duration of VR activity increased significantly (p VR activity such as cycle length, rostrocaudal delay and episode duration

  11. Multiple mechanisms promote the retained expression of gene duplicates in the tetraploid frog Xenopus laevis.

    Directory of Open Access Journals (Sweden)

    2006-04-01

    Full Text Available Gene duplication provides a window of opportunity for biological variants to persist under the protection of a co-expressed copy with similar or redundant function. Duplication catalyzes innovation (neofunctionalization, subfunction degeneration (subfunctionalization, and genetic buffering (redundancy, and the genetic survival of each paralog is triggered by mechanisms that add, compromise, or do not alter protein function. We tested the applicability of three types of mechanisms for promoting the retained expression of duplicated genes in 290 expressed paralogs of the tetraploid clawed frog, Xenopus laevis. Tests were based on explicit expectations concerning the ka/ks ratio, and the number and location of nonsynonymous substitutions after duplication. Functional constraints on the majority of paralogs are not significantly different from a singleton ortholog. However, we recover strong support that some of them have an asymmetric rate of nonsynonymous substitution: 6% match predictions of the neofunctionalization hypothesis in that (1 each paralog accumulated nonsynonymous substitutions at a significantly different rate and (2 the one that evolves faster has a higher ka/ks ratio than the other paralog and than a singleton ortholog. Fewer paralogs (3% exhibit a complementary pattern of substitution at the protein level that is predicted by enhancement or degradation of different functional domains, and the remaining 13% have a higher average ka/ks ratio in both paralogs that is consistent with altered functional constraints, diversifying selection, or activity-reducing mutations after duplication. We estimate that these paralogs have been retained since they originated by genome duplication between 21 and 41 million years ago. Multiple mechanisms operate to promote the retained expression of duplicates in the same genome, in genes in the same functional class, over the same period of time following duplication, and sometimes in the same pair of

  12. Translation of maternal TATA-binding protein mRNA potentiates basal but not activated transcription in Xenopus embryos at the midblastula transition.

    NARCIS (Netherlands)

    Veenstra, G.J.C.; Destree, O.H.; Wolff, A.P.

    1999-01-01

    Early embryonic development in Xenopus laevis is characterized by transcriptional repression which is relieved at the midblastula stage (MBT). Here we show that the relative abundance of TATA-binding protein (TBP) increases robustly at the MBT and that the mechanism underlying this increase is

  13. Increased rate of capping of concanavalin a receptors during early Xenopus development is related to changes in protein and lipid mobility

    NARCIS (Netherlands)

    Laat, S.W. de; Gadenne, M.; Zoelen, E.J.J. van; Tencer, R.

    1984-01-01

    Abstract The mobility characteristics of plasma membrane constituents were studied in dissociated cells from embryos of Xenopus laevis at various stages of development from early blastula until neurulation. An increased rate of fluorescein isothiocyanate-concanavalin A induced patching and capping

  14. Effects of strain on contractile force and number of sarcomeres in series of Xenopus laevis single muscle fibres during long-term culture

    NARCIS (Netherlands)

    Jaspers, R.T.; Feenstra, Hiske; Verheyen, A.K.; van der Laarse, W.J.; Huijing, P.A.J.B.M.

    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

  15. Effects of strain on contractile force and number of sarcomeres in series of Xenopus laevis single muscle fibres during long-term culture

    NARCIS (Netherlands)

    Jaspers, R.T.; Feenstra, H.M.; Verheyen, A.K.; van der Laarse, W.J.; Huijing, P.A.J.B.M.

    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

  16. Calbindin-D28k immunoreactivity in the spinal cord of Xenopus laevis and its participation in ascending and descending projections.

    NARCIS (Netherlands)

    Morona, R.; Moreno, N.; Lopez, J.M.; Munoz, M.; Donkelaar, H.J. ten; Gonzalez, A.

    2005-01-01

    Immunohistochemistry for calbindin-D28k (CB) revealed that the spinal cord of Xenopus laevis possess a large number of CB-containing neurons widely distributed in both the dorsal and ventral horns, including areas which possess long ascending projections to supraspinal structures. In addition, the

  17. Cas de necrose cutanée chez des grenouilles africaines à griffes Xenopus laevis suite à une application topique d'eugénol.

    OpenAIRE

    Ross, Andréanne; Guénette, Sarah Annie; Hélie, Pierre; Vachon, Pascal

    2006-01-01

    Case of cutaneous necrosis in African Clawed frogs Xenopus laevis after the topical application of eugenol. African Clawed frogs showed necrotic cutaneous lesions after a topical application of high concentrations of eugenol, an analgesic and anesthetic agent. Microscopically, ulceration of the epidermis, a loss of mucous and serous glands as well as an infiltration of inflammatory cells were observed.

  18. 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

  19. 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...

  20. HUMAN PAPILLOMA VIRUS — ONCOGENIC VIRUS

    Directory of Open Access Journals (Sweden)

    A.N. Mayansky

    2010-01-01

    Full Text Available The lecture is devoted to oncogenic viruses, particularly human papilloma virus. Papilloma viral infection is found in all parts of the globe and highly contagious. In addition to exhaustive current data on classification, specifics of papilloma viruses composition and epidemiology, the author describes in great detail the malignization mechanisms of papilloma viruses pockets. Also, issues of diagnostics and specific prevention and treatment of diseases caused by this virus are illustrated. Key words: oncogenic viruses, papilloma viruses, prevention, vaccination. (Pediatric Pharmacology. – 2010; 7(4:48-55

  1. Xenopus Smad4beta is the co-Smad component of developmentally regulated transcription factor complexes responsible for induction of early mesodermal genes.

    Science.gov (United States)

    Howell, M; Itoh, F; Pierreux, C E; Valgeirsdottir, S; Itoh, S; ten Dijke, P; Hill, C S

    1999-10-15

    Smad4 is defined as the common-mediator Smad (co-Smad) required for transducing signals for all TGF-beta superfamily members. This paper describes two Smad4s in Xenopus: XSmad4alpha, which is probably the Xenopus orthologue of human Smad4, and a distinct family member, XSmad4beta, which differs primarily at the extreme N-terminus and in the linker region. Both XSmad4s act as co-Smads, forming ligand-dependent complexes with receptor-regulated Smads 1 and 2 and synergizing with them to activate transcription of mesodermal genes in Xenopus embryos. The two XSmad4 genes have reciprocal temporal expression patterns in Xenopus embryos and are expressed in varying ratios in adult tissues, suggesting distinct functional roles in vivo. XSmad4beta is the predominant maternal co-Smad and we go on to demonstrate its role in the transcriptional regulation of early mesodermal genes. We have identified two distinct nuclear complexes that bind the activin-responsive element of the Xenopus Mix.2 promoter: one formed in response to high levels of activin signaling and the other activated by endogenous signaling pathways. Using specific antisera we demonstrate the presence of endogenous XSmad4beta and also XSmad2 in both of these complexes, and our data indicate that the DNA-binding components of the complexes are different. Furthermore, we show that the presence of these complexes in the nucleus perfectly correlates with the transcriptional activity of the target gene, Mix.2, and we show that one of the XSmad4beta-containing transcription factor complexes undergoes a developmentally regulated nuclear translocation. Copyright 1999 Academic Press.

  2. Worldwide occurrence of virus-infections in filamentous marine brown algae

    Science.gov (United States)

    Müller, D. G.; Stache, B.

    1992-03-01

    Virus infections were detected in Ectocarpus siliculosus and Ectocarpus fasciculatus on the coasts of Ireland, California, Peru, southern South America, Australia and New Zealand; in three Feldmannia species on the coasts of Ireland, continental Chile and Archipelago Juan Fernandez (Chile); and in Leptonematella from Antarctica. Natural populations on the Irish coast contained 3% infected plants in E. fasciculatus, and less than 1% in Feldmannia simplex. On the Californian coast, 15 to 25% of Ectocarpus isolates were infected. Virus symptoms were absent in E. siliculosus from Peru, but appeared after meiosis in laboratory cultures. The virus particles in E. fasciculatus are identical in size and capsid structure to those reported for E. siliculosus, while the virus in F. simplex is smaller and has a different envelope. Our findings suggest that virus infections are a common and worldwide phenomenon in filamentous brown algae.

  3. Efficient cell culture system for hepatitis C virus genotype 5A

    DEFF Research Database (Denmark)

    2013-01-01

    of in vitro transcripts in Huh7.5 cells, production of infectious viruses was delayed. However, in subsequent viral passages efficient spread of infection and HCV RNA titers as high as for J6/JFH were obtained. Infectivity titers were at all time points analyzed comparable to J6/JFH control virus. Sequence...... analysis of recovered 5a/2a recombinants from 2 serial passages and subsequent reverse genetic studies revealed adaptive mutations in p7, NS2 and/or NS3. Infectivity of the 5a/2a viruses was CD81 and SR-BI dependant, and the recombinant viruses could be neutralized by chronic phase sera from patients...... infected with genotype 5a. Conclusion: The developed 5a/2a viruses provide a robust in vitro tool for research in HCV genotype 5, including vaccine studies and functional analyses of an increasingly important genotype in South Africa and Europe...

  4. Oropuche virus: A virus present but ignored

    Directory of Open Access Journals (Sweden)

    Salim Mattar V.

    2015-09-01

    Full Text Available Bunyaviruses are RNA viruses that affect animals and plants; they have five genera and four of them affect humans: Orthobunyavirus, Nairovirus, Phlebovirus and Hantavirus. All of them are Arbovirus, except Hantavirus. The Orthobunyaviruses comprise Oropouche, Tahyna, La Crosse virus, California encephalitis virus and Heartland virus recently discovered (1. Except for Heartland virus which is transmitted by ticks of the genus Amblyoma, these Phleboviruses have as vectors mosquitoes, which bite small mammals which are able to be as reservoirs amplifiers.

