The Mexican Axolotl in Schools

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Thomas, R. M.

1976-01-01

Suggests and describes laboratory activities in which the Mexican axolotl (Ambystoma mexicanum Shaw) is used, including experiments in embryology and early development, growth and regeneration, neoteny and metamorphosis, genetics and coloration, anatomy and physiology, and behavior. Discusses care and maintenance of animals. (CS)

A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors

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Donald M. Bryant

2017-01-01

Full Text Available Mammals have extremely limited regenerative capabilities; however, axolotls are profoundly regenerative and can replace entire limbs. The mechanisms underlying limb regeneration remain poorly understood, partly because the enormous and incompletely sequenced genomes of axolotls have hindered the study of genes facilitating regeneration. We assembled and annotated a de novo transcriptome using RNA-sequencing profiles for a broad spectrum of tissues that is estimated to have near-complete sequence information for 88% of axolotl genes. We devised expression analyses that identified the axolotl orthologs of cirbp and kazald1 as highly expressed and enriched in blastemas. Using morpholino anti-sense oligonucleotides, we find evidence that cirbp plays a cytoprotective role during limb regeneration whereas manipulation of kazald1 expression disrupts regeneration. Our transcriptome and annotation resources greatly complement previous transcriptomic studies and will be a valuable resource for future research in regenerative biology.

Ambiguities in the relationship between gonadal steroids and reproduction in axolotls (Ambystoma mexicanum).

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Eisthen, Heather L; Krause, Brianne Chung

2012-05-01

Axolotls (Ambystoma mexicanum) are aquatic salamanders that are widely used in research. Axolotls have been bred in laboratories for nearly 150 years, yet little is known about the basic biology of reproduction in these animals. We investigated the effects of changing day length, time of year, and food availability on levels of circulating estradiol and androgens in adult female and male axolotls, respectively. In addition, we examined the effects of these variables on the mass of ovaries, oviducts, and eggs in females and on mass of testes in males relative to each individual's body weight, to calculate a form of gonadosomatic index (GSI). In both sexes, GSI was not correlated with levels of circulating steroids. In female axolotls, estradiol levels were influenced by food availability, changes in day length, and season, even when animals were held at a constant temperature and day length was decorrelated with calendar date. In addition, the mass of ovaries, oviducts, and eggs varied seasonally, peaking in the winter months and declining during the summer months, even though our animals were not breeding and shedding eggs. In males, levels of androgens appeared to vary independently of external conditions, but GSI varied dramatically with changes in day length. These results suggest that reproduction in axolotls may vary seasonally, as it does in many other ambystomid species, although both male and female axolotls are capable of reproducing several times each year. The physiological basis of this ability remains enigmatic, given the indications of seasonality contained in our data. Copyright © 2012 Elsevier Inc. All rights reserved.

Evolutionary genetics of metamorphic failure using wild-caught vs. laboratory axolotls (Ambystoma mexicanum).

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Voss, S R; Shaffer, H B

2000-09-01

In many organisms metamorphosis allows for an ecologically important habitat-shift from water to land. However, in some salamanders an adaptive life cycle mode has evolved that is characterized by metamorphic failure (paedomorphosis); these species remain in the aquatic habitat throughout the life cycle. Perhaps the most famous example of metamorphic failure is the Mexican axolotl (Ambystoma mexicanum), which has become a focal species for developmental biology since it was introduced into laboratory culture in the 1800s. Our previous genetic linkage mapping analysis, using an interspecific crossing design, demonstrated that a major gene effect underlies the expression of metamorphic failure in laboratory stocks of the Mexican axolotl. Here, we repeated this experiment using A. mexicanum that were sampled directly from their natural habitat at Lake Xochimilco, Mexico. We found no significant association between the major gene and metamorphic failure when wild-caught axolotls were used in the experimental design, although there is evidence of a smaller genetic effect. Thus, there appears to be genetic variation among Mexican axolotls (and possibly A. tigrinum tigrinum) at loci that contribute to metamorphic failure. This result suggests a role for more than one mutation and possibly artificial selection in the evolution of the major gene effect in the laboratory Mexican axolotl.

Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

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Flowers, G. Parker; Timberlake, Andrew T.; Mclean, Kaitlin C.; Monaghan, James R.; Crews, Craig M.

2014-01-01

Among tetrapods, only urodele salamanders, such as the axolotl Ambystoma mexicanum, can completely regenerate limbs as adults. The mystery of why salamanders, but not other animals, possess this ability has for generations captivated scientists seeking to induce this phenomenon in other vertebrates. Although many recent advances in molecular biology have allowed limb regeneration and tissue repair in the axolotl to be investigated in increasing detail, the molecular toolkit for the study of this process has been limited. Here, we report that the CRISPR-Cas9 RNA-guided nuclease system can efficiently create mutations at targeted sites within the axolotl genome. We identify individual animals treated with RNA-guided nucleases that have mutation frequencies close to 100% at targeted sites. We employ this technique to completely functionally ablate EGFP expression in transgenic animals and recapitulate developmental phenotypes produced by loss of the conserved gene brachyury. Thus, this advance allows a reverse genetic approach in the axolotl and will undoubtedly provide invaluable insight into the mechanisms of salamanders' unique regenerative ability. PMID:24764077

Courtship Pheromone Use in a Model Urodele, the Mexican Axolotl (Ambystoma mexicanum).

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Maex, Margo; Van Bocxlaer, Ines; Mortier, Anneleen; Proost, Paul; Bossuyt, Franky

2016-02-04

Sex pheromones have been shown to constitute a crucial aspect of salamander reproduction. Until now, courtship pheromones of Salamandridae and Plethodontidae have been intensively studied, but information on chemical communication in other urodelan families is essentially lacking. The axolotl (Ambystoma mexicanum, Ambystomatidae) has a courtship display that suggests a key role for chemical communication in the orchestration of its sexual behavior, but no sex pheromones have yet been characterized from this species. Here we combined whole transcriptome analyses of the male cloaca with proteomic analyses of water in which axolotls were allowed to court to show that male axolotls secrete multiple ca. 20 kDa glycosylated sodefrin precursor-like factor (SPF) proteins during courtship. In combination with phylogenetic analyses, our data show that the male cloaca essentially secretes a courtship-specific clade of SPF proteins that is orthologous to salamandrid courtship pheromones. In addition, we identified an SPF protein for which no orthologs have been described from other salamanders so far. Overall, our study advocates a central role for SPF proteins during the courtship display of axolotls and adds knowledge on pheromone use in a previously unexplored deep evolutionary branch of salamander evolution.

Role of Myofibril-Inducing RNA in cardiac TnT expression in developing Mexican axolotl

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Sferrazza, Gian-Franco; Zhang, Chi; Jia, Pingping; Lemanski, Sharon L.; Athauda, Gagani; Stassi, Alyssa; Halager, Kristine; Maier, Jennifer A.; Rueda-de-Leon, Elena; Gupta, Amit; Dube, Syamalima; Huang, Xupei; Prentice, Howard M.; Dube, Dipak K.; Lemanski, Larry F.

2007-01-01

The Mexican axolotl, Ambystoma mexicanum, has been a useful animal model to study heart development and cardiac myofibrillogenesis. A naturally-occurring recessive mutant, gene “c”, for cardiac non-function in the Mexican axolotl causes a failure of myofibrillogenesis due to a lack of tropomyosin expression in homozygous mutant (c/c) embryonic hearts.. Myofibril-Inducing RNA (MIR) rescues mutant hearts in vitro by promoting tropomyosin expression and myofibril formation thereafter. We have studied the effect of MIR on the expression of various isoforms of cardiac Troponin-T (cTnT), a component of the thin filament that binds with tropomyosin. Four alternatively spliced cTnT isoforms have been characterized from developing axolotl heart. The expression of various cTnT isoforms in normal, mutant, and mutant hearts corrected with MIR, is evaluated by real-time RT-PCR using isoform specific primer pairs; MIR affects the total transcription as well as the splicing of the cTnT in axolotl heart PMID:17408593

Experimentally induced metamorphosis in axolotls reduces regenerative rate and fidelity

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Stier, Adrian C.; Michonneau, François; Smith, Matthew D.; Pasch, Bret; Maden, Malcolm

2014-01-01

Abstract While most tetrapods are unable to regenerate severed body parts, amphibians display a remarkable ability to regenerate an array of structures. Frogs can regenerate appendages as larva, but they lose this ability around metamorphosis. In contrast, salamanders regenerate appendages as larva, juveniles, and adults. However, the extent to which fundamental traits (e.g., metamorphosis, body size, aging, etc.) restrict regenerative ability remains contentious. Here we utilize the ability of normally paedomorphic adult axolotls (Ambystoma mexicanum) to undergo induced metamorphosis by thyroxine exposure to test how metamorphosis and body size affects regeneration in age‐matched paedomorphic and metamorphic individuals. We show that body size does not affect regeneration in adult axolotls, but metamorphosis causes a twofold reduction in regeneration rate, and lead to carpal and digit malformations. Furthermore, we find evidence that metamorphic blastemal cells may take longer to traverse the cell cycle and display a lower proliferative rate. This study identifies the axolotl as a powerful system to study how metamorphosis restricts regeneration independently of developmental stage, body size, and age; and more broadly how metamorphosis affects tissue‐specific changes. PMID:27499857

Esterases activity in the axolotl Ambystoma mexicanum exposed to chlorpyrifos and its implication to motor activity.

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Robles-Mendoza, Cecilia; Zúñiga-Lagunes, Sebastian R; Ponce de León-Hill, Claudia A; Hernández-Soto, Jesús; Vanegas-Pérez, Cecilia

2011-10-01

The axolotl Ambystoma mexicanum is a neotenic salamander considered a good biological model due to its ability to regenerate limbs, tail, brain and heart cells. Nevertheless, severe reduction of A. mexicanum wild populations in the lacustrine area of Xochimilco, the natural habitat of the axolotl, could be related to several environmental pressures as the presence of organophosphate pesticides (OPPs), intensively applied in agricultural activities in Xochimilco. Thus the aim of this study was to evaluate the effect of environmentally realistic chlorpyrifos (CPF) concentrations, a OPP commonly used in this zone, on esterases activity (acetylcholinesterase and carboxylesterase) and bioconcentration of CPF and to relate them with the motor activity of A. mexicanum juveniles. Axolotls were exposed 48 h to 0.05 and 0.1mg CPF/L, and the responses were evaluated at the end of the CPF exposure. Results suggest that CPF is bioconcentrated into axolotls and that the CPF internal concentrations are related with the observed inhibition activity of AChE (>50%) and CbE (≈ 50%). CPF concentration responsible of the inhibition of the 50% of AChE activity (IC50) was estimated in 0.04 mg CPF/L; however IC50 for CbE activity was not possible to calculate since inhibition levels were lower than 50%, results that suggest a higher resistance of CbE enzymatic activity to CPF. However, motor activity was a more sensitive endpoint to CPF poisoning since time that axolotls spent active and walking, frequency and speed of swimming, frequency of prey attack were reduced >90% of control groups. The motor activity alterations in the axolotl could be related with the registered esterases inhibition. Thus important alterations on axolotls were identified even at short time and low concentrations of CPF exposure. Also, it was possible to link biochemical responses as esterases activity with higher levels of biological organization as behavior. This study provides tools for the regulation of the

Propofol (2,6-diisopropylphenol) is an applicable immersion anesthetic in the axolotl with potential uses in hemodynamic and neurophysiological experiments

DEFF Research Database (Denmark)

Thygesen, Mathias; Rasmussen, Mikkel Mylius; Madsen, Jesper Guldsmed

2017-01-01

The Mexican axolotl (Ambystoma mexicanum) is an important model species in regenerative biology. Traditionally, axolotls are anesthetized using benzocaine or MS-222, both of which act to inhibit voltage gated sodium channels thereby preventing action potential propagation. In some neurophysiologi......The Mexican axolotl (Ambystoma mexicanum) is an important model species in regenerative biology. Traditionally, axolotls are anesthetized using benzocaine or MS-222, both of which act to inhibit voltage gated sodium channels thereby preventing action potential propagation. In some...... neurophysiological experiments this is not desirable; therefore we tested propofol as an alternative anesthetic in the axolotl. We evaluated benzocaine, MS-222, and propofol's cardiovascular effects, effects on action potential propagation in the spinal cord, and gross limb regenerative effects. We found...

Evidence for balancing selection at the DAB locus in the axolotl, Ambystoma mexicanum.

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Richman, A D; Herrera, G; Reynoso, V H; Méndez, G; Zambrano, L

2007-12-01

The axolotl (Ambystoma mexicanum) has been characterized as immunodeficient, and the absence of major histocompatibility complex (MHC) class II polymorphism has been cited as a possible explanation. Here we present evidence for considerable allelic polymorphism at the MHC class II DAB locus for a sample of wild-caught axolotls. Evidence that these sequences are the product of balancing selection for disease resistance is discussed.

Injection of an antibody against a p21 c-Ha-ras protein inhibits cleavage in axolotl eggs.

OpenAIRE

Baltus, E; Hanocq-Quertier, J; Hanocq, F; Brachet, J

1988-01-01

The presence of a ras protein was demonstrated in cleaving axolotl eggs by selective immunoprecipitation with a polyclonal antibody against a peptide encoded by the c-Ha-ras oncogene, cellular homolog of the v-Ha-ras oncogene of Harvey rat sarcoma virus. Injection of this antibody into axolotl oocytes subjected to progesterone treatment does not prevent meiotic maturation. Injection of the same antibody into a blastomere of axolotl eggs at the 2- or 4-cell stage causes cleavage arrest in the ...

Data on chemical activation of Wnt/β-catenin during axolotl limb regeneration

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Sabina Wischin

2017-04-01

Full Text Available Limb amputation in axolotls was performed to obtain data demonstrating that a chemical agonist of Wnt (int-related protein/β-catenin signalling can have a role in axolotl limb regeneration (Wischin et al., 2017 [1]. The data revealed that active β-catenin protein was present during limb regeneration in some Leydig cells in the epithelium; after the chemical treatment, it was observed in more Leydig cells. In addition, the chemical agonist of Wnt generated distinct limb malformation.

Gallium nitrate: effects on cartilage during limb regeneration in the axolotl, Ambystoma mexicanum.

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Tassava, Roy A; Mendenhall, Luciara; Apseloff, Glen; Gerber, Nicholas

2002-09-01

Gallium nitrate, a drug shown to have efficacy in Paget's disease of bone, hypercalcemia of malignancy, and a variety of experimental autoimmune diseases, also inhibits the growth of some types of cancer. We examined dose and timing of administration of gallium nitrate on limb regeneration in the Mexican axolotl, Ambystoma mexicanum. Administered by intraperitoneal injection, gallium nitrate inhibited limb regeneration in a dose-dependent manner. Gallium nitrate initially suppressed epithelial wound healing and subsequently distorted both anterior-posterior and proximo-distal chondrogenic patterns. Gallium nitrate given at three days after amputation severely inhibited regeneration at high doses (6.25 mg/axolotl) and altered the normal patterning of the regenerates at low doses (3.75 mg/axolotl). Administration of 6.25 mg of gallium nitrate at four or 14 days prior to amputation also inhibited regeneration. In amputated limbs of gallium-treated axolotls, the chondrocytes were lost from inside the radius/ulna. Limbs that regenerated after gallium treatment was terminated showed blastema formation preferentially over the ulna. New cartilage of the regenerate often attached to the sides of the existing radius/ulna proximally into the stump and less so to the distal cut ends. J. Exp. Zool. 293:384-394, 2002. Copyright 2002 Wiley-Liss, Inc.

Highly restricted diversity of TCR delta chains of the amphibian Mexican axolotl (Ambystoma mexicanum) in peripheral tissues.

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André, Sébastien; Kerfourn, Fabienne; Affaticati, Pierre; Guerci, Aline; Ravassard, Philippe; Fellah, Julien S

2007-06-01

Gammadelta T cells localize at mammalian epithelial surfaces to exert both protective and regulatory roles in response to infections. We have previously characterized the Mexican axolotl (Ambystoma mexicanum) T cell receptor delta (TRD) chain. In this study, TRD repertoires in spleen, liver, intestine and skin from larvae, pre-adult and adult axolotls were examined and compared to the thymic TRD repertoire. A TRDV transcript without N/D diversity, TRDV1S1-TRDJ1, dominates the TRD repertoires until sexual maturation. In adult tissues, this canonical transcript is replaced by another dominant TRDV1S1-TRDJ1 transcript. In the thymus, these two transcripts are detected early in development. Our results suggest that gammadelta T cells that express the canonical TRDV1S1-TRDJ1 transcript emerge from the thymus and colonize the peripheral tissues, where they are selectively expanded by recurrent ligands. This particular situation is probably related to the neotenic state and the slow development of the axolotl. In thymectomized axolotls, TRD repertoires appear different from those of normal axolotls, suggesting that extrathymic gammadelta T cell differentiation could occur. Gene expression analysis showed the importance of the gut in T cell development.

Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration.

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Fei, Ji-Feng; Schuez, Maritta; Knapp, Dunja; Taniguchi, Yuka; Drechsel, David N; Tanaka, Elly M

2017-11-21

Salamanders exhibit extensive regenerative capacities and serve as a unique model in regeneration research. However, due to the lack of targeted gene knockin approaches, it has been difficult to label and manipulate some of the cell populations that are crucial for understanding the mechanisms underlying regeneration. Here we have established highly efficient gene knockin approaches in the axolotl ( Ambystoma mexicanum ) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged Cherry-ER T2 -Cre-ER T2 (∼5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5-15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl limb regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl regeneration.

Transgenesis in axolotl (Ambystoma mexicanum).

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Khattak, Shahryar; Tanaka, Elly M

2015-01-01

Transgenic animals have been indispensable in elucidating and deciphering mechanisms underlying various biological phenomena. In regeneration, transgenic animals expressing fluorescent protein genes have been crucial for identifying the source cells for regeneration and the mechanism of blastema formation. Animals are usually generated by manipulating their genome using various techniques at/in one cell embryo/fertilized egg stage. Here, we describe the generation of germline transgenic axolotls (Ambystoma mexicanum) using the I-SceI meganuclease and Tol2 transposase.

Proteinaceous Pheromone Homologs Identified from the Cloacal Gland Transcriptome of a Male Axolotl, Ambystoma mexicanum.

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Kevin W Hall

Full Text Available Pheromones play an important role in modifying vertebrate behavior, especially during courtship and mating. Courtship behavior in urodele amphibians often includes female exposure to secretions from the cloacal gland, as well as other scent glands. The first vertebrate proteinaceous pheromone discovered, the decapeptide sodefrin, is a female attracting pheromone secreted by the cloacal gland of male Cynops pyrrhogaster. Other proteinaceous pheromones in salamanders have been shown to elicit responses from females towards conspecific males. The presence and levels of expression of proteinaceous pheromones have not been identified in the family Ambystomatidae, which includes several important research models. The objective of this research was therefore to identify putative proteinaceous pheromones from male axolotls, Ambystoma mexicanum, as well as their relative expression levels. The results indicate that axolotls possess two different forms of sodefrin precursor-like factor (alpha and beta, as well as a putative ortholog of plethodontid modulating factor. The beta form of sodefrin precursor-like factor was amongst the most highly expressed transcripts within the cloacal gland. The ortholog of plethodontid modulating factor was expressed at a level equivalent to the beta sodefrin precursor-like factor. The results are from a single male axolotl; therefore, we are unable to assess how representative our results may be. Nevertheless, the presence of these highly expressed proteinaceous pheromones suggests that male axolotls use multiple chemical cues to attract female conspecifics. Behavioral assays would indicate whether the putative protein pheromones elicit courtship activity from female axolotls.

Proteinaceous Pheromone Homologs Identified from the Cloacal Gland Transcriptome of a Male Axolotl, Ambystoma mexicanum.

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Hall, Kevin W; Eisthen, Heather L; Williams, Barry L

2016-01-01

Pheromones play an important role in modifying vertebrate behavior, especially during courtship and mating. Courtship behavior in urodele amphibians often includes female exposure to secretions from the cloacal gland, as well as other scent glands. The first vertebrate proteinaceous pheromone discovered, the decapeptide sodefrin, is a female attracting pheromone secreted by the cloacal gland of male Cynops pyrrhogaster. Other proteinaceous pheromones in salamanders have been shown to elicit responses from females towards conspecific males. The presence and levels of expression of proteinaceous pheromones have not been identified in the family Ambystomatidae, which includes several important research models. The objective of this research was therefore to identify putative proteinaceous pheromones from male axolotls, Ambystoma mexicanum, as well as their relative expression levels. The results indicate that axolotls possess two different forms of sodefrin precursor-like factor (alpha and beta), as well as a putative ortholog of plethodontid modulating factor. The beta form of sodefrin precursor-like factor was amongst the most highly expressed transcripts within the cloacal gland. The ortholog of plethodontid modulating factor was expressed at a level equivalent to the beta sodefrin precursor-like factor. The results are from a single male axolotl; therefore, we are unable to assess how representative our results may be. Nevertheless, the presence of these highly expressed proteinaceous pheromones suggests that male axolotls use multiple chemical cues to attract female conspecifics. Behavioral assays would indicate whether the putative protein pheromones elicit courtship activity from female axolotls.

Diffusion tensor tractography reveals muscle reconnection during axolotl limb regeneration.

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Cheng-Han Wu

Full Text Available Axolotls have amazing ability to regenerate their lost limbs. Our previous works showed that after amputation the remnant muscle ends remained at their original location whilst sending satellite cells into the regenerating parts to develop into early muscle fibers in the late differentiation stage. The parental and the newly formed muscle fibers were not connected until very late stage. The present study used non-invasive diffusion tensor imaging (DTI to monitor weekly axolotl upper arm muscles after amputation of their upper arms. DTI tractography showed that the regenerating muscle fibers became visible at 9-wpa (weeks post amputation, but a gap was observed between the regenerating and parental muscles. The gap was filled at 10-wpa, indicating reconnection of the fibers of both muscles. This was confirmed by histology. The DTI results indicate that 23% of the muscle fibers were reconnected at 10-wpa. In conclusion, DTI can be used to visualize axolotls' skeletal muscles and the results of muscle reconnection were in accordance with our previous findings. This non-invasive technique will allow researchers to identify the timeframe in which muscle fiber reconnection takes place and thus enable the study of the mechanisms underlying this reconnection.

Identification of Conserved and Novel MicroRNAs during Tail Regeneration in the Mexican Axolotl

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Micah D. Gearhart

2015-09-01

Full Text Available The Mexican axolotl salamander (Ambystoma mexicanum is one member of a select group of vertebrate animals that have retained the amazing ability to regenerate multiple body parts. In addition to being an important model system for regeneration, the axolotl has also contributed extensively to studies of basic development. While many genes known to play key roles during development have now been implicated in various forms of regeneration, much of the regulatory apparatus controlling the underlying molecular circuitry remains unknown. In recent years, microRNAs have been identified as key regulators of gene expression during development, in many diseases and also, increasingly, in regeneration. Here, we have used deep sequencing combined with qRT-PCR to undertake a comprehensive identification of microRNAs involved in regulating regeneration in the axolotl. Specifically, among the microRNAs that we have found to be expressed in axolotl tissues, we have identified 4564 microRNA families known to be widely conserved among vertebrates, as well as 59,811 reads of putative novel microRNAs. These findings support the hypothesis that microRNAs play key roles in managing the precise spatial and temporal patterns of gene expression that ensures the correct regeneration of missing tissues.

Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum.

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Woodcock, M Ryan; Vaughn-Wolfe, Jennifer; Elias, Alexandra; Kump, D Kevin; Kendall, Katharina Denise; Timoshevskaya, Nataliya; Timoshevskiy, Vladimir; Perry, Dustin W; Smith, Jeramiah J; Spiewak, Jessica E; Parichy, David M; Voss, S Randal

2017-01-31

The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning-candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a ) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

Evaluation of the anesthetic effects of MS222 in the adult Mexican axolotl (Ambystoma mexicanum

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Zullian C

2016-01-01

Full Text Available Chiara Zullian,1 Aurore Dodelet-Devillers,1 Stéphane Roy,2 Pascal Vachon1 1Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, 2Département de Stomatologie, Faculté de Médecine Dentaire, Montréal, Québec, Canada Abstract: The Mexican axolotl (Ambystoma mexicanum is a unique research model in several fields of medicine, where surgical and invasive procedures may be required. As yet, little is known about the efficacy of MS222 (tricaine methanesulfonate, which is the most commonly used anesthetic agent in amphibians. The main objectives of this study were to evaluate the anesthetic effects and physiological changes in adult axolotls following a 20-minute immersion bath, containing progressive MS222 concentrations starting at 0.1%. Depth of anesthesia and physiological changes were evaluated every 15 minutes post-MS222 exposure with the following parameters: righting behavior, withdrawal reflex, acetic acid test response, heart rate, and blood oxygen saturation, as well as cloacal and body surface temperatures. A 20-minute exposure in a 0.1% MS222 immersion bath (n=6 animals had no anesthetic effects on adult axolotls after 20 minutes of exposure. With a 0.2% MS222 solution, all axolotls (n=9 were deeply anesthetized at 15 minutes, and 80% were still unresponsive at 30 minutes postexposure. Blood oxygen saturation and heart rate were slightly, but significantly, increased when compared with the baseline value and remained stable up to recovery. There was no significant increase in surface and cloaca temperatures, compared with baseline. With the 0.4% MS222 solution, the duration of anesthesia lasted for 90 minutes to at least 120 minutes (n=3 animals and this concentration was deemed too high. In conclusion, a 20-minute immersion bath with 0.2% MS222 may be used for short procedures (15–30 minutes requiring anesthesia of adult axolotls. Keywords: Ambystoma mexicanum

Propofol (2,6-diisopropylphenol) is an applicable immersion anesthetic in the axolotl with potential uses in hemodynamic and neurophysiological experiments

DEFF Research Database (Denmark)

Thygesen, Mathias; Rasmussen, Mikkel Mylius; Madsen, Jesper Guldsmed

2017-01-01

The Mexican axolotl (Ambystoma mexicanum) is an important model species in regenerative biology. Traditionally, axolotls are anesthetized using benzocaine or MS-222, both of which act to inhibit voltage gated sodium channels thereby preventing action potential propagation. In some...... neurophysiological experiments this is not desirable; therefore we tested propofol as an alternative anesthetic in the axolotl. We evaluated benzocaine, MS-222, and propofol's cardiovascular effects, effects on action potential propagation in the spinal cord, and gross limb regenerative effects. We found...... that propofol is applicable as a general anesthetic in the axolotl allowing for neurophysiological experiments and yielding a stable anesthesia with significantly less cardiovascular effect than both benzocaine and MS-222. Additionally, propofol did not affect gross limb regeneration. In conclusion we suggest...

Organophosphorus pesticides effect on early stages of the axolotl Ambystoma mexicanum (Amphibia: Caudata).

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Robles-Mendoza, C; García-Basilio, C; Cram-Heydrich, S; Hernández-Quiroz, M; Vanegas-Pérez, C

2009-02-01

Ambystoma mexicanum is an endemic salamander of Xochimilco, a wetland of the basin of Mexico valley. Nowadays, axolotl populations are decreasing due environmental stressors. Particularly, studies about organophosphorus pesticides (OPPs; i.e. chlorpyrifos and malathion) toxicity are of great importance due to their intensive use in agricultural activities in Xochimilco. Thus, the aim of this study was to evaluate under controlled conditions the toxicity of chlorpyrifos (CPF) and malathion (MLT) on embryos and larvae (stage 44 and 54) of A. mexicanum. Embryos and larvae were exposure 96h from 0.5 to 3mg CPFL(-1) and from 10 to 30mg MLTL(-1) in independent tests. Embryos at the end of this period were maintained 9d without pesticide in order to identify possible recuperation. Differences obtained in mortality, hatching success, development, morphological abnormalities, behaviour and activity, suggest that toxicity of CPF and MLT differs in embryos and larval stages. Embryos were less sensitive to OPPs acute exposure than axolotl larvae; additionally, toxicity of CPF in larval stages was greater than MLT. On the other hand, data obtained in axolotl embryos during the period of recuperation to CPF in particular as delay and inhibition of development, malformations and success of hatching, indicated that these responses turned out more sensitive than mortality. This study allowed to identify the toxicological potential of OPPs on early developmental stages of A. mexicanum and it is a valuable contribution for the future management of the axolotl wild population.

Expression of Msx genes in regenerating and developing limbs of axolotl.

