Identification of immune and non-immune cells in regenerating axolotl limbs by single-cell sequencing.

Immune cells are known to be critical for successful limb regeneration in the axolotl (Ambystoma mexicanum), but many details regarding their identity, behavior, and function are yet to be resolved. We isolated peripheral leukocytes from the blood of adult axolotls and then created two samples for single-cell sequencing: 1) peripheral leukocytes (N = 7889) and 2) peripheral leukocytes with presumptive macrophages from the intraperitoneal cavity (N = 4998). Using k-means clustering, we identified 6 cell populations from each sample that presented gene expression patterns indicative of erythrocyte, thrombocyte, neutrophil, B-cell, T-cell, and myeloid cell populations. A seventh, presumptive macrophage cell population was identified uniquely from sample 2. We then isolated cells from amputated axolotl limbs at 1 and 6 days post-amputation (DPA) and performed single cell sequencing (N = 8272 and 9906 cells respectively) to identify immune and non-immune cell populations. Using k-means clustering, we identified 8 cell populations overall, with the majority of cells expressing erythrocyte-specific genes. Even though erythrocytes predominated, we used an unbiased approach to identify infiltrating neutrophil, macrophage, and lymphocyte populations at both time points. Additionally, populations expressing genes for epidermal cells, fibroblast-like cells, and endothelial cells were also identified. Consistent with results from previous experimental studies, neutrophils were more abundant at 1 DPA than 6 DPA, while macrophages and non-immune cells exhibited inverse abundance patterns. Of note, we identified a small population of fibroblast-like cells at 1 DPA that was represented by considerably more cells at 6 DPA. We hypothesize that these are early progenitor cells that give rise to the blastema. The enriched gene sets from our work will aid future single-cell investigations of immune cell diversity and function during axolotl limb regeneration.

Thyroid hormone responsive QTL and the evolution of paedomorphic salamanders.

The transformation of ancestral phenotypes into novel traits is poorly understood for many examples of evolutionary novelty. Ancestrally, salamanders have a biphasic life cycle with an aquatic larval stage, a brief and pronounced metamorphosis, followed by a terrestrial adult stage. Repeatedly during evolution, metamorphic timing has been delayed to exploit growth-permissive environments, resulting in paedomorphic salamanders that retain larval traits as adults. We used thyroid hormone (TH) to rescue metamorphic phenotypes in paedomorphic salamanders and then identified quantitative trait loci (QTL) for life history traits that are associated with amphibian life cycle evolution: metamorphic timing and adult body size. We demonstrate that paedomorphic tiger salamanders (Ambystoma tigrinum complex) carry alleles at three moderate effect QTL (met1-3) that vary in responsiveness to TH and additively affect metamorphic timing. Salamanders that delay metamorphosis attain significantly larger body sizes as adults and met2 explains a significant portion of this variation. Thus, substitution of alleles at TH-responsive loci suggests an adaptive pleiotropic basis for two key life-history traits in amphibians: body size and metamorphic timing. Our study demonstrates a likely pathway for the evolution of novel paedomorphic species from metamorphic ancestors via selection of TH-response alleles that delay metamorphic timing and increase adult body size.

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

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 (p<0.05, fold change >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.

Intrinsic repair of full-thickness articular cartilage defects in the axolotl salamander.

OBJECTIVE: The ability to fully regenerate lost limbs has made the axolotl salamander (Ambystoma mexicanum) a valuable model for studies of tissue regeneration. The current experiments investigate the ability of these vertebrates to repair large articular cartilage defects and restore normal hyaline cartilage and joint structure independent of limb amputation. METHODS: Full-thickness articular cartilage defects were made by resection of the medial femoral condyle to the level of the metaphysis. At 0, 2 days, 1, 2, 3, 4, 6, 8, 12, 18, 24, 36 and 48 weeks post-surgery, the repair process was analyzed on H&E and Safranin-O stained 7 µm tissue sections. Symmetric Kullback-Leibler (SKL) divergences were used to assess proteoglycan staining intensities. Immunohistochemistry was performed for collagen types I and II. RESULTS: A fibrous "interzone-like" tissue occupies the intraarticular space of the axolotl femorotibial joint and no evidence of joint cavitation was observed. By 4 weeks post-surgery, cells within the defect site exhibited morphological similarities to those of the interzone-like tissue. At 24 weeks, joint structure and cartilaginous tissue repair were confirmed by immunohistochemistry for collagen types I and II. Quantitation of Safranin-O staining indicated restoration of proteoglycan content by 18 weeks. CONCLUSIONS: The axolotl femorotibial joint has morphological similarities to the developing mammalian diarthrodial joint. Cells in the intraarticular space may be homologous to the interzone tissue and contribute to intrinsic repair of full-thickness articular cartilage defects. Taken together, these results suggest that the axolotl may serve as a valuable model for the investigation of cellular and molecular mechanisms that achieve full articular cartilage repair.