  5. Co-expression of Argonaute2 enhances short hairpin RNA-induced RNA interference in Xenopus CNS neurons in vivo

    Directory of Open Access Journals (Sweden)

    Chih-ming Chen

    2009-07-01

    Full Text Available RNA interference (RNAi is an evolutionarily conserved mechanism for sequence-specific gene silencing. Recent advances in our understanding of RNAi machinery make it possible to reduce protein expression by introducing short hairpin RNA (shRNA into cells of many systems, however, the efficacy of RNAi-mediated protein knockdown can be quite variable, especially in intact animals, and this limits its application. We built adaptable molecular tools, pSilencer (pSi and pReporter (pRe constructs, to evaluate the impact of different promoters, shRNA structures and overexpression of Ago2, the key enzyme in the RNA-induced silencing complex (RISC, on the efficiency of RNAi. The magnitude of RNAi knockdown was evaluated in cultured cells and intact animals by comparing fluorescence intensity levels of GFP, the RNAi target, relative to mCherry, which was not targeted. Co-expression of human Ago2 with shRNA significantly enhanced efficiency of GFP knockdown in cell lines and in neurons of intact Xenopus tadpoles. Human H1- and U6-promotors alone or the U6-promotor with an enhancer element were equally effective at driving GFP knockdown. shRNA derived from the microRNA-30 design (shRNAmir30 enhanced the efficiency of GFP knockdown. Expressing pSi containing Ago2 with shRNA increased knockdown efficiency of an endogenous neuronal protein, the GluR2 subunit of the AMPA receptor, functionally accessed by recording AMPA receptor-mediated spontaneous synaptic currents in Xenopus CNS neurons. Our data suggest that co-expression of Ago2 and shRNA is a simple method to enhance RNAi in intact animals. While morpholino antisense knockdown is effective in Xenopus and Zebrafish, a principle advantage of the RNAi method is the possibility of spatial and temporal control of protein knockdown by use of cell type specific and regulatable pol II promoters to drive shRNA and Ago2. This should extend the application of RNAi to study gene function of intact brain circuits.

  6. Mengenal Hanta Virus

    OpenAIRE

    Wijayanti, Tri

    2009-01-01

    Virus Hanta kurang infeksius, kecuali di dalam lingkungan tertentu. Lamanya waktu virus ini dapat bertahan di lingkungan, setelah keluar dari tubuh tikus tidaklah diketahui secara pasti. Tetapi percobaan laboratorium menunjukkan bahwa, daya infektifitasnya tidak dijumpai setelah dua hari pengeringan. Genus hanta virus terdiri dari 22 spesies virus, dapat menyebabkan hemorrhagic fever with renal syndrome (HFRS) dan hanta virus pulmonary syndrome (HPS).

  7. Viruses of hyperthermophilic Crenarchaea

    DEFF Research Database (Denmark)

    Prangishvili, D.; Garrett, R. A.

    2005-01-01

    , when one examines the archaeal viruses, the picture appears complex. Most viruses that are known to infect members of the kingdom Euryarchaeota resemble bacterial viruses, whereas those associated with the kingdom Crenarchaeota show little resemblance to either bacterial or eukaryal viruses....... This review summarizes our current knowledge of this group of exceptional and highly diverse archaeal viruses....

  8. Triton X-100 inhibits agonist-induced currents and suppresses benzodiazepine modulation of GABA(A) receptors in Xenopus oocytes

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Ebert, Bjarke; Klaerke, Dan

    2009-01-01

    effects on gramicidin channel A appearance rate and lifetime in artificial lipid bilayers. In the present study, the pharmacological action of Triton-X 100 on GABA(A) receptors expressed in Xenopus laevis oocytes was examined. Triton-X 100 inhibited GABA(A) alpha(1)beta(3)gamma(2S) receptor currents...... by flunitrazepam at alpha(1)beta(3)gamma(2S) receptors. All effects were independent of the presence of a gamma(2S) subunit in the GABA(A) receptor complex. The present study suggests that Triton X-100 may stabilize open and desensitized states of the GABA(A) receptor through changes in lipid bilayer elasticity....... in a noncompetitive, time- and voltage-dependent manner and increased the apparent rate and extent of desensitization at 10 muM, which is 30 fold below the critical micelle concentration. In addition, Triton X-100 induced picrotoxin-sensitive GABA(A) receptor currents and suppressed allosteric modulation...

  9. To swim or not to swim: A population-level model of Xenopus tadpole decision making and locomotor behaviour.

    Science.gov (United States)

    Borisyuk, Roman; Merrison-Hort, Robert; Soffe, Steve R; Koutsikou, Stella; Li, Wen-Chang

    2017-11-01

    We present a detailed computational model of interacting neuronal populations that mimic the hatchling Xenopus tadpole nervous system. The model includes four sensory pathways, integrators of sensory information, and a central pattern generator (CPG) network. Sensory pathways of different modalities receive inputs from an "environment"; these inputs are then processed and integrated to select the most appropriate locomotor action. The CPG populations execute the selected action, generating output in motor neuron populations. Thus, the model describes a detailed and biologically plausible chain of information processing from external signals to sensors, sensory pathways, integration and decision-making, action selection and execution and finally, generation of appropriate motor activity and behaviour. We show how the model produces appropriate behaviours in response to a selected scenario, which consists of a sequence of "environmental" signals. These behaviours might be relatively complex due to noisy sensory pathways and the possibility of spontaneous actions. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  10. The fertilization layer mediated block to polyspermy in Xenopus laevis: isolation of the cortical granule lectin ligand.

    Science.gov (United States)

    Quill, T A; Hedrick, J L

    1996-09-15

    The fertilization layer of Xenopus laevis eggs is formed by the cortical granule lectin binding to its ligand. The binding requires Ca2+, is specific for galactose, and functionally establishes a block to polyspermy at fertilization. We have designed a new enzyme-linked lectin assay for the cortical granule lectin (CGL) ligand which can detect the presence of the CGL ligand at a sensitivity of 1-2 ng/ml. This assay is specifically inhibited with galactose, 50% inhibition at 9.9 mM, and produces a linear response between 2 and 20 ng of jelly adsorbed to the microtiter plate. Using this assay, the CGL ligand was purified through gel filtration, anion-exchange, and CGL affinity chromatography. Hydrolysis of the purified CGL ligand with a series of exoglycosidases showed that a terminal alpha-galactose is the ligand structure required for recognition by CGL.

  11. Expression of pluripotency factors in larval epithelia of the frog Xenopus: Evidence for the presence of cornea epithelial stem cells

    Science.gov (United States)

    Perry, Kimberly J.; Thomas, Alvin G.; Henry, Jonathan J.

    2013-01-01

    Understanding the biology of somatic stem cells in self renewing tissues represents an exciting field of study, especially given the potential to harness these cells for tissue regeneration and repair in treating injury and disease. The mammalian cornea contains a population of basal epithelial stem cells involved in cornea homeostasis and repair. Research has been restricted to mammalian systems and little is known about the presence or function of these stem cells in other vertebrates. Therefore, we carried out studies to characterize frog cornea epithelium. Careful examination shows that the Xenopus larval cornea epithelium consists of three distinct layers that include an outer epithelial layer and underlying basal epithelium, in addition to a deeper fibrous layer that contains the main sensory nerve trunks that give rise to numerous branches that extend into these epithelia. These nerves convey sensory and presumably also autonomic innervation to those tissues. The sensory nerves are all derived as branches of the trigeminal nerve/ganglion similar to the situation encountered in mammals, though there appear to be some potentially interesting differences, which are detailed in this paper. We show further that numerous pluripotency genes are expressed by cells in the cornea epithelium, including: sox2, p63, various oct4 homologs, c-myc, klf4 and many others. Antibody localization revealed that p63, a well known mammalian epithelial stem cell marker, was localized strictly to all cells in the basal cornea epithelium. c-myc, was visualized in a smaller subset of basal epithelial cells and adjacent stromal tissue predominately at the periphery of the cornea (limbal zone). Finally, sox2 protein was found to be present throughout all cells of both the outer and basal epithelia, but was much more intensely expressed in a distinct subset of cells that appeared to be either multinucleate or possessed multi-lobed nuclei that are normally located at the periphery of the

  12. Zika Virus.

    Science.gov (United States)

    Musso, Didier; Gubler, Duane J

    2016-07-01

    Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) in the genus Flavivirus and the family Flaviviridae. ZIKV was first isolated from a nonhuman primate in 1947 and from mosquitoes in 1948 in Africa, and ZIKV infections in humans were sporadic for half a century before emerging in the Pacific and the Americas. ZIKV is usually transmitted by the bite of infected mosquitoes. The clinical presentation of Zika fever is nonspecific and can be misdiagnosed as other infectious diseases, especially those due to arboviruses such as dengue and chikungunya. ZIKV infection was associated with only mild illness prior to the large French Polynesian outbreak in 2013 and 2014, when severe neurological complications were reported, and the emergence in Brazil of a dramatic increase in severe congenital malformations (microcephaly) suspected to be associated with ZIKV. Laboratory diagnosis of Zika fever relies on virus isolation or detection of ZIKV-specific RNA. Serological diagnosis is complicated by cross-reactivity among members of the Flavivirus genus. The adaptation of ZIKV to an urban cycle involving humans and domestic mosquito vectors in tropical areas where dengue is endemic suggests that the incidence of ZIKV infections may be underestimated. There is a high potential for ZIKV emergence in urban centers in the tropics that are infested with competent mosquito vectors such as Aedes aegypti and Aedes albopictus. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation.

    Science.gov (United States)

    Yamagishi, Yuka; Abe, Hiroshi

    2015-12-01

    We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation. © 2015 Yamagishi and Abe. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  14. Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition.

    Science.gov (United States)

    Amodeo, Amanda A; Jukam, David; Straight, Aaron F; Skotheim, Jan M

    2015-03-10

    During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development.