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Koshiba, K; Kuroiwa, A; Yamamoto, H; Tamura, K; Ide, H

1998-12-15

Msx genes, homeobox-containing genes, have been isolated as homologues of the Drosophila msh gene and are thought to play important roles in the development of chick or mouse limb buds. We isolated two Msx genes, Msx1 and Msx2, from regenerating blastemas of axolotl limbs and examined their expression patterns using Northern blot and whole mount in situ hybridization during regeneration and development. Northern blot analysis revealed that the expression level of both Msx genes increased during limb regeneration. The Msx2 expression level increased in the blastema at the early bud stage, and Msx1 expression level increased at the late bud stage. Whole mount in situ hybridization revealed that Msx2 was expressed in the distal mesenchyme and Msx1 in the entire mesenchyme of the blastema at the late bud stage. In the developing limb bud, Msx1 was expressed in the entire mesenchyme, while Msx2 was expressed in the distal and peripheral mesenchyme. The expression patterns of Msx genes in the blastemas and limb buds of the axolotl were different from those reported for chick or mouse limb buds. These expression patterns of axolotl Msx genes are discussed in relation to the blastema or limb bud morphology and their possible roles in limb patterning.

Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum.

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Zhang, C; Pietras, K M; Sferrazza, G F; Jia, P; Athauda, G; Rueda-de-Leon, E; Rveda-de-Leon, E; Maier, J A; Dube, D K; Lemanski, S L; Lemanski, L F

2007-01-01

The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full-length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development. 2006 Wiley-Liss, Inc.

Myofibril-Inducing RNA (MIR is essential for tropomyosin expression and myofibrillogenesis in axolotl hearts

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Lemanski Sharon L

2009-09-01

Full Text Available Abstract The Mexican axolotl, Ambystoma mexicanum, carries the naturally-occurring recessive mutant gene 'c' that results in a failure of homozygous (c/c embryos to form hearts that beat because of an absence of organized myofibrils. Our previous studies have shown that a noncoding RNA, Myofibril-Inducing RNA (MIR, is capable of promoting myofibrillogenesis and heart beating in the mutant (c/c axolotls. The present study demonstrates that the MIR gene is essential for tropomyosin (TM expression in axolotl hearts during development. Gene expression studies show that mRNA expression of various tropomyosin isoforms in untreated mutant hearts and in normal hearts knocked down with double-stranded MIR (dsMIR are similar to untreated normal. However, at the protein level, selected tropomyosin isoforms are significantly reduced in mutant and dsMIR treated normal hearts. These results suggest that MIR is involved in controlling the translation or post-translation of various TM isoforms and subsequently of regulating cardiac contractility.

Regulation of antibody synthesis in the X-irradiated Mexican axolotl

International Nuclear Information System (INIS)

Charlemagne, J.

1981-01-01

The effects of X-irradiation were studied on the Mexican axolotl antibody synthesis. To reduce the anti-horse red blood cell (HRBC) antibody titers, 150 rd and smaller doses are ineffective, 200-450 rd are increasingly effective, and 700 rd are maximally effective (and lethal). A significant enhancement of the anti-HRBC titers was observed in low doses (50-150 rd X-irradiated animals). This enhancement was also observed when a low X-ray dose was applied only on the thymic areas. In whole body, but thymus area-shielded, 100 rd X-irradiated animals, the anti-HRBC titers were similar to those of the nonirradiated, HRBC-immunized control group. To explain these phenomena, it is suggested that a radiosensitive, adult thymectomy-sensitive and hydrocortisone-sensitive suppressor T cell subpopulation regulates the antibody synthesis in the axolotl. (orig.) [de

Regulation of antibody synthesis in the X-irradiated Mexican axolotl

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Charlemagne, J.

1981-09-01

The effects of X-irradiation were studied on the Mexican axolotl antibody synthesis. To reduce the anti-horse red blood cell (HRBC) antibody titers, 150 rd and smaller doses are ineffective, 200-450 rd are increasingly effective, and 700 rd are maximally effective (and lethal). A significant enhancement of the anti-HRBC titers was observed in low doses (50-150 rd X-irradiated animals). This enhancement was also observed when a low X-ray dose was applied only on the thymic areas. In whole body, but thymus area-shielded, 100 rd X-irradiated animals, the anti-HRBC titers were similar to those of the nonirradiated, HRBC-immunized control group. To explain these phenomena, it is suggested that a radiosensitive, adult thymectomy-sensitive and hydrocortisone-sensitive suppressor T cell subpopulation regulates the antibody synthesis in the axolotl.

Terminal-Nerve-Derived Neuropeptide Y Modulates Physiological Responses in the Olfactory Epithelium of Hungry Axolotls (Ambystoma mexicanum)

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Mousley, Angela; Polese, Gianluca; Marks, Nikki J.; Eisthen, Heather L.

2007-01-01

The vertebrate brain actively regulates incoming sensory information, effectively filtering input and focusing attention toward environmental stimuli that are most relevant to the animal's behavioral context or physiological state. Such centrifugal modulation has been shown to play an important role in processing in the retina and cochlea, but has received relatively little attention in olfaction. The terminal nerve, a cranial nerve that extends underneath the lamina propria surrounding the olfactory epithelium, displays anatomical and neurochemical characteristics that suggest that it modulates activity in the olfactory epithelium. Using immunocytochemical techniques, we demonstrate that neuropeptide Y (NPY) is abundantly present in the terminal nerve in the axolotl (Ambystoma mexicanum), an aquatic salamander. Because NPY plays an important role in regulating appetite and hunger in many vertebrates, we investigated the possibility that NPY modulates activity in the olfactory epithelium in relation to the animal's hunger level. We therefore characterized the full length NPY gene from axolotls to enable synthesis of authentic axolotl NPY for use in electrophysiological experiments. We find that axolotl NPY modulates olfactory epithelial responses evoked by L-glutamic acid, a food-related odorant, but only in hungry animals. Similarly, whole-cell patch-clamp recordings demonstrate that bath application of axolotl NPY enhances the magnitude of a tetrodotoxin-sensitive inward current, but only in hungry animals. These results suggest that expression or activity of NPY receptors in the olfactory epithelium may change with hunger level, and that terminal nerve-derived peptides modulate activity in the olfactory epithelium in response to an animal's changing behavioral and physiological circumstances. PMID:16855098

Terminal nerve-derived neuropeptide y modulates physiological responses in the olfactory epithelium of hungry axolotls (Ambystoma mexicanum).

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Mousley, Angela; Polese, Gianluca; Marks, Nikki J; Eisthen, Heather L

2006-07-19

The vertebrate brain actively regulates incoming sensory information, effectively filtering input and focusing attention toward environmental stimuli that are most relevant to the animal's behavioral context or physiological state. Such centrifugal modulation has been shown to play an important role in processing in the retina and cochlea, but has received relatively little attention in olfaction. The terminal nerve, a cranial nerve that extends underneath the lamina propria surrounding the olfactory epithelium, displays anatomical and neurochemical characteristics that suggest that it modulates activity in the olfactory epithelium. Using immunocytochemical techniques, we demonstrate that neuropeptide Y (NPY) is abundantly present in the terminal nerve in the axolotl (Ambystoma mexicanum), an aquatic salamander. Because NPY plays an important role in regulating appetite and hunger in many vertebrates, we investigated the possibility that NPY modulates activity in the olfactory epithelium in relation to the animal's hunger level. We therefore characterized the full-length NPY gene from axolotls to enable synthesis of authentic axolotl NPY for use in electrophysiological experiments. We find that axolotl NPY modulates olfactory epithelial responses evoked by l-glutamic acid, a food-related odorant, but only in hungry animals. Similarly, whole-cell patch-clamp recordings demonstrate that bath application of axolotl NPY enhances the magnitude of a tetrodotoxin-sensitive inward current, but only in hungry animals. These results suggest that expression or activity of NPY receptors in the olfactory epithelium may change with hunger level, and that terminal nerve-derived peptides modulate activity in the olfactory epithelium in response to an animal's changing behavioral and physiological circumstances.

Novel axolotl cardiac function analysis method using magnetic resonance imaging

NARCIS (Netherlands)

Sanches, Pedro Gomes; Op 't Veld, Roel C.; de Graaf, Wolter; Strijkers, Gustav J.; Grüll, Holger

2017-01-01

The salamander axolotl is capable of complete regeneration of amputated heart tissue. However, non-invasive imaging tools for assessing its cardiac function were so far not employed. In this study, cardiac magnetic resonance imaging is introduced as a non-invasive technique to image heart function

Novel axolotl cardiac function analysis method using magnetic resonance imaging

NARCIS (Netherlands)

Sanches, P.G.; Op ‘t Veld, R.C.; de Graaf, W.; Strijkers, G.J.; Grüll, H.

2017-01-01

The salamander axolotl is capable of complete regeneration of amputated heart tissue. However, non-invasive imaging tools for assessing its cardiac function were so far not employed. In this study, cardiac magnetic resonance imaging is introduced as a noninvasive technique to image heart function of

Comparative RNA-seq analysis in the unsequenced axolotl: the oncogene burst highlights early gene expression in the blastema.

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Ron Stewart

Full Text Available The salamander has the remarkable ability to regenerate its limb after amputation. Cells at the site of amputation form a blastema and then proliferate and differentiate to regrow the limb. To better understand this process, we performed deep RNA sequencing of the blastema over a time course in the axolotl, a species whose genome has not been sequenced. Using a novel comparative approach to analyzing RNA-seq data, we characterized the transcriptional dynamics of the regenerating axolotl limb with respect to the human gene set. This approach involved de novo assembly of axolotl transcripts, RNA-seq transcript quantification without a reference genome, and transformation of abundances from axolotl contigs to human genes. We found a prominent burst in oncogene expression during the first day and blastemal/limb bud genes peaking at 7 to 14 days. In addition, we found that limb patterning genes, SALL genes, and genes involved in angiogenesis, wound healing, defense/immunity, and bone development are enriched during blastema formation and development. Finally, we identified a category of genes with no prior literature support for limb regeneration that are candidates for further evaluation based on their expression pattern during the regenerative process.

Neuregulin-1 signaling is essential for nerve-dependent axolotl limb regeneration.

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Farkas, Johanna E; Freitas, Polina D; Bryant, Donald M; Whited, Jessica L; Monaghan, James R

2016-08-01

The Mexican axolotl (Ambystoma mexicanum) is capable of fully regenerating amputated limbs, but denervation of the limb inhibits the formation of the post-injury proliferative mass called the blastema. The molecular basis behind this phenomenon remains poorly understood, but previous studies have suggested that nerves support regeneration via the secretion of essential growth-promoting factors. An essential nerve-derived factor must be found in the blastema, capable of rescuing regeneration in denervated limbs, and its inhibition must prevent regeneration. Here, we show that the neuronally secreted protein Neuregulin-1 (NRG1) fulfills all these criteria in the axolotl. Immunohistochemistry and in situ hybridization of NRG1 and its active receptor ErbB2 revealed that they are expressed in regenerating blastemas but lost upon denervation. NRG1 was localized to the wound epithelium prior to blastema formation and was later strongly expressed in proliferating blastemal cells. Supplementation by implantation of NRG1-soaked beads rescued regeneration to digits in denervated limbs, and pharmacological inhibition of NRG1 signaling reduced cell proliferation, blocked blastema formation and induced aberrant collagen deposition in fully innervated limbs. Taken together, our results show that nerve-dependent NRG1/ErbB2 signaling promotes blastemal proliferation in the regenerating limb and may play an essential role in blastema formation, thus providing insight into the longstanding question of why nerves are required for axolotl limb regeneration. © 2016. Published by The Company of Biologists Ltd.

Ultrastructure of the renal juxtaglomerular complex and peripolar cells in the axolotl (Ambystoma mexicanum) and toad (Bufo marinus).

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Hanner, R H; Ryan, G B

1980-01-01

Renal juxtaglomerular regions were examined in the axolotl (Ambystoma mexicanum and toad (Bufo marinus). Prominent granulated peripolar epithelial cells were found surrounding the origin of the glomerular tuft in the axolotl. These cells resembled the peripolar cells recently discovered in mammalian species. They contained multiple electron-dense cytoplasmic granules, some of which showed a paracrystalline substructure and signs of exocytoxic activity. Such cells were difficult to find and smaller in the toad. In contrast, granulated juxtaglomerular arteriolar myoephithelial cells were much more readily found and larger in the toad than in the axolotl. No consistent differences were noted in juxtaglomerular cells or their granules in response to changes in environmental chloride concentration. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:7410189

Regeneration of limb joints in the axolotl (Ambystoma mexicanum).

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Lee, Jangwoo; Gardiner, David M

2012-01-01

In spite of numerous investigations of regenerating salamander limbs, little attention has been paid to the details of how joints are reformed. An understanding of the process and mechanisms of joint regeneration in this model system for tetrapod limb regeneration would provide insights into developing novel therapies for inducing joint regeneration in humans. To this end, we have used the axolotl (Mexican Salamander) model of limb regeneration to describe the morphology and the expression patterns of marker genes during joint regeneration in response to limb amputation. These data are consistent with the hypothesis that the mechanisms of joint formation whether it be development or regeneration are conserved. We also have determined that defects in the epiphyseal region of both forelimbs and hind limbs in the axolotl are regenerated only when the defect is small. As is the case with defects in the diaphysis, there is a critical size above which the endogenous regenerative response is not sufficient to regenerate the joint. This non-regenerative response in an animal that has the ability to regenerate perfectly provides the opportunity to screen for the signaling pathways to induce regeneration of articular cartilage and joints.

Regeneration of limb joints in the axolotl (Ambystoma mexicanum.

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Jangwoo Lee

Full Text Available In spite of numerous investigations of regenerating salamander limbs, little attention has been paid to the details of how joints are reformed. An understanding of the process and mechanisms of joint regeneration in this model system for tetrapod limb regeneration would provide insights into developing novel therapies for inducing joint regeneration in humans. To this end, we have used the axolotl (Mexican Salamander model of limb regeneration to describe the morphology and the expression patterns of marker genes during joint regeneration in response to limb amputation. These data are consistent with the hypothesis that the mechanisms of joint formation whether it be development or regeneration are conserved. We also have determined that defects in the epiphyseal region of both forelimbs and hind limbs in the axolotl are regenerated only when the defect is small. As is the case with defects in the diaphysis, there is a critical size above which the endogenous regenerative response is not sufficient to regenerate the joint. This non-regenerative response in an animal that has the ability to regenerate perfectly provides the opportunity to screen for the signaling pathways to induce regeneration of articular cartilage and joints.

Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

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Holman, Edna C.; Campbell, Leah J.; Hines, John; Crews, Craig M.

2012-01-01

Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes. PMID:23028429

Microarray analysis of microRNA expression during axolotl limb regeneration.

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Edna C Holman

Full Text Available Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum ("Amex" miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3'UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes.

Network based transcription factor analysis of regenerating axolotl limbs

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Cameron Jo Ann

2011-03-01

Full Text Available Abstract Background Studies on amphibian limb regeneration began in the early 1700's but we still do not completely understand the cellular and molecular events of this unique process. Understanding a complex biological process such as limb regeneration is more complicated than the knowledge of the individual genes or proteins involved. Here we followed a systems biology approach in an effort to construct the networks and pathways of protein interactions involved in formation of the accumulation blastema in regenerating axolotl limbs. Results We used the human orthologs of proteins previously identified by our research team as bait to identify the transcription factor (TF pathways and networks that regulate blastema formation in amputated axolotl limbs. The five most connected factors, c-Myc, SP1, HNF4A, ESR1 and p53 regulate ~50% of the proteins in our data. Among these, c-Myc and SP1 regulate 36.2% of the proteins. c-Myc was the most highly connected TF (71 targets. Network analysis showed that TGF-β1 and fibronectin (FN lead to the activation of these TFs. We found that other TFs known to be involved in epigenetic reprogramming, such as Klf4, Oct4, and Lin28 are also connected to c-Myc and SP1. Conclusions Our study provides a systems biology approach to how different molecular entities inter-connect with each other during the formation of an accumulation blastema in regenerating axolotl limbs. This approach provides an in silico methodology to identify proteins that are not detected by experimental methods such as proteomics but are potentially important to blastema formation. We found that the TFs, c-Myc and SP1 and their target genes could potentially play a central role in limb regeneration. Systems biology has the potential to map out numerous other pathways that are crucial to blastema formation in regeneration-competent limbs, to compare these to the pathways that characterize regeneration-deficient limbs and finally, to identify stem

Neural crest does not contribute to the neck and shoulder in the axolotl (Ambystoma mexicanum).

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Epperlein, Hans-Henning; Khattak, Shahryar; Knapp, Dunja; Tanaka, Elly M; Malashichev, Yegor B

2012-01-01

A major step during the evolution of tetrapods was their transition from water to land. This process involved the reduction or complete loss of the dermal bones that made up connections to the skull and a concomitant enlargement of the endochondral shoulder girdle. In the mouse the latter is derived from three separate embryonic sources: lateral plate mesoderm, somites, and neural crest. The neural crest was suggested to sustain the muscle attachments. How this complex composition of the endochondral shoulder girdle arose during evolution and whether it is shared by all tetrapods is unknown. Salamanders that lack dermal bone within their shoulder girdle were of special interest for a possible contribution of the neural crest to the endochondral elements and muscle attachment sites, and we therefore studied them in this context. We grafted neural crest from GFP+ fluorescent transgenic axolotl (Ambystoma mexicanum) donor embryos into white (d/d) axolotl hosts and followed the presence of neural crest cells within the cartilage of the shoulder girdle and the connective tissue of muscle attachment sites of the neck-shoulder region. Strikingly, neural crest cells did not contribute to any part of the endochondral shoulder girdle or to the connective tissue at muscle attachment sites in axolotl. Our results in axolotl suggest that neural crest does not serve a general function in vertebrate shoulder muscle attachment sites as predicted by the "muscle scaffold theory," and that it is not necessary to maintain connectivity of the endochondral shoulder girdle to the skull. Our data support the possibility that the contribution of the neural crest to the endochondral shoulder girdle, which is observed in the mouse, arose de novo in mammals as a developmental basis for their skeletal synapomorphies. This further supports the hypothesis of an increased neural crest diversification during vertebrate evolution.

Neural crest does not contribute to the neck and shoulder in the axolotl (Ambystoma mexicanum.

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Hans-Henning Epperlein

Full Text Available BACKGROUND: A major step during the evolution of tetrapods was their transition from water to land. This process involved the reduction or complete loss of the dermal bones that made up connections to the skull and a concomitant enlargement of the endochondral shoulder girdle. In the mouse the latter is derived from three separate embryonic sources: lateral plate mesoderm, somites, and neural crest. The neural crest was suggested to sustain the muscle attachments. How this complex composition of the endochondral shoulder girdle arose during evolution and whether it is shared by all tetrapods is unknown. Salamanders that lack dermal bone within their shoulder girdle were of special interest for a possible contribution of the neural crest to the endochondral elements and muscle attachment sites, and we therefore studied them in this context. RESULTS: We grafted neural crest from GFP+ fluorescent transgenic axolotl (Ambystoma mexicanum donor embryos into white (d/d axolotl hosts and followed the presence of neural crest cells within the cartilage of the shoulder girdle and the connective tissue of muscle attachment sites of the neck-shoulder region. Strikingly, neural crest cells did not contribute to any part of the endochondral shoulder girdle or to the connective tissue at muscle attachment sites in axolotl. CONCLUSIONS: Our results in axolotl suggest that neural crest does not serve a general function in vertebrate shoulder muscle attachment sites as predicted by the "muscle scaffold theory," and that it is not necessary to maintain connectivity of the endochondral shoulder girdle to the skull. Our data support the possibility that the contribution of the neural crest to the endochondral shoulder girdle, which is observed in the mouse, arose de novo in mammals as a developmental basis for their skeletal synapomorphies. This further supports the hypothesis of an increased neural crest diversification during vertebrate evolution.

Expression of Msx-2 during development, regeneration, and wound healing in axolotl limbs.

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Carlson, M R; Bryant, S V; Gardiner, D M

1998-12-15

Msx genes are transcription factors that are expressed during embryogenesis of developing appendages in regions of epithelial-mesenchymal interactions. Various lines of evidence indicate that these genes function to maintain embryonic tissues in an undifferentiated, proliferative state. We have identified the axolotl homolog of Msx-2, and investigated its expression during limb development, limb regeneration, and wound healing. As in limb buds of higher vertebrates, axolotl Msx-2 is expressed in the apical epidermis and mesenchyme; however, its expression domain is more extensive, reflecting the broader region of the apical epidermal cap in amphibians. Msx-2 expression is downregulated at late stages of limb development, but is reexpressed within one hour after limb amputation. Msx-2 is also reexpressed during wound healing, and may be essential in the early stages of initiation of the limb regeneration cascade.

Analysis of embryonic development in the unsequenced axolotl: waves of transcriptomic upheaval and stability

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Jiang, Peng; Nelson, Jeffrey D.; Leng, Ning; Collins, Michael; Swanson, Scott; Dewey, Colin N.; Thomson, James A.; Stewart, Ron

2016-01-01

The axolotl (Ambystoma mexicanum) has long been the subject of biological research, primarily owing to its outstanding regenerative capabilities. However, the gene expression programs governing its embryonic development are particularly underexplored, especially when compared to other amphibian model species. Therefore, we performed whole transcriptome polyA+ RNA sequencing experiments on 17 stages of embryonic development. As the axolotl genome is unsequenced and its gene annotation is incomplete, we built de novo transcriptome assemblies for each stage and garnered functional annotation by comparing expressed contigs with known genes in other organisms. In evaluating the number of differentially expressed genes over time, we identify three waves of substantial transcriptome upheaval each followed by a period of relative transcriptome stability. The first wave of upheaval is between the one and two cell stage. We show that the number of differentially expressed genes per unit time is higher between the one and two cell stage than it is across the mid-blastula transition (MBT), the period of zygotic genome activation. We use total RNA sequencing to demonstrate that the vast majority of genes with increasing polyA+ signal between the one and two cell stage result from polyadenylation rather than de novo transcription. The first stable phase begins after the two cell stage and continues until the mid-blastula transition, corresponding with the pre-MBT phase of transcriptional quiescence in amphibian development. Following this is a peak of differential gene expression corresponding with the activation of the zygotic genome and a phase of transcriptomic stability from stages 9 to 11. We observe a third wave of transcriptomic change between stages 11 and 14, followed by a final stable period. The last two stable phases have not been documented in amphibians previously and correspond to times of major morphogenic change in the axolotl embryo: gastrulation and

Linking vertebral number to performance of aquatic escape responses in the axolotl (Ambystoma mexicanum).

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Ackerly, Kerri L; Ward, Andrea B

2015-12-01

Environmental conditions during early development in ectothermic vertebrates can lead to variation in vertebral number among individuals of the same species. It is often seen that individuals of a species raised at cooler temperatures have more vertebrae than individuals raised at warmer temperatures, although the functional consequences of this variation in vertebral number on swimming performance are relatively unclear. To investigate this relationship, we tested how vertebral number in axolotls (Ambystoma mexicanum) affected performance of aquatic escape responses (C-starts). Axolotls were reared at four temperatures (12-24°C) encompassing their natural thermal range and then transitioned to a mean temperature (18°C) three months before C-starts were recorded. Our results showed variation in vertebral number, but that variation was not significantly affected by developmental temperature. C-start performance among axolotls was significantly correlated with caudal vertebral number, and individuals with more caudal vertebrae were able to achieve greater curvature more quickly during their responses than individuals with fewer vertebrae. However, our results show that these individuals did not achieve greater displacements or velocities, and that developmental temperature did not have any effect on C-start performance. We highlight that the most important aspects of escape swim performance (i.e., how far individuals get from a threat and how quickly they move the most important parts of the body away from that threat) are consistent across individuals regardless of developmental temperature and morphological variation. Copyright © 2015 Elsevier GmbH. All rights reserved.

Axolotl cells and tissues enhances cutaneous wound healing in mice

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DEMIRCAN, Turan; KESKIN, Ilknur; GUNAL, Yalcin; ILHAN, Ayse Elif; KOLBASI, Bircan; OZTURK, Gurkan

2017-01-01

Adult mammalian skin wound repair is defective due to loss of the regulation in balancing the complete epithelial regeneration and excessive connective tissue production, and this repair process commonly results in scar tissue formation. However, unlike mammals, adult salamanders repair the wounds by regeneration compared to scarring. To elucidate the healing capability of a salamander, Axolotl, in a different species, here we addressed this question by treating the wounds in mice with Axolot...

Hyperbaric oxygen therapy in a true regenerative environment, the regenerating limb of the axolotl

DEFF Research Database (Denmark)

Hansen, Kasper; Lauridsen, Henrik; Pedersen, Michael

2012-01-01

vertebrates such as the urodele amphibians (salamanders and newts), are excellent animal models for regenerative studies. The iconic Mexican axolotl (Ambystoma mexicanum) is capable of regenerating whole limbs, tail, jaw, and many inner organs, by dedifferentiation of cells to form a blastema (collection...

Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

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Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng

2013-11-01

Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs

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Finnson Kenneth

2010-02-01

Full Text Available Abstract Background Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration. Results The expression of BMP-2 and SOX-9 in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration but did not affect the expression of BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division. Conclusion The expression of BMP-2 and its target SOX-9 was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has

Multiple regeneration from axolotl limb stumps bearing cross-transplanted minced muscle regenerates : brief note

NARCIS (Netherlands)

Carlson, Bruce M.

Flexor and extensor muscles in the upper arms of axolotls were minced and cross-transplanted. The limbs were amputated 5 and 30 days after mincing. In each experiment a high percentage of the regenerates consisted of multiple limbs. This demonstrates that the morphogenetic information which produces

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

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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.

Comparison of tissue processing methods for microvascular visualization in axolotls.

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Montoro, Rodrigo; Dickie, Renee

2017-01-01

The vascular system, the pipeline for oxygen and nutrient delivery to tissues, is essential for vertebrate development, growth, injury repair, and regeneration. With their capacity to regenerate entire appendages throughout their lifespan, axolotls are an unparalleled model for vertebrate regeneration, but they lack many of the molecular tools that facilitate vascular imaging in other animal models. The determination of vascular metrics requires high quality image data for the discrimination of vessels from background tissue. Quantification of the vasculature using perfused, cleared specimens is well-established in mammalian systems, but has not been widely employed in amphibians. The objective of this study was to optimize tissue preparation methods for the visualization of the microvascular network in axolotls, providing a basis for the quantification of regenerative angiogenesis. To accomplish this aim, we performed intracardiac perfusion of pigment-based contrast agents and evaluated aqueous and non-aqueous clearing techniques. The methods were verified by comparing the quality of the vascular images and the observable vascular density across treatment groups. Simple and inexpensive, these tissue processing techniques will be of use in studies assessing vascular growth and remodeling within the context of regeneration. Advantages of this method include: •Higher contrast of the vasculature within the 3D context of the surrounding tissue •Enhanced detection of microvasculature facilitating vascular quantification •Compatibility with other labeling techniques.

Using Ambystoma mexicanum (Mexican axolotl) embryos, chemical genetics, and microarray analysis to identify signaling pathways associated with tissue regeneration.

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Ponomareva, Larissa V; Athippozhy, Antony; Thorson, Jon S; Voss, S Randal

2015-12-01

Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies, and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here, we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration. Copyright © 2015 Elsevier Inc. All rights reserved.

Abdominal Distension Associated with Luminal Fungi in the Intestines of Axolotl Larvae

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Chiara Zullian

2015-01-01

Full Text Available Axolotls show a remarkable regeneration capacity compared with higher vertebrates, regenerating missing appendages such as limbs and tail as well as other body parts (i.e., apex of the heart, forebrain, and jaw after amputations which makes this animal a very interesting research model for tissue regeneration mechanisms. Larvae are individually housed in a 20% Holtfreter’s solution within clear plastic containers. The photoperiod light : darkness cycle is 12 : 12 h. Larvae with a total body length of less than 5 cm are fed once a day with large brine shrimp and blood worm. Albino larvae appeared to have a tendency to exhibit abdominal distention. No clinical signs of illness seemed to be associated with the condition; however, these animals exhibit a relatively slower growth rate. To better characterize this condition, we performed histological sectioning for cross sectional slide preparation on wild type and albino axolotl larvae following euthanasia. The only lesion seen in the albino larvae was a thickened gut wall and the presence of fungi within the intestines. We hypothesize that this may be due to a lower efficacy of the albino larvae’s immune system.