Amphibian sex determination: segregation and linkage analysis using members of the tiger salamander species complex (Ambystoma mexicanum and A. t. tigrinum).

Little is known about the genetic basis of sex determination in vertebrates though considerable progress has been made in recent years. In this study, segregation analysis and linkage mapping were performed to localize an amphibian sex-determining locus (ambysex) in the tiger salamander (Ambystoma) genome. Segregation of sex phenotypes (male and female) among the second generation individuals of interspecific crosses (Ambystoma mexicanum x Ambystoma tigrinum tigrinum) was consistent with Mendelian expectations, although a slight female bias was observed. Individuals from these same crosses were typed for single-nucleotide polymorphisms distributed throughout the genome to identify molecular markers for ambysex. A marker (E24C3) was identified approximately 5.9 cM from ambysex. Linkage of E24C3 to ambysex was independently validated in a second, intraspecific cross (A. mexicanum). Interestingly, ambysex locates to the tip of one of the larger linkage groups of the Ambystoma meiotic map. Considering that this location does not show reduced recombination, we speculate that the ambysex locus may have arisen quite recently, within the last few million years. Localization of ambysex sets the stage for gene identification and provides important tools for studying the effect of sex in laboratory and natural populations of this model amphibian system.

A comprehensive expressed sequence tag linkage map for tiger salamander and Mexican axolotl: enabling gene mapping and comparative genomics in Ambystoma.

Expressed sequence tag (EST) markers were developed for Ambystoma tigrinum tigrinum (Eastern tiger salamander) and for A. mexicanum (Mexican axolotl) to generate the first comprehensive linkage map for these model amphibians. We identified 14 large linkage groups (125.5-836.7 cM) that presumably correspond to the 14 haploid chromosomes in the Ambystoma genome. The extent of genome coverage for these linkage groups is apparently high because the total map size (5251 cM) falls within the range of theoretical estimates and is consistent with independent empirical estimates. Unlike most vertebrate species, linkage map size in Ambystoma is not strongly correlated with chromosome arm number. Presumably, the large physical genome size ( approximately 30 Gbp) is a major determinant of map size in Ambystoma. To demonstrate the utility of this resource, we mapped the position of two historically significant A. mexicanum mutants, white and melanoid, and also met, a quantitative trait locus (QTL) that contributes to variation in metamorphic timing. This new collection of EST-based PCR markers will better enable the Ambystoma system by facilitating development of new molecular probes, and the linkage map will allow comparative studies of this important vertebrate group.

Evolution of salamander life cycles: a major-effect quantitative trait locus contributes to discrete and continuous variation for metamorphic timing.

The evolution of alternate modes of development may occur through genetic changes in metamorphic timing. This hypothesis was examined by crossing salamanders that express alternate developmental modes: metamorphosis vs. paedomorphosis. Three strains were used in the crossing design: Ambystoma tigrinum tigrinum (Att; metamorph), wild-caught A. mexicanum (Am; paedomorph), and laboratory Am (paedomorph). Att/Am hybrids were created for each Am strain and then backcrossed to their respective Am line. Previous studies have shown that a dominant allele from Att (met(Att)) and a recessive allele from lab Am (met(lab)) results in metamorphosis in Att/Am hybrids, and met(Att)/met(lab) and met(lab)/met(lab) backcross genotypes are strongly associated with metamorphosis and paedomorphosis, respectively. We typed a molecular marker (contig325) linked to met and found that met(Att)/met(lab) and met(Att)/met(wild) were associated with metamorphosis in 99% of the cases examined. However, the frequency of paedomorphosis was 4.5 times higher for met(lab)/met(lab) than for met(wild)/met(wild). We also found that met(Att)/met(wild) and met(wild)/met(wild) genotypes discriminated distributions of early and late metamorphosing individuals. Two forms of phenotypic variation are contributed by met: continuous variation of metamorphic age and expression of discrete, alternate morphs. We suggest that the evolution of paedomorphosis is associated with genetic changes that delay metamorphic timing in biphasic life cycles.

Candidate gene analysis of metamorphic timing in ambystomatid salamanders.