  15. Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

    Science.gov (United States)

    Reifenberger, Matthew S; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Alli, Ahmed A; Eaton, Douglas C; Alli, Abdel A

    2014-07-01

    Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. Copyright © 2014 the American Physiological Society.

  16. Direct intertectal inputs are an integral component of the bilateral sensorimotor circuit for behavior in Xenopus tadpoles.

    Science.gov (United States)

    Gambrill, Abigail C; Faulkner, Regina L; Cline, Hollis T

    2018-02-14

    The circuit controlling visually-guided behavior in non-mammalian vertebrates, like Xenopus tadpoles, includes retinal projections to the contralateral optic tectum, where visual information is processed, and tectal motor outputs projecting ipsilaterally to hindbrain and spinal cord. Tadpoles have an intertectal commissure whose function is unknown, but it might transfer information between the tectal lobes. Differences in visual experience between the two eyes have profound effects on the development and function of visual circuits in animals with binocular vision, but the effects on animals with fully-crossed retinal projections are not clear. We tested the effect of monocular visual experience on the visuomotor circuit in Xenopus tadpoles. We show that cutting the intertectal commissure or providing visual experience to one eye (monocular visual experience) are both sufficient to disrupt tectally-mediated visual avoidance behavior. Monocular visual experience induces asymmetry in tectal circuit activity across the midline. Repeated exposure to monocular visual experience drives maturation of the stimulated retinotectal synapses, seen as increased AMPA/NMDA ratios, induces synaptic plasticity in intertectal synaptic connections and induces bilaterally asymmetric changes in the tectal excitation/inhibition ratio (E/I). We show that unilateral expression of peptides that interfere with AMPA or GABAA receptor trafficking alters E/I in the transfected tectum and is sufficient to degrade visuomotor behavior. Our study demonstrates that monocular visual experience in animals with fully-crossed visual systems produces asymmetric circuit function across the midline and degrades visuomotor behavior. The data further suggest that intertectal inputs are an integral component of a bilateral visuomotor circuit critical for behavior.

  17. Effect of PCMBS on CO2 permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant.

    Science.gov (United States)

    Cooper, G J; Boron, W F

    1998-12-01

    A recent study on Xenopus oocytes [N. L. Nakhoul, M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 (Cell Physiol. 43): C543-548, 1998] injected with carbonic anhydrase showed that expressing aquaporin 1 (AQP1) increases by approximately 40% the rate at which exposing the cell to CO2 causes intracellular pH to fall. This observation is consistent with several interpretations. Overexpressing AQP1 might increase apparent CO2 permeability by 1) allowing CO2 to pass through AQP1, 2) stimulating injected carbonic anhydrase, 3) enhancing the CO2 solubility of the membrane's lipid, or 4) increasing the expression of a native "gas channel." The purpose of the present study was to distinguish among these possibilities. We found that expressing the H2O channel AQP1 in Xenopus oocytes increases the CO2 permeability of oocytes in an expression-dependent fashion, whereas expressing the K+ channel ROMK1 has no effect. The mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS), which inhibits the H2O movement through AQP1, also blocks the AQP1-dependent increase in CO2 permeability. The mercury-insensitive C189S mutant of AQP1 increases the CO2 permeability of the oocyte to the same extent as does the wild-type channel. However, the C189S-dependent increase in CO2 permeability is unaffected by treatment with PCMBS. These data rule out options 2-4 listed above. Thus our results suggest that CO2 passes through the pore of AQP1 and are the first data to demonstrate that a gas can enter a cell by a means other than diffusing through the membrane lipid.

  18. Agglutination of Jelly Coat and Cortical Granule Components and the Block to Polyspermy in the Amphibian Xenopus laevis

    Science.gov (United States)

    Wyrick, Ron E.; Nishihara, Tatsuro; Hedrick, Jerry L.

    1974-01-01

    A block to polyspermy in amphibians is established at fertilization by the conversion of the vitelline envelope to the fertilization envelope. In Xenopus laevis a major ultrastructural change in the envelope at fertilization is the appearance of an electron-dense layer, termed the F layer, between the envelope and the inner-most jelly coat layer, J1. The F layer is derived, at least in part, from materials released from the cortical granules. Further definition of the origin and chemical nature of the F layer was sought by using isolated cortical granule (CG) exudate and jelly coat layer J1. In double diffusion experiments, the isolated components interacted in an agglutination reaction producing a band of precipitation. The agglutination involved α-galactoside residues and metal ions (Ca++). Employing chemically modified jelly, we demonstrated that sulfhydryl-disulfide interchanges were not involved in the agglutination and, with 35S-labeled jelly, that the agglutinating J1 component possessed sulfate esters. Both the CG exudate and the J1 components contained carbohydrate, as evidenced by their lectin reactivity. A number of ionic polymers, both natural and synthetic, were tested as chemical analogs of CG exudate and J1; none gave an agglutination band. Dissolved jelly coat material from eggs of two different species of frogs agglutinated with CG exudate, while jelly from sea urchin eggs and hyaluronic acid from mammalian eggs did not. Thus, the agglutination reaction was chemically and phylogenetically specific. An electron-dense layer, similar to the F layer, formed on the outer of the vitelline envelope when jellied unfertilized eggs were immersed in CG exudate; such eggs were not fertilizable. We suggest that in Xenopus laevis, and perhaps other organisms as well, an agglutination type of reaction between cortical granule components and egg integuments is a participant in the structural and molecular events establishing a block to polyspermy. Images PMID

  19. Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

    Science.gov (United States)

    Nakayama, Takuya; Fisher, Marilyn; Nakajima, Keisuke; Odeleye, Akinleye O; Zimmerman, Keith B; Fish, Margaret B; Yaoita, Yoshio; Chojnowski, Jena L; Lauderdale, James D; Netland, Peter A; Grainger, Robert M

    2015-12-15

    Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Xenopus TRPN1 (NOMPC) localizes to microtubule-based cilia in epithelial cells, including inner-ear hair cells.

    Science.gov (United States)

    Shin, Jung-Bum; Adams, Dany; Paukert, Martin; Siba, Maria; Sidi, Samuel; Levin, Michael; Gillespie, Peter G; Gründer, Stefan

    2005-08-30

    In vertebrates, the senses of hearing and balance depend on hair cells, which transduce sounds with their hair bundles, containing actin-based stereocilia and microtubule-based kinocilia. A longstanding question in auditory science is the identity of the mechanically sensitive transduction channel of hair cells, thought to be localized at the tips of their stereocilia. Experiments in zebrafish implicated the transient receptor potential (TRP) channel NOMPC (drTRPN1) in this role; TRPN1 is absent from the genomes of higher vertebrates, however, and has not been localized in hair cells. Another candidate for the transduction channel, TRPA1, apparently is required for transduction in mammalian and nonmammalian vertebrates. This discrepancy raises the question of the relative contribution of TRPN1 and TRPA1 to transduction in nonmammalian vertebrates. To address this question, we cloned the TRPN1 ortholog from the amphibian Xenopus laevis, generated an antibody against the protein, and determined the protein's cellular and subcellular localization. We found that TRPN1 is prominently located in lateral-line hair cells, auditory hair cells, and ciliated epidermal cells of developing Xenopus embryos. In ciliated epidermal cells TRPN1 staining was enriched at the tips and bases of the cilia. In saccular hair cells, TRPN1 was located prominently in the kinocilial bulb, a component of the mechanosensory hair bundles. Moreover, we observed redistribution of TRPN1 upon treatment of hair cells with calcium chelators, which disrupts the transduction apparatus. This result suggests that although TRPN1 is unlikely to be the transduction channel of stereocilia, it plays an essential role, functionally related to transduction, in the kinocilium.

  1. TRESK background K(+ channel is inhibited by PAR-1/MARK microtubule affinity-regulating kinases in Xenopus oocytes.

    Directory of Open Access Journals (Sweden)

    Gabriella Braun

    Full Text Available TRESK (TWIK-related spinal cord K(+ channel, KCNK18 is a major background K(+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K(+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K(+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279 in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel.

  2. Xaml1/Runx1 is required for the specification of Rohon-Beard sensory neurons in Xenopus.

    Science.gov (United States)

    Park, Byung-Yong; Hong, Chang-Soo; Weaver, Jamie R; Rosocha, Elizabeth M; Saint-Jeannet, Jean-Pierre

    2012-02-01

    Lower vertebrates develop a unique set of primary sensory neurons located in the dorsal spinal cord. These cells, known as Rohon-Beard (RB) sensory neurons, innervate the skin and mediate the response to touch during larval stages. Here we report the expression and function of the transcription factor Xaml1/Runx1 during RB sensory neurons formation. In Xenopus embryos Runx1 is specifically expressed in RB progenitors at the end of gastrulation. Runx1 expression is positively regulated by Fgf and canonical Wnt signaling and negatively regulated by Notch signaling, the same set of factors that control the development of other neural plate border cell types, i.e. the neural crest and cranial placodes. Embryos lacking Runx1 function fail to differentiate RB sensory neurons and lose the mechanosensory response to touch. At early stages Runx1 knockdown results in a RB progenitor-specific loss of expression of Pak3, a p21-activated kinase that promotes cell cycle withdrawal, and of N-tub, a neuronal-specific tubulin. Interestingly, the pro-neural gene Ngnr1, an upstream regulator of Pak3 and N-tub, is either unaffected or expanded in these embryos, suggesting the existence of two distinct regulatory pathways controlling sensory neuron formation in Xenopus. Consistent with this possibility Ngnr1 is not sufficient to activate Runx1 expression in the ectoderm. We propose that Runx1 function is critically required for the generation of RB sensory neurons, an activity reminiscent of that of Runx1 in the development of the mammalian dorsal root ganglion nociceptive sensory neurons. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character

    Science.gov (United States)

    Fisher, Marilyn; Hirsch, Nicolas; Cox, Amanda; Reeder, Rollin; Carruthers, Samantha; Hall, Amanda; Stemple, Derek L.; Grainger, Robert M.