Germline Transgenic Methods for Tracking Cells and Testing Gene Function during Regeneration in the Axolotl

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Khattak, Shahryar; Schuez, Maritta; Richter, Tobias; Knapp, Dunja; Haigo, Saori L.; Sandoval-Guzmán, Tatiana; Hradlikova, Kristyna; Duemmler, Annett; Kerney, Ryan; Tanaka, Elly M.

2013-01-01

The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum (axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly overexpress the cell-cycle inhibitor p16INK4a, which negatively regulates spinal cord regeneration. These tissue-specific germline axolotl lines and tightly inducible Cre drivers and LoxP reporter lines render this classical regeneration model molecularly accessible. PMID:24052945

Live Imaging of Axolotl Digit Regeneration Reveals Spatiotemporal Choreography of Diverse Connective Tissue Progenitor Pools.

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Currie, Joshua D; Kawaguchi, Akane; Traspas, Ricardo Moreno; Schuez, Maritta; Chara, Osvaldo; Tanaka, Elly M

2016-11-21

Connective tissues-skeleton, dermis, pericytes, fascia-are a key cell source for regenerating the patterned skeleton during axolotl appendage regeneration. This complexity has made it difficult to identify the cells that regenerate skeletal tissue. Inability to identify these cells has impeded a mechanistic understanding of blastema formation. By tracing cells during digit tip regeneration using brainbow transgenic axolotls, we show that cells from each connective tissue compartment have distinct spatial and temporal profiles of proliferation, migration, and differentiation. Chondrocytes proliferate but do not migrate into the regenerate. In contrast, pericytes proliferate, then migrate into the blastema and give rise solely to pericytes. Periskeletal cells and fibroblasts contribute the bulk of digit blastema cells and acquire diverse fates according to successive waves of migration that choreograph their proximal-distal and tissue contributions. We further show that platelet-derived growth factor signaling is a potent inducer of fibroblast migration, which is required to form the blastema. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

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Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

2013-08-01

Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian.

On the participation of irradiated tissues in the formation of limb regenerate in axolotls

International Nuclear Information System (INIS)

Tuchkova, S.Ya.

1976-01-01

The aim of the study was to obtain further information on the participation of irradiated tissue cells in formation of regenerated limbs after X-irradiation of axolotls and experimental restoration of the regenerational ability. Cells of irradiated tissues were labeled with H 3 -thymidine; the presence of the label in regenerated tissues would be indicative of participation of irradiated cells in the regeneration process. Irradiation dose was 700 R. 30 axolotls with irradiated limbs were intramuscularly injected with rat muscle homogenate into the right limb once a day beginning from the day of treatment. 15 similarly irradiated animals which did not receive homogenate served as a control. The authors concluded that the presence of highly labeled cells in regenerated tissues was likely to indicate the participation of irradiated tissue cells in regeneration of the limb. However, the quantitative contribution of such cells was impossible to determine since remaining irradiated tissues of the organ contained mostly unlabeled cells. It was also impossible to rule out the possibility of cell migration from non-irradiated tissues [ru

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.

De novo transcriptome sequencing of axolotl blastema for identification of differentially expressed genes during limb regeneration

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2013-01-01

Background Salamanders are unique among vertebrates in their ability to completely regenerate amputated limbs through the mediation of blastema cells located at the stump ends. This regeneration is nerve-dependent because blastema formation and regeneration does not occur after limb denervation. To obtain the genomic information of blastema tissues, de novo transcriptomes from both blastema tissues and denervated stump ends of Ambystoma mexicanum (axolotls) 14 days post-amputation were sequenced and compared using Solexa DNA sequencing. Results The sequencing done for this study produced 40,688,892 reads that were assembled into 307,345 transcribed sequences. The N50 of transcribed sequence length was 562 bases. A similarity search with known proteins identified 39,200 different genes to be expressed during limb regeneration with a cut-off E-value exceeding 10-5. We annotated assembled sequences by using gene descriptions, gene ontology, and clusters of orthologous group terms. Targeted searches using these annotations showed that the majority of the genes were in the categories of essential metabolic pathways, transcription factors and conserved signaling pathways, and novel candidate genes for regenerative processes. We discovered and confirmed numerous sequences of the candidate genes by using quantitative polymerase chain reaction and in situ hybridization. Conclusion The results of this study demonstrate that de novo transcriptome sequencing allows gene expression analysis in a species lacking genome information and provides the most comprehensive mRNA sequence resources for axolotls. The characterization of the axolotl transcriptome can help elucidate the molecular mechanisms underlying blastema formation during limb regeneration. PMID:23815514

Excretory nitrogen metabolism in the juvenile axolotl Ambystoma mexicanum: differences in aquatic and terrestrial environments.

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Loong, Ai M; Chew, Shit F; Ip, Yuen K

2002-01-01

The fully grown but nonmetamorphosed (juvenile) axolotl Ambystoma mexicanum was ureogenic and primarily ureotelic in water. A complete ornithine-urea cycle (OUC) was present in the liver. Aerial exposure impeded urea (but not ammonia) excretion, leading to a decrease in the percentage of nitrogen excreted as urea in the first 24 h. However, urea and not ammonia accumulated in the muscle, liver, and plasma during aerial exposure. By 48 h, the rate of urea excretion recovered fully, probably due to the greater urea concentration gradient in the kidney. It is generally accepted that an increase in carbamoyl phosphate synthetase activity is especially critical in the developmental transition from ammonotelism to ureotelism in the amphibian. Results from this study indicate that such a transition in A. mexicanum would have occurred before migration to land. Aerial exposure for 72 h exhibited no significant effect on carbamoyl phosphate synthetase-I activity or that of other OUC enzymes (with the exception of ornithine transcarbamoylase) from the liver of the juvenile A. mexicanum. This supports our hypothesis that the capacities of OUC enzymes present in the liver of the aquatic juvenile axolotl were adequate to prepare it for its invasion of the terrestrial environment. The high OUC capacity was further supported by the capability of the juvenile A. mexicanum to survive in 10 mM NH(4)Cl without accumulating amino acids in its body. The majority of the accumulating endogenous and exogenous ammonia was detoxified to urea, which led to a greater than twofold increase in urea levels in the muscle, liver, and plasma and a significant increase in urea excretion by hour 96. Hence, it can be concluded that the juvenile axolotl acquired ureotelism while submerged in water, and its hepatic capacity of urea synthesis was more than adequate to handle the toxicity of endogenous ammonia during migration to land.

Cutaneous mastocytomas in the neotenic caudate amphibians Ambystoma mexicanum (axolotl) and Ambystoma tigrinun (tiger salamander)

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Harshbarger, J.C.; Chang, S.C.; DeLanney, L.E.; Rose, F.L.; Green, D.E.

1999-01-01

Spontaneous mastocytomas studied in 18 axolotls (Ambystoma mexicanum) and six tiger salamanders (Ambystoma tigrinum) were gray-white, uni- to multilobular cutaneous protrusions from 2mm to 2cm in diameter. Tumors were moderately cellular unencapsulated masses that usually infiltrated the dermis and hypodermis with the destruction of intervening tissues. Some tumors were invading superficial bundles of the underlying skeletal muscle. Tumors consisted of mitotically active cells derived from a single lineage but showing a range of differentiation. Immature cells had nearly smooth to lightly cleft or folded basophilic nuclei bordered by a band of cytoplasm with few cytoplasmic processes and containing a few small uniform eccentric granules. Mature cells had basophilic nuclei with deep clefts or folds and abundant eosinophilic cytoplasm with multiple long intertwining cytoplasmic extensions packed with metachromatic granules. The axolotls were old individuals from an inbred laboratory colony. The tiger salamanders were wild animals from a single polluted pond. They could have been old and inbred. Both groups were neotenic. These are the first mastocytomas discovered in cold-blooded animals.

Probability of Regenerating a Normal Limb After Bite Injury in the Mexican Axolotl (Ambystoma mexicanum).

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Thompson, Sierra; Muzinic, Laura; Muzinic, Christopher; Niemiller, Matthew L; Voss, S Randal

2014-06-01

Multiple factors are thought to cause limb abnormalities in amphibian populations by altering processes of limb development and regeneration. We examined adult and juvenile axolotls ( Ambystoma mexicanum ) in the Ambystoma Genetic Stock Center (AGSC) for limb and digit abnormalities to investigate the probability of normal regeneration after bite injury. We observed that 80% of larval salamanders show evidence of bite injury at the time of transition from group housing to solitary housing. Among 717 adult axolotls that were surveyed, which included solitary-housed males and group-housed females, approximately half presented abnormalities, including examples of extra or missing digits and limbs, fused digits, and digits growing from atypical anatomical positions. Bite injury likely explains these limb defects, and not abnormal development, because limbs with normal anatomy regenerated after performing rostral amputations. We infer that only 43% of AGSC larvae will present four anatomically normal looking adult limbs after incurring a bite injury. Our results show regeneration of normal limb anatomy to be less than perfect after bite injury.

Effect of thyroid hormone concentration on the transcriptional response underlying induced metamorphosis in the Mexican axolotl (Ambystoma).

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Page, Robert B; Voss, Stephen R; Samuels, Amy K; Smith, Jeramiah J; Putta, Srikrishna; Beachy, Christopher K

2008-02-11

Thyroid hormones (TH) induce gene expression programs that orchestrate amphibian metamorphosis. In contrast to anurans, many salamanders do not undergo metamorphosis in nature. However, they can be induced to undergo metamorphosis via exposure to thyroxine (T4). We induced metamorphosis in juvenile Mexican axolotls (Ambystoma mexicanum) using 5 and 50 nM T4, collected epidermal tissue from the head at four time points (Days 0, 2, 12, 28), and used microarray analysis to quantify mRNA abundances. Individuals reared in the higher T4 concentration initiated morphological and transcriptional changes earlier and completed metamorphosis by Day 28. In contrast, initiation of metamorphosis was delayed in the lower T4 concentration and none of the individuals completed metamorphosis by Day 28. We identified 402 genes that were statistically differentially expressed by > or = two-fold between T4 treatments at one or more non-Day 0 sampling times. To complement this analysis, we used linear and quadratic regression to identify 542 and 709 genes that were differentially expressed by > or = two-fold in the 5 and 50 nM T4 treatments, respectively. We found that T4 concentration affected the timing of gene expression and the shape of temporal gene expression profiles. However, essentially all of the identified genes were similarly affected by 5 and 50 nM T4. We discuss genes and biological processes that appear to be common to salamander and anuran metamorphosis, and also highlight clear transcriptional differences. Our results show that gene expression in axolotls is diverse and precise, and that axolotls provide new insights about amphibian metamorphosis.

MRI tracking of SPIO labelled stem cells in a true regenerative environment, the regenerating limb of the axolotl

DEFF Research Database (Denmark)

Lauridsen, Henrik; Foldager, Casper Bindzus; Hagensen, Mette

are generally restricted by their limited regenerative potential. Conversely, excellent animal models for regenerative studies exist in lower vertebrates such as the urodele amphibians (salamanders and newts), exemplified in the iconic Mexican axolotl (Ambystoma mexicanum) capable of regenerating whole limbs...

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum.

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Shishikura, Fumio; Takeuchi, Hiro-aki; Nagai, Takatoshi

2005-11-01

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.

Effect of thyroid hormone concentration on the transcriptional response underlying induced metamorphosis in the Mexican axolotl (Ambystoma

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Samuels Amy K

2008-02-01

Full Text Available Abstract Background Thyroid hormones (TH induce gene expression programs that orchestrate amphibian metamorphosis. In contrast to anurans, many salamanders do not undergo metamorphosis in nature. However, they can be induced to undergo metamorphosis via exposure to thyroxine (T4. We induced metamorphosis in juvenile Mexican axolotls (Ambystoma mexicanum using 5 and 50 nM T4, collected epidermal tissue from the head at four time points (Days 0, 2, 12, 28, and used microarray analysis to quantify mRNA abundances. Results Individuals reared in the higher T4 concentration initiated morphological and transcriptional changes earlier and completed metamorphosis by Day 28. In contrast, initiation of metamorphosis was delayed in the lower T4 concentration and none of the individuals completed metamorphosis by Day 28. We identified 402 genes that were statistically differentially expressed by ≥ two-fold between T4 treatments at one or more non-Day 0 sampling times. To complement this analysis, we used linear and quadratic regression to identify 542 and 709 genes that were differentially expressed by ≥ two-fold in the 5 and 50 nM T4 treatments, respectively. Conclusion We found that T4 concentration affected the timing of gene expression and the shape of temporal gene expression profiles. However, essentially all of the identified genes were similarly affected by 5 and 50 nM T4. We discuss genes and biological processes that appear to be common to salamander and anuran metamorphosis, and also highlight clear transcriptional differences. Our results show that gene expression in axolotls is diverse and precise, and that axolotls provide new insights about amphibian metamorphosis.

Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl

OpenAIRE

Zhu, Wei; Pao, Gerald M; Satoh, Akira; Cummings, Gillian; Monaghan, James R; Harkins, Timothy T; Bryant, Susan V; Voss, S Randal; Gardiner, David M; Hunter, Tony

2012-01-01

The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration-wound healing, cellular dedifferentiation and blastemal formation-will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of ce...

Axolotl/Bichos Raros Crónica

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Susana Chávez-Silverman

2012-11-01

Full Text Available In this crónica, I pay homage (and talk back to! one of my favourite authors, Julio Cortázar, who I had the great privilege and pleasure of befriending in 1980, when he was a visiting professor at UC-Berkeley. I have been obsessed with time-travel, doubling, and interstitiality since I was very young; even the most casual Cortázar reader (if such a thing is possible will immediately recognise these as recurrent themes in his work. Here, faced with several actual axolotl in a Buenos Aires aquarium, I explore and comment on Cortázar’s strangely mesmerising meditation on identity and transformation. My personal connection is (as in much of my writing concerned with aspects of gender and sexuality suppressed or (more likely ignored in Cortázar’s version. I identify, too, with a poignant in-betweenness and ambiguity I read in the figure of the axolotl—and in the work of Cortázar and Alejandra Pizarnik.

The effects of rotation and positional change of stump tissues upon morphogenesis of the regenerating axolotl limb

NARCIS (Netherlands)

Carlson, Bruce M.

1972-01-01

Rotation of a skin cuff 180° around the proximodistal axis of the upper arm in the axolotl results in the formation of multiple regenerates in about 80° of cases after amputation of the limb through the rotated skin. Rotation of the dermis or the flexor and extensor muscles folowed by amputation

Induction of Metamorphosis Causes Differences in Sex-Specific Allocation Patterns in Axolotls (Ambystoma mexicanum) that Have Different Growth Histories.

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Clarkson, Pamela M; Beachy, Christopher K

2015-12-01

We tested the hypothesis that salamanders growing at different rates would have allocation patterns that differ among male and female metamorphic and larval salamanders. We raised individual axolotls, Ambystoma mexicanum , on four food regimes: constant high growth (throughout the experiment), constant low growth (restricted throughout the experiment), high growth switched to low growth (ad libitum switched after 140 d to restricted), and low growth switched to high growth (restricted switched after 140 d to ad libitum). Because axolotls are obligate paedomorphs, we exposed half of the salamanders to thyroid hormone to induce metamorphosis. We assayed growth and dissected and weighed gonads and fat bodies. Salamanders that were switched from restricted to ad libitum food regime delayed metamorphosis. In all treatment groups, females had larger gonads than males and males had larger fat bodies than females. The association between storage and reproduction differed between larvae and metamorphs and depended on sex.

Functional characterization of the vertebrate primary ureter: Structure and ion transport mechanisms of the pronephric duct in axolotl larvae (Amphibia)

DEFF Research Database (Denmark)

Haugan, Birgitte M; Halberg, Kenneth Agerlin; Jespersen, Åse

2010-01-01

whether the duct is involved in urine modification using larvae of the freshwater amphibian Ambystoma mexicanum (axolotl) as model. Results We investigated structural as well as physiological properties of the pronephric duct. The key elements of our methodology were: using histology, light...

Pathological changes of Golgi complex in hemocytoblasts of spleen of young axolotls after x-irradiation

Energy Technology Data Exchange (ETDEWEB)

Turska, R.

1975-01-01

The data available up to now concerning the structure of Golgi complex in different types of normal and pathologically changed cells are very divergent. Results are reported from detailed studies on changes occurring within the Golgi complex in the spleen of three-month old axolotls after x-ray irradiation with a single dose of 1,200 R and killed by decapitation 15, 30, 60 minutes and 3, 6, and 12 hours after irradiation.

Resegmentation in the Mexican axolotl, Ambystoma mexicanum.

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Piekarski, Nadine; Olsson, Lennart

2014-02-01

The segmental series of somites in the vertebrate embryo gives rise to the axial skeleton. In amniote models, single vertebrae are derived from the sclerotome of two adjacent somites. This process, known as resegmentation, is well-studied using the quail-chick chimeric system, but the presumed generality of resegmentation across vertebrates remains poorly evaluated. Resegmentation has been questioned in anamniotes, given that the sclerotome is much smaller and lacks obvious differentiation between cranial and caudal portions. Here, we provide the first experimental evidence that resegmentation does occur in a species of amphibian. Fate mapping of individual somites in the Mexican axolotl (Ambystoma mexicanum) revealed that individual vertebrae receive cells from two adjacent somites as in the chicken. These findings suggest that large size and segmentation of the sclerotome into distinct cranial and caudal portions are not requirements for resegmentation. Our results, in addition to those for zebrafish, indicate that resegmentation is a general process in building the vertebral column in vertebrates, although it may be achieved in different ways in different groups. Copyright © 2013 Wiley Periodicals, Inc.

Regulation of Regenerative Responses by Factors in the Extracellular Matrix during Axolotl (Ambystoma mexicanum) Limb Regeneration

OpenAIRE

Phan, Anne Quy

2014-01-01

Salamanders are unique among adult vertebrates in their ability to regenerate complex body structures after traumatic injury. Axolotl limb regeneration is a stepwise sequence of three requisite processes: (1) scarless wound healing to generate a regenerative wound epithelium, (2) blastema formation by migration, proliferation and dedifferentiation to create a mass of multipotent regeneration-competent progenitor cells, and (3) induction of pattern formation by interaction of cells with opposi...

Ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum with reference to ionic transport.

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Jarial, M S; Wilkins, J H

2003-10-01

The ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum, has been examined using conventional transmission electron microscopy to elucidate its role in ionic transport. Four cell types are identified in the gill filament and primary gill bar epithelium. These are granular, ciliated, Leydig and basal cells. A fifth cell type, the flat mitochondria-rich cell is only found in the gill bar epithelium. The predominant granular cells display microvilli at their surface and their cytoplasm contains abundant mitochondria, rough endoplasmic reticulum, Golgi complexes, vesicles and PAS+ secretory granules that are extruded at the surface, which along with secretions from the Leydig cells form a mucous coat. The granular cells are joined apically by junctional complexes consisting of zonulae occludens, zonulae adherens and desmosomes. The lateral membranes of granular cells enclose large intercellular spaces that are closed at the apical ends but remain open at the basal ends adjoining capillaries. In AgNO3-treated axolotl, the gills become darkly stained, the silver grains penetrate apical membranes and appear in the cytoplasm, accumulating near the lateral membranes and also enter the intercellular spaces. These findings are consistent with the dual role of the gill epithelium in mucus production and active ionic transport.

Early regulation of axolotl limb regeneration.

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Makanae, Aki; Satoh, Akira

2012-10-01

Amphibian limb regeneration has been studied for a long time. In amphibian limb regeneration, an undifferentiated blastema is formed around the region damaged by amputation. The induction process of blastema formation has remained largely unknown because it is difficult to study the induction of limb regeneration. The recently developed accessory limb model (ALM) allows the investigation of limb induction and reveals early events of amphibian limb regeneration. The interaction between nerves and wound epidermis/epithelium is an important aspect of limb regeneration. During early limb regeneration, neurotrophic factors act on wound epithelium, leading to development of a functional epidermis/epithelium called the apical epithelial cap (AEC). AEC and nerves create a specific environment that inhibits wound healing and induces regeneration through blastema formation. It is suggested that FGF-signaling and MMP activities participate in creating a regenerative environment. To understand why urodele amphibians can create such a regenerative environment and humans cannot, it is necessary to identify the similarities and differences between regenerative and nonregenerative animals. Here we focus on ALM to consider limb regeneration from a new perspective and we also reported that focal adhesion kinase (FAK)-Src signaling controlled fibroblasts migration in axolotl limb regeneration. Copyright © 2012 Wiley Periodicals, Inc.

Visualisation of axolotl blastema cells and pig endothelial progenitor cells using very small super paramagnetic iron oxide particles in MRI: A technique with applications for non invasive visualisation of regenerative processes

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Lauridsen, Henrik; Kjær, N.B.; Bek, Maria

oxide particles (VSOP) in animal cells enable non invasive cell tracking using magnetic resonance imaging (MRI) and can prove useful, when visualising regenerative processes. This study examines the possibility of labelling limited numbers of axolotl blastema cells (aBC) and pig endothelial progenitor...... implanted in live axolotl tail and dead porcine heart, respectively. Cellular iron uptake was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Results: T2*-weighted 2D gradient-echo sequences on samples of 10˄5 cells yielded at significant linear correlations between...

Low submetamorphic doses of dexamethasone and thyroxine induce complete metamorphosis in the axolotl (Ambystoma mexicanum) when injected together.

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Kühn, Eduard R; De Groef, Bert; Grommen, Sylvia V H; Van der Geyten, Serge; Darras, Veerle M

2004-06-01

Entanglement of functions between the adrenal (or interrenal) and thyroid axis has been well described for all vertebrates and can be tracked down up to the level of gene expression. Both thyroid hormones and corticosteroids may induce morphological changes leading to metamorphosis climax in the neotenic Mexican axolotl (Ambystoma mexicanum). In a first series of experiments, metamorphosis was induced with an injection of 25 microg T(4) on three alternate days as judged by a decrease in body weight and tail height together with complete gill resorption. This injection also resulted in elevated plasma concentrations of T(3) and corticosterone. Previous results have indicated that the same dose of dexamethasone (DEX) is ineffective in this regard (Gen. Comp. Endocrinol. 127 (2002) 157). In a second series of experiments low doses of T(4) (0.5 microg) or DEX (5 microg) were ineffective to induce morphological changes. However, when these submetamorphic doses were injected together, morphological changes were observed within one week leading to complete metamorphosis. It is concluded that thyroid hormones combined with corticosteroids are essential for metamorphosis in the axolotl and that only high doses of either thyroid hormone or corticosteroid can induce morphological changes when injected separately.

[Specific growth rate and the rate of energy metabolism in the ontogenesis of axolotl, Ambystoma mexicanum (Amphibia: Ambystomatidae)].

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Vladimirova, I G; Kleĭmenov, S Iu; Alekseeva, T A; Radzinskaia, L I

2003-01-01

Concordant changes in the rate of energy metabolism and specific growth rate of axolotls have been revealed. Several periods of ontogeny are distinguished, which differ in the ratio of energy metabolism to body weight and, therefore, are described by different allometric equations. It is suggested that the specific growth rate of an animal determines the type of dependence of energy metabolism on body weight.

Absence of mutation at the 5'-upstream promoter region of the TPM4 gene from cardiac mutant axolotl (Ambystoma mexicanum).

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Denz, Christopher R; Zhang, Chi; Jia, Pingping; Du, Jianfeng; Huang, Xupei; Dube, Syamalima; Thomas, Anish; Poiesz, Bernard J; Dube, Dipak K

2011-09-01

Tropomyosins are a family of actin-binding proteins that show cell-specific diversity by a combination of multiple genes and alternative RNA splicing. Of the 4 different tropomyosin genes, TPM4 plays a pivotal role in myofibrillogenesis as well as cardiac contractility in amphibians. In this study, we amplified and sequenced the upstream regulatory region of the TPM4 gene from both normal and mutant axolotl hearts. To identify the cis-elements that are essential for the expression of the TPM4, we created various deletion mutants of the TPM4 promoter DNA, inserted the deleted segments into PGL3 vector, and performed promoter-reporter assay using luciferase as the reporter gene. Comparison of sequences of the promoter region of the TPM4 gene from normal and mutant axolotl revealed no mutations in the promoter sequence of the mutant TPM4 gene. CArG box elements that are generally involved in controlling the expression of several other muscle-specific gene promoters were not found in the upstream regulatory region of the TPM4 gene. In deletion experiments, loss of activity of the reporter gene was noted upon deletion which was then restored upon further deletion suggesting the presence of both positive and negative cis-elements in the upstream regulatory region of the TPM4 gene. We believe that this is the first axolotl promoter that has ever been cloned and studied with clear evidence that it functions in mammalian cell lines. Although striated muscle-specific cis-acting elements are absent from the promoter region of TPM4 gene, our results suggest the presence of positive and negative cis-elements in the promoter region, which in conjunction with positive and negative trans-elements may be involved in regulating the expression of TPM4 gene in a tissue-specific manner.

Pathological features of olfactory neuroblastoma in an axolotl (Ambystoma mexicanum).

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Shioda, Chieko; Uchida, Kazuyuki; Nakayama, Hiroyuki

2011-08-01

A one-year-old, female Mexican axolotl (Ambystoma mexicanum) had a rough-surfaced, polypoid, pink tumor mass of approximately 10 mm in diameter in the oral cavity. Histologically, the tumor extended from the ethmoturbinate region and into the oral cavity and had replaced some of the maxillary bone tissue. The tumor mass was composed of a lobular architecture of small round-shaped tumor cells with occasional Flexner-Wintersteiner-like rosette formation. There were no metastatic lesions in the other organs. Immunohistochemically, the tumor cells were partly positive for several neural markers (class III beta-tubulin, S-100 protein, and doublecortin) and intensely positive for an epithelial marker (cytokeratin AE1/AE3). These results suggest that the present tumor originated from neuroectodermal tissue. Considering the location and histological and immunohistochemical features of the tumor, a diagnosis of olfactory neuroblastoma was made.

Lineage tracing of genome-edited alleles reveals high fidelity axolotl limb regeneration.

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Flowers, Grant Parker; Sanor, Lucas D; Crews, Craig M

2017-09-16

Salamanders are unparalleled among tetrapods in their ability to regenerate many structures, including entire limbs, and the study of this ability may provide insights into human regenerative therapies. The complex structure of the limb poses challenges to the investigation of the cellular and molecular basis of its regeneration. Using CRISPR/Cas, we genetically labelled unique cell lineages within the developing axolotl embryo and tracked the frequency of each lineage within amputated and fully regenerated limbs. This allowed us, for the first time, to assess the contributions of multiple low frequency cell lineages to the regenerating limb at once. Our comparisons reveal that regenerated limbs are high fidelity replicas of the originals even after repeated amputations.

The axolotl limb blastema: cellular and molecular mechanisms driving blastema formation and limb regeneration in tetrapods

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McCusker, Catherine; Bryant, Susan V.

2015-01-01

Abstract The axolotl is one of the few tetrapods that are capable of regenerating complicated biological structures, such as complete limbs, throughout adulthood. Upon injury the axolotl generates a population of regeneration‐competent limb progenitor cells known as the blastema, which will grow, establish pattern, and differentiate into the missing limb structures. In this review we focus on the crucial early events that occur during wound healing, the neural−epithelial interactions that drive the formation of the early blastema, and how these mechanisms differ from those of other species that have restricted regenerative potential, such as humans. We also discuss how the presence of cells from the different axes of the limb is required for the continued growth and establishment of pattern in the blastema as described in the polar coordinate model, and how this positional information is reprogrammed in blastema cells during regeneration. Multiple cell types from the mature limb stump contribute to the blastema at different stages of regeneration, and we discuss the contribution of these types to the regenerate with reference to whether they are “pattern‐forming” or “pattern‐following” cells. Lastly, we explain how an engineering approach will help resolve unanswered questions in limb regeneration, with the goal of translating these concepts to developing better human regenerative therapies. PMID:27499868

Metamorfosis: La animalidad y el mito en La Metamorfosis de Kafka y Axolotl de Cortázar

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Catalina Villalobos Díaz

2017-07-01

Full Text Available El presente artículo propone una lectura de la animalidad, principalmente de la metamorfosis, por medio de dos relatos de dos grandes escritores de la literatura: Axolotl, de Julio Cortázar, y La Metamorfosis, de Franz Kafka. A través de estos textos, se plantea un análisis con respecto a lo que representa una transformación y el mito tras la metamorfosis como tal.