Although much is known about the ecological significance of metamorphosis and metamorphic timing, few studies have examined the underlying genetic architecture of these traits, and no study has attempted to associate phenotypic variation to molecular variation in specific genes. Here we report on a candidate gene approach (CGA) to test specific loci for a statistical contribution to variation in metamorphic timing. Three segregating populations (SP1, SP2 and SP3) were constructed utilizing three species of paedomorphic Mexican ambystomatid salamander, including the axolotl, Ambystoma mexicanum. We used these replicated species to test the hypothesis that inheritance of alternate genotypes at two thyroid hormone receptor loci (TRalpha, TRbeta) affects metamorphic timing in ambystomatid salamanders. A significant TRalpha*SP effect indicated that variation in metamorphic timing may be influenced by TRalpha genotype, however, the effect was not a simple one, as both the magnitude and direction of the phenotypic effect depended upon the genetic background. These are the first data to implicate a specific gene in contributing to variation in metamorphic timing. In general, candidate gene approaches can be extended to any number of loci and to any organism where simple genetic crosses can be performed to create segregating populations. The approach is thus of particular value in ecological studies where target genes have been identified but the study organism is not one of the few well-characterized model systems that dominate genetic research.

Conserved vertebrate chromosome segments in the large salamander genome.

Urodele amphibians (salamanders) are important models for embryological, physiological, and natural history research and are also a biomedically important group because they are the only vertebrates capable of regenerating entire organ systems. To enhance the utility of salamanders for biomedical research and for understanding genome evolution, genetic linkage analysis was used to identify chromosome segments that are homologous between ambystomatid salamanders and distantly related vertebrate model organisms. A total of 347 loci (AFLPs, RAPDs, and protein-coding loci) were mapped using an interspecific meiotic mapping panel (Ambystoma mexicanum and A. tigrinum tigrinum; family Ambystomatidae). Genome size in Ambystoma was estimated to be 7291 cM, the largest linkage map estimate reported for any organism. However, the relatively large size of the salamander genome did not hinder efforts to map and identify conserved syntenies from a small sample of 24 protein-coding loci. Chromosomal segments that are conserved between fishes and mammals are also conserved in these salamanders. Thus, comparative gene mapping appears to be an efficient strategy for identifying orthologous loci between ambystomatid salamanders and genomically well-characterized vertebrate model organisms.

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

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.

Candidate gene analysis of thyroid hormone receptors in metamorphosing vs. nonmetamorphosing salamanders.

We used two different experimental approaches to test the hypothesis that thyroid hormone receptor (TR) variation is associated with alternate life cycles modes in ambystomatid salamanders. In the first experiment, the inheritance of TRalpha and TRbeta genotypes was determined for metamorphic and non metamorphic offspring from backcrosses between Ambystoma mexicanum (an obligate metamorphic-failure species) and metamorphic F1 hybrids (A. mexicanum x A. tigrinum tigrinum). The segregation of TR genotype was independent of the expression of life cycle mode phenotype, and neither TR locus was linked to DNA markers that flank a major-effect locus for life cycle mode. In the second experiment, a portion of the ligand-binding domain of TRalpha and TRbeta was cloned and sequenced for DNA samples from 14 different ambystomatid salamander populations, including obligate metamorphic, facultative metamorphic, and obligate metamorphic-failure taxa. Nucleotide sequence variation was found for both TRalpha and TRbeta, with several nonsynonomous substitutions that presumably code for nonconservative amino acid replacements. However, no general relationship was found between TR allelic variation and life cycle mode among populations or species. These data do not implicate TRs as candidate loci involved in the current maintenance or past evolution of alternate life cycle modes in members of the tiger salamander complex.

The genetics of amphibian declines: population substructure and molecular differentiation in the yosemite toad, Bufo canorus (Anura, bufonidae) based on single-strand conformation polymorphism analysis (SSCP) and mitochondrial DNA sequence data.

We present a comprehensive survey of genetic variation across the range of the narrowly distributed endemic Yosemite toad Bufo canorus, a declining amphibian restricted to the Sierra Nevada of California. Based on 322 bp of mitochondrial cytochrome b sequence data, we found limited support for the monophyly of B. canorus and its closely related congener B. exsul to the exclusion of the widespread western toad B. boreas. However, B. exsul was always phylogenetically nested within B. canorus, suggesting that the latter may not be monophyletic. SSCP (single-strand conformation polymorphism) analysis of 372 individual B. canorus from 28 localities in Yosemite and Kings Canyon National Parks revealed no shared haplotypes among these two regions and lead us to interpret these two parks as distinct management units for B. canorus