    2014-01-01

    SUMMARY The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant. PMID:25224223

  4. Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition

    Science.gov (United States)

    Amodeo, Amanda A.; Jukam, David; Straight, Aaron F.; Skotheim, Jan M.

    2015-01-01

    During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development. PMID:25713373

  5. 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.

  6. Zika Virus and Pregnancy

    Medline Plus

    Full Text Available ... Education & Events Advocacy For Patients About ACOG Zika Virus and Pregnancy Home For Patients Zika Virus and ... Education Pamphlets - Spanish Share: PEV002, September 2016 Zika Virus and Pregnancy There are risks to your fetus ...

  7. Zika Virus and Pregnancy

    Science.gov (United States)

    ... Management Education & Events Advocacy For Patients About ACOG Zika Virus and Pregnancy Home For Patients Zika Virus and ... Patient Education Pamphlets - Spanish Share: PEV002, September 2016 Zika Virus and Pregnancy There are risks to your fetus ...

  8. Zika Virus and Pregnancy

    Medline Plus

    Full Text Available ... Management Education & Events Advocacy For Patients About ACOG Zika Virus and Pregnancy Home For Patients Zika Virus and ... Patient Education Pamphlets - Spanish Share: PEV002, September 2016 Zika Virus and Pregnancy There are risks to your fetus ...

  9. Computer Viruses: An Overview.

    Science.gov (United States)

    Marmion, Dan

    1990-01-01

    Discusses the early history and current proliferation of computer viruses that occur on Macintosh and DOS personal computers, mentions virus detection programs, and offers suggestions for how libraries can protect themselves and their users from damage by computer viruses. (LRW)

  10. Virus Ebola Asia

    OpenAIRE

    Wuryadi, Suharyono

    1996-01-01

    Virus Marburg dan Ebola diklasifikasikan sebagai virus yang sangat menular dan dimasukkan dalam klasifikasi sebagai virus/pathogen dengan derajat biosafety 4, sehingga untuk menanganinya diperlukan laboratorium khusus tingkat 4.

  11. Zika Virus and Pregnancy

    Medline Plus

    Full Text Available ... Advocacy For Patients About ACOG Zika Virus and Pregnancy Home For Patients Zika Virus and Pregnancy Page ... Spanish Share: PEV002, September 2016 Zika Virus and Pregnancy There are risks to your fetus if you ...

  12. Zika Virus and Pregnancy

    Medline Plus

    Full Text Available ... Management Education & Events Advocacy For Patients About ACOG Zika Virus and Pregnancy Home For Patients Zika Virus ... Patient Education Pamphlets - Spanish Share: PEV002, September 2016 Zika Virus and Pregnancy There are risks to your ...

  13. Computer Virus and Trends

    OpenAIRE

    Tutut Handayani; Soenarto Usna,Drs.MMSI

    2004-01-01

    Since its appearance the first time in the mid-1980s, computer virus has invited various controversies that still lasts to this day. Along with the development of computer systems technology, viruses komputerpun find new ways to spread itself through a variety of existing communications media. This paper discusses about some things related to computer viruses, namely: the definition and history of computer viruses; the basics of computer viruses; state of computer viruses at this time; and ...

  14. Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

    Science.gov (United States)

    Dubińska-Magiera, Magda; Chmielewska, Magdalena; Kozioł, Katarzyna; Machowska, Magdalena; Hutchison, Christopher J; Goldberg, Martin W; Rzepecki, Ryszard

    2016-05-01

    Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

  15. PNPLA 3 I148M genetic variant associates with insulin resistance and baseline viral load in HCV genotype 2 but not in genotype 3 infection

    Directory of Open Access Journals (Sweden)

    Rembeck Karolina

    2012-09-01

    Full Text Available Abstract Background Hepatic steatosis in HCV patients has been postulated as a risk factor associated with a higher frequency of fibrosis and cirrhosis. A single genetic variant, PNPLA3 I148M, has been widely associated with increased hepatic steatosis. Previous studies of the PNPLA3 I148M sequence variant in HCV infected individuals have reported an association between this variant and prevalence of steatosis, fibrosis, and cirrhosis. To evaluate the impact of PNPLA3 I148M variant on metabolic traits and treatment response in HCV genotype 2 and 3 infected patients. Methods Three hundred and eighty-two treatment naïve HCV genotype 2 or 3 infected patients were included in a phase III, open label, randomized, multicenter, investigator-initiated trial (the NORDynamIC study, in which pretreatment liver biopsies were mandatory. PNPLA3I148M genotyping was performed in a total of 359 Caucasian patients. Results In HCV genotype 2 infected patients carrying the PNPLA3 148M allele, there was significantly increased insulin resistance (P = 0.023 and lower viral load (P = 0.005 at baseline as well as the first seven days of antiviral treatment. These results were not observed in HCV genotype 3 infected patients. Conclusions Our results suggest a possible association between the PNPLA3 148M allele and insulin resistance as well as baseline viral load in HCV genotype 2, but not in genotype 3.

  16. Validation of the sperm chromatin dispersion (SCD) test in the Amphibian Xenopus laevis using in situ nick translation and comet assay

    OpenAIRE

    Pollock, K; Gosálvez, J; Arroyo, F; López-Fernández, C; Guille, M; Noble, A; Johnston, S D

    2014-01-01

    The integrity of sperm DNA is becoming increasingly recognised as an important parameter of semen quality, but there are no published reports of this procedure for any amphibian. The primary aim of this study was to apply a modified sperm chromatin dispersion (SCD) test (Halomax) to an amphibian sperm model (African clawed frog; Xenopus laevis) and to validate the assay against in situ nick translation (ISNT) and the double-comet assay procedure. Inactivated spermatozoa were collected from fr...

  17. Genome-wide expression profile of the response to spinal cord injury in Xenopus laevis reveals extensive differences between regenerative and non-regenerative stages

    Science.gov (United States)

    2014-01-01

    Background Xenopus laevis has regenerative and non-regenerative stages. As a tadpole, it is fully capable of functional recovery after a spinal cord injury, while its juvenile form (froglet) loses this capability during metamorphosis. We envision that comparative studies between regenerative and non-regenerative stages in Xenopus could aid in understanding why spinal cord regeneration fails in human beings. Results To identify the mechanisms that allow the tadpole to regenerate and inhibit regeneration in the froglet, we obtained a transcriptome-wide profile of the response to spinal cord injury in Xenopus regenerative and non-regenerative stages. We found extensive transcriptome changes in regenerative tadpoles at 1 day after injury, while this was only observed by 6 days after injury in non-regenerative froglets. In addition, when comparing both stages, we found that they deployed a very different repertoire of transcripts, with more than 80% of them regulated in only one stage, including previously unannotated transcripts. This was supported by gene ontology enrichment analysis and validated by RT-qPCR, which showed that transcripts involved in metabolism, response to stress, cell cycle, development, immune response and inflammation, neurogenesis, and axonal regeneration were regulated differentially between regenerative and non-regenerative stages. Conclusions We identified differences in the timing of the transcriptional response and in the inventory of regulated transcripts and biological processes activated in response to spinal cord injury when comparing regenerative and non-regenerative stages. These genes and biological processes provide an entry point to understand why regeneration fails in mammals. Furthermore, our results introduce Xenopus laevis as a genetic model organism to study spinal cord regeneration. PMID:24885550

  18. In Situ Hybridization Localization of TRH Precursor and TRH Receptor mRNAs in the Brain and Pituitary of Xenopus laevis

    NARCIS (Netherlands)

    Galas, L.; Bidaud, I.; Bulant, M.; Jenks, B.G.; Ouwens, D.T.; Jegou, S.; Ladram, A.; Roubos, E.W.; Nicolas, P.; Tonon, M.C.; Vaudry, H.

    2005-01-01

    We examined the distribution of the mRNAs encoding proTRH and the three TRH receptor subtypes (xTRHR1, xTRHR2, and xTRHR3) in the Xenopus laevis CNS and pituitary. A positive correlation was generally observed between the expression patterns of proTRH and xTRHR mRNAs. xTRHRs were widely expressed in

  19. The proteins of Vent-family and their mRNAs are located in different areas of the tails of Zebrafish and Xenopus embryos.

    Science.gov (United States)

    Pshennikova, Elena S; Voronina, Anna S

    2016-10-01

    In molecular embryology, by tacit consent, a presence or absence of the specific mRNA in the cell indicates the presence or absence of the corresponding protein. However, there are lots of evidences that mRNA may be associated with inactive non-translated ribonucleoprotein complexes. Here we for the first time compared the temporal and spatial distribution of the Vent-family transcription factors and their mRNAs in the tails of embryos of two biological species-Xenopus laevis and Zebrafish (Danio reria). We have shown that Xvent-2 mRNA was not active in tails of Xenopus embryos at the early tail bud stage. Although the Xvent-2 mRNA of Xenopus and Vox mRNA of Zebrafish embryos were detected in the tips of the tails, the Xvent-2 and Vox proteins were revealed in other regions of the tails-along axial structures and around somites. We propose that when the translation of masked Xvent-2 (or Vox) mRNA is activated in a tail bud cell, this transcription factor changes activity of target genes. As a result, the cell comes to differentiation. Copyright © 2016. Published by Elsevier Ltd.

  20. Long-term effect on in vitro cloning efficiency after treatment of somatic cells with Xenopus egg extract in the pig.

    Science.gov (United States)

    Liu, Ying; Ostrup, Olga; Li, Rong; Li, Juan; Vajta, Gábor; Kragh, Peter M; Schmidt, Mette; Purup, Stig; Hyttel, Poul; Klærke, Dan; Callesen, Henrik

    2014-08-01

    In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus egg extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus egg extract on donor cells was maintained for several cell passages. The extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7-22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus egg extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.