Dual embryonic origin and patterning of the pharyngeal skeleton in the axolotl (Ambystoma mexicanum).

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Sefton, Elizabeth M; Piekarski, Nadine; Hanken, James

2015-01-01

The impressive morphological diversification of vertebrates was achieved in part by innovation and modification of the pharyngeal skeleton. Extensive fate mapping in amniote models has revealed a primarily cranial neural crest derivation of the pharyngeal skeleton. Although comparable fate maps of amphibians produced over several decades have failed to document a neural crest derivation of ventromedial elements in these vertebrates, a recent report provides evidence of a mesodermal origin of one of these elements, basibranchial 2, in the axolotl. We used a transgenic labeling protocol and grafts of labeled cells between GFP+ and white embryos to derive a fate map that describes contributions of both cranial neural crest and mesoderm to the axolotl pharyngeal skeleton, and we conducted additional experiments that probe the mechanisms that underlie mesodermal patterning. Our fate map confirms a dual embryonic origin of the pharyngeal skeleton in urodeles, including derivation of basibranchial 2 from mesoderm closely associated with the second heart field. Additionally, heterotopic transplantation experiments reveal lineage restriction of mesodermal cells that contribute to pharyngeal cartilage. The mesoderm-derived component of the pharyngeal skeleton appears to be particularly sensitive to retinoic acid (RA): administration of exogenous RA leads to loss of the second basibranchial, but not the first. Neural crest was undoubtedly critical in the evolution of the vertebrate pharyngeal skeleton, but mesoderm may have played a central role in forming ventromedial elements, in particular. When and how many times during vertebrate phylogeny a mesodermal contribution to the pharyngeal skeleton evolved remain to be resolved. © 2015 Wiley Periodicals, Inc.

Collection of gametes from live axolotl, Ambystoma mexicanum, and standardization of in vitro fertilization.

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Mansour, N; Lahnsteiner, F; Patzner, R A

2011-01-15

This study established the first protocol for collection of gametes from live axolotl, Ambystoma mexicanum, by gentle abdominal massage and in vitro fertilization. To stimulate spermiation and ovulation, human chorionic gonadotrophin (hCG) and Ovopel pellets, which are commercially used to stimulate spawning in fish, were tested. The hCG was more effective than Ovopel pellets and yielded a higher semen volume in the injected males and a shorter response time in the females. Collected semen by this method was already motile and fertile. Fertile eggs could be collected in 3-4 successive collection times after the female has started the typical spawning behaviour. The fertilization condition that yielded the highest hatching rate was mixing semen with eggs before the addition of a fertilization saline solution (20 mmol/l NaCl, 1 mmol/l KCl, 1 mmol/l Mg(2)SO(4), 1 mmol Ca(2)Cl, 3 mmol NaHCO(3), 10 mmol/l Tris, pH 8.5 - Osmolality = 65 mosmol/kg). When the pH of the fertilization solution was increased to ≥ 10, the hatching rate was significantly increased. The use of fertilization solutions with osmolalities of ≥ 150 and ≥ 182 were accompanied with a significant decrease in hatching rates and the appearance of deformed larvae, respectively. In conclusion, a reliable protocol for gamete collection from live axolotl is established as a laboratory model of in vitro fertilization for urodele amphibians. This protocol may be transferable to endangered urodeles. Copyright © 2011 Elsevier Inc. All rights reserved.

Proliferation zones in the axolotl brain and regeneration of the telencephalon

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Maden Malcolm

2013-01-01

Full Text Available Abstract Background Although the brains of lower vertebrates are known to exhibit somewhat limited regeneration after incisional or stab wounds, the Urodele brain exhibits extensive regeneration after massive tissue removal. Discovering whether and how neural progenitor cells that reside in the ventricular zones of Urodeles proliferate to mediate tissue repair in response to injury may produce novel leads for regenerative strategies. Here we show that endogenous neural progenitor cells resident to the ventricular zone of Urodeles spontaneously proliferate, producing progeny that migrate throughout the telencephalon before terminally differentiating into neurons. These progenitor cells appear to be responsible for telencephalon regeneration after tissue removal and their activity may be up-regulated by injury through an olfactory cue. Results There is extensive proliferation of endogenous neural progenitor cells throughout the ventricular zone of the adult axolotl brain. The highest levels are observed in the telencephalon, especially the dorsolateral aspect, and cerebellum. Lower levels are observed in the mesencephalon and rhombencephalon. New cells produced in the ventricular zone migrate laterally, dorsally and ventrally into the surrounding neuronal layer. After migrating from the ventricular zone, the new cells primarily express markers of neuronal differentiative fates. Large-scale telencephalic tissue removal stimulates progenitor cell proliferation in the ventricular zone of the damaged region, followed by proliferation in the tissue that surrounds the healing edges of the wound until the telencephalon has completed regeneration. The proliferative stimulus appears to reside in the olfactory system, because telencephalic regeneration does not occur in the brains of olfactory bulbectomized animals in which the damaged neural tissue simply heals over. Conclusion There is a continual generation of neuronal cells from neural progenitor cells

Role of thyroid hormones in the normal and glucocorticosteroid hormone-induced evolution of carbamoyl-phosphate synthase (ammonia) activity in axolotl liver

NARCIS (Netherlands)

Lamers, W. H.; Vink, C.; Charles, R.

1978-01-01

1. In axolotl liver, the activity of carbamoyl-phosphate synthase (ammonia), expressed per mg liver protein, decreases to a minimum at 5 months of age, then increases to a maximum at 8 months of age which is followed by a decrease again. The initial decrease between 3 and 5 months of age appears to

The Axolotl Fibula as a Model for the Induction of Regeneration across Large Segment Defects in Long Bones of the Extremities

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Chen, Xiaoping; Song, Fengyu; Jhamb, Deepali; Li, Jiliang; Bottino, Marco C.; Palakal, Mathew J.; Stocum, David L.

2015-01-01

We tested the ability of the axolotl (Ambystoma mexicanum) fibula to regenerate across segment defects of different size in the absence of intervention or after implant of a unique 8-braid pig small intestine submucosa (SIS) scaffold, with or without incorporated growth factor combinations or tissue protein extract. Fractures and defects of 10% and 20% of the total limb length regenerated well without any intervention, but 40% and 50% defects failed to regenerate after either simple removal of bone or implanting SIS scaffold alone. By contrast, scaffold soaked in the growth factor combination BMP-4/HGF or in protein extract of intact limb tissue promoted partial or extensive induction of cartilage and bone across 50% segment defects in 30%-33% of cases. These results show that BMP-4/HGF and intact tissue protein extract can promote the events required to induce cartilage and bone formation across a segment defect larger than critical size and that the long bones of axolotl limbs are an inexpensive model to screen soluble factors and natural and synthetic scaffolds for their efficacy in stimulating this process. PMID:26098852

The Axolotl Fibula as a Model for the Induction of Regeneration across Large Segment Defects in Long Bones of the Extremities.

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Xiaoping Chen

Full Text Available We tested the ability of the axolotl (Ambystoma mexicanum fibula to regenerate across segment defects of different size in the absence of intervention or after implant of a unique 8-braid pig small intestine submucosa (SIS scaffold, with or without incorporated growth factor combinations or tissue protein extract. Fractures and defects of 10% and 20% of the total limb length regenerated well without any intervention, but 40% and 50% defects failed to regenerate after either simple removal of bone or implanting SIS scaffold alone. By contrast, scaffold soaked in the growth factor combination BMP-4/HGF or in protein extract of intact limb tissue promoted partial or extensive induction of cartilage and bone across 50% segment defects in 30%-33% of cases. These results show that BMP-4/HGF and intact tissue protein extract can promote the events required to induce cartilage and bone formation across a segment defect larger than critical size and that the long bones of axolotl limbs are an inexpensive model to screen soluble factors and natural and synthetic scaffolds for their efficacy in stimulating this process.

CRISPR-Mediated Genomic Deletion of Sox2 in the Axolotl Shows a Requirement in Spinal Cord Neural Stem Cell Amplification during Tail Regeneration

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Ji-Feng Fei

2014-09-01

Full Text Available The salamander is the only tetrapod that functionally regenerates all cell types of the limb and spinal cord (SC and thus represents an important regeneration model, but the lack of gene-knockout technology has limited molecular analysis. We compared transcriptional activator-like effector nucleases (TALENs and clustered regularly interspaced short palindromic repeats (CRISPRs in the knockout of three loci in the axolotl and find that CRISPRs show highly penetrant knockout with less toxic effects compared to TALENs. Deletion of Sox2 in up to 100% of cells yielded viable F0 larvae with normal SC organization and ependymoglial cell marker expression such as GFAP and ZO-1. However, upon tail amputation, neural stem cell proliferation was inhibited, resulting in spinal-cord-specific regeneration failure. In contrast, the mesodermal blastema formed normally. Sox3 expression during development, but not regeneration, most likely allowed embryonic survival and the regeneration-specific phenotype. This analysis represents the first tissue-specific regeneration phenotype from the genomic deletion of a gene in the axolotl.

Fine structure of the epidermal Leydig cells in the axolotl Ambystoma mexicanum in relation to their function.

Science.gov (United States)

Jarial, M S

1989-01-01

The fine structure of the Leydig cells in the epidermis of the strictly aquatic adult axolotl Ambystoma mexicanum resembles that of similar cells in larval salamanders. The major finding of this study is that the mucous secretion of the Leydig cells is released into the intercellular spaces from which it is discharged through pores onto the surface of the epidermis where it forms a mucous layer to protect the skin. Images Figs. 1-2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Figs. 11-13 PMID:2630544

Comparative pelvic development of the axolotl (Ambystoma mexicanum) and the Australian lungfish (Neoceratodus forsteri): conservation and innovation across the fish-tetrapod transition.

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Boisvert, Catherine Anne; Joss, Jean Mp; Ahlberg, Per E

2013-01-23

The fish-tetrapod transition was one of the major events in vertebrate evolution and was enabled by many morphological changes. Although the transformation of paired fish fins into tetrapod limbs has been a major topic of study in recent years, both from paleontological and comparative developmental perspectives, the interest has focused almost exclusively on the distal part of the appendage and in particular the origin of digits. Relatively little attention has been paid to the transformation of the pelvic girdle from a small unipartite structure to a large tripartite weight-bearing structure, allowing tetrapods to rely mostly on their hindlimbs for locomotion. In order to understand how the ischium and the ilium evolved and how the acetabulum was reoriented during this transition, growth series of the Australian lungfish Neoceratodus forsteri and the Mexican axolotl Ambystoma mexicanum were cleared and stained for cartilage and bone and immunostained for skeletal muscles. In order to understand the myological developmental data, hypotheses about the homologies of pelvic muscles in adults of Latimeria, Neoceratodus and Necturus were formulated based on descriptions from the literature of the coelacanth (Latimeria), the Australian Lungfish (Neoceratodus) and a salamander (Necturus). In the axolotl and the lungfish, the chondrification of the pelvic girdle starts at the acetabula and progresses anteriorly in the lungfish and anteriorly and posteriorly in the salamander. The ilium develops by extending dorsally to meet and connect to the sacral rib in the axolotl. Homologous muscles develop in the same order with the hypaxial musculature developing first, followed by the deep, then the superficial pelvic musculature. Development of the pelvic endoskeleton and musculature is very similar in Neoceratodus and Ambystoma. If the acetabulum is seen as being a fixed landmark, the evolution of the ischium only required pubic pre-chondrogenic cells to migrate posteriorly. It

Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum).

Science.gov (United States)

Phan, Anne Q; Lee, Jangwoo; Oei, Michelle; Flath, Craig; Hwe, Caitlyn; Mariano, Rachele; Vu, Tiffany; Shu, Cynthia; Dinh, Andrew; Simkin, Jennifer; Muneoka, Ken; Bryant, Susan V; Gardiner, David M

2015-08-01

Urodele amphibians are unique among adult vertebrates in their ability to regenerate complex body structures after traumatic injury. In salamander regeneration, the cells maintain a memory of their original position and use this positional information to recreate the missing pattern. We used an in vivo gain-of-function assay to determine whether components of the extracellular matrix (ECM) have positional information required to induce formation of new limb pattern during regeneration. We discovered that salamander limb ECM has a position-specific ability to either inhibit regeneration or induce de novo limb structure, and that this difference is dependent on heparan sulfates that are associated with differential expression of heparan sulfate sulfotransferases. We also discovered that an artificial ECM containing only heparan sulfate was sufficient to induce de novo limb pattern in salamander limb regeneration. Finally, ECM from mouse limbs is capable of inducing limb pattern in axolotl blastemas in a position-specific, developmental-stage-specific, and heparan sulfate-dependent manner. This study demonstrates a mechanism for positional information in regeneration and establishes a crucial functional link between salamander regeneration and mammals.

Corticotropin-releasing hormone-mediated metamorphosis in the neotenic axolotl Ambystoma mexicanum: synergistic involvement of thyroxine and corticoids on brain type II deiodinase.

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Kühn, Eduard R; De Groef, Bert; Van der Geyten, Serge; Darras, Veerle M

2005-08-01

In the present study, morphological changes leading to complete metamorphosis have been induced in the neotenic axolotl Ambystoma mexicanum using a submetamorphic dose of T(4) together with an injection of corticotropin-releasing hormone (CRH). An injection of CRH alone is ineffective in this regard presumably due to a lack of thyrotropic stimulation. Using this low hormone profile for induction of metamorphosis, the deiodinating enzymes D2 and D3 known to be present in amphibians were measured in liver and brain 24h following an intraperitoneal injection. An injection of T(4) alone did not influence liver nor brain D2 and D3, but dexamethasone (DEX) or CRH alone or in combination with T(4) decreased liver D2 and D3. Brain D2 activity was slightly increased with a higher dose of DEX, though CRH did not have this effect. A profound synergistic effect occurred when T(4) and DEX or CRH were injected together, in the dose range leading to metamorphosis, increasing brain D2 activity more than fivefold. This synergistic effect was not found in the liver. It is concluded that brain T(3) availability may play an important role for the onset of metamorphosis in the neotenic axolotl.

The yeast two hybrid system in a screen for proteins interacting with axolotl (Ambystoma mexicanum) Msx1 during early limb regeneration.

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Abuqarn, Mehtap; Allmeling, Christina; Amshoff, Inga; Menger, Bjoern; Nasser, Inas; Vogt, Peter M; Reimers, Kerstin

2011-07-01

Urodele amphibians are exceptional in their ability to regenerate complex body structures such as limbs. Limb regeneration depends on a process called dedifferentiation. Under an inductive wound epidermis terminally differentiated cells transform to pluripotent progenitor cells that coordinately proliferate and eventually redifferentiate to form the new appendage. Recent studies have developed molecular models integrating a set of genes that might have important functions in the control of regenerative cellular plasticity. Among them is Msx1, which induced dedifferentiation in mammalian myotubes in vitro. Herein, we screened for interaction partners of axolotl Msx1 using a yeast two hybrid system. A two hybrid cDNA library of 5-day-old wound epidermis and underlying tissue containing more than 2×10⁶ cDNAs was constructed and used in the screen. 34 resulting cDNA clones were isolated and sequenced. We then compared sequences of the isolated clones to annotated EST contigs of the Salamander EST database (BLASTn) to identify presumptive orthologs. We subsequently searched all no-hit clone sequences against non redundant NCBI sequence databases using BLASTx. It is the first time, that the yeast two hybrid system was adapted to the axolotl animal model and successfully used in a screen for proteins interacting with Msx1 in the context of amphibian limb regeneration. 2011 Elsevier B.V. All rights reserved.

Role of cranial neural crest cells in visceral arch muscle positioning and morphogenesis in the Mexican axolotl, Ambystoma mexicanum.

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Ericsson, Rolf; Cerny, Robert; Falck, Pierre; Olsson, Lennart

2004-10-01

The role of cranial neural crest cells in the formation of visceral arch musculature was investigated in the Mexican axolotl, Ambystoma mexicanum. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine, perchlorate) labeling and green fluorescent protein (GFP) mRNA injections combined with unilateral transplantations of neural folds showed that neural crest cells contribute to the connective tissues but not the myofibers of developing visceral arch muscles in the mandibular, hyoid, and branchial arches. Extirpations of individual cranial neural crest streams demonstrated that neural crest cells are necessary for correct morphogenesis of visceral arch muscles. These do, however, initially develop in their proper positions also in the absence of cranial neural crest. Visceral arch muscles forming in the absence of neural crest cells start to differentiate at their origins but fail to extend toward their insertions and may have a frayed appearance. Our data indicate that visceral arch muscle positioning is controlled by factors that do not have a neural crest origin. We suggest that the cranial neural crest-derived connective tissues provide directional guidance important for the proper extension of the cranial muscles and the subsequent attachment to the insertion on the correct cartilage. In a comparative context, our data from the Mexican axolotl support the view that the cranial neural crest plays a fundamental role in the development of not only the skeleton of the vertebrate head but also in the morphogenesis of the cranial muscles and that this might be a primitive feature of cranial development in vertebrates. 2004 Wiley-Liss, Inc.

Leucine-enkephalin-like immunoreactivity is localized in luteinizing hormone-producing cells in the axolotl (Ambystoma mexicanum) pituitary.

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Suzuki, Hirohumi; Yamamoto, Toshiharu

2014-02-01

In this study, we used immunohistochemical techniques to determine the cell type of leucine-enkephalin (Leu-ENK)-immunoreactive cells in the axolotl (Ambystoma mexicanum) pituitary. Immunoreactive cells were scattered throughout the pars distalis except for the dorso-caudal portion. These cells were immuno-positive for luteinizing hormone (LH), but they were immuno-negative for adrenocorticotrophic, growth, and thyroid-stimulating hormones, as well as prolactin. Immunoelectron microscopy demonstrated that Leu-ENK-like substance and LH co-localized within the same secretory granules. Leu-ENK secreted from gonadotrophs may participate in LH secretion in an autocrine fashion, and/or may participate in the release of sex steroids together with LH. Copyright © 2013 Elsevier Ltd. All rights reserved.

Severe necrotizing myocarditis caused by serratia marcescens infection in an axolotl (Ambystoma mexicanum).

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Del-Pozo, J; Girling, S; Pizzi, R; Mancinelli, E; Else, R W

2011-05-01

This report provides the first account of the pathological changes associated with infection by Serratia marcescens in an adult male axolotl. The infection resulted in septicaemia with severe multifocal necrotizing myocarditis. The latter lesion evolved to cardiac rupture, haemopericardium and death resulting from cardiac tamponade. This animal was exposed to higher than usual temperatures (24-25 °C) 2 weeks before the onset of disease and this may have resulted in immunocompromise and opportunistic bacterial infection. S. marcescens was isolated from the coelomic and pericardial cavity. Both isolates were identical and were resistant to β-lactam antibiotics, but not to aminoglycosides or fluoroquinolones. The production of red prodigiosin pigment by the bacterium suggested an environmental origin. Overall, the clinical and histopathological presentation suggests that S. marcescens should be included in the list of aetiological agents of the 'red-leg'/bacterial dermatosepticaemia syndrome of amphibians. Copyright © 2010 Elsevier Ltd. All rights reserved.

Localization of amylin-like immunoreactivity in melanocyte-stimulating hormone-containing cells of the pars intermedia but not those of the pars distalis in the axolotl (Ambystoma mexicanum) pituitary.

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Suzuki, Hirohumi; Yamamoto, Toshiharu

2016-04-01

Immunohistochemical techniques were employed to investigate the distribution of amylin-like immunoreactivity in the axolotl (Ambystoma mexicanum) pituitary. Amylin-immunoreactive cells were observed in the pars intermedia, and these cells were found to be immunoreactive for α-melanocyte-stimulating hormone (αMSH) as well. In contrast, αMSH-immunoreactive cells in the pars distalis were immuno-negaitive for amylin. These light microscopic findings were confirmed by immunoelectron microscopy. Amylin-immunoreactive signals were located on the haloes of presumable secretory granules in association with αMSH-immunoreactive signals in the amylin-positive cells. However, in the pars distalis, the αMSH-positive cells did not contain amylin-immunoreactive secretory granules. Western blot analysis of axolotl pituitary extracts revealed the labeling of a protein band at approximately 10.5-kDa by the anti-rat amylin serum, which was not labeled by the anti-αMSH antibody. These findings indicate that amylin secreted from MSH-producing cells in the pars intermedia may modulate MSH secretion in an autocrine fashion and may participate in MSH functions such as fatty homeostasis together with MSH. Copyright © 2016 Elsevier GmbH. All rights reserved.

Oxygen consumption and cytochrome exidase activity of axolotl limbs muscle tissue in restoration of regenerative ability suprressed by X-irradiation

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Teplits, N.A.

1975-01-01

The rate of oxygen use and activity of cytochrome oxidase in a homogenate of mitochondria and nuclei of muscle tissue of axolotl limbs after suppression of their regenerative capability by x irradiation and their restoration was studied experimentally. With suppression of the regenative capability the use of oxygen was depressed. Cytochrome oxidase activity in the homogenate and mitochondria decreased compared to that of the intact limb, in the nuclei of muscle tissue it was the same or greater. With restoration of the regenerative capability of the limbs the respiration rate of the homogenate and the mitochondria increased, accompanied by increased cytochrome oxidase activity. In the nuclei the cytochrome oxidase activity did not change in the blastema stage and sharply decreased in the limb formation state. (E.T.)

Oxygen consumption and cytochrome exidase activity of axolotl limbs muscle tissue in restoration of regenerative ability suppressed by X-irradiation

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Teplits, N A [AN SSSR, Moscow. Inst. Biologii Razvitiya

1975-01-01

The rate of oxygen use and activity of cytochrome oxidase in a homogenate of mitochondria and nuclei of muscle tissue of axolotl limbs after suppression of their regenerative capability by x irradiation and their restoration was studied experimentally. With suppression of the regenative capability the use of oxygen was depressed. Cytochrome oxidase activity in the homogenate and mitochondria decreased compared to that of the intact limb, in the nuclei of muscle tissue it was the same or greater. With restoration of the regenerative capability of the limbs the respiration rate of the homogenate and the mitochondria increased, accompanied by increased cytochrome oxidase activity. In the nuclei the cytochrome oxidase activity did not change in the blastema stage and sharply decreased in the limb formation state.

Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin.

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Ferris, Donald R; Satoh, Akira; Mandefro, Berhan; Cummings, Gillian M; Gardiner, David M; Rugg, Elizabeth L

2010-10-01

Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs. Regeneration is a stepwise process that requires interactions between keratinocytes, nerves and fibroblasts. The formation of a wound epithelium covering the amputation site is an early and necessary event in the process but the molecular mechanisms that underlie the role of the wound epithelium in regeneration remain unclear. We have developed an ex vivo model that recapitulates many features of in vivo wound healing. The model comprises a circular explant of axolotl (Ambystoma mexicanum) limb skin with a central circular, full thickness wound. Re-epithelialization of the wound area is rapid (typically <11 h) and is dependent on metalloproteinase activity. The ex vivo wound epithelium is viable, responds to neuronal signals and is able to participate in ectopic blastema formation and limb regeneration. This ex vivo model provides a reproducible and tractable system in which to study the cellular and molecular events that underlie wound healing and regeneration. © 2010 The Authors. Journal compilation © 2010 Japanese Society of Developmental Biologists.

Comparative transcriptional profiling of the axolotl limb identifies a tripartite regeneration-specific gene program.

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Dunja Knapp

Full Text Available Understanding how the limb blastema is established after the initial wound healing response is an important aspect of regeneration research. Here we performed parallel expression profile time courses of healing lateral wounds versus amputated limbs in axolotl. This comparison between wound healing and regeneration allowed us to identify amputation-specific genes. By clustering the expression profiles of these samples, we could detect three distinguishable phases of gene expression - early wound healing followed by a transition-phase leading to establishment of the limb development program, which correspond to the three phases of limb regeneration that had been defined by morphological criteria. By focusing on the transition-phase, we identified 93 strictly amputation-associated genes many of which are implicated in oxidative-stress response, chromatin modification, epithelial development or limb development. We further classified the genes based on whether they were or were not significantly expressed in the developing limb bud. The specific localization of 53 selected candidates within the blastema was investigated by in situ hybridization. In summary, we identified a set of genes that are expressed specifically during regeneration and are therefore, likely candidates for the regulation of blastema formation.

Ontogeny of the VIP system in the gastro-intestinal tract of the Axolotl, Ambystoma mexicanum: successive appearance of co-existing PACAP and NOS.

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Badawy, Gamal; Reinecke, Manfred

2003-03-01

Evidence for the presence and potential co-existence of vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP) and nitric oxide synthase (NOS) in gastro-intestinal endocrine cells and/or nerve fibers is conflicting and very few results exist on development. This immunofluorescence study aims to clarify the appearance and localization of VIP, PACAP and NOS in the gastro-intestinal tract of the Axolotl, Ambystoma mexicanum, during ontogeny. VIP-immunoreactivity appeared in nerve fibers as early as on day 3 after hatching likely indicating a particular role, such as a trophic action, of VIP in very early development. PACAP-immunoreactivity was observed 3 days later within the VIP-immunoreactive (-IR) fibers. From this time on, VIP- and PACAP-immunoreactivity exhibited complete co-existence. VIP/PACAP-IR fibers were found throughout the gastro-intestinal tract. They were most prominent in the myenteric plexus and the muscle layers and less frequent in the submucosa. NOS-immunoreactivity appeared as late as at the 1st (64 days) juvenile stage in a subpopulation of the VIP/PACAP-IR fibers that contacted submucosal arteries. We found only very few VIP/PACAP-IR perikarya, indicating that part of the VIP/PACAP-IR fibers is of extrinsic origin. On day 12 and in the 1st and 2nd (104 days) juvenile stage, infrequent PACAP-IR entero-endocrine cells were noted, while neither VIP- nor NOS-immunoreactivity occurred in endocrine cells at any stage of development. The complete coexistence of neuronal PACAP- and VIP-immunoreactivities and their very early appearance in ontogeny may suggest important and coordinated roles of both peptides in the control of Axolotl gastro-intestinal activity, while the VIP/ PACAP/NOS-IR fibers may be involved in the regulation of submucosal blood flow.

Patterns of spatial and temporal visceral arch muscle development in the Mexican axolotl (Ambystoma mexicanum).

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Ericsson, Rolf; Olsson, Lennart

2004-08-01

Vertebrate head development is a classical topic that has received renewed attention during the last decade. Most reports use one of a few model organisms (chicken, mouse, zebrafish) and have focused on molecular mechanisms and the role of the neural crest, while cranial muscle development has received less attention. Here we describe cranial muscle differentiation and morphogenesis in the Mexican axolotl, Ambystoma mexicanum. To determine the onset of differentiation we use antibodies against desmin and optical sectioning using confocal laser scanning microscopy on whole-mount immunostained embryos. This technique makes it possible to document the cranial muscle in three dimensions while keeping the specimens intact. Desmin expression starts almost simultaneously in the first, second, and third visceral arch muscles (as in other amphibians studied). Muscle anlagen divide up early into the different elements which constitute the larval cranial musculature. We extend and refine earlier findings, e.g., by documenting a clear division between interhyoideus and interhyoideus posterior. The timing of cranial muscle differentiation differs among vertebrate groups, but seems to be constant within each group. This study provides a morphological foundation for further studies of muscle cell fate and early differentiation. Copyright 2004 Wiley-Liss, Inc.

DNA Methylation Dynamics Regulate the Formation of a Regenerative Wound Epithelium during Axolotl Limb Regeneration.

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Cristian Aguilar

Full Text Available The formation of a blastema during regeneration of an axolotl limb involves important changes in the behavior and function of cells at the site of injury. One of the earliest events is the formation of the wound epithelium and subsequently the apical epidermal cap, which involves in vivo dedifferentiation that is controlled by signaling from the nerve. We have investigated the role of epigenetic modifications to the genome as a possible mechanism for regulating changes in gene expression patterns of keratinocytes of the wound and blastema epithelium that are involved in regeneration. We report a modulation of the expression DNMT3a, a de novo DNA methyltransferase, within the first 72 hours post injury that is dependent on nerve signaling. Treatment of skin wounds on the upper forelimb with decitabine, a DNA methyltransferase inhibitor, induced changes in gene expression and cellular behavior associated with a regenerative response. Furthermore, decitabine-treated wounds were able to participate in regeneration while untreated wounds inhibited a regenerative response. Elucidation of the specific epigenetic modifications that mediate cellular dedifferentiation likely will lead to insights for initiating a regenerative response in organisms that lack this ability.