  1. What's West Nile Virus?

    Science.gov (United States)

    ... OK for Kids? Your Teeth Heart Murmurs What's West Nile Virus? KidsHealth > For Kids > What's West Nile Virus? Print A A A en español ¿Qué es ... Virus del Nilo Occidental? What exactly is the West Nile virus? And why is everyone talking about mosquitoes ? Even ...

  2. Viruses infecting maize

    OpenAIRE

    Krstić, Branka; Stanković, Ivana; Bulajić, Aleksandra

    2014-01-01

    Over 40 plant viruses has been known to cause diseases of maize, but economically the most important yield looses, which in certain years can be total, are caused by viruses from Potyvirus genera, known to be aphid-transmitted in a non-persistant maner. The most important viruses, pathogens of maize, sugar cane and sorghum are considered to be Maize dwarf mosaic virus (MDMV), Sorghum mosaic virus (SrMV), Sugarcane mosaic virus (SCMV), and Johnsongrass mosaic virus (JGMV). In Serbia, the prese...

  3. Viruses in cancer treatment.

    Science.gov (United States)

    Alemany, R

    2013-03-01

    Soon after the discovery that viruses cause human disease, started the idea of using viruses to treat cancer. After the initial indiscriminate use, crude preparations of each novel virus in the early twentieth century, a second wave of virotherapy blossomed in the 60s with purified and selected viruses. Responses were rare and short-lived. Immune rejection of the oncolytic viruses was identified as the major problem and virotherapy was abandoned. During the past two decades virotherapy has re-emerged with engineered viruses, with a trend towards using them as tumor-debulking immunostimulatory agents combined with radio or chemotherapy. Currently, oncolytic Reovirus, Herpes, and Vaccinia virus are in late phase clinical trials. Despite the renewed hope, efficacy will require improving systemic tumor targeting, overcoming stroma barriers for virus spread, and selectively stimulating immune responses against tumor antigens but not against the virus. Virotherapy history, viruses, considerations for clinical trials, and hurdles are briefly overviewed.

  4. MENGENAL HANTA VIRUS

    Directory of Open Access Journals (Sweden)

    Tri Wijayanti

    2012-11-01

    Full Text Available Virus Hanta kurang infeksius, kecuali di dalam lingkungan tertentu. Lamanya waktu virus ini dapat bertahan di lingkungan, setelah keluar dari tubuh tikus tidaklah diketahui secara pasti. Tetapi percobaan laboratorium menunjukkan bahwa, daya infektifitasnya tidak dijumpai setelah dua hari pengeringan. Genus hanta virus terdiri dari 22 spesies virus, dapat menyebabkan hemorrhagic fever with renal syndrome (HFRS dan hanta virus pulmonary syndrome (HPS.

  5. Modulation of thyroid hormone-dependent gene expression in Xenopus laevis by INhibitor of Growth (ING) proteins.

    Science.gov (United States)

    Helbing, Caren C; Wagner, Mary J; Pettem, Katherine; Johnston, Jill; Heimeier, Rachel A; Veldhoen, Nik; Jirik, Frank R; Shi, Yun-Bo; Browder, Leon W

    2011-01-01

    INhibitor of Growth (ING) proteins belong to a large family of plant homeodomain finger-containing proteins important in epigenetic regulation and carcinogenesis. We have previously shown that ING1 and ING2 expression is regulated by thyroid hormone (TH) during metamorphosis of the Xenopus laevis tadpole. The present study investigates the possibility that ING proteins modulate TH action. Tadpoles expressing a Xenopus ING2 transgene (Trans(ING2)) were significantly smaller than tadpoles not expressing the transgene (Trans(GFP)). When exposed to 10 nM 3,5,3'-triiodothyronine (T(3)), premetamorphic Trans(ING2) tadpoles exhibited a greater reduction in tail, head, and brain areas, and a protrusion of the lower jaw than T(3)-treated Trans(GFP) tadpoles. Quantitative real time polymerase chain reaction (QPCR) demonstrated elevated TH receptor β (TRβ) and TH/bZIP transcript levels in Trans(ING2) tadpole tails compared to Trans(GFP) tadpoles while TRα mRNAs were unaffected. In contrast, no difference in TRα, TRβ or insulin-like growth factor (IGF2) mRNA abundance was observed in the brain between Trans(ING2) and Trans(GFP) tadpoles. All of these transcripts, except for TRα mRNA in the brain, were inducible by the hormone in both tissues. Oocyte transcription assays indicated that ING proteins enhanced TR-dependent, T(3)-induced TRβ gene promoter activity. Examination of endogenous T(3)-responsive promoters (TRβ and TH/bZIP) in the tail by chromatin immunoprecipitation assays showed that ING proteins were recruited to TRE-containing regions in T(3)-dependent and independent ways, respectively. Moreover, ING and TR proteins coimmunoprecipitated from tail protein homogenates derived from metamorphic climax animals. We show for the first time that ING proteins modulate TH-dependent responses, thus revealing a novel role for ING proteins in hormone signaling. This has important implications for understanding hormone influenced disease states and suggests that the

  6. Sequences coding for the ribosomal protein L14 in Xenopus laevis and Xenopus tropicalis; homologies in the 5' untranslated region are shared with other r-protein mRNAs.

    Science.gov (United States)

    Beccari, E; Mazzetti, P; Mileo, A; Bozzoni, I; Pierandrei-Amaldi, P; Amaldi, F

    1986-01-01

    In the haploid genome of Xenopus laevis there are two genes coding for the r-protein L14. It is not known if they are located on the same chromosome. cDNA clones deriving from the transcripts of the two genes have been isolated from an oocyte messenger cDNA bank showing that they are both expressed. We have studied the structure of one of the L14 genes by Electron Microscopy, restriction mapping and sequencing. An allelic form of the L14 gene was also isolated. It contains a large deletion covering the 5' end region up to the middle of the third intron. The 5' end of the X. laevis L14 gene was compared to that of the corresponding gene in the closely related species X. tropicalis and found to be highly conserved. The L14 gene has multiple initiation sites, but the large majority of the transcripts start in the middle of a pyrimidine tract not preceded by a canonical TATA box as in other eukaryotic housekeeping genes. The X. laevis L1 and L14 genes have a common decanucleotide in the first exon in the same position with regard to the initiator ATG which just precedes the first intron. The decanucleotide shows homology with the X. laevis 18S rRNA. Images PMID:3774540

  7. Identification of a novel conserved mixed-isoform B56 regulatory subunit and spatiotemporal regulation of protein phosphatase 2A during Xenopus laevis development

    Directory of Open Access Journals (Sweden)

    Seeling Joni M

    2007-12-01

    Full Text Available Abstract Background Wnt signaling is a key regulator of development and tumorigenesis. Protein phosphatase 2A (PP2A, which consists of a catalytic C, a structural A, and a regulatory B subunit, plays diverse roles in Wnt signaling through its B56 subunits. B56 is a multigene family encoding for proteins with a conserved core domain and divergent amino- and carboxy-termini. Ectopic B56α and B56γ reduce β-catenin abundance and B56α reduces Wnt-dependent transcription, suggesting that B56α and B56γ inhibit Wnt signaling. In contrast, B56ε is required for Wnt signaling. Knowledge of where and when B56 subunits are expressed during Xenopus development will aid in our understanding of their roles in Wnt signaling. Results We have undertaken expression analyses of B56α and B56γ in Xenopus laevis. We cloned Xenopus B56α; it is 88% identical to human B56α. Xenopus B56γ is 94% identical with human B56γ, however, a novel evolutionarily conserved mixed-isoform transcript was identified that contains a B56δ-like amino-terminal domain and a B56γ core domain. The B56δ-like variable domain exon is located upstream of the B56γ variable domain exon at the human B56γ locus, suggesting that the mixed-isoform transcript is due to alternative splicing. B56γ transcripts with different 3' ends were identified that lack or possess a 35 base pair sequence, resulting in either a transcript similar to human B56γ1, or an uncharacterized evolutionarily conserved sequence. Real time RT-PCR analyses revealed that B56α is expressed at moderate levels before the midblastula transition (MBT, at reduced levels during gastrulation and neurulation, and at high levels during organogenesis, while B56γ is expressed at low levels until organogenesis. B56α is enriched in the ventral hemisphere pre-MBT, while B56γ is ventrally enriched post-MBT. Aα, Aβ, Cα and Cβ are expressed in early Xenopus development, suggesting the presence of a functional heterotrimer

  8. Viruses Infecting Reptiles

    Science.gov (United States)

    Marschang, Rachel E.

    2011-01-01

    A large number of viruses have been described in many different reptiles. These viruses include arboviruses that primarily infect mammals or birds as well as viruses that are specific for reptiles. Interest in arboviruses infecting reptiles has mainly focused on the role reptiles may play in the epidemiology of these viruses, especially over winter. Interest in reptile specific viruses has concentrated on both their importance for reptile medicine as well as virus taxonomy and evolution. The impact of many viral infections on reptile health is not known. Koch’s postulates have only been fulfilled for a limited number of reptilian viruses. As diagnostic testing becomes more sensitive, multiple infections with various viruses and other infectious agents are also being detected. In most cases the interactions between these different agents are not known. This review provides an update on viruses described in reptiles, the animal species in which they have been detected, and what is known about their taxonomic positions. PMID:22163336

  9. Viruses Infecting Reptiles

    Directory of Open Access Journals (Sweden)

    Rachel E. Marschang

    2011-11-01

    Full Text Available A large number of viruses have been described in many different reptiles. These viruses include arboviruses that primarily infect mammals or birds as well as viruses that are specific for reptiles. Interest in arboviruses infecting reptiles has mainly focused on the role reptiles may play in the epidemiology of these viruses, especially over winter. Interest in reptile specific viruses has concentrated on both their importance for reptile medicine as well as virus taxonomy and evolution. The impact of many viral infections on reptile health is not known. Koch’s postulates have only been fulfilled for a limited number of reptilian viruses. As diagnostic testing becomes more sensitive, multiple infections with various viruses and other infectious agents are also being detected. In most cases the interactions between these different agents are not known. This review provides an update on viruses described in reptiles, the animal species in which they have been detected, and what is known about their taxonomic positions.

  10. Viruses infecting reptiles.

    Science.gov (United States)

    Marschang, Rachel E

    2011-11-01

    A large number of viruses have been described in many different reptiles. These viruses include arboviruses that primarily infect mammals or birds as well as viruses that are specific for reptiles. Interest in arboviruses infecting reptiles has mainly focused on the role reptiles may play in the epidemiology of these viruses, especially over winter. Interest in reptile specific viruses has concentrated on both their importance for reptile medicine as well as virus taxonomy and evolution. The impact of many viral infections on reptile health is not known. Koch's postulates have only been fulfilled for a limited number of reptilian viruses. As diagnostic testing becomes more sensitive, multiple infections with various viruses and other infectious agents are also being detected. In most cases the interactions between these different agents are not known. This review provides an update on viruses described in reptiles, the animal species in which they have been detected, and what is known about their taxonomic positions.

  11. Alteration of structure and penetrability of the vitelline envelope after passage of eggs from coelom to oviduct in Xenopus laevis.

    Science.gov (United States)

    Grey, R D; Working, P K; Hedrick, J L

    1977-07-01

    The vitelline envelope (VE) that surrounds an egg released from the ovary into the coelom of Xenopus laevis differs markedly, in structure and penetrability, from the VE surrounding an oviposited egg. In a coelomic egg, the filaments that form the VE are arranged in distinct fascicles or bundles. The exterior surface of the VE is irregular in contour and is permeated by channels. In an oviposited egg, the filaments are evenly dispersed and lack a fasciculated arrangement; the exterior surface is smooth and no channels are present. The fascicular arrangement of fibrils in the coelomic VE is maintained only at neutral pH, and is not visibly altered by the cortical reaction. VEs from coelomic eggs retain their fasciculated morphology after isolation from the egg. In an in vitro test system, sperm penetrated VEs isolated from oviposited eggs, but failed to penetrate VEs isolated from coelomic eggs. The structural transformation of the VE from the coelomic type to the oviposited type occurs in the first 1-cm segment of the oviduct, prior to addition of jelly to the egg. Neither intact jelly, solubilized jelly, nor jelly extracts were capable of altering the structural organization of coelomic VEs, suggesting that the structural transformation of the VE is effected by some oviducal factor other than jelly.

  12. Xenopus cytoplasmic linker–associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules

    Science.gov (United States)

    Marx, Astrid; Godinez, William J.; Tsimashchuk, Vasil; Bankhead, Peter; Rohr, Karl; Engel, Ulrike

    2013-01-01

    Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker–associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin–microtubule interactions. PMID:23515224

  13. Cytoskeleton and gravity at work in the establishment of dorso-ventral polarity in the egg of Xenopus laevis

    Science.gov (United States)

    Ubbels, Geertje A.; Brom, Tim G.

    The establishment of polarities during early embryogenesis is essential for normal development. Amphibian eggs are appropriate models for studies on embryonic pattern formation. The animal-vegetal axis of the axially symmetrical amphibian egg originates during oogenesis and foreshadows the main body axis of the embryo. The dorso-ventral polarity is epigenetically established before first cleavage. Recent experiments strongly suggest that in the monospermic eggs of the anuran Xenopus laevis both the cytoskeleton and gravity act in the determination of the dorso-ventral polarity. In order to test the role of gravity in this process, eggs will be fertilized under microgravity conditions during the SL-D1 flight in 1985. In a fully automatic experiment container eggs will be kept under well-defined conditions and artificially fertilized as soon as microgravity is reached; eggs and embryos at different stages will then be fixed for later examination. Back on earth the material will be analysed and we will know whether fertilization under microgravity conditions is possible. If so, the relation of the dorso-ventral axis to the former sperm entry point will be determined on the whole embryos; in addition eggs and embryos will be analysed cytologically.

  14. Thylacine 1 is expressed segmentally within the paraxial mesoderm of the Xenopus embryo and interacts with the Notch pathway.

    Science.gov (United States)

    Sparrow, D B; Jen, W C; Kotecha, S; Towers, N; Kintner, C; Mohun, T J

    1998-06-01

    The presomitic mesoderm of vertebrates undergoes a process of segmentation in which cell-cell interactions mediated by the Notch family of receptors and their associated ligands are involved. The vertebrate homologues of Drosophila &Dgr ; are expressed in a dynamic, segmental pattern within the presomitic mesoderm, and alterations in the function of these genes leads to a perturbed pattern of somite segmentation. In this study we have characterised Thylacine 1 which encodes a basic helix-loop-helix class transcription activator. Expression of Thylacine is restricted to the presomitic mesoderm, localising to the anterior half of several somitomeres in register with domains of X-Delta-2 expression. Ectopic expression of Thylacine in embryos causes segmentation defects similar to those seen in embryos in which Notch signalling is altered, and these embryos also show severe disruption in the expression patterns of the marker genes X-Delta-2 and X-ESR5 within the presomitic mesoderm. Finally, the expression of Thylacine is altered in embryos when Notch signalling is perturbed. These observations suggest strongly that Thylacine 1 has a role in the segmentation pathway of the Xenopus embryo, by interacting with the Notch signalling pathway.

  15. Functional coupling between human E-type Ca2+ channels and mu opioid receptors expressed in Xenopus oocytes.

    Science.gov (United States)

    Ottolia, M; Platano, D; Qin, N; Noceti, F; Birnbaumer, M; Toro, L; Birnbaumer, L; Stefani, E; Olcese, R

    1998-05-01

    Neuronal alpha1E Ca2+ channels were expressed in Xenopus laevis oocytes alone and in combination with the mu opioid receptor. Macroscopic currents were recorded under voltage clamp conditions. The stimulation of the morphine receptor by the synthetic [D-Ala2,N-Me-Phe4,Gly-ol5] enkephalin (DAMGO) produced a 20% reduction in the alpha1E ionic current. This effect was associated with a large change in the decay phase of the Ba2+ current. The effect of 1 microM DAMGO was fully antagonized by the universal mu opioid receptor antagonist naloxone and by the selective antagonist beta-funaltrexamine. The ionic current inhibition induced by DAMGO was partially recovered by preceding strong depolarizations. The injection of the catalytic subunit of pertussis toxin (A-protomer) abolished the effect of DAMGO, suggesting the involvement of a GTP binding protein in the alpha1E modulation. The coexpression of the regulatory beta2a Ca2a channel subunit, together with the alpha1E subunit and the mu opioid receptor, prevented the reduction of the ionic current following the receptor stimulation with DAMGO, whereas the coexpression with the beta3 subunit reduced by approximately 50% the modulatory effect of DAMGO. The effect produced by the stimulation of the opioid receptor could be mimicked by coexpressing the alpha1E channel with the G-protein betagamma subunits.

  16. Abundant and dynamically expressed miRNAs, piRNAs, and other small RNAs in the vertebrate Xenopus tropicalis.

    Science.gov (United States)

    Armisen, Javier; Gilchrist, Michael J; Wilczynska, Anna; Standart, Nancy; Miska, Eric A

    2009-10-01

    Small regulatory RNAs have recently emerged as key regulators of eukaryotic gene expression. Here we used high-throughput sequencing to determine small RNA populations in the germline and soma of the African clawed frog Xenopus tropicalis. We identified a number of miRNAs that were expressed in the female germline. miRNA expression profiling revealed that miR-202-5p is an oocyte-enriched miRNA. We identified two novel miRNAs that were expressed in the soma. In addition, we sequenced large numbers of Piwi-associated RNAs (piRNAs) and other endogenous small RNAs, likely representing endogenous siRNAs (endo-siRNAs). Of these, only piRNAs were restricted to the germline, suggesting that endo-siRNAs are an abundant class of small RNAs in the vertebrate soma. In the germline, both endogenous small RNAs and piRNAs mapped to many high copy number loci. Furthermore, endogenous small RNAs mapped to the same specific subsets of repetitive elements in both the soma and the germline, suggesting that these RNAs might act to silence repetitive elements in both compartments. Data presented here suggest a conserved role for miRNAs in the vertebrate germline. Furthermore, this study provides a basis for the functional analysis of small regulatory RNAs in an important vertebrate model system.

  17. Synergistic interaction of glycoalkaloids alpha-chaconine and alpha-solanine on developmental toxicity in Xenopus embryos.

    Science.gov (United States)

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

    1995-12-01

    The embryo toxicities of two major potato glycoalkaloids, alpha-chaconine and alpha-solanine, were examined individually and in mixtures using the frog embryo teratogenesis assay-Xenopus. Calculations of toxic units (TUs) were used to assess possible antagonism, synergism or response addition of several mixtures ranging from approximately 3:1 to 1:20 TUs of alpha-chaconine to alpha-solanine. Some combinations exhibited strong synergism in the following measures of developmental toxicity: (a) 96-hr LC50, defined as the median concentration causing 50% embryo lethality; (b) 96-hr EC50 (malformation), defined as the concentration causing 50% malformation of the surviving embryos; and (c) teratogenic index which is equal to LC50/EC50 (malformation). The results indicated that each of the mixtures caused synergistic mortality or malformation. Furthermore, these studies suggested that the synergism observed for a specific mixture cannot be used to predict possible synergism of other mixtures with different ratios of the two glycoalkaloids; toxicities observed for individual glycoalkaloids may not be able to predict toxicities of mixtures; and specific combinations found in different potato varieties need to be tested to assess the safety of a particular cultivar.