Neurotrophic regulation of fibroblast dedifferentiation during limb skeletal regeneration in the axolotl (Ambystoma mexicanum).

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Satoh, Akira; Cummings, Gillian M C; Bryant, Susan V; Gardiner, David M

2010-01-15

The ability of animals to repair tissue damage is widespread and impressive. Among tissues, the repair and remodeling of bone occurs during growth and in response to injury; however, loss of bone above a threshold amount is not regenerated, resulting in a "critical-size defect" (CSD). The development of therapies to replace or regenerate a CSD is a major focus of research in regenerative medicine and tissue engineering. Adult urodeles (salamanders) are unique in their ability to regenerate complex tissues perfectly, yet like mammals do not regenerate a CSD. We report on an experimental model for the regeneration of a CSD in the axolotl (the Excisional Regeneration Model) that allows for the identification of signals to induce fibroblast dedifferentiation and skeletal regeneration. This regenerative response is mediated in part by BMP signaling, as is the case in mammals; however, a complete regenerative response requires the induction of a population of undifferentiated, regeneration-competent cells. These cells can be induced by signaling from limb amputation to generate blastema cells that can be grafted to the wound, as well as by signaling from a nerve and a wound epithelium to induce blastema cells from fibroblasts within the wound environment. Copyright 2009 Elsevier Inc. All rights reserved.

Crystal structure of axolotl (Ambystoma mexicanum) liver bile acid-binding protein bound to cholic and oleic acid.

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Capaldi, Stefano; Guariento, Mara; Perduca, Massimiliano; Di Pietro, Santiago M; Santomé, José A; Monaco, Hugo L

2006-07-01

The family of the liver bile acid-binding proteins (L-BABPs), formerly called liver basic fatty acid-binding proteins (Lb-FABPs) shares fold and sequence similarity with the paralogous liver fatty acid-binding proteins (L-FABPs) but has a different stoichiometry and specificity of ligand binding. This article describes the first X-ray structure of a member of the L-BABP family, axolotl (Ambystoma mexicanum) L-BABP, bound to two different ligands: cholic and oleic acid. The protein binds one molecule of oleic acid in a position that is significantly different from that of either of the two molecules that bind to rat liver FABP. The stoichiometry of binding of cholate is of two ligands per protein molecule, as observed in chicken L-BABP. The cholate molecule that binds buried most deeply into the internal cavity overlaps well with the analogous bound to chicken L-BABP, whereas the second molecule, which interacts with the first only through hydrophobic contacts, is more external and exposed to the solvent. (c) 2006 Wiley-Liss, Inc.

Proteomic analysis of blastema formation in regenerating axolotl limbs

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Nye Holly LD

2009-11-01

Full Text Available Abstract Background Following amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa through the mid-tibia/fibula of axolotl hind limbs. Results We identified 309 unique proteins with significant fold change relative to controls (0 dpa, representing 10 biological process categories: (1 signaling, (2 Ca2+ binding and translocation, (3 transcription, (4 translation, (5 cytoskeleton, (6 extracellular matrix (ECM, (7 metabolism, (8 cell protection, (9 degradation, and (10 cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5, a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation. Conclusion Our data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR. Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to

Dual embryonic origin of the hyobranchial apparatus in the Mexican axolotl (Ambystoma mexicanum).

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Davidian, Asya; Malashichev, Yegor

2013-01-01

Traditionally, the cartilaginous viscerocranium of vertebrates is considered as neural crest (NC)-derived. Morphological work carried out on amphibian embryos in the first half of the XX century suggested potentially mesodermal origin for some hyobranchial elements. Since then, the embryonic sources of the hyobranchial apparatus in amphibians has not been investigated due to lack of an appropriate long-term labelling system. We performed homotopic transplantations of neural folds along with the majority of cells of the presumptive NC, and/or fragments of the head lateral plate mesoderm (LPM) from transgenic GFP+ into white embryos. In these experiments, the NC-derived GFP+ cells contributed to all hyobranchial elements, except for basibranchial 2, whereas the grafting of GFP+ head mesoderm led to a reverse labelling result. The grafting of only the most ventral part of the head LPM resulted in marking of the basibranchial 2 and the heart myocardium, implying their origin from a common mesodermal region. This is the first evidence of contribution of LPM of the head to cranial elements in any vertebrate. If compared to fish, birds, and mammals, in which all branchial skeletal elements are NC-derived, the axolotl (probably this is true for all amphibians) demonstrates an evolutionary deviation, in which the head LPM replaces NC cells in a hyobranchial element. This implies that cells of different embryonic origin may have the same developmental program, leading to the formation of identical (homologous) elements of the skeleton.

Regulation of Axolotl (Ambystoma mexicanum) Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

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Lehrberg, Jeffrey; Gardiner, David M

2015-01-01

We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

Regulation of Axolotl (Ambystoma mexicanum Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

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Jeffrey Lehrberg

Full Text Available We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

Effect of water quality on the feeding ecology of axolotl Ambystoma mexicanum

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Diego de Jesus Chaparro-Herrera

2013-10-01

Full Text Available Ambystoma mexicanum, a highly endangered species, is endemic to lake Xochimilco (Mexico City, Mexico which currently is being negatively affected by the introduction of Oreochromis niloticus (Tilapia and water pollution. During the first weeks of development, when mortality is the highest, Ambystoma mexicanumdepends on a diet of zooplankton. The aim of this study was to check whether contamination levels in lake Xochimilco influence zooplankton consumption by similar size classes of A. mexicanum and Oreochromis niloticus. In this study, we analysed changes in the functional responses and prey preference of A. mexicanum and larval Tilapia in two media, one with filtered lake Xochimilco water and another one with reconstituted water. As prey we used cladocerans (Moina macrocopa, Alona glabra, Macrothrix triserialis and Simocephalus vetulus and ostracods (Heterocypris incongruens. Zooplankton was offered in 5 different densities, 10, 20, 40, 80, 160 ind./mL. Prey consumption by A. mexicanum varied in relation to the species offered and age of the larvae. From the first week to the eighth week prey consumption by A. mexicanum increased by 57%. Our functional response tests showed that regardless of the prey type, prey consumption by A. mexicanum was lower in the contaminated water from lake Xochimilco. Among the zooplankton offered in the contaminated environment predators preferred smaller and slower moving microcrustaceans such as Alona glabra and Heterocypris incongruens. Furthermore, O. niloticus preferred prey such as Moina macrocopa and Macrothrix triserialis in the contaminated medium and was more voracious than the axolotl. Our results indicate that both water quality of the lake and the presence of the more resistant exotic fish adversely impact the survival of this endangered amphibian.

Neurotrophic regulation of epidermal dedifferentiation during wound healing and limb regeneration in the axolotl (Ambystoma mexicanum).

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Satoh, A; Graham, G M C; Bryant, S V; Gardiner, D M

2008-07-15

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. In the present study, we have utilized the ALM to identity the buttonhead-like zinc-finger transcription factor, Sp9, as being involved in the formation of the regeneration epithelium. Sp9 expression is induced in basal keratinocytes of the apical blastema epithelium in a pattern that is comparable to its expression in developing limb buds, and it thus is an important marker for dedifferentiation of the epidermis. Induction of Sp9 expression is nerve-dependent, and we have identified KGF as an endogenous nerve factor that induces expression of Sp9 in the regeneration epithelium.

Ion channel signaling influences cellular proliferation and phagocyte activity during axolotl tail regeneration.

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Franklin, Brandon M; Voss, S Randal; Osborn, Jeffrey L

2017-08-01

Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, K V 2.1, K V 2.2, L-type Ca V channels and H/K ATPases) or completely (GlyR, GABA A R, K V 1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in tail muscle and mesenchymal regions. Inhibition of anoctamin 1/2 also altered the post-amputation transcriptional response of p44/42 MAPK signaling pathway genes, including decreased expression of erk1/erk2. We also found that complete inhibition via voltage gated K + channel blockade was associated with diminished phagocyte recruitment to the amputation site. The identification of H + pumps as required for axolotl tail regeneration supports findings in Xenopus and Planaria models, and more generally, the conservation of ion channels as regulators of tissue regeneration. This study provides a preliminary framework for an in-depth investigation of the mechanistic role of ion channels and their potential involvement in regulating cellular proliferation and other processes essential to wound healing, appendage regeneration, and tissue repair. Copyright © 2017 Elsevier B.V. All rights reserved.

Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl

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Zhu, Wei; Pao, Gerald M; Satoh, Akira; Cummings, Gillian; Monaghan, James R; Harkins, Timothy T; Bryant, Susan V; Voss, S Randal; Gardiner, David M; Hunter, Tony

2013-01-01

The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration – wound healing, cellular dedifferentiation and blastemal formation – will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration. PMID:22841627

Identification of differentially expressed thyroid hormone responsive genes from the brain of the Mexican Axolotl (Ambystoma mexicanum).

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Huggins, P; Johnson, C K; Schoergendorfer, A; Putta, S; Bathke, A C; Stromberg, A J; Voss, S R

2012-01-01

The Mexican axolotl (Ambystoma mexicanum) presents an excellent model to investigate mechanisms of brain development that are conserved among vertebrates. In particular, metamorphic changes of the brain can be induced in free-living aquatic juveniles and adults by simply adding thyroid hormone (T4) to rearing water. Whole brains were sampled from juvenile A. mexicanum that were exposed to 0, 8, and 18 days of 50 nM T4, and these were used to isolate RNA and make normalized cDNA libraries for 454 DNA sequencing. A total of 1,875,732 high quality cDNA reads were assembled with existing ESTs to obtain 5884 new contigs for human RefSeq protein models, and to develop a custom Affymetrix gene expression array (Amby_002) with approximately 20,000 probe sets. The Amby_002 array was used to identify 303 transcripts that differed statistically (p1.5) as a function of days of T4 treatment. Further statistical analyses showed that Amby_002 performed concordantly in comparison to an existing, small format expression array. This study introduces a new A. mexicanum microarray resource for the community and the first lists of T4-responsive genes from the brain of a salamander amphibian. Copyright © 2011 Elsevier Inc. All rights reserved.

Positive in vitro wound healing effects of functional inclusion bodies of a lipoxygenase from the Mexican axolotl.

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Stamm, Anne; Strauß, Sarah; Vogt, Peter; Scheper, Thomas; Pepelanova, Iliyana

2018-04-07

AmbLOXe is a lipoxygenase, which is up-regulated during limb-redevelopment in the Mexican axolotl, Ambystoma mexicanum, an animal with remarkable regeneration capacity. Previous studies have shown that mammalian cells transformed with the gene of this epidermal lipoxygenase display faster migration and wound closure rate during in vitro wound healing experiments. In this study, the gene of AmbLOXe was codon-optimized for expression in Escherichia coli and was produced in the insoluble fraction as protein aggregates. These inclusion bodies or nanopills were shown to be reservoirs containing functional protein during in vitro wound healing assays. For this purpose, functional inclusion bodies were used to coat cell culture surfaces prior cell seeding or were added directly to the medium after cells reached confluence. In both scenarios, AmbLOXe inclusion bodies led to faster migration rate and wound closure, in comparison to controls containing either no AmbLOXe or GFP inclusion bodies. Our results demonstrate that AmbLOXe inclusion bodies are functional and may serve as stable reservoirs of this enzyme. Nevertheless, further studies with soluble enzyme are also necessary in order to start elucidating the exact molecular substrates of AmbLOXe and the biochemical pathways involved in the wound healing effect.

Identification of Differentially Expressed Thyroid Hormone Responsive Genes from the Brain of the Mexican Axolotl (Ambystoma mexicanum) ✧

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Huggins, P; Johnson, CK; Schoergendorfer, A; Putta, S; Bathke, AC; Stromberg, AJ; Voss, SR

2011-01-01

The Mexican axolotl (Ambystoma mexicanum) presents an excellent model to investigate mechanisms of brain development that are conserved among vertebrates. In particular, metamorphic changes of the brain can be induced in free-living aquatic juveniles and adults by simply adding thyroid hormone (T4) to rearing water. Whole brains were sampled from juvenile A. mexicanum that were exposed to 0, 8, and 18 days of 50 nM T4, and these were used to isolate RNA and make normalized cDNA libraries for 454 DNA sequencing. A total of 1,875,732 high quality cDNA reads were assembled with existing ESTs to obtain 5,884 new contigs for human RefSeq protein models, and to develop a custom Affymetrix gene expression array (Amby_002) with approximately 20,000 probe sets. The Amby_002 array was used to identify 303 transcripts that differed statistically (p 1.5) as a function of days of T4 treatment. Further statistical analyses showed that Amby_002 performed concordantly in comparison to an existing, small format expression array. This study introduces a new A. mexicanum microarray resource for the community and the first lists of T4-responsive genes from the brain of a salamander amphibian. PMID:21457787

Glucose transporter distribution in the vessels of the central nervous system of the axolotl Ambystoma mexicanum (Urodela: Ambystomatidae).

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Lazzari, Maurizio; Bettini, Simone; Ciani, Franco; Franceschini, Valeria

2008-10-01

The GLUT-1 isoform of the glucose transporter is commonly considered a reliable molecular marker of blood-brain barrier endothelia in the neural vasculature organized in a three-dimensional network of single vessels. The central nervous system of the axolotl Ambystoma mexicanum is characterized by a vascular architecture that contains both single and paired vessels. The presence and distribution of the GLUT-1 transporter are studied in this urodele using both immunoperoxidase histochemistry and immunogold technique. Light microscopy reveals immunopositivity in both parenchymal and meningeal vessels. The transverse-sectioned pairs of vessels do not show the same size. Furthermore, in the same pair, the two elements often differ in diameter. The main regions of the central nervous system show a different percentage of the paired structures. Only immunogold cytochemistry reveals different staining intensity in the two adjoined elements of a vascular pair. Colloidal gold particles show an asymmetric distribution in the endothelia of both single and paired vessels. These particles are more numerous on the abluminal surface than on the luminal one. The particle density is calculated in both vascular types. The different values could indicate functional differences between single and paired vessels and between the two adjoined elements of a pair, regarding glucose transport.

Muscular derivatives of the cranialmost somites revealed by long-term fate mapping in the Mexican axolotl (Ambystoma mexicanum).

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Piekarski, Nadine; Olsson, Lennart

2007-01-01

The fate of single somites has not been analyzed from a comparative perspective with modern cell-marking methods. Most of what we know is based on work using quail-chick chimeras. Consequently, to what degree cell fate has been conserved despite the anatomical differences among vertebrates is unknown. We have analyzed the cell fate of the cranialmost somites, with the focus on somite two, in the Mexican axolotl (Ambystoma mexicanum). Somite cells were marked by injection of dextran-fluorescein and detected using immunofluorescence after 2 months of development in paraffin sections. Our data confirm and extend earlier studies based on classical histology in salamanders. We show that somite two contributes to different muscles, skeletal elements, and connective tissues of the head and cranial trunk region. Cells from somites two and three migrate latero-ventrally and contribute to the hypobranchial muscles mm. geniohyoideus and rectus cervicis. We provide evidence that the specific formation of the hypobranchial musculature from ventral processes of the somites might be variable in different classes of vertebrates. We further demonstrate that mm. cucullaris and dilatator laryngis, which were earlier thought to have a branchial origin, arise from somitic material in a manner very similar to the findings in quail-chick chimeras. Our findings indicate that the pattern of somitic derivatives is highly conserved within tetrapods.

The Center for Regenerative Biology and Medicine at Mount Desert Island Biological Laboratory

Science.gov (United States)

2015-08-01

TERMS limb regeneration Positional Memory Code Axolotl microRNAs Zebrafish Polypterus 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18...injury induced regeneration of limbs/appendage tissues in Ambystoma ( axolotl ) and Polypterus animals. The defining feature of limb/appendage...downregulated in UPREGULATED DOWNREGULATED Figure 1: Venn diagram of UniProt protein sequence IDs among Axolotl and Polypterus contigs that

Analysis of the expression and function of Wnt-5a and Wnt-5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs.

Science.gov (United States)

Ghosh, Sukla; Roy, Stéphane; Séguin, Carl; Bryant, Susan V; Gardiner, David M

2008-05-01

Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non-canonical Wnt genes, Wnt-5a and Wnt-5b. We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl (Ambystoma mexicanum), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt-5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration-competent cells of the blastema.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum.

Science.gov (United States)

Saltman, Anna J; Barakat, May; Bryant, Donald M; Brodovskaya, Anastasia; Whited, Jessica L

2017-06-16

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1'-Dioctadecy-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl's blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

Ecological Risk Assessment of Perchlorate in Avian Species, Rodents, Amphibians and Fish

Science.gov (United States)

2003-04-01

http://www .indiana.edu/- axolotl ). 10.0 JUSTIFICATION OF TEST SYSTEM Perchlorate occurs in ground and surface waters in 44 states in the USA... axolotl ). * Sequentially numbered in order of the date that the change is effective Dept. of Biological Sciences (DBS) Box 43131 Lubbock, TX 79409...KCl, 0.025 giL; CaCh2 H20, 0.65 g/L; MgS04·7H20, 0.1 giL (http://www.indiana.edu/~ axolotl ). *Sequentially numbered in order of the date that the

Genic regions of a large salamander genome contain long introns and novel genes

Directory of Open Access Journals (Sweden)

Bryant Susan V

2009-01-01

Full Text Available Abstract Background The basis of genome size variation remains an outstanding question because DNA sequence data are lacking for organisms with large genomes. Sixteen BAC clones from the Mexican axolotl (Ambystoma mexicanum: c-value = 32 × 109 bp were isolated and sequenced to characterize the structure of genic regions. Results Annotation of genes within BACs showed that axolotl introns are on average 10× longer than orthologous vertebrate introns and they are predicted to contain more functional elements, including miRNAs and snoRNAs. Loci were discovered within BACs for two novel EST transcripts that are differentially expressed during spinal cord regeneration and skin metamorphosis. Unexpectedly, a third novel gene was also discovered while manually annotating BACs. Analysis of human-axolotl protein-coding sequences suggests there are 2% more lineage specific genes in the axolotl genome than the human genome, but the great majority (86% of genes between axolotl and human are predicted to be 1:1 orthologs. Considering that axolotl genes are on average 5× larger than human genes, the genic component of the salamander genome is estimated to be incredibly large, approximately 2.8 gigabases! Conclusion This study shows that a large salamander genome has a correspondingly large genic component, primarily because genes have incredibly long introns. These intronic sequences may harbor novel coding and non-coding sequences that regulate biological processes that are unique to salamanders.

Profiling neurotransmitter receptor expression in the Ambystoma mexicanum brain.

Science.gov (United States)

Reyes-Ruiz, Jorge Mauricio; Limon, Agenor; Korn, Matthew J; Nakamura, Paul A; Shirkey, Nicole J; Wong, Jamie K; Miledi, Ricardo

2013-03-22

Ability to regenerate limbs and central nervous system (CNS) is unique to few vertebrates, most notably the axolotl (Ambystoma sp.). However, despite the fact the neurotransmitter receptors are involved in axonal regeneration, little is known regarding its expression profile. In this project, RT-PCR and qPCR were performed to gain insight into the neurotransmitter receptors present in Ambystoma. Its functional ability was studied by expressing axolotl receptors in Xenopus laevis oocytes by either injection of mRNA or by direct microtransplantation of brain membranes. Oocytes injected with axolotl mRNA expressed ionotropic receptors activated by GABA, aspartate+glycine and kainate, as well as metabotropic receptors activated by acetylcholine and glutamate. Interestingly, we did not see responses following the application of serotonin. Membranes from the axolotl brain were efficiently microtransplanted into Xenopus oocytes and two types of native GABA receptors that differed in the temporal course of their responses and affinities to GABA were observed. Results of this study are necessary for further characterization of axolotl neurotransmitter receptors and may be useful for guiding experiments aimed at understanding activity-dependant limb and CNS regeneration. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

A Formal Approach to Discourse Anaphora

Science.gov (United States)

1978-05-01

was given a nut by Wendy, and Druce did 0 too. 0 = ? 41. The aardvark was given two peanuts for its birthday, and the axolotl was 0 too. 0...given two peanuts for its birthday 42. Wendy avoidf the aardvark, and the axolotl was 0 too. 0 = 7 In the case of multiple passives (such as are...the axolotl was 0 too. 0 = given an apple 45. Bert is a super dentist and his father was 0 too. 0 = (be) a super dentist Moreover, it appeals to me

Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer

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Villiard Éric

2007-09-01

Full Text Available Abstract Background Urodele amphibians like the axolotl are unique among vertebrates in their ability to regenerate and their resistance to develop cancers. It is unknown whether these traits are linked at the molecular level. Results Blocking p53 signaling in axolotls using the p53 inhibitor, pifithrin-α, inhibited limb regeneration and the expression of p53 target genes such as Mdm2 and Gadd45, suggesting a link between tumor suppression and regeneration. To understand this relationship we cloned the p53 gene from axolotl. When comparing its sequence with p53 from other organisms, and more specifically human we observed multiple amino acids changes found in human tumors. Phylogenetic analysis of p53 protein sequences from various species is in general agreement with standard vertebrate phylogeny; however, both mice-like rodents and teleost fishes are fast evolving. This leads to long branch attraction resulting in an artefactual basal emergence of these groups in the phylogenetic tree. It is tempting to assume a correlation between certain life style traits (e.g. lifespan and the evolutionary rate of the corresponding p53 sequences. Functional assays of the axolotl p53 in human or axolotl cells using p53 promoter reporters demonstrated a temperature sensitivity (ts, which was further confirmed by performing colony assays at 37°C. In addition, axolotl p53 was capable of efficient transactivation at the Hmd2 promoter but has moderate activity at the p21 promoter. Endogenous axolotl p53 was activated following UV irradiation (100 j/m2 or treatment with an alkylating agent as measured using serine 15 phosphorylation and the expression of the endogenous p53 target Gadd45. Conclusion Urodele p53 may play a role in regeneration and has evolved to contain multiple amino acid changes predicted to render the human protein defective in tumor suppression. Some of these mutations were probably selected to maintain p53 activity at low temperature. However

Microarray analysis of a salamander hopeful monster reveals transcriptional signatures of paedomorphic brain development

Science.gov (United States)

2010-01-01

Background The Mexican axolotl (Ambystoma mexicanum) is considered a hopeful monster because it exhibits an adaptive and derived mode of development - paedomorphosis - that has evolved rapidly and independently among tiger salamanders. Unlike related tiger salamanders that undergo metamorphosis, axolotls retain larval morphological traits into adulthood and thus present an adult body plan that differs dramatically from the ancestral (metamorphic) form. The basis of paedomorphic development was investigated by comparing temporal patterns of gene transcription between axolotl and tiger salamander larvae (Ambystoma tigrinum tigrinum) that typically undergo a metamorphosis. Results Transcript abundances from whole brain and pituitary were estimated via microarray analysis on four different days post hatching (42, 56, 70, 84 dph) and regression modeling was used to independently identify genes that were differentially expressed as a function of time in both species. Collectively, more differentially expressed genes (DEGs) were identified as unique to the axolotl (n = 76) and tiger salamander (n = 292) than were identified as shared (n = 108). All but two of the shared DEGs exhibited the same temporal pattern of expression and the unique genes tended to show greater changes later in the larval period when tiger salamander larvae were undergoing anatomical metamorphosis. A second, complementary analysis that directly compared the expression of 1320 genes between the species identified 409 genes that differed as a function of species or the interaction between time and species. Of these 409 DEGs, 84% exhibited higher abundances in tiger salamander larvae at all sampling times. Conclusions Many of the unique tiger salamander transcriptional responses are probably associated with metamorphic biological processes. However, the axolotl also showed unique patterns of transcription early in development. In particular, the axolotl showed a genome-wide reduction in mRNA abundance

Microarray analysis of a salamander hopeful monster reveals transcriptional signatures of paedomorphic brain development

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Putta Srikrishna

2010-06-01

Full Text Available Abstract Background The Mexican axolotl (Ambystoma mexicanum is considered a hopeful monster because it exhibits an adaptive and derived mode of development - paedomorphosis - that has evolved rapidly and independently among tiger salamanders. Unlike related tiger salamanders that undergo metamorphosis, axolotls retain larval morphological traits into adulthood and thus present an adult body plan that differs dramatically from the ancestral (metamorphic form. The basis of paedomorphic development was investigated by comparing temporal patterns of gene transcription between axolotl and tiger salamander larvae (Ambystoma tigrinum tigrinum that typically undergo a metamorphosis. Results Transcript abundances from whole brain and pituitary were estimated via microarray analysis on four different days post hatching (42, 56, 70, 84 dph and regression modeling was used to independently identify genes that were differentially expressed as a function of time in both species. Collectively, more differentially expressed genes (DEGs were identified as unique to the axolotl (n = 76 and tiger salamander (n = 292 than were identified as shared (n = 108. All but two of the shared DEGs exhibited the same temporal pattern of expression and the unique genes tended to show greater changes later in the larval period when tiger salamander larvae were undergoing anatomical metamorphosis. A second, complementary analysis that directly compared the expression of 1320 genes between the species identified 409 genes that differed as a function of species or the interaction between time and species. Of these 409 DEGs, 84% exhibited higher abundances in tiger salamander larvae at all sampling times. Conclusions Many of the unique tiger salamander transcriptional responses are probably associated with metamorphic biological processes. However, the axolotl also showed unique patterns of transcription early in development. In particular, the axolotl showed a genome

Ion transport by the amphibian primary ureter

DEFF Research Database (Denmark)

Møbjerg, Nadja

2008-01-01

putative ion transport mechanisms in the primary ureter of the freshwater amphibian Ambystoma mexicanum (axolotl). Primary ureters isolated from axolotl larvae were perfused in vitro and single cells were impaled across the basal cell membrane with glass microelectrodes. In 42 cells the membrane potential......+] steps from 3 to 20 mmol/l and a hyperpolarization of Vm upon lowering [Na+] from 102 to 2 mmol/l, indicating the presence of luminal K+ and Na+ conductances. This study provides the first functional data on the vertebrate primary ureter. The data show that the primary ureter of axolotl larvae...

Transcriptome dynamics along axolotl regenerative development are consistent with an extensive reduction in gene expression heterogeneity in dedifferentiated cells

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Carlos Díaz-Castillo

2017-11-01

Full Text Available Although in recent years the study of gene expression variation in the absence of genetic or environmental cues or gene expression heterogeneity has intensified considerably, many basic and applied biological fields still remain unaware of how useful the study of gene expression heterogeneity patterns might be for the characterization of biological systems and/or processes. Largely based on the modulator effect chromatin compaction has for gene expression heterogeneity and the extensive changes in chromatin compaction known to occur for specialized cells that are naturally or artificially induced to revert to less specialized states or dedifferentiate, I recently hypothesized that processes that concur with cell dedifferentiation would show an extensive reduction in gene expression heterogeneity. The confirmation of the existence of such trend could be of wide interest because of the biomedical and biotechnological relevance of cell dedifferentiation-based processes, i.e., regenerative development, cancer, human induced pluripotent stem cells, or plant somatic embryogenesis. Here, I report the first empirical evidence consistent with the existence of an extensive reduction in gene expression heterogeneity for processes that concur with cell dedifferentiation by analyzing transcriptome dynamics along forearm regenerative development in Ambystoma mexicanum or axolotl. Also, I briefly discuss on the utility of the study of gene expression heterogeneity dynamics might have for the characterization of cell dedifferentiation-based processes, and the engineering of tools that afforded better monitoring and modulating such processes. Finally, I reflect on how a transitional reduction in gene expression heterogeneity for dedifferentiated cells can promote a long-term increase in phenotypic heterogeneity following cell dedifferentiation with potential adverse effects for biomedical and biotechnological applications.