  18. Perfluoroalkylated Substance Effects in Xenopus laevis A6 Kidney Epithelial Cells Determined by ATR-FTIR Spectroscopy and Chemometric Analysis

    Science.gov (United States)

    2016-01-01

    The effects of four perfluoroalkylated substances (PFASs), namely, perfluorobutanesulfonate (PFBS), perfluorooctanoic acid (PFOA), perfluorooctanesulfonate (PFOS), and perfluorononanoic acid (PFNA) were assessed in Xenopus laevis A6 kidney epithelial cells by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and chemometric analysis. Principal component analysis–linear discriminant analysis (PCA-LDA) was used to visualize wavenumber-related alterations and ANOVA-simultaneous component analysis (ASCA) allowed data processing considering the underlying experimental design. Both analyses evidenced a higher impact of low-dose PFAS-treatments (10–9 M) on A6 cells forming monolayers, while there was a larger influence of high-dose PFAS-treatments (10–5 M) on A6 cells differentiated into dome structures. The observed dose–response PFAS-induced effects were to some extent related to their cytotoxicity: the EC50-values of most influential PFAS-treatments increased (PFOS < PFNA < PFOA ≪ PFBS), and higher-doses of these chemicals induced a larger impact. Major spectral alterations were mainly attributed to DNA/RNA, secondary protein structure, lipids, and fatty acids. Finally, PFOS and PFOA caused a decrease in A6 cell numbers compared to controls, whereas PFBS and PFNA did not significantly change cell population levels. Overall, this work highlights the ability of PFASs to alter A6 cells, whether forming monolayers or differentiated into dome structures, and the potential of PFOS and PFOA to induce cell death. PMID:27078751

  19. 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

  20. 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.

  1. Meiotic maturation induces animal-vegetal asymmetric distribution of aPKC and ASIP/PAR-3 in Xenopus oocytes.

    Science.gov (United States)

    Nakaya, M; Fukui, A; Izumi, Y; Akimoto, K; Asashima, M; Ohno, S

    2000-12-01

    The asymmetric distribution of cellular components is an important clue for understanding cell fate decision during embryonic patterning and cell functioning after differentiation. In C. elegans embryos, PAR-3 and aPKC form a complex that colocalizes to the anterior periphery of the one-cell embryo, and are indispensable for anterior-posterior polarity that is formed prior to asymmetric cell division. In mammals, ASIP (PAR-3 homologue) and aPKCgamma form a complex and colocalize to the epithelial tight junctions, which play critical roles in epithelial cell polarity. Although the mechanism by which PAR-3/ASIP and aPKC regulate cell polarization remains to be clarified, evolutionary conservation of the PAR-3/ASIP-aPKC complex suggests their general role in cell polarity organization. Here, we show the presence of the protein complex in Xenopus laevis. In epithelial cells, XASIP and XaPKC colocalize to the cell-cell contact region. To our surprise, they also colocalize to the animal hemisphere of mature oocytes, whereas they localize uniformly in immature oocytes. Moreover, hormonal stimulation of immature oocytes results in a change in the distribution of XaPKC 2-3 hours after the completion of germinal vesicle breakdown, which requires the kinase activity of aPKC. These results suggest that meiotic maturation induces the animal-vegetal asymmetry of aPKC.

  2. The nodal target gene Xmenf is a component of an FGF-independent pathway of ventral mesoderm induction in Xenopus.

    Science.gov (United States)

    Kumano, Gaku; Smith, William C

    2002-10-01

    The interplay of fibroblast growth factor (FGF) and nodal signaling in the Xenopus gastrula marginal zone specifies distinct populations of presumptive mesodermal cells. Cells in the vegetal marginal zone, making up the presumptive leading edge mesoderm, are exposed to nodal signaling, as evidenced by SMAD2 activation, but do not appear to be exposed to FGF signaling, as evidenced by the lack of MAP kinase (MAPK) activation. However, in the animal marginal zone, activation of both SMAD2 and MAPK occurs. The differential activation of these two signaling pathways in the marginal zone results in the vegetal and animal marginal zones expressing different genes at gastrulation, and subsequently having different fates, with the vegetal marginal zone contributing to ventral mesoderm (e.g. ventral blood island) and the animal marginal zone giving rise to dorsal fates (e.g. notochord and somite). We report here the cloning of a cDNA encoding a novel nuclear protein, Xmenf, that is expressed in the vegetal marginal zone. The expression of Xmenf is induced by nodal signaling and negatively regulated by FGF signaling. Results from animal cap studies indicate that Xmenf plays a role in the pathway of ventral mesoderm induction in the vegetal marginal zone.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Wnt11b is involved in cilia-mediated symmetry breakage during Xenopus left-right development.

    Directory of Open Access Journals (Sweden)

    Peter Walentek

    Full Text Available Breakage of bilateral symmetry in amphibian embryos depends on the development of a ciliated epithelium at the gastrocoel roof during early neurulation. Motile cilia at the gastrocoel roof plate (GRP give rise to leftward flow of extracellular fluids. Flow is required for asymmetric gene expression and organ morphogenesis. Wnt signaling has previously been involved in two steps, Wnt/ß-catenin mediated induction of Foxj1, a regulator of motile cilia, and Wnt/planar cell polarity (PCP dependent cilia polarization to the posterior pole of cells. We have studied Wnt11b in the context of laterality determination, as this ligand was reported to activate canonical and non-canonical Wnt signaling. Wnt11b was found to be expressed in the so-called superficial mesoderm (SM, from which the GRP derives. Surprisingly, Foxj1 was only marginally affected in loss-of-function experiments, indicating that another ligand acts in this early step of laterality specification. Wnt11b was required, however, for polarization of GRP cilia and GRP morphogenesis, in line with the known function of Wnt/PCP in cilia-driven leftward flow. In addition Xnr1 and Coco expression in the lateral-most GRP cells, which sense flow and generate the first asymmetric signal, was attenuated in morphants, involving Wnt signaling in yet another process related to symmetry breakage in Xenopus.

  5. 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.

  6. Cotransport of water by Na¿-K¿-2Cl¿ cotransporters expressed in Xenopus oocytes

    DEFF Research Database (Denmark)

    Zeuthen, Thomas; Macaulay, Nanna

    2012-01-01

    conclude that NKCC1 plays a direct role for water balance in most cell types, while NKCC2 fulfils its role in the kidney of transporting ions but not water. The different behaviour of NKCC1 and NKCC2 is discussed on the basis of recent molecular models based on studies of structural and molecular dynamics.......The NKCC1 and NKCC2 isoforms of the mammalian Na¿–K¿–2Cl¿ cotransporter were expressed in Xenopus oocytes and the relation between external ion concentration and water fluxes determined.Water fluxes were determined from changes in the oocytes volume and ion fluxes from 86Rb+ uptake. Isotonic...... increases in external K¿ concentration elicited abrupt inward water fluxes in NKCC1; the K¿ dependence obeyed one-site kinetics with a K0.5 of 7.5 mM. The water fluxes were blocked by bumetanide, had steep temperature dependence and could proceed uphill against an osmotic gradient of 20 mosmol l...

  7. Structural and functional divergence of growth hormone-releasing hormone receptors in early sarcopterygians: lungfish and Xenopus.

    Directory of Open Access Journals (Sweden)

    Janice K V Tam

    Full Text Available The evolutionary trajectories of growth hormone-releasing hormone (GHRH receptor remain enigmatic since the discovery of physiologically functional GHRH-GHRH receptor (GHRHR in non-mammalian vertebrates in 2007. Interestingly, subsequent studies have described the identification of a GHRHR(2 in chicken in addition to the GHRHR and the closely related paralogous receptor, PACAP-related peptide (PRP receptor (PRPR. In this article, we provide information, for the first time, on the GHRHR in sarcopterygian fish and amphibians by the cloning and characterization of GHRHRs from lungfish (P. dolloi and X. laevis. Sequence alignment and phylogenetic analyses demonstrated structural resemblance of lungfish GHRHR to their mammalian orthologs, while the X. laevis GHRHR showed the highest homology to GHRHR(2 in zebrafish and chicken. Functionally, lungfish GHRHR displayed high affinity towards GHRH in triggering intracellular cAMP and calcium accumulation, while X. laevis GHRHR(2 was able to react with both endogenous GHRH and PRP. Tissue distribution analyses showed that both lungfish GHRHR and X. laevis GHRHR(2 had the highest expression in brain, and interestingly, X. laevis(GHRHR2 also had high abundance in the reproductive organs. These findings, together with previous reports, suggest that early in the Sarcopterygii lineage, GHRHR and PRPR have already established diverged and specific affinities towards their cognate ligands. GHRHR(2, which has only been found in xenopus, zebrafish and chicken hitherto, accommodates both GHRH and PRP.

  8. 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.

  9. Regulation of the insulin-Akt signaling pathway and glycolysis during dehydration stress in the African clawed frog Xenopus laevis.

    Science.gov (United States)

    Wu, Cheng-Wei; Tessier, Shannon N; Storey, Kenneth B

    2017-12-01

    Estivation is an adaptive stress response utilized by some amphibians during periods of drought in the summer season. In this study, we examine the regulation of the insulin signaling cascade and glycolysis pathway in the African clawed frog Xenopus laevis during the dehydration stress induced state of estivation. We show that in the brain and heart of X. laevis, dehydration reduces the phosphorylation of the insulin growth factor-1 receptor (IGF-1R), and this is followed by similar reductions in the phosphorylation of the Akt and mechanistic target of rapamycin (mTOR) kinase. Interestingly, phosphorylation levels of IGF-1R and mTOR were not affected in the kidney, and phosphorylation levels of P70S6K and the ribosomal S6 protein were elevated during dehydration stress. Animals under estivation are also susceptible to periods of hypoxia, suggesting that glycolysis may also be affected. We observed that protein levels of many glycolytic enzymes remained unchanged during dehydration; however, the hypoxia response factor-1 alpha (HIF-1α) protein was elevated by greater than twofold in the heart during dehydration. Overall, we provide evidence that shows that the insulin signaling pathway in X. laevis is regulated in a tissue-specific manner during dehydration stress and suggests an important role for this signaling cascade in mediating the estivation response.