Transcriptome dynamics along axolotl regenerative development are consistent with an extensive reduction in gene expression heterogeneity in dedifferentiated cells

Science.gov (United States)

2017-01-01

Although in recent years the study of gene expression variation in the absence of genetic or environmental cues or gene expression heterogeneity has intensified considerably, many basic and applied biological fields still remain unaware of how useful the study of gene expression heterogeneity patterns might be for the characterization of biological systems and/or processes. Largely based on the modulator effect chromatin compaction has for gene expression heterogeneity and the extensive changes in chromatin compaction known to occur for specialized cells that are naturally or artificially induced to revert to less specialized states or dedifferentiate, I recently hypothesized that processes that concur with cell dedifferentiation would show an extensive reduction in gene expression heterogeneity. The confirmation of the existence of such trend could be of wide interest because of the biomedical and biotechnological relevance of cell dedifferentiation-based processes, i.e., regenerative development, cancer, human induced pluripotent stem cells, or plant somatic embryogenesis. Here, I report the first empirical evidence consistent with the existence of an extensive reduction in gene expression heterogeneity for processes that concur with cell dedifferentiation by analyzing transcriptome dynamics along forearm regenerative development in Ambystoma mexicanum or axolotl. Also, I briefly discuss on the utility of the study of gene expression heterogeneity dynamics might have for the characterization of cell dedifferentiation-based processes, and the engineering of tools that afforded better monitoring and modulating such processes. Finally, I reflect on how a transitional reduction in gene expression heterogeneity for dedifferentiated cells can promote a long-term increase in phenotypic heterogeneity following cell dedifferentiation with potential adverse effects for biomedical and biotechnological applications. PMID:29134148

Dicty_cDB: Contig-U11847-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available 00512_699( AE000512 |pid:none) Thermotoga maritima MSB8, comple... 42 0.065 JC2551( JC2551 ) tropomyosin alpha chain - axolotl...AltName: Full=Myosin heavy chai... 42 0.11 JC6199( JC6199 ) alpha-tropomyosin S-1 - axolotl &U33450_1(U33450

Intracellular Signalling in Retinal Ischemia

Science.gov (United States)

1990-07-01

spatial buffering capacity of the Miller cells of axolotl retina indicated that gap junctional coupling can increase the capacity of the Muller cell to...1974. 27 33. Mobbs P, Brew H, and Atwell D: A quantitative analysis of glial cell coupling in the retina of the axolotl (Ambystoma mexicanum). Brain

Electrically Mediated Trauma Repair

Science.gov (United States)

1994-07-07

potential (TEP) (Johnston and Hoshiko, 1971; Rick et al., 1988; Shi and Borgens, 1994). In the axolotl neurulae, a gradient of 10 - 20 mV/mm is...reach 60-80 mV/mm. A more shallow voltage gradient is observed in the transverse plane of axolotl neurulae associated with mirror image outwardly

Functional characterization of the vertebrate primary ureter: Structure and ion transport mechanisms of the pronephric duct in axolotl larvae (Amphibia

Directory of Open Access Journals (Sweden)

Prehn Lea R

2010-05-01

Full Text Available Abstract Background Three kidney systems appear during vertebrate development: the pronephroi, mesonephroi and metanephroi. The pronephric duct is the first or primary ureter of these kidney systems. Its role as a key player in the induction of nephrogenic mesenchyme is well established. Here we investigate whether the duct is involved in urine modification using larvae of the freshwater amphibian Ambystoma mexicanum (axolotl as model. Results We investigated structural as well as physiological properties of the pronephric duct. The key elements of our methodology were: using histology, light and transmission electron microscopy as well as confocal laser scanning microscopy on fixed tissue and applying the microperfusion technique on isolated pronephric ducts in combination with single cell microelectrode impalements. Our data show that the fully differentiated pronephric duct is composed of a single layered epithelium consisting of one cell type comparable to the principal cell of the renal collecting duct system. The cells are characterized by a prominent basolateral labyrinth and a relatively smooth apical surface with one central cilium. Cellular impalements demonstrate the presence of apical Na+ and K+ conductances, as well as a large K+ conductance in the basolateral cell membrane. Immunolabeling experiments indicate heavy expression of Na+/K+-ATPase in the basolateral labyrinth. Conclusions We propose that the pronephric duct is important for the subsequent modification of urine produced by the pronephros. Our results indicate that it reabsorbs sodium and secretes potassium via channels present in the apical cell membrane with the driving force for ion movement provided by the Na+/K+ pump. This is to our knowledge the first characterization of the pronephric duct, the precursor of the collecting duct system, which provides a model of cell structure and basic mechanisms for ion transport. Such information may be important in understanding

Senescence gives insights into the morphogenetic evolution of anamniotes

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Éric Villiard

2017-06-01

Full Text Available Senescence represents a mechanism to avoid undesired cell proliferation that plays a role in tumor suppression, wound healing and embryonic development. In order to gain insight on the evolution of senescence, we looked at its presence in developing axolotls (urodele amphibians and in zebrafish (teleost fish, which are both anamniotes. Our data indicate that cellular senescence is present in various developing structures in axolotls (pronephros, olfactory epithelium of nerve fascicles, lateral organs, gums and in zebrafish (epithelium of the yolk sac and in the lower part of the gut. Senescence was particularly associated with transient structures (pronephros in axolotls and yolk sac in zebrafish suggesting that it may play a role in the elimination of these tissues. Our data supports the notion that cellular senescence evolved early in vertebrate evolution to influence embryonic development.

Anatomy of the pectoral and forelimb muscles of wildtype and green fluorescent protein-transgenic axolotls and comparison with other tetrapods including humans: a basis for regenerative, evolutionary and developmental studies

Science.gov (United States)

Diogo, R; Tanaka, E M

2012-01-01

The axolotl Ambystoma mexicanum is one of the most used model organisms in evolutionary, developmental and regenerative studies, particularly because it can reconstitute a fully functional and complete forelimb/hindlimb. Surprisingly, there is no publication that describes all the pectoral and forelimb muscles of this species or provides a comparative framework between these muscles and those of other model organisms and of modern humans. In the present paper we describe and illustrate all these muscles in A. mexicanum and provide the first report about the myology of adults of a model organism that is based on analyses and dissections of both wildtype animals and transgenic animals that express green fluorescent protein (GFP) in muscle fibers. On the one hand, the inclusion of GFP-transgenic animals allows us to show the muscles as more commonly seen, and thus easier to understand, by current developmental and regenerative biologists. On the other hand, by including wildtype and GFP-transgenic animals and by visualizing these latter animals with and without a simultaneous transmission laser light, we were able to obtain a more complete and clearer understanding of the exact limit of the fleshy and tendinous parts of the muscles and their specific connections with the skeletal elements. This in turn allowed us to settle some controversies in previous anatomical and comparative studies. As most developmental, regenerative and evolutionary biologists are interested in comparing their observations of A. mexicanum with observations in other model organisms, and ultimately in using this information to increase the understanding of human evolution and medicine, we also provide tables showing the homologies between the pectoral and forelimb muscles of axolotls, of model organisms such as mice, frogs and chicken, and of Homo sapiens. An example illustrating the outcomes of using our methodology and of our observations is that they revealed that, contrary to what is often

Anatomy of the pectoral and forelimb muscles of wildtype and green fluorescent protein-transgenic axolotls and comparison with other tetrapods including humans: a basis for regenerative, evolutionary and developmental studies.

Science.gov (United States)

Diogo, R; Tanaka, E M

2012-12-01

The axolotl Ambystoma mexicanum is one of the most used model organisms in evolutionary, developmental and regenerative studies, particularly because it can reconstitute a fully functional and complete forelimb/hindlimb. Surprisingly, there is no publication that describes all the pectoral and forelimb muscles of this species or provides a comparative framework between these muscles and those of other model organisms and of modern humans. In the present paper we describe and illustrate all these muscles in A. mexicanum and provide the first report about the myology of adults of a model organism that is based on analyses and dissections of both wildtype animals and transgenic animals that express green fluorescent protein (GFP) in muscle fibers. On the one hand, the inclusion of GFP-transgenic animals allows us to show the muscles as more commonly seen, and thus easier to understand, by current developmental and regenerative biologists. On the other hand, by including wildtype and GFP-transgenic animals and by visualizing these latter animals with and without a simultaneous transmission laser light, we were able to obtain a more complete and clearer understanding of the exact limit of the fleshy and tendinous parts of the muscles and their specific connections with the skeletal elements. This in turn allowed us to settle some controversies in previous anatomical and comparative studies. As most developmental, regenerative and evolutionary biologists are interested in comparing their observations of A. mexicanum with observations in other model organisms, and ultimately in using this information to increase the understanding of human evolution and medicine, we also provide tables showing the homologies between the pectoral and forelimb muscles of axolotls, of model organisms such as mice, frogs and chicken, and of Homo sapiens. An example illustrating the outcomes of using our methodology and of our observations is that they revealed that, contrary to what is often

An Aryl Hydrocarbon Receptor from the Salamander Ambystoma mexicanum Exhibits Low Sensitivity to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

Science.gov (United States)

Shoots, Jenny; Fraccalvieri, Domenico; Franks, Diana G; Denison, Michael S; Hahn, Mark E; Bonati, Laura; Powell, Wade H

2015-06-02

Structural features of the aryl hydrocarbon receptor (AHR) can underlie species- and population-specific differences in its affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These differences often explain variations in TCDD toxicity. Frogs are relatively insensitive to dioxin, and Xenopus AHRs bind TCDD with low affinity. Weak TCDD binding results from the combination of three residues in the ligand-binding domain: A354 and A370, and N325. Here we sought to determine whether this mechanism of weak TCDD binding is shared by other amphibian AHRs. We isolated an AHR cDNA from the Mexican axolotl (Ambystoma mexicanum). The encoded polypeptide contains identical residues at positions that confer low TCDD affinity to X. laevis AHRs (A364, A380, and N335), and homology modeling predicts they protrude into the binding cavity. Axolotl AHR bound one-tenth the TCDD of mouse AHR in velocity sedimentation analysis, and in transactivation assays, the EC50 for TCDD was 23 nM, similar to X. laevis AHR1β (27 nM) and greater than AHR containing the mouse ligand-binding domain (0.08 nM). Sequence, modeled structure, and function indicate that axolotl AHR binds TCDD weakly, predicting that A. mexicanum lacks sensitivity toTCDD toxicity. We hypothesize that this characteristic of axolotl and Xenopus AHRs arose in a common ancestor of the Caudata and Anura.

Virtual Genome Walking across the 32 Gb Ambystoma mexicanum genome; assembling gene models and intronic sequence.

Science.gov (United States)

Evans, Teri; Johnson, Andrew D; Loose, Matthew

2018-01-12

Large repeat rich genomes present challenges for assembly using short read technologies. The 32 Gb axolotl genome is estimated to contain ~19 Gb of repetitive DNA making an assembly from short reads alone effectively impossible. Indeed, this model species has been sequenced to 20× coverage but the reads could not be conventionally assembled. Using an alternative strategy, we have assembled subsets of these reads into scaffolds describing over 19,000 gene models. We call this method Virtual Genome Walking as it locally assembles whole genome reads based on a reference transcriptome, identifying exons and iteratively extending them into surrounding genomic sequence. These assemblies are then linked and refined to generate gene models including upstream and downstream genomic, and intronic, sequence. Our assemblies are validated by comparison with previously published axolotl bacterial artificial chromosome (BAC) sequences. Our analyses of axolotl intron length, intron-exon structure, repeat content and synteny provide novel insights into the genic structure of this model species. This resource will enable new experimental approaches in axolotl, such as ChIP-Seq and CRISPR and aid in future whole genome sequencing efforts. The assembled sequences and annotations presented here are freely available for download from https://tinyurl.com/y8gydc6n . The software pipeline is available from https://github.com/LooseLab/iterassemble .

Hibridez y metamorfosis en Juan Villoro: el universo mágico-mitológico del ajolote

Directory of Open Access Journals (Sweden)

An Van Hecke

2014-06-01

This study focuses on the symbolic meanings of the axolotl in Juan Villoro’s novel Materia dispuesta and the way the author approaches the concept of identity from both, the adolescent protagonist and Mexicans in general. This novel will be read in light of Roger Bartra’s essay, La jaula de la melancolía. Bartra uses the metaphor of the axolotl to analyse the concept of metamorphosis in the Mexican identity. Thereafter, Villoro’s novel will be compared with Julio Cortazar’s short story “Axolotl”, and Octavio Paz’s poem “Salamandra”. This comparison will allow to amplify the perspective and deepen the analysis of this hybrid animal by Villoro. The study of the axolotl demonstrates that Villoro revives the ancient aztec mythology about this amphibian, that was the representation of the god Xolotl, and that he introduces at the same time new meanings. It is in that combination of both perspectives that the richness of Villoro’s literary universe will be revealed.

Radioautographic investigation of retinal growth in mature amphibians

International Nuclear Information System (INIS)

Svistunov, S.A.; Mitashov, V.I.

1986-01-01

Growth of the retina was studied in mature intact amphibians, tritons, axolotls, ambystomas and clawed frogs, for six months using multiple injection of 3 H-thymidine. It was established that the source of replenishment of the retina by new cells is its terminal zone in all animals investigated. This is attested to by the gradual migration of labeled cells from the growth zone into differentiated layers of the retina. The most intensely labeled cells occupy a distal position relative to other labeled cells, therefore marking the boundary between the initial part of the retina, not containing labeled nuclei, and the part being augmented. For each species studied, a level of proliferative activity is characteristic for cells of the terminal zone, which decreases in the order axolotl-clawed frog-triton -ambystoma. In the axolotl and additional growth zone is noted in the retina, in addition to the terminal, which is located in the area of the unclosed section of the embryonic fissure. Results obtained serve as a basis for the regenerative potentials of eye tissues revealed previously in these amphibian species

Immunocytochemical localization and immunochemical characterization of an insulin-related peptide in the pancreas of the urodele amphibian, Ambystoma mexicanum

DEFF Research Database (Denmark)

Hansen, G N; Hansen, B L; Jørgensen, P N

1989-01-01

The pancreas of the axolotl, Ambystoma mexicanum, was investigated by immunocytochemical methods for the presence of immunoreactivity to a number of antisera raised against mammalian insulins. All anti-insulin antisera tested revealed substantial amounts of reaction products confined solely...

Enzyme clusters during the metamorphic period of Ambystoma mexicanum: role of thyroid hormone

NARCIS (Netherlands)

Lamers, W. H.; Mooren, P. G.; de Graaf, A.

1982-01-01

Enzyme activities and DNA content have been measure in axolotl liver during the metamorphic period (4-8 months after spawning). Three different types of enzyme activity profiles were observed. In the type I profile (carbamoyl-phosphate synthase, arginase, ornithine transcarbamoylase, and glutamate

Development and regulation of response properties in spinal cord motoneurons

DEFF Research Database (Denmark)

Perrier, J F; Hounsgaard, J

2000-01-01

vertebrates in terms of both phylogeny and ontogeny. Spinal motoneurons in adults are remarkably similar in many respects ranging from the resting membrane potential to pacemaker properties. Apart from the axolotls, spinal motoneurons from all species investigated have latent intrinsic response properties...

Cartilage and bone cells do not participate in skeletal regeneration in Ambystoma mexicanum limbs.

Science.gov (United States)

McCusker, Catherine D; Diaz-Castillo, Carlos; Sosnik, Julian; Q Phan, Anne; Gardiner, David M

2016-08-01

The Mexican Axolotl is one of the few tetrapod species that is capable of regenerating complete skeletal elements in injured adult limbs. Whether the skeleton (bone and cartilage) plays a role in the patterning and contribution to the skeletal regenerate is currently unresolved. We tested the induction of pattern formation, the effect on cell proliferation, and contributions of skeletal tissues (cartilage, bone, and periosteum) to the regenerating axolotl limb. We found that bone tissue grafts from transgenic donors expressing GFP fail to induce pattern formation and do not contribute to the newly regenerated skeleton. Periosteum tissue grafts, on the other hand, have both of these activities. These observations reveal that skeletal tissue does not contribute to the regeneration of skeletal elements; rather, these structures are patterned by and derived from cells of non-skeletal connective tissue origin. Copyright © 2016 Elsevier Inc. All rights reserved.

TRACKING STEM CELLS IN AN INHERENTLY REGENRATIVE ENVIRONMENT

DEFF Research Database (Denmark)

Lauridsen, Henrik; Foldager, Casper Bindzus; Hagensen, Mette

2012-01-01

of such therapies. The objective of this study was to non-invasively evaluate regeneration over time in a truly regenerative process, the regeneration of an axolotl limb, employing superparamagnetic iron oxide particles (SPIO) contrast agents for stem cell tracking in MRI. Materials and Methods: Amputation of one...... in conjugation with the transfection agent poly-L-lysin (PLL) was tested on cultures of axolotl blastema cells from 7 animals in vitro. PicoGreen-DNA quantification following 3 weeks of culturing was performed to quantify cell viability. MRI-tracking of SPIO labelled blastema cells in the regenerating limb of 5....... Results: SPIO labelling with neither VSOP-C200, Resovist nor Resovist/PLL had any significant effect on blastema cell viability in vitro. Labelled tissue was clearly detectable in vivo 49 days after amputation using MRI (Fig. 1) and a significant decline in signal intensity of labelled limbs versus sham...

Cranial muscle development in the model organism ambystoma mexicanum: implications for tetrapod and vertebrate comparative and evolutionary morphology and notes on ontogeny and phylogeny.

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Ziermann, Janine M; Diogo, Rui

2013-07-01

There is still confusion about the homology of several cranial muscles in salamanders with those of other vertebrates. This is true, in part, because of the fact that many muscles present in early ontogeny of amphibians disappear during development and specifically during metamorphosis. Resolving this confusion is important for the understanding of the comparative and evolutionary morphology of vertebrates and tetrapods because amphibians are the phylogenetically most plesiomorphic tetrapods, concerning for example their myology, and include two often used model organisms, Xenopus laevis (anuran) and Ambystoma mexicanum (urodele). Here we provide the first detailed report of the cranial muscle development in axolotl from early ontogenetic stages to the adult stage. We describe different and complementary types of general muscle morphogenetic gradients in the head: from anterior to posterior, from lateral to medial, and from origin to insertion. Furthermore, even during the development of neotenic salamanders such as axolotls, various larval muscles become indistinct, contradicting the commonly accepted view that during ontogeny the tendency is mostly toward the differentiation of muscles. We provide an updated comparison between these muscles and the muscles of other vertebrates, a discussion of the homologies and evolution, and show that the order in which the muscles appear during axolotl ontogeny is in general similar to their appearance in phylogeny (e.g. differentiation of adductor mandibulae muscles from one anlage to four muscles), with only a few remarkable exceptions, as for example the dilatator laryngis that appears evolutionary later but in the development before the intermandibularis. Copyright © 2013 Wiley Periodicals, Inc.

Visualisation of animal anatomy using MRI and CT

DEFF Research Database (Denmark)

Lauridsen, Henrik; Hansen, Kasper; Pedersen, Michael

imaging (MRI) and CT. Various species (tarantula, horseshoe crab, carp, haddock, lungfish, axolotl) were subjected to multi-slice MRI and CT protocols to produce 2D images of body slices, followed by volume rendering producing 3D digital models of animal anatomy with applications for visualising specific...

Evolution of the head-trunk interface in tetrapod vertebrates

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Sefton, Elizabeth M; Bhullar, Bhart-Anjan S; Mohaddes, Zahra; Hanken, James

2016-01-01

Vertebrate neck musculature spans the transition zone between head and trunk. The extent to which the cucullaris muscle is a cranial muscle allied with the gill levators of anamniotes or is instead a trunk muscle is an ongoing debate. Novel computed tomography datasets reveal broad conservation of the cucullaris in gnathostomes, including coelacanth and caecilian, two sarcopterygians previously thought to lack it. In chicken, lateral plate mesoderm (LPM) adjacent to occipital somites is a recently identified embryonic source of cervical musculature. We fate-map this mesoderm in the axolotl (Ambystoma mexicanum), which retains external gills, and demonstrate its contribution to posterior gill-levator muscles and the cucullaris. Accordingly, LPM adjacent to the occipital somites should be regarded as posterior cranial mesoderm. The axial position of the head-trunk border in axolotl is congruent between LPM and somitic mesoderm, unlike in chicken and possibly other amniotes. DOI: http://dx.doi.org/10.7554/eLife.09972.001 PMID:27090084

The MHC molecules of nonmammalian vertebrates

DEFF Research Database (Denmark)

Kaufman, J; Skjoedt, K; Salomonsen, J

1990-01-01

class II distribution. The axolotl has a very poor immune response (as though there are no helper T cells), a wide class II distribution and, for most animals, no cell surface class I molecule. It would be enlightening to understand both the mechanisms for the regulation of the MHC molecules during...

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

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

2010-01-01

In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy. Copyright © 2010 Elsevier Inc. All rights reserved.

Spatial distribution of prominin-1 (CD133-positive cells within germinative zones of the vertebrate brain.

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József Jászai

Full Text Available In mammals, embryonic neural progenitors as well as adult neural stem cells can be prospectively isolated based on the cell surface expression of prominin-1 (CD133, a plasma membrane glycoprotein. In contrast, characterization of neural progenitors in non-mammalian vertebrates endowed with significant constitutive neurogenesis and inherent self-repair ability is hampered by the lack of suitable cell surface markers. Here, we have investigated whether prominin-1-orthologues of the major non-mammalian vertebrate model organisms show any degree of conservation as for their association with neurogenic geminative zones within the central nervous system (CNS as they do in mammals or associated with activated neural progenitors during provoked neurogenesis in the regenerating CNS.We have recently identified prominin-1 orthologues from zebrafish, axolotl and chicken. The spatial distribution of prominin-1-positive cells--in comparison to those of mice--was mapped in the intact brain in these organisms by non-radioactive in situ hybridization combined with detection of proliferating neural progenitors, marked either by proliferating cell nuclear antigen or 5-bromo-deoxyuridine. Furthermore, distribution of prominin-1 transcripts was investigated in the regenerating spinal cord of injured axolotl.Remarkably, a conserved association of prominin-1 with germinative zones of the CNS was uncovered as manifested in a significant co-localization with cell proliferation markers during normal constitutive neurogenesis in all species investigated. Moreover, an enhanced expression of prominin-1 became evident associated with provoked, compensatory neurogenesis during the epimorphic regeneration of the axolotl spinal cord. Interestingly, significant prominin-1-expressing cell populations were also detected at distinct extraventricular (parenchymal locations in the CNS of all vertebrate species being suggestive of further, non-neurogenic neural function

Amphioxus and lamprey AP-2 genes: implications for neural crest evolution and migration patterns

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Meulemans, Daniel; Bronner-Fraser, Marianne

2002-01-01

The neural crest is a uniquely vertebrate cell type present in the most basal vertebrates, but not in cephalochordates. We have studied differences in regulation of the neural crest marker AP-2 across two evolutionary transitions: invertebrate to vertebrate, and agnathan to gnathostome. Isolation and comparison of amphioxus, lamprey and axolotl AP-2 reveals its extensive expansion in the vertebrate dorsal neural tube and pharyngeal arches, implying co-option of AP-2 genes by neural crest cells early in vertebrate evolution. Expression in non-neural ectoderm is a conserved feature in amphioxus and vertebrates, suggesting an ancient role for AP-2 genes in this tissue. There is also common expression in subsets of ventrolateral neurons in the anterior neural tube, consistent with a primitive role in brain development. Comparison of AP-2 expression in axolotl and lamprey suggests an elaboration of cranial neural crest patterning in gnathostomes. However, migration of AP-2-expressing neural crest cells medial to the pharyngeal arch mesoderm appears to be a primitive feature retained in all vertebrates. Because AP-2 has essential roles in cranial neural crest differentiation and proliferation, the co-option of AP-2 by neural crest cells in the vertebrate lineage was a potentially crucial event in vertebrate evolution.

FGF and BMP derived from dorsal root ganglia regulate blastema induction in limb regeneration in Ambystoma mexicanum.

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Satoh, Akira; Makanae, Aki; Nishimoto, Yurie; Mitogawa, Kazumasa

2016-09-01

Urodele amphibians have a remarkable organ regeneration ability that is regulated by neural inputs. The identification of these neural inputs has been a challenge. Recently, Fibroblast growth factor (Fgf) and Bone morphogenic protein (Bmp) were shown to substitute for nerve functions in limb and tail regeneration in urodele amphibians. However, direct evidence of Fgf and Bmp being secreted from nerve endings and regulating regeneration has not yet been shown. Thus, it remained uncertain whether they were the nerve factors responsible for successful limb regeneration. To gather experimental evidence, the technical difficulties involved in the usage of axolotls had to be overcome. We achieved this by modifying the electroporation method. When Fgf8-AcGFP or Bmp7-AcGFP was electroporated into the axolotl dorsal root ganglia (DRG), GFP signals were detectable in the regenerating limb region. This suggested that Fgf8 and Bmp7 synthesized in neural cells in the DRG were delivered to the limbs through the long axons. Further knockdown experiments with double-stranded RNA interference resulted in impaired limb regeneration ability. These results strongly suggest that Fgf and Bmp are the major neural inputs that control the organ regeneration ability. Copyright © 2016 Elsevier Inc. All rights reserved.

Open Source Software Projects Needing Security Investments

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2015-06-19

modtls, BouncyCastle, gpg, otr, axolotl. 7. Static analyzers: Clang, Frama-C. 8. Nginx. 9. OpenVPN . It was noted that the funding model may be similar...to OpenSSL, where consulting funds the company. It was also noted that OpenVPN needs to correctly use OpenSSL in order to be secure, so focusing on...Dovecot 4. Other high-impact network services: OpenSSH, OpenVPN , BIND, ISC DHCP, University of Delaware NTPD 5. Core infrastructure data parsers

Irradiation inhibits the regeneration of aneurogenic limbs

International Nuclear Information System (INIS)

Wallace, H.; Maden, M.

1976-01-01

The developing arms of axolotl larvae from the 2-digit stage onward and the aneurogenic arms of surgically denervated larvae maintained in parabiosis are able to regenerate after amputation. Such regeneration is uniformly inhibited by local irradiation of the arm, whether innervated or not. This demonstration refutes a recent hypothesis that x-rays interfere with a special activity of nerves required for regeneration, and supports the earlier concept that x-rays act directly on those cells which must proliferate to form the regenerated tissues

Carbamoyl-phosphate synthase (ammonia) of rat and axolotl liver: determination of immunological cross-reactivity without purification of the axolotl enzyme

NARCIS (Netherlands)

Lamers, W. H.; de Graaf, A.; Mooren, P. G.; Moorman, A. F.; Charles, R.

1982-01-01

A method has been developed to establish the degree of cross-reactivity of an antiserum raised against purified carbamoyl-phosphate synthase (ammonia) from adult rat liver, toward a homologous enzyme from another species without purification of the latter enzyme. For that purpose the ratio between

entre la pirámide y el axolotl

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Paula Andrea Marín Colorado

2006-01-01

Full Text Available En este artículo se presenta un acercamiento a la novela Materia dispuesta, del escritor mexicano Juan Villoro. En esta aproximación analítica se pretende dilucidar la conexión entre la obra literaria y el medio en el que se gesta, así como con el momento histórico recreado en la anécdota de la novela (el terremoto ocurrido en Ciudad de México en 1985; lo anterior con el objetivo de establecer la propuesta estética del autor en esta obra y su relación con la visión que construye Materia dispuesta sobre los procesos de Modernidad y Postmodernidad en Latinoamérica. Para tal propósito se recurrirá a las investigaciones literarias y sociológicas de Mijail Bajtín, Zigmunt Bauman, Carlos Monsiváis y Octavio Paz, entre otros.

Retrotransposon long interspersed nucleotide element-1 (LINE-1) is activated during salamander limb regeneration

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Zhu, Wei; Kuo, Dwight; Nathanson, Jason; Satoh, Akira; Pao, Gerald M.; Yeo, Gene W.; Bryant, Susan V.; Voss, S. Randal; Gardiner, David M.; Hunter, Tony

2012-01-01

Salamanders possess an extraordinary capacity for tissue and organ regeneration when compared to mammals. In our effort to characterize the unique transcriptional fingerprint emerging during the early phase of salamander limb regeneration, we identified transcriptional activation of some germline-specific genes within the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells into a germline-like state. In this work, we focus on one of these genes, the long interspersed nucleotide element-1 (LINE-1) retrotransposon, which is usually active in germ cells and silent in most of the somatic tissues in other organisms. LINE-1 was found to be dramatically upregulated during regeneration. In addition, higher genomic LINE-1 content was also detected in the limb regenerate when compared to that before amputation indicating that LINE-1 retrotransposition is indeed active during regeneration. Active LINE-1 retrotransposition has been suggested to have a potentially deleterious impact on genomic integrity. Silencing of activated LINE-1 by small RNAs has been reported to be part of the machinery aiming to maintain genomic integrity. Indeed, we were able to identify putative LINE-1-related piRNAs in the limb blastema. Transposable element-related piRNAs have been identified frequently in the germline in other organisms. Thus, we present here a scenario in which a unique germline-like state is established during axolotl limb regeneration, and the re-activation of LINE-1 may serve as a marker for cellular dedifferentiation in the early-stage of limb regeneration. PMID:22913491

Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration

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Bryant Susan V

2009-01-01

Full Text Available Abstract Background Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR and denervated (DL forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. Results Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa. Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST contigs from the Ambystoma EST database more than doubled (3935 to 9411 the number of non-redundant human-A. mexicanum orthologous sequences. Conclusion Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

Precise control of miR-125b levels is required to create a regeneration-permissive environment after spinal cord injury: a cross-species comparison between salamander and rat

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Juan Felipe Diaz Quiroz

2014-06-01

Full Text Available Most spinal cord injuries lead to permanent paralysis in mammals. By contrast, the remarkable regenerative abilities of salamanders enable full functional recovery even from complete spinal cord transections. The molecular differences underlying this evolutionary divergence between mammals and amphibians are poorly understood. We focused on upstream regulators of gene expression as primary entry points into this question. We identified a group of microRNAs (miRNAs that are conserved between the Mexican axolotl salamander (Ambystoma mexicanum and mammals but show marked cross-species differences in regulation patterns following spinal cord injury. We found that precise post-injury levels of one of these miRNAs (miR-125b is essential for functional recovery, and guides correct regeneration of axons through the lesion site in a process involving the direct downstream target Sema4D in axolotls. Translating these results to a mammalian model, we increased miR-125b levels in the rat through mimic treatments following spinal cord transection. These treatments downregulated Sema4D and other glial-scar-related genes, and enhanced the animal’s functional recovery. Our study identifies a key regulatory molecule conserved between salamander and mammal, and shows that the expression of miR-125b and Sema4D must be carefully controlled in the right cells at the correct level to promote regeneration. We also show that these molecular components of the salamander’s regeneration-permissive environment can be experimentally harnessed to improve treatment outcomes for mammalian spinal cord injuries.

Ectopic Fgf signaling induces the intercalary response in developing chicken limb buds.

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Makanae, Aki; Satoh, Akira

2018-01-01

Intercalary pattern formation is an important regulatory step in amphibian limb regeneration. Amphibian limb regeneration is composed of multiple steps, including wounding, blastema formation, and intercalary pattern formation. Attempts have been made to transfer insights from regeneration-competent animals to regeneration-incompetent animalsat each step in the regeneration process. In the present study, we focused on the intercalary mechanism in chick limb buds. In amphibian limb regeneration, a proximodistal axis is organized as soon as a regenerating blastema is induced. Intermediate structures are subsequently induced (intercalated) between the established proximal and distal identities. Intercalary tissues are derived from proximal tissues. Fgf signaling mediates the intercalary response in amphibian limb regeneration. We attempted to transfer insights into intercalary regeneration from amphibian models to the chick limb bud. The zeugopodial part was dissected out, and the distal and proximal parts were conjunct at st. 24. Delivering ectopic Fgf2 + Fgf8 between the distal and proximal parts resulted in induction of zeugopodial elements. Examination of HoxA11 expression, apoptosis, and cell proliferation provides insights to compare with those in the intercalary mechanism of amphibian limb regeneration. Furthermore, the cellular contribution was investigated in both the chicken intercalary response and that of axolotl limb regeneration. We developed new insights into cellular contribution in amphibian intercalary regeneration, and found consistency between axolotl and chicken intercalary responses. Our findings demonstrate that the same principal of limb regeneration functions between regeneration-competent and -incompetent animals. In this context, we propose the feasibility of the induction of the regeneration response in amniotes.

Buoyancy disorders in pet axolotls Ambystoma mexicanum: three cases.

Science.gov (United States)

Takami, Yoshinori; Une, Yumi

2018-01-31

As far as we are aware, there are no previous reports on the pathologic conditions of buoyancy disorders in Ambystoma mexicanum. Herein, we describe various clinical test results, clinical outcomes, and the pathological findings of an experimental pneumonectomy procedure in 3 A. mexicanum exhibiting abnormal buoyancy. The 3 pet A. mexicanum were adults, and their respective ages and body weights were 1, 5, and 6 yr and 48, 55, and 56 g. Two of these cases were confirmed via radiographic examination to have free air within the body cavity, and all 3 cases were found via ultrasonography to have an acoustic shadow within the body cavity and were diagnosed with pneumocoelom. Lung perforations were detected macroscopically in 2 of the cases, and all 3 cases had fibrosis in the caudal ends of the lungs. Removal of the lung lesions eliminated the abnormal buoyancy in all 3 cases. We concluded that air had leaked into the body cavity from the lungs, and we propose that lung lesions are an important cause of buoyancy disorders in A. mexicanum.

Homology of the cranial vault in birds: new insights based on embryonic fate-mapping and character analysis

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Maddin, Hillary C.; Piekarski, Nadine; Sefton, Elizabeth M.; Hanken, James

2016-08-01

Bones of the cranial vault appear to be highly conserved among tetrapod vertebrates. Moreover, bones identified with the same name are assumed to be evolutionarily homologous. However, recent developmental studies reveal a key difference in the embryonic origin of cranial vault bones between representatives of two amniote lineages, mammals and birds, thereby challenging this view. In the mouse, the frontal is derived from cranial neural crest (CNC) but the parietal is derived from mesoderm, placing the CNC-mesoderm boundary at the suture between these bones. In the chicken, this boundary is located within the frontal. This difference and related data have led several recent authors to suggest that bones of the avian cranial vault are misidentified and should be renamed. To elucidate this apparent conflict, we fate-mapped CNC and mesoderm in axolotl to reveal the contributions of these two embryonic cell populations to the cranial vault in a urodele amphibian. The CNC-mesoderm boundary in axolotl is located between the frontal and parietal bones, as in the mouse but unlike the chicken. If, however, the avian frontal is regarded instead as a fused frontal and parietal (i.e. frontoparietal) and the parietal as a postparietal, then the cranial vault of birds becomes developmentally and topologically congruent with those of urodeles and mammals. This alternative hypothesis of cranial vault homology is also phylogenetically consistent with data from the tetrapod fossil record, where frontal, parietal and postparietal bones are present in stem lineages of all extant taxa, including birds. It further implies that a postparietal may be present in most non-avian archosaurs, but fused to the parietal or supraoccipital as in many extant mammals.

Regulation of Injury-Induced Ovarian Regeneration by Activation of Oogonial Stem Cells.

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Erler, Piril; Sweeney, Alexandra; Monaghan, James R

2017-01-01

Some animals have the ability to generate large numbers of oocytes throughout life. This raises the question whether persistent adult germline stem cell populations drive continuous oogenesis and whether they are capable of mounting a regenerative response after injury. Here we demonstrate the presence of adult oogonial stem cells (OSCs) in the adult axolotl salamander ovary and show that ovarian injury induces OSC activation and functional regeneration of the ovaries to reproductive capability. Cells that have morphological similarities to germ cells were identified in the developing and adult ovaries via histological analysis. Genes involved in germ cell maintenance including Vasa, Oct4, Sox2, Nanog, Bmp15, Piwil1, Piwil2, Dazl, and Lhx8 were expressed in the presumptive OSCs. Colocalization of Vasa protein with H3 mitotic marker showed that both oogonial and spermatogonial adult stem cells were mitotically active. Providing evidence of stemness and viability of adult OSCs, enhanced green fluorescent protein (EGFP) adult OSCs grafted into white juvenile host gonads gave rise to EGFP OSCs, and oocytes. Last, the axolotl ovaries completely regenerated after partial ovariectomy injury. During regeneration, OSC activation resulted in rapid differentiation into new oocytes, which was demonstrated by Vasa + /BrdU + coexpression. Furthermore, follicle cell proliferation promoted follicle maturation during ovarian regeneration. Overall, these results show that adult oogenesis occurs via proliferation of endogenous OSCs in a tetrapod and mediates ovarian regeneration. This study lays the foundations to elucidate mechanisms of ovarian regeneration that will assist regenerative medicine in treating premature ovarian failure and reduced fertility. Stem Cells 2017;35:236-247. © 2016 AlphaMed Press.

Distinct and conserved prominin-1/CD133-positive retinal cell populations identified across species.

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József Jászai

2011-03-01

Full Text Available Besides being a marker of various somatic stem cells in mammals, prominin-1 (CD133 plays a role in maintaining the photoreceptor integrity since mutations in the PROM1 gene are linked with retinal degeneration. In spite of that, little information is available regarding its distribution in eyes of non-mammalian vertebrates endowed with high regenerative abilities. To address this subject, prominin-1 cognates were isolated from axolotl, zebrafish and chicken, and their retinal compartmentalization was investigated and compared to that of their mammalian orthologue. Interestingly, prominin-1 transcripts--except for the axolotl--were not strictly restricted to the outer nuclear layer (i.e., photoreceptor cells, but they also marked distinct subdivisions of the inner nuclear layer (INL. In zebrafish, where the prominin-1 gene is duplicated (i.e., prominin-1a and prominin-1b, a differential expression was noted for both paralogues within the INL being localized either to its vitreal or scleral subdivision, respectively. Interestingly, expression of prominin-1a within the former domain coincided with Pax-6-positive cells that are known to act as progenitors upon injury-induced retino-neurogenesis. A similar, but minute population of prominin-1-positive cells located at the vitreal side of the INL was also detected in developing and adult mice. In chicken, however, prominin-1-positive cells appeared to be aligned along the scleral side of the INL reminiscent of zebrafish prominin-1b. Taken together our data indicate that in addition to conserved expression of prominin-1 in photoreceptors, significant prominin-1-expressing non-photoreceptor retinal cell populations are present in the vertebrate eye that might represent potential sources of stem/progenitor cells for regenerative therapies.

Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole.

Science.gov (United States)

Taniguchi, Yuka; Watanabe, Kenji; Mochii, Makoto

2014-06-18

Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans.

The urodelean Mauthner cell. Morphology of the afferent synapses to the M-cell of larval Salamandra salamandra

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Cioni, C.; De Palma, F.; De Vito, L.; Stefanelli, A. [Rome, Univ. (Italy). Dipt. di Biologia Animale e dell`Uomo

1997-12-31

In the present work the fine morphology and the distribution of the afferent synapses to the Mauthner cell of larval Salamandra salamandra are described. The aim of the study is to characterize the synaptic bed in the larvae of this terrestrial salamander in order to compare it with that of larval axolotl and larval anurans. Four main types of afferent endings have been identified: myelinated club endings, round-vesicle end bulbs, flattened-vesicle end bulbs and spiral fibers endings. The M-cell afferent synaptology of larval stages of terrestrial amphibians is quite similar to that previously observed in larval stages of aquatic species. This fact can be related to the fundamental similarities between the larval lifestyles.

The urodelean Mauthner cell. Morphology of the afferent synapses to the M-cell of larval Salamandra salamandra

Energy Technology Data Exchange (ETDEWEB)

Cioni, C; De Palma, F; De Vito, L; Stefanelli, A [Rome, Univ. (Italy). Dipt. di Biologia Animale e dell` Uomo

1998-12-31

In the present work the fine morphology and the distribution of the afferent synapses to the Mauthner cell of larval Salamandra salamandra are described. The aim of the study is to characterize the synaptic bed in the larvae of this terrestrial salamander in order to compare it with that of larval axolotl and larval anurans. Four main types of afferent endings have been identified: myelinated club endings, round-vesicle end bulbs, flattened-vesicle end bulbs and spiral fibers endings. The M-cell afferent synaptology of larval stages of terrestrial amphibians is quite similar to that previously observed in larval stages of aquatic species. This fact can be related to the fundamental similarities between the larval lifestyles.

How x rays inhibit amphibian limb regeneration

International Nuclear Information System (INIS)

Maden, M.; Wallace, H.

1976-01-01

The effects of an inhibiting dose of 2,000 rad of x-rays on the regenerating limbs of axolotl larvae have been examined in a histological and cytological study. Particular attention was paid to the mitotic indices of normal and irradiated epidermal and blastemal cells. Both the characteristic pattern of epidermal mitotic stimulation which normally follows amputation and the later increase in blastemal mitoses are suppressed by irradiation. In most cells the effects are permanent, but in a small proportion a mitotic delay is induced and upon subsequent division chromosome damage in the form of micronuclei is revealed. Thus irradiated cells which do divide almost certainly die. These results are discussed in relation to other theories of x-ray inhibition of regeneration with particular reference to the view that irradiated cells can be reactivated

Collagen reconstitution is inversely correlated with induction of limb regeneration in Ambystoma mexicanum.

Science.gov (United States)

Satoh, Akira; Hirata, Ayako; Makanae, Aki

2012-03-01

Amphibians can regenerate missing body parts, including limbs. The regulation of collagen has been considered to be important in limb regeneration. Collagen deposition is suppressed during limb regeneration, so we investigated collagen deposition and apical epithelial cap (AEC) formation during axolotl limb regeneration. The accessory limb model (ALM) has been developed as an alternative model for studying limb regeneration. Using this model, we investigated the relationship between nerves, epidermis, and collagen deposition. We found that Sp-9, an AEC marker gene, was upregulated by direct interaction between nerves and epidermis. However, collagen deposition hindered this interaction, and resulted in the failure of limb regeneration. During wound healing, an increase in deposition of collagen caused a decrease in the blastema induction rate in ALM. Wound healing and limb regeneration are alternate processes.

Development and regulation of response properties in spinal cord motoneurons

DEFF Research Database (Denmark)

Perrier, J F; Hounsgaard, J

2000-01-01

The intrinsic response properties of spinal motoneurons determine how converging premotor neuronal input is translated into the final motor command transmitted to muscles. From the patchy data available it seems that these properties and their underlying currents are highly conserved in terrestrial...... vertebrates in terms of both phylogeny and ontogeny. Spinal motoneurons in adults are remarkably similar in many respects ranging from the resting membrane potential to pacemaker properties. Apart from the axolotls, spinal motoneurons from all species investigated have latent intrinsic response properties...... mediated by L-type Ca2+ channels. This mature phenotype is reached gradually during development through phases in which A-type potassium channels and T-type calcium channels are transiently expressed. The intrinsic response properties of mature spinal motoneurons are subject to short-term adjustments via...

Mobility narratives that move me

DEFF Research Database (Denmark)

Lysgaard, Jonas Andreasen; Kronlid, David

and fiction. By opposing the classic anthropocentric ontological split between human (discourse) and object, fiction and Reality, OOO opens up for a new understanding of the role of narratives as independent objects on the same scale as grains of sand, universities, axolotls and planets. In short...... of speed, acceleration, the rhythm of technogenic moving and mooring, which can be translated into an understanding of our own movements and moorings through life and how we engage with new things, such as mediating new information through a certain pace, rhythm, movement, acceleration, slowing down....... This fictive/Real example of a narrative object illustrates that regardless of the metaphysical status of what/who we encounter in education and throughout life, if this experience becomes meaningful to us, we are navigating around them, towards them, and enmesh with them in the same principle ways...

Gene expression patterns specific to the regenerating limb of the Mexican axolotl

Directory of Open Access Journals (Sweden)

James R. Monaghan

2012-07-01

Salamander limb regeneration is dependent upon tissue interactions that are local to the amputation site. Communication among limb epidermis, peripheral nerves, and mesenchyme coordinate cell migration, cell proliferation, and tissue patterning to generate a blastema, which will form missing limb structures. An outstanding question is how cross-talk between these tissues gives rise to the regeneration blastema. To identify genes associated with epidermis-nerve-mesenchymal interactions during limb regeneration, we examined histological and transcriptional changes during the first week following injury in the wound epidermis and subjacent cells between three injury types; 1 a flank wound on the side of the animal that will not regenerate a limb, 2 a denervated limb that will not regenerate a limb, and 3 an innervated limb that will regenerate a limb. Early, histological and transcriptional changes were similar between the injury types, presumably because a common wound-healing program is employed across anatomical locations. However, some transcripts were enriched in limbs compared to the flank and are associated with vertebrate limb development. Many of these genes were activated before blastema outgrowth and expressed in specific tissue types including the epidermis, peripheral nerve, and mesenchyme. We also identified a relatively small group of transcripts that were more highly expressed in innervated limbs versus denervated limbs. These transcripts encode for proteins involved in myelination of peripheral nerves, epidermal cell function, and proliferation of mesenchymal cells. Overall, our study identifies limb-specific and nerve-dependent genes that are upstream of regenerative growth, and thus promising candidates for the regulation of blastema formation.

Swept source optical coherence tomography of objects with arbitrary reflectivity profiles

Science.gov (United States)

Mezgebo, Biniyam; Nagib, Karim; Fernando, Namal; Kordi, Behzad; Sherif, Sherif

2018-03-01

Swept Source optical coherence tomography (SS-OCT) has become a well established imaging modality for both medical and industrial diagnostic applications. A cross-sectional SS-OCT image is obtained by applying an inverse discrete Fourier transform (DFT) to axial interferogram measured in the frequency domain (k-space). Fourier inversion of the obtained interferogram typically produces a potentially overlapping conjugate mirror image, whose overlap could be avoided by restricting the object to have its highest reflectivity at its surface. However, this restriction may not be fulfilled when imaging a very thin object that is placed on a highly reflective surface, or imaging an object containing a contrast agent with high reflectivity. In this paper, we show that oversampling of the SS-OCT signal in k-space would overcome the need for such restriction on the object. Our result is demonstrated using SS-OCT images of Axolotl salamander eggs.

Regulation of proximal-distal intercalation during limb regeneration in the axolotl (Ambystoma mexicanum).

Science.gov (United States)

Satoh, Akira; Cummings, Gillian M C; Bryant, Susan V; Gardiner, David M

2010-12-01

Intercalation is the process whereby cells located at the boundary of a wound interact to stimulate proliferation and the restoration of the structures between the boundaries that were lost during wounding. Thus, intercalation is widely considered to be the mechanism of regeneration. When a salamander limb is amputated, the entire cascade of regeneration events is activated, and the missing limb segments and their boundaries (joints) as well as the structures within each segment are regenerated. Therefore, in an amputated limb it is not possible to distinguish between intersegmental regeneration (formation of new segments/joints) and intrasegmental regeneration (formation of structures within a given segment), and it is not possible to study the differential regulation of these two processes. We have used two models for regeneration that allow us to study these two processes independently, and report that inter- and intrasegmental regeneration are different processes regulated by different signaling pathways. New limb segments/joints can be regenerated from cells that dedifferentiate to form blastema cells in response to signaling that is mediated in part by fibroblast growth factor. © 2010 The Authors. Journal compilation © 2010 Japanese Society of Developmental Biologists.

Characterization of glycosaminoglycans during tooth development and mineralization in the axolotl, Ambystoma mexicanum.

Science.gov (United States)

Wistuba, J; Völker, W; Ehmcke, J; Clemen, G

2003-10-01

Glycosaminoglycans (GAGs) involved in the formation of the teeth of Ambystoma mexicanum were located and characterized with the cuprolinic blue (CB) staining method and transmission electron microscopy (TEM). Glycosaminoglycan-cuprolinic blue precipitates (GAGCB) were found in different compartments of the mineralizing tissue. Various populations of elongated GAGCB could be discriminated both according to their size and their preferential distribution in the extracellular matrix (ECM). GAGCB populations that differ in their composition could be attributed not only to the compartments of the ECM but also to different zones and to different tooth types (early-larval and transformed). Larger precipitates were only observed within the dentine matrix of the shaft of the early-larval tooth. The composition of the populations differed significantly between the regions of the transformed tooth: pedicel, shaft and dividing zone. In later stages of tooth formation, small-sized GAGCBs were seen as intracellular deposits in the ameloblasts. It is concluded that the composition of GAGCB populations seems to play a role in the mineralization processes during tooth development in A. mexicanum and influence qualitative characteristics of the mineral in different tooth types and zones, and it is suggested that GAGs might be resorbed by the enamel epithelium during the late phase of enamel formation.

Expression of a novel cardiac-specific tropomyosin isoform in humans

International Nuclear Information System (INIS)

Denz, Christopher R.; Narshi, Aruna; Zajdel, Robert W.; Dube, Dipak K.

2004-01-01

Tropomyosins are a family of actin binding proteins encoded by a group of highly conserved genes. Humans have four tropomyosin-encoding genes: TPM1, TPM2, TPM3, and TPM4, each of which is known to generate multiple isoforms by alternative splicing, promoters, and 3 ' end processing. TPM1 is the most versatile and encodes a variety of tissue specific isoforms. The TPM1 isoform specific to striated muscle, designated TPM1α, consists of 10 exons: 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b. In this study, using RT-PCR with adult and fetal human RNAs, we present evidence for the expression of a novel isoform of the TPM1 gene that is specifically expressed in cardiac tissues. The new isoform is designated TPM1κ and contains exon 2a instead of 2b. Ectopic expression of human GFP.TPM1κ fusion protein can promote myofibrillogenesis in cardiac mutant axolotl hearts that are lacking in tropomyosin

The scarless heart and the MRL mouse.

Science.gov (United States)

Heber-Katz, Ellen; Leferovich, John; Bedelbaeva, Khamilia; Gourevitch, Dmitri; Clark, Lise

2004-05-29

The ability to regenerate tissues and limbs in its most robust form is seen in many non-mammalian species. The serendipitous discovery that the MRL mouse has a profound capacity for regeneration in some ways rivalling the classic newt and axolotl species raises the possibility that humans, too, may have an innate regenerative ability. The adult MRL mouse regrows cartilage, skin, hair follicles and myocardium with near perfect fidelity and without scarring. This is seen in the ability to close through-and-through ear holes, which are generally used for lifelong identification of mice, and the anatomic and functional recovery of myocardium after a severe cryo-injury. We present histological, biochemical and genetic data indicating that the enhanced breakdown of scar-like tissue may be an underlying factor in the MRL regenerative response. Studies as to the source of the cells in the regenerating MRL tissue are discussed. Such studies appear to support multiple mechanisms for cell replacement.

Functional and structural regeneration in the axolotl heart (Ambystoma mexicanum) after partial ventricular amputation.

Science.gov (United States)

Cano-Martínez, Agustina; Vargas-González, Alvaro; Guarner-Lans, Verónica; Prado-Zayago, Esteban; León-Oleda, Martha; Nieto-Lima, Betzabé

2010-01-01

"In the present study we evaluated the effect of partial ventricular amputation (PVA) in the heart of the adult urodele amphibian (Ambystoma mexicanum) in vivo on spontaneous heart contractile activity recorded in vitro in association to the structural recovery at one, five, 30 and 90 days after injury. One day after PVA, ventricular-tension (VT) (16 ± 3%), atrium-tension (AT) (46 ± 4%) and heart rate (HR) (58+10%) resulted lower in comparison to control hearts. On days five, 30 and 90 after damage, values achieved a 61 ± 5, 93 ± 3, and 98 ± 5% (VT), 60 ± 4, 96 ± 3 and 99 ± 5% (AT) and 74 ± 5, 84 ± 10 and 95 ± 10% (HR) of the control values, respectively. Associated to contractile activity recovery we corroborated a gradual tissue restoration by cardiomyocyte proliferation. Our results represent the first quantitative evidence about the recovery of heart of A. mexicanum restores its functional capacity concomitantly to the structural recovery of the myocardium by proliferation of cardiomyocytes after PVA. These properties make the heart of A. mexicanum a potential model to study the mechanisms underlying heart regeneration in adult vertebrates in vivo.

A novel protein involved in heart development in Ambystoma mexicanum is localized in endoplasmic reticulum.

Science.gov (United States)

Jia, P; Zhang, C; Huang, X P; Poda, M; Akbas, F; Lemanski, S L; Erginel-Unaltuna, N; Lemanski, L F

2008-11-01

The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.

ISOLATION AND CHARACTERIZATION OF AXOLOTL NPDC-1 AND ITS EFFECTS ON RETINOIC ACID RECEPTOR SIGNALING

OpenAIRE

Theodosiou, Maria; Monaghan, James R; Spencer, Michael L; Voss, S Randal; Noonan, Daniel J

2007-01-01

Retinoic acid, a key morphogen in early vertebrate development and tissue regeneration, mediates its effects through the binding of receptors that act as ligand-induced transcription factors. These binding events function to recruit an array of transcription co-regulatory proteins to specific gene promoters. One such co-regulatory protein, neuronal proliferation and differentiation control-1 (NPDC-1), is broadly expressed during mammalian development and functions as an in vitro repressor of ...

Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum).

Science.gov (United States)

McCusker, Catherine D; Gardiner, David M

2013-01-01

The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP), to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment.

Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum.

Directory of Open Access Journals (Sweden)

Catherine D McCusker

Full Text Available The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP, to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment.

Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

Science.gov (United States)

Brew, Helen; Attwell, David

1987-06-01

Glutamate is taken up avidly by glial cells in the central nervous system1. Glutamate uptake may terminate the transmitter action of glutamate released from neurons1, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique2. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.

Terminal-Nerve-Derived Neuropeptide Y Modulates Physiological Responses in the Olfactory Epithelium of Hungry Axolotls (Ambystoma mexicanum)

OpenAIRE

Mousley, Angela; Polese, Gianluca; Marks, Nikki J.; Eisthen, Heather L.

2006-01-01

The vertebrate brain actively regulates incoming sensory information, effectively filtering input and focusing attention toward environmental stimuli that are most relevant to the animal's behavioral context or physiological state. Such centrifugal modulation has been shown to play an important role in processing in the retina and cochlea, but has received relatively little attention in olfaction. The terminal nerve, a cranial nerve that extends underneath the lamina propria surrounding the o...

Nerve signaling regulates basal keratinocyte proliferation in the blastema apical epithelial cap in the axolotl (Ambystoma mexicanum).

Science.gov (United States)

Satoh, Akira; Bryant, Susan V; Gardiner, David M

2012-06-15

The ability of adult vertebrates to repair tissue damage is widespread and impressive; however, the ability to regenerate structurally complex organs such as the limb is limited largely to the salamanders. The fact that most of the tissues of the limb can regenerate has led investigators to question and identify the barriers to organ regeneration. From studies in the salamander, it is known that one of the earliest steps required for successful regeneration involves signaling between nerves and the wound epithelium/apical epithelial cap (AEC). In this study we confirm an earlier report that the keratinocytes of the AEC acquire their function coincident with exiting the cell cycle. We have discovered that this unique, coordinated behavior is regulated by nerve signaling and is associated with the presence of gap junctions between the basal keratinocytes of the AEC. Disruption of nerve signaling results in a loss of gap junction protein, the reentry of the cells into the cell cycle, and regenerative failure. Finally, coordinated exit from the cell cycle appears to be a conserved behavior of populations of cells that function as signaling centers during both development and regeneration. Copyright © 2012 Elsevier Inc. All rights reserved.

Inhibition of the Processes of Growth and Differentiation in the Embryonic Development of the Axolotl (Ambystma mexicanum)

NARCIS (Netherlands)

Stolk, Anth.

1954-01-01

The problem of the retardation of the processes of growth and differentiation is certainly as important as the processes of growth and differentiation themselves. It is striking, therefore, that whereas the analysis of growth has been carried out for a considerable period of time already, the

Calcium waves.

Science.gov (United States)

Jaffe, Lionel F

2008-04-12

Waves through living systems are best characterized by their speeds at 20 degrees C. These speeds vary from those of calcium action potentials to those of ultraslow ones which move at 1-10 and/or 10-20 nm s(-1). All such waves are known or inferred to be calcium waves. The two classes of calcium waves which include ones with important morphogenetic effects are slow waves that move at 0.2-2 microm s(-1) and ultraslow ones. Both may be propagated by cycles in which the entry of calcium through the plasma membrane induces subsurface contraction. This contraction opens nearby stretch-sensitive calcium channels. Calcium entry through these channels propagates the calcium wave. Many slow waves are seen as waves of indentation. Some are considered to act via cellular peristalsis; for example, those which seem to drive the germ plasm to the vegetal pole of the Xenopus egg. Other good examples of morphogenetic slow waves are ones through fertilizing maize eggs, through developing barnacle eggs and through axolotl embryos during neural induction. Good examples of ultraslow morphogenetic waves are ones during inversion in developing Volvox embryos and across developing Drosophila eye discs. Morphogenetic waves may be best pursued by imaging their calcium with aequorins.

Microarray analysis identifies keratin loci as sensitive biomarkers for thyroid hormone disruption in the salamander Ambystoma mexicanum.