  10. High variability of expression profiles of homeologous genes for Wnt, Hh, Notch, and Hippo signaling pathways in Xenopus laevis.

    Science.gov (United States)

    Michiue, Tatsuo; Yamamoto, Takayoshi; Yasuoka, Yuuri; Goto, Toshiyasu; Ikeda, Takafumi; Nagura, Kei; Nakayama, Takuya; Taira, Masanori; Kinoshita, Tsutomu

    2017-06-15

    Cell signaling pathways, such as Wnt, Hedgehog (Hh), Notch, and Hippo, are essential for embryogenesis, organogenesis, and tissue homeostasis. In this study, we analyzed 415 genes involved in these pathways in the allotetraploid frog, Xenopus laevis. Most genes are retained in two subgenomes called L and S (193 homeologous gene pairs and 29 singletons). This conservation rate of homeologs is much higher than that of all genes in the X. laevis genome (86.9% vs 60.2%). Among singletons, 24 genes are retained in the L subgenome, a rate similar to the average for all genes (82.8% vs 74.6%). In addition, as general components of signal transduction, we also analyzed 32 heparan sulfate proteoglycan (HSPG)-related genes and eight TLE/Groucho transcriptional corepressors-related genes. In these gene sets, all homeologous pairs have been retained. Transcriptome analysis using RNA-seq data from developmental stages and adult tissues demonstrated that most homeologous pairs of signaling components have variable expression patterns, in contrast to the conservative expression profiles of homeologs for transcription factors. Our results indicate that homeologous gene pairs for cell signaling regulation have tended to become subfunctionalized after allotetraploidization. Diversification of signaling pathways by subfunctionalization of homeologs may enhance environmental adaptability. These results provide insights into the evolution of signaling pathways after polyploidization. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Modulation by extracellular pH of GABAA receptors expressed in Xenopus oocytes injected with rat brain mRNA.

    Science.gov (United States)

    Robello, M; Balduzzi, R; Cupello, A

    2000-01-01

    Rat brain poly(A)(+) mRNA was injected into Xenopus oocytes. After 72-96 hr, GABA(A) receptors expressed in this heterologous system were studied by perfusion of GABA and recording of GABA evoked chloride current under voltage-clamp conditions. The GABA activated currents were blocked by bicuculline and enhanced by flunitrazepam. Acidic (6.4) extracellular pH (pH(e) ) augmented, whereas basic pH (8.4) decreased the current evoked by 100 microM GABA in the respect of the current evoked at pH 7.4. Concentration-response curves for GABA evoked chloride currents were built at the three pHs. These data showed that acidic pH does not change the EC50 for GABA but it increases significantly I(max) in comparison to pH 7.4. At pH 8.4 there was a significant decrease of EC50 for GABA. However, there was also a very strong decrease of I(max), so that the overall effect at 100 microM GABA was a decrease of GABA activated chloride current in the respect of the one activated at neutral pH. These data may indicate that on average brain GABA(A) receptors are positively modulated by extracellular acidosis. The opposite may occur in extracellular alcalosis.

  12. Cranial neural crest contributes to the bony skull vault in adult Xenopus laevis: insights from cell labeling studies.

    Science.gov (United States)

    Gross, Joshua B; Hanken, James

    2005-03-15

    As a step toward resolving the developmental origin of the ossified skull in adult anurans, we performed a series of cell labeling and grafting studies of the cranial neural crest (CNC) in the clawed frog, Xenopus laevis. We employ an indelible, fixative-stable fluorescent dextran as a cell marker to follow migration of the three embryonic streams of cranial neural crest and to directly assess their contributions to the bony skull vault, which forms weeks after hatching. The three streams maintain distinct boundaries in the developing embryo. Their cells proliferate widely through subsequent larval (tadpole) development, albeit in regionally distinct portions of the head. At metamorphosis, each stream contributes to the large frontoparietal bone, which is the primary constituent of the skull vault in adult anurans. The streams give rise to regionally distinct portions of the bone, thereby preserving their earlier relative position anteroposteriorly within the embryonic neural ridge. These data, when combined with comparable experimental observations from other model species, provide insights into the ancestral pattern of cranial development in tetrapod vertebrates as well as the origin of differences reported between birds and mammals. Copyright 2005 Wiley-Liss, Inc.

  13. Voltage-clamp study of the activation currents and fast block to polyspermy in the egg of Xenopus laevis.

    Science.gov (United States)

    Glahn, David; Nuccitelli, Richard

    2003-04-01

    Voltage-clamped mature, jelly-intact Xenopus eggs were used to carefully examine the ionic currents crossing the plasma membrane before, during, and after fertilization. The bulk of the fertilization current was transient, of large amplitude, and reversed at the predicted Cl- reversal potential. However, the large amplitude fertilization current was preceded by a small, step-like increase in holding current. This small increase in holding current is referred to in this paper as Ion to acknowledge its qualitative similarity to the Ion current previously described in the sea urchin. It was observed in both fertilized and artificially activated eggs, and was found to be unaffected by 10 mm tetra-ethyl ammonium (TEA), a concentration found to block K+ currents in Rana pipiens. Current-voltage relationships are presented for the large fertilization potential, and show that the fertilization currents have a marked outward rectification and are voltage sensitive. These properties are in contrast to the total lack of rectification and slight voltage sensitivity seen before or after the fertilization currents. The time required for sperm to fertilize the egg was found to be voltage dependent with a relatively more depolarized voltage requiring a longer time for fertilization to occur. The percentage of eggs blocked with varying potential levels was determined and this information was fitted to a modified Boltzmann equation having a midpoint of -9 mV.

  14. 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.

  15. Integrin alpha5beta1 function is regulated by XGIPC/kermit2 mediated endocytosis during Xenopus laevis gastrulation.

    Science.gov (United States)

    Spicer, Erin; Suckert, Catherine; Al-Attar, Hyder; Marsden, Mungo

    2010-05-17

    During Xenopus gastrulation alpha5beta1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the alpha5 subunit and regulates the activity of alpha5beta1 integrin. The interaction of kermit2 with alpha5beta1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis.

  16. Integrin alpha5beta1 function is regulated by XGIPC/kermit2 mediated endocytosis during Xenopus laevis gastrulation.

    Directory of Open Access Journals (Sweden)

    Erin Spicer

    Full Text Available During Xenopus gastrulation alpha5beta1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the alpha5 subunit and regulates the activity of alpha5beta1 integrin. The interaction of kermit2 with alpha5beta1 is essential for fibronectin (FN matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis.

  17. Integrin α5β1 Function Is Regulated by XGIPC/kermit2 Mediated Endocytosis during Xenopus laevis Gastrulation

    Science.gov (United States)

    Spicer, Erin; Suckert, Catherine; Al-Attar, Hyder; Marsden, Mungo

    2010-01-01

    During Xenopus gastrulation α5β1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the α5 subunit and regulates the activity of α5β1 integrin. The interaction of kermit2 with α5β1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates α5β1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the α5β1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the α5β1 integrin through receptor endocytosis. PMID:20498857

  18. Yolk platelets in Xenopus oocytes maintain an acidic internal pH which may be essential for sodium accumulation

    Science.gov (United States)

    1994-01-01

    Yolk platelets constitute an embryonic endocytic compartment that stores maternally synthesized nutrients. The pH of Xenopus yolk platelets, measured by photometry on whole oocytes which had endocytosed FITC-vitellogenin, was found to be acidic (around pH 5.6). Experiments on digitonin-permeabilized oocytes showed that acidification was due to the activity of an NEM- and bafilomycin A1- sensitive vacuolar proton-ATPase. Proton pumping required chloride, but was not influenced by potassium or sodium. Passive proton leakage was slow, probably due to the buffer capacity of the yolk, and was dependent on the presence of cytoplasmic monovalent cations. In particular, sodium could drive proton efflux through an amiloride- sensitive Na+/H+ exchanger. 8-Bromo-cyclic-AMP was found to increase acidification, suggesting that pH can be regulated by intracellular second messengers. The moderately acidic pH does not promote degradation of the yolk platelets, which in oocytes are stable for weeks, but it is likely to be required to maintain the integrity of these organelles. Furthermore, the pH gradient created by the proton pump, when coupled with the Na+/H+ exchanger, is probably responsible for the accumulation and storage of sodium into the yolk platelets during oogenesis. PMID:8195288

  19. HDAC3 but not HDAC2 mediates visual experience-dependent radial glia proliferation in the developing Xenopus tectum

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    Juanmei Gao

    2016-09-01

    Full Text Available Radial glial cells (RGs are one of the important progenitor cells that can differentiate into neurons or glia to form functional neural circuits in the developing central nervous system (CNS. Histone deacetylases (HDACs has been associated with visual activity dependent changes in BrdU-positive progenitor cells in the developing brain. We previously have shown that HDAC1 is involved in the experience-dependent proliferation of RGs. However, it is less clear whether two other members of class I HDACs, HDAC2 and HDAC3, are involved in the regulation of radial glia proliferation. Here, we reported that HDAC2 and HDAC3 expression were developmentally regulated in tectal cells, especially in the ventricular layer of the BLBP-positive RGs. Pharmacological blockade using an inhibitor of class I HDACs, MS-275 decreased the number of BrdU-positive dividing progenitor cells. Specific knockdown of HDAC3 but not HDAC2 decreased the number of BrdU- and BLBP-labeled cells, suggesting that the proliferation of radial glia was selectively mediated by HDAC3. Visual deprivation induced selective augmentation of histone H4 acetylation at lysine 16 in BLBP-positive cells. Furthermore, the visual deprivation-induced increase in BrdU-positive cells was partially blocked by HDAC3 downregulation but not by HDAC2 knockdown at stage 49 tadpoles. These data revealed a specific role of HDAC3 in experience-dependent radial glia proliferation during the development of Xenopus tectum.

  20. 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

    2015-01-01

    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. PMID:26225989