Science.gov (United States)

Page, Robert B; Monaghan, James R; Samuels, Amy K; Smith, Jeramiah J; Beachy, Christopher K; Voss, S Randal

2007-02-01

Ambystomatid salamanders offer several advantages for endocrine disruption research, including genomic and bioinformatics resources, an accessible laboratory model (Ambystoma mexicanum), and natural lineages that are broadly distributed among North American habitats. We used microarray analysis to measure the relative abundance of transcripts isolated from A. mexicanum epidermis (skin) after exogenous application of thyroid hormone (TH). Only one gene had a >2-fold change in transcript abundance after 2 days of TH treatment. However, hundreds of genes showed significantly different transcript levels at days 12 and 28 in comparison to day 0. A list of 123 TH-responsive genes was identified using statistical, BLAST, and fold level criteria. Cluster analysis identified two groups of genes with similar transcription patterns: up-regulated versus down-regulated. Most notably, several keratins exhibited dramatic (1000 fold) increases or decreases in transcript abundance. Keratin gene expression changes coincided with morphological remodeling of epithelial tissues. This suggests that keratin loci can be developed as sensitive biomarkers to assay temporal disruptions of larval-to-adult gene expression programs. Our study has identified the first collection of loci that are regulated during TH-induced metamorphosis in a salamander, thus setting the stage for future investigations of TH disruption in the Mexican axolotl and other salamanders of the genus Ambystoma.

Initial characterization of the large genome of the salamander Ambystoma mexicanum using shotgun and laser capture chromosome sequencing.

Science.gov (United States)

Keinath, Melissa C; Timoshevskiy, Vladimir A; Timoshevskaya, Nataliya Y; Tsonis, Panagiotis A; Voss, S Randal; Smith, Jeramiah J

2015-11-10

Vertebrates exhibit substantial diversity in genome size, and some of the largest genomes exist in species that uniquely inform diverse areas of basic and biomedical research. For example, the salamander Ambystoma mexicanum (the Mexican axolotl) is a model organism for studies of regeneration, development and genome evolution, yet its genome is ~10× larger than the human genome. As part of a hierarchical approach toward improving genome resources for the species, we generated 600 Gb of shotgun sequence data and developed methods for sequencing individual laser-captured chromosomes. Based on these data, we estimate that the A. mexicanum genome is ~32 Gb. Notably, as much as 19 Gb of the A. mexicanum genome can potentially be considered single copy, which presumably reflects the evolutionary diversification of mobile elements that accumulated during an ancient episode of genome expansion. Chromosome-targeted sequencing permitted the development of assemblies within the constraints of modern computational platforms, allowed us to place 2062 genes on the two smallest A. mexicanum chromosomes and resolves key events in the history of vertebrate genome evolution. Our analyses show that the capture and sequencing of individual chromosomes is likely to provide valuable information for the systematic sequencing, assembly and scaffolding of large genomes.

Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms.

Science.gov (United States)

Mundell, Nathan A; Beier, Kevin T; Pan, Y Albert; Lapan, Sylvain W; Göz Aytürk, Didem; Berezovskii, Vladimir K; Wark, Abigail R; Drokhlyansky, Eugene; Bielecki, Jan; Born, Richard T; Schier, Alexander F; Cepko, Constance L

2015-08-01

Current limitations in technology have prevented an extensive analysis of the connections among neurons, particularly within nonmammalian organisms. We developed a transsynaptic viral tracer originally for use in mice, and then tested its utility in a broader range of organisms. By engineering the vesicular stomatitis virus (VSV) to encode a fluorophore and either the rabies virus glycoprotein (RABV-G) or its own glycoprotein (VSV-G), we created viruses that can transsynaptically label neuronal circuits in either the retrograde or anterograde direction, respectively. The vectors were investigated for their utility as polysynaptic tracers of chicken and zebrafish visual pathways. They showed patterns of connectivity consistent with previously characterized visual system connections, and revealed several potentially novel connections. Further, these vectors were shown to infect neurons in several other vertebrates, including Old and New World monkeys, seahorses, axolotls, and Xenopus. They were also shown to infect two invertebrates, Drosophila melanogaster, and the box jellyfish, Tripedalia cystophora, a species previously intractable for gene transfer, although no clear evidence of transsynaptic spread was observed in these species. These vectors provide a starting point for transsynaptic tracing in most vertebrates, and are also excellent candidates for gene transfer in organisms that have been refractory to other methods. © 2015 Wiley Periodicals, Inc.

Scaled nonuniform Fourier transform for image reconstruction in swept source optical coherence tomography

Science.gov (United States)

Mezgebo, Biniyam; Nagib, Karim; Fernando, Namal; Kordi, Behzad; Sherif, Sherif

2018-02-01

Swept Source optical coherence tomography (SS-OCT) is an important imaging modality for both medical and industrial diagnostic applications. A cross-sectional SS-OCT image is obtained by applying an inverse discrete Fourier transform (DFT) to axial interferograms measured in the frequency domain (k-space). This inverse DFT is typically implemented as a fast Fourier transform (FFT) that requires the data samples to be equidistant in k-space. As the frequency of light produced by a typical wavelength-swept laser is nonlinear in time, the recorded interferogram samples will not be uniformly spaced in k-space. Many image reconstruction methods have been proposed to overcome this problem. Most such methods rely on oversampling the measured interferogram then use either hardware, e.g., Mach-Zhender interferometer as a frequency clock module, or software, e.g., interpolation in k-space, to obtain equally spaced samples that are suitable for the FFT. To overcome the problem of nonuniform sampling in k-space without any need for interferogram oversampling, an earlier method demonstrated the use of the nonuniform discrete Fourier transform (NDFT) for image reconstruction in SS-OCT. In this paper, we present a more accurate method for SS-OCT image reconstruction from nonuniform samples in k-space using a scaled nonuniform Fourier transform. The result is demonstrated using SS-OCT images of Axolotl salamander eggs.

Multi-scale finite element modeling allows the mechanics of amphibian neurulation to be elucidated

International Nuclear Information System (INIS)

Chen Xiaoguang; Wayne Brodland, G

2008-01-01

The novel multi-scale computational approach introduced here makes possible a new means for testing hypotheses about the forces that drive specific morphogenetic movements. A 3D model based on this approach is used to investigate neurulation in the axolotl (Ambystoma mexicanum), a type of amphibian. The model is based on geometric data from 3D surface reconstructions of live embryos and from serial sections. Tissue properties are described by a system of cell-based constitutive equations, and parameters in the equations are determined from physical tests. The model includes the effects of Shroom-activated neural ridge reshaping and lamellipodium-driven convergent extension. A typical whole-embryo model consists of 10 239 elements and to run its 100 incremental time steps requires 2 days. The model shows that a normal phenotype does not result if lamellipodium forces are uniform across the width of the neural plate; but it can result if the lamellipodium forces decrease from a maximum value at the mid-sagittal plane to zero at the plate edge. Even the seemingly simple motions of neurulation are found to contain important features that would remain hidden, they were not studied using an advanced computational model. The present model operates in a setting where data are extremely sparse and an important outcome of the study is a better understanding of the role of computational models in such environments

Endogenous electric fields in embryos during development, regeneration and wound healing

International Nuclear Information System (INIS)

Nuccitelli, R.

2003-01-01

All embryos that have been investigated drive ionic currents through themselves and these currents will generate internal electric fields. Here, those examples in which such fields have been measured directly are discussed. The first such measurements were made in chick embryos and about 20 mV mm -1 was measured near the posterier intestinal portal in 2-4-day-old embryos. This electric field is important for the development of tail structures because reducing its magnitude results in abnormal tail development. The second embryonic electric field measured directly was in the axolotl, where a rostral-caudal field of about the same magnitude was detected. Modification of this field during neurulation but not gastrulation caused developmental abnormalities. Most recently, the development of left-right asymmetry in frog and chick embryos was found to require a voltage difference between blastomeres at a very early developmental stage. This field was measured in the chick embryo to be 10-20 mV mm -1 across the primitive streak. Mammalian skin wounds generate 150 mV mm -1 fields lateral to the wound and corneal epidermal wounds exhibit lateral fields of 40 mV mm -1 . The presence of these endogenous fields would suggest that exposures to external electric fields should be limited to magnitudes of less than 0.1 V m -1 . (author)

Endogenous electric fields in embryos during development, regeneration and wound healing

Energy Technology Data Exchange (ETDEWEB)

Nuccitelli, R

2003-07-01

All embryos that have been investigated drive ionic currents through themselves and these currents will generate internal electric fields. Here, those examples in which such fields have been measured directly are discussed. The first such measurements were made in chick embryos and about 20 mV mm^-1 was measured near the posterier intestinal portal in 2-4-day-old embryos. This electric field is important for the development of tail structures because reducing its magnitude results in abnormal tail development. The second embryonic electric field measured directly was in the axolotl, where a rostral-caudal field of about the same magnitude was detected. Modification of this field during neurulation but not gastrulation caused developmental abnormalities. Most recently, the development of left-right asymmetry in frog and chick embryos was found to require a voltage difference between blastomeres at a very early developmental stage. This field was measured in the chick embryo to be 10-20 mV mm^-1 across the primitive streak. Mammalian skin wounds generate 150 mV mm^-1 fields lateral to the wound and corneal epidermal wounds exhibit lateral fields of 40 mV mm^-1. The presence of these endogenous fields would suggest that exposures to external electric fields should be limited to magnitudes of less than 0.1 V m^-1. (author)

Multi-scale finite element modeling allows the mechanics of amphibian neurulation to be elucidated

Science.gov (United States)

Chen, Xiaoguang; Brodland, G. Wayne

2008-03-01

The novel multi-scale computational approach introduced here makes possible a new means for testing hypotheses about the forces that drive specific morphogenetic movements. A 3D model based on this approach is used to investigate neurulation in the axolotl (Ambystoma mexicanum), a type of amphibian. The model is based on geometric data from 3D surface reconstructions of live embryos and from serial sections. Tissue properties are described by a system of cell-based constitutive equations, and parameters in the equations are determined from physical tests. The model includes the effects of Shroom-activated neural ridge reshaping and lamellipodium-driven convergent extension. A typical whole-embryo model consists of 10 239 elements and to run its 100 incremental time steps requires 2 days. The model shows that a normal phenotype does not result if lamellipodium forces are uniform across the width of the neural plate; but it can result if the lamellipodium forces decrease from a maximum value at the mid-sagittal plane to zero at the plate edge. Even the seemingly simple motions of neurulation are found to contain important features that would remain hidden, they were not studied using an advanced computational model. The present model operates in a setting where data are extremely sparse and an important outcome of the study is a better understanding of the role of computational models in such environments.

Gain-of-function assays in the axolotl (Ambystoma mexicanum) to identify signaling pathways that induce and regulate limb regeneration.

Science.gov (United States)

Lee, Jangwoo; Aguilar, Cristian; Gardiner, David

2013-01-01

The adult salamander has been studied as a model for regeneration of complex tissues for many decades. Only recently with the development of gain-of-function assays for regeneration, has it been possible to screen for and assay the function of the multitude of signaling factors that have been identified in studies of embryonic development and tumorigenesis. Given the conservation of function of these regulatory pathways controlling growth and pattern formation, it is now possible to use the functional assays in the salamander to test the ability of endogenous as well as small-molecule signaling factors to induce a regenerative response.

Regeneration of soft tissues is promoted by MMP1 treatment after digit amputation in mice.

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Xiaodong Mu

Full Text Available The ratio of matrix metalloproteinases (MMPs to the tissue inhibitors of metalloproteinases (TIMPs in wounded tissues strictly control the protease activity of MMPs, and therefore regulate the progress of wound closure, tissue regeneration and scar formation. Some amphibians (i.e. axolotl/newt demonstrate complete regeneration of missing or wounded digits and even limbs; MMPs play a critical role during amphibian regeneration. Conversely, mammalian wound healing re-establishes tissue integrity, but at the expense of scar tissue formation. The differences between amphibian regeneration and mammalian wound healing can be attributed to the greater ratio of MMPs to TIMPs in amphibian tissue. Previous studies have demonstrated the ability of MMP1 to effectively promote skeletal muscle regeneration by favoring extracellular matrix (ECM remodeling to enhance cell proliferation and migration. In this study, MMP1 was administered to the digits amputated at the mid-second phalanx of adult mice to observe its effect on digit regeneration. Results indicated that the regeneration of soft tissue and the rate of wound closure were significantly improved by MMP1 administration, but the elongation of the skeletal tissue was insignificantly affected. During digit regeneration, more mutipotent progenitor cells, capillary vasculature and neuromuscular-related tissues were observed in MMP1 treated tissues; moreover, there was less fibrotic tissue formed in treated digits. In summary, MMP1 was found to be effective in promoting wound healing in amputated digits of adult mice.

Genomics of a Metamorphic Timing QTL: met1 Maps to a Unique Genomic Position and Regulates Morph and Species-Specific Patterns of Brain Transcription

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Page, Robert B.; Boley, Meredith A.; Kump, David K.; Voss, Stephen R.

2013-01-01

Very little is known about genetic factors that regulate life history transitions during ontogeny. Closely related tiger salamanders (Ambystoma species complex) show extreme variation in metamorphic timing, with some species foregoing metamorphosis altogether, an adaptive trait called paedomorphosis. Previous studies identified a major effect quantitative trait locus (met1) for metamorphic timing and expression of paedomorphosis in hybrid crosses between the biphasic Eastern tiger salamander (Ambystoma tigrinum tigrinum) and the paedomorphic Mexican axolotl (Ambystoma mexicanum). We used existing hybrid mapping panels and a newly created hybrid cross to map the met1 genomic region and determine the effect of met1 on larval growth, metamorphic timing, and gene expression in the brain. We show that met1 maps to the position of a urodele-specific chromosome rearrangement on linkage group 2 that uniquely brought functionally associated genes into linkage. Furthermore, we found that more than 200 genes were differentially expressed during larval development as a function of met1 genotype. This list of differentially expressed genes is enriched for proteins that function in the mitochondria, providing evidence of a link between met1, thyroid hormone signaling, and mitochondrial energetics associated with metamorphosis. Finally, we found that met1 significantly affected metamorphic timing in hybrids, but not early larval growth rate. Collectively, our results show that met1 regulates species and morph-specific patterns of brain transcription and life history variation. PMID:23946331

Tooth development in Ambystoma mexicanum: phosphatase activities, calcium accumulation and cell proliferation in the tooth-forming tissues.

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Wistuba, Joachim; Ehmcke, Jens; Clemen, Günter

2003-06-01

Prerequisites of tooth formation, cell proliferation in the tooth-forming tissues, calcium accumulation and the enzymatic activities of alkaline (ALP) and acid phosphatases (ACP) were investigated by immunohistochemical and histochemical methods in various developmental stages of the Mexican Axolotl, Ambystoma mexicanum. During the growth of replacement teeth, the tooth-forming tissues continually recruit cells from the surrounding regions. The basal layer of the oral epithelium, the dental lamina and sometimes even the outer enamel epithelium provide cells for the differentiated inner enamel epithelium, in which the active ameloblasts are localized. The differentiating odontoblasts are derived from proliferating cells situated basally to the replacement teeth in the mesenchymal tissue. When differentiation has started and the cells have become functional, proliferative activity can no longer be observed. Calcium is accumulated close to the site of mineralization in the inner enamel epithelium and in the odontoblasts as it is in mammals, elasmobranchii and teleostei. The activities of ACP and ALP related to the mineralization of the replacement teeth are separated spatially and not sequentially as they are in mammals. However, the results indicate a similar function of these enzymatic components in relation to tooth formation and maturation of mineral deposition. Most of the substantial processes related to tooth formation reported from other vertebrates occur in a manner similar to that in Ambystoma mexicanum, but there also seem to be basic mechanisms present that are realised in a unique way in this urodele.

Multiplicity of Buc copies in Atlantic salmon contrasts with loss of the germ cell determinant in primates, rodents and axolotl

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Skugor, Adrijana; Tveiten, Helge; Johnsen, Hanne; Andersen, Øivind

2016-01-01

Background The primordial germ cells (PGCs) giving rise to gametes are determined by two different mechanisms in vertebrates. While the germ cell fate in mammals and salamanders is induced by zygotic signals, maternally delivered germ cell determinants specify the PGCs in birds, frogs and teleost fish. Assembly of the germ plasm in the oocyte is organized by the single Buc in zebrafish, named Velo1 in Xenopus, and by Oskar in Drosophila. Secondary loss of oskar in several insect lineages coin...

Neuropeptide Y enhances olfactory mucosa responses to odorant in hungry rats.

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Negroni, Julia; Meunier, Nicolas; Monnerie, Régine; Salesse, Roland; Baly, Christine; Caillol, Monique; Congar, Patrice

2012-01-01

Neuropeptide Y (NPY) plays an important role in regulating appetite and hunger in vertebrates. In the hypothalamus, NPY stimulates food intake under the control of the nutritional status. Previous studies have shown the presence of NPY and receptors in rodent olfactory system, and suggested a neuroproliferative role. Interestingly, NPY was also shown to directly modulate olfactory responses evoked by a food-related odorant in hungry axolotls. We have recently demonstrated that another nutritional cue, insulin, modulates the odorant responses of the rat olfactory mucosa (OM). Therefore, the aim of the present study was to investigate the potential effect of NPY on rat OM responses to odorants, in relation to the animal's nutritional state. We measured the potential NPY modulation of OM responses to odorant, using electro-olfactogram (EOG) recordings, in fed and fasted adult rats. NPY application significantly and transiently increased EOG amplitudes in fasted but not in fed rats. The effects of specific NPY-receptor agonists were similarly quantified, showing that NPY operated mainly through Y1 receptors. These receptors appeared as heterogeneously expressed by olfactory neurons in the OM, and western blot analysis showed that they were overexpressed in fasted rats. These data provide the first evidence that NPY modulates the initial events of odorant detection in the rat OM. Because this modulation depends on the nutritional status of the animal, and is ascribed to NPY, the most potent orexigenic peptide in the central nervous system, it evidences a strong supplementary physiological link between olfaction and nutritional processes.

An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries

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Habermann, Bianca; Bebin, Anne-Gaelle; Herklotz, Stephan; Volkmer, Michael; Eckelt, Kay; Pehlke, Kerstin; Epperlein, Hans Henning; Schackert, Hans Konrad; Wiebe, Glenis; Tanaka, Elly M

2004-01-01

Background The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. Results Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. Conclusions Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online. PMID:15345051

Origin of amphibian and avian chromosomes by fission, fusion, and retention of ancestral chromosomes

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Voss, Stephen R.; Kump, D. Kevin; Putta, Srikrishna; Pauly, Nathan; Reynolds, Anna; Henry, Rema J.; Basa, Saritha; Walker, John A.; Smith, Jeramiah J.

2011-01-01

Amphibian genomes differ greatly in DNA content and chromosome size, morphology, and number. Investigations of this diversity are needed to identify mechanisms that have shaped the evolution of vertebrate genomes. We used comparative mapping to investigate the organization of genes in the Mexican axolotl (Ambystoma mexicanum), a species that presents relatively few chromosomes (n = 14) and a gigantic genome (>20 pg/N). We show extensive conservation of synteny between Ambystoma, chicken, and human, and a positive correlation between the length of conserved segments and genome size. Ambystoma segments are estimated to be four to 51 times longer than homologous human and chicken segments. Strikingly, genes demarking the structures of 28 chicken chromosomes are ordered among linkage groups defining the Ambystoma genome, and we show that these same chromosomal segments are also conserved in a distantly related anuran amphibian (Xenopus tropicalis). Using linkage relationships from the amphibian maps, we predict that three chicken chromosomes originated by fusion, nine to 14 originated by fission, and 12–17 evolved directly from ancestral tetrapod chromosomes. We further show that some ancestral segments were fused prior to the divergence of salamanders and anurans, while others fused independently and randomly as chromosome numbers were reduced in lineages leading to Ambystoma and Xenopus. The maintenance of gene order relationships between chromosomal segments that have greatly expanded and contracted in salamander and chicken genomes, respectively, suggests selection to maintain synteny relationships and/or extremely low rates of chromosomal rearrangement. Overall, the results demonstrate the value of data from diverse, amphibian genomes in studies of vertebrate genome evolution. PMID:21482624

From biomedicine to natural history research: EST resources for ambystomatid salamanders

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Bryant Susan V

2004-08-01

Full Text Available Abstract Background Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (Ambystoma mexicanum and Eastern tiger salamander (A. tigrinum tigrinum, species with deep and diverse research histories. Results Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the A. t. tigrinum ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human – Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species. Conclusions Our study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research.

From biomedicine to natural history research: EST resources for ambystomatid salamanders

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Putta, Srikrishna; Smith, Jeramiah J; Walker, John A; Rondet, Mathieu; Weisrock, David W; Monaghan, James; Samuels, Amy K; Kump, Kevin; King, David C; Maness, Nicholas J; Habermann, Bianca; Tanaka, Elly; Bryant, Susan V; Gardiner, David M; Parichy, David M; Voss, S Randal

2004-01-01

Background Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (Ambystoma mexicanum) and Eastern tiger salamander (A. tigrinum tigrinum), species with deep and diverse research histories. Results Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the A. t. tigrinum ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human – Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species. Conclusions Our study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research. PMID:15310388

Neural crest contributions to the lamprey head

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McCauley, David W.; Bronner-Fraser, Marianne

2003-01-01

The neural crest is a vertebrate-specific cell population that contributes to the facial skeleton and other derivatives. We have performed focal DiI injection into the cranial neural tube of the developing lamprey in order to follow the migratory pathways of discrete groups of cells from origin to destination and to compare neural crest migratory pathways in a basal vertebrate to those of gnathostomes. The results show that the general pathways of cranial neural crest migration are conserved throughout the vertebrates, with cells migrating in streams analogous to the mandibular and hyoid streams. Caudal branchial neural crest cells migrate ventrally as a sheet of cells from the hindbrain and super-pharyngeal region of the neural tube and form a cylinder surrounding a core of mesoderm in each pharyngeal arch, similar to that seen in zebrafish and axolotl. In addition to these similarities, we also uncovered important differences. Migration into the presumptive caudal branchial arches of the lamprey involves both rostral and caudal movements of neural crest cells that have not been described in gnathostomes, suggesting that barriers that constrain rostrocaudal movement of cranial neural crest cells may have arisen after the agnathan/gnathostome split. Accordingly, neural crest cells from a single axial level contributed to multiple arches and there was extensive mixing between populations. There was no apparent filling of neural crest derivatives in a ventral-to-dorsal order, as has been observed in higher vertebrates, nor did we find evidence of a neural crest contribution to cranial sensory ganglia. These results suggest that migratory constraints and additional neural crest derivatives arose later in gnathostome evolution.

Epigenetic reprogramming of breast cancer cells with oocyte extracts

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Kumari Rajendra

2011-01-01

Full Text Available Abstract Background Breast cancer is a disease characterised by both genetic and epigenetic alterations. Epigenetic silencing of tumour suppressor genes is an early event in breast carcinogenesis and reversion of gene silencing by epigenetic reprogramming can provide clues to the mechanisms responsible for tumour initiation and progression. In this study we apply the reprogramming capacity of oocytes to cancer cells in order to study breast oncogenesis. Results We show that breast cancer cells can be directly reprogrammed by amphibian oocyte extracts. The reprogramming effect, after six hours of treatment, in the absence of DNA replication, includes DNA demethylation and removal of repressive histone marks at the promoters of tumour suppressor genes; also, expression of the silenced genes is re-activated in response to treatment. This activity is specific to oocytes as it is not elicited by extracts from ovulated eggs, and is present at very limited levels in extracts from mouse embryonic stem cells. Epigenetic reprogramming in oocyte extracts results in reduction of cancer cell growth under anchorage independent conditions and a reduction in tumour growth in mouse xenografts. Conclusions This study presents a new method to investigate tumour reversion by epigenetic reprogramming. After testing extracts from different sources, we found that axolotl oocyte extracts possess superior reprogramming ability, which reverses epigenetic silencing of tumour suppressor genes and tumorigenicity of breast cancer cells in a mouse xenograft model. Therefore this system can be extremely valuable for dissecting the mechanisms involved in tumour suppressor gene silencing and identifying molecular activities capable of arresting tumour growth. These applications can ultimately shed light on the contribution of epigenetic alterations in breast cancer and advance the development of epigenetic therapies.

The rise of the boy-genius: psychological neoteny, science and modern life.

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Charlton, Bruce G

2006-01-01

The mid-20th century saw the rise of the boy-genius, probably because a personality type characterized by prolonged youthfulness is advantageous both in science and modern life generally. This is the evolution of 'psychological neoteny', in which ever-more people retain for ever-longer the characteristic behaviours and attitudes of earlier developmental stages. Whereas traditional societies are characterized by initiation ceremonies marking the advent of adulthood, these have now dwindled and disappeared. In a psychological sense, some contemporary individuals never actually become adults. A child-like flexibility of attitudes, behaviours and knowledge is probably adaptive in modern society because people need repeatedly to change jobs, learn new skills, move to new places and make new friends. It seems that this adaptation is achieved by the expedient of postponing cognitive maturation - a process that could be termed psychological neoteny. ('Neoteny' refers to the biological phenomenon whereby development is delayed such that juvenile characteristics are retained into maturity.) Psychological neoteny is probably caused by the prolonged average duration of formal education, since students' minds are in a significant sense 'unfinished'. Since modern cultures favour cognitive flexibility, 'immature' people tend to thrive and succeed, and have set the tone of contemporary life: the greatest praise of an elderly person is to state that they retain the characteristics of youth. But the faults of youth are retained with well as its virtues: short attention span, sensation- and novelty-seeking, short cycles of arbitrary fashion and a sense of cultural shallowness. Nonetheless, as health gets better and cosmetic technologies improve, future humans may become somewhat like an axolotl - the cave-dwelling salamander which retains its larval form until death.

A new and improved algorithm for the quantification of chromatin condensation from microscopic data shows decreased chromatin condensation in regenerating axolotl limb cells.

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Julian Sosnik

Full Text Available The nuclear landscape plays an important role in the regulation of tissue and positional specific genes in embryonic and developing cells. Changes in this landscape can be dynamic, and are associated with the differentiation of cells during embryogenesis, and the de-differentiation of cells during induced pluripotent stem cell (iPSC formation and in many cancers. However, tools to quantitatively characterize these changes are limited, especially in the in vivo context, where numerous tissue types are present and cells are arranged in multiple layers. Previous tools have been optimized for the monolayer nature of cultured cells. Therefore, we present a new algorithm to quantify the condensation of chromatin in two in vivo systems. We first developed this algorithm to quantify changes in chromatin compaction and validated it in differentiating spermatids in zebrafish testes. Our algorithm successfully detected the typical increase in chromatin compaction as these cells differentiate. We then employed the algorithm to quantify the changes that occur in amphibian limb cells as they participate in a regenerative response. We observed that the chromatin in the limb cells de-compacts as they contribute to the regenerating organ. We present this new tool as an open sourced software that can be readily accessed and optimized to quantify chromatin compaction in complex multi-layered samples.

A Conserved MicroRNA Regulatory Circuit Is Differentially Controlled during Limb/Appendage Regeneration.

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Benjamin L King

Full Text Available Although regenerative capacity is evident throughout the animal kingdom, it is not equally distributed throughout evolution. For instance, complex limb/appendage regeneration is muted in mammals but enhanced in amphibians and teleosts. The defining characteristic of limb/appendage regenerative systems is the formation of a dedifferentiated tissue, termed blastema, which serves as the progenitor reservoir for regenerating tissues. In order to identify a genetic signature that accompanies blastema formation, we employ next-generation sequencing to identify shared, differentially regulated mRNAs and noncoding RNAs in three different, highly regenerative animal systems: zebrafish caudal fins, bichir pectoral fins and axolotl forelimbs.These studies identified a core group of 5 microRNAs (miRNAs that were commonly upregulated and 5 miRNAs that were commonly downregulated, as well as 4 novel tRNAs fragments with sequences conserved with humans. To understand the potential function of these miRNAs, we built a network of 1,550 commonly differentially expressed mRNAs that had functional relationships to 11 orthologous blastema-associated genes. As miR-21 was the most highly upregulated and most highly expressed miRNA in all three models, we validated the expression of known target genes, including the tumor suppressor, pdcd4, and TGFβ receptor subunit, tgfbr2 and novel putative target genes such as the anti-apoptotic factor, bcl2l13, Choline kinase alpha, chka and the regulator of G-protein signaling, rgs5.Our extensive analysis of RNA-seq transcriptome profiling studies in three regenerative animal models, that diverged in evolution ~420 million years ago, reveals a common miRNA-regulated genetic network of blastema genes. These comparative studies extend our current understanding of limb/appendage regeneration by identifying previously unassociated blastema genes and the extensive regulation by miRNAs, which could serve as a foundation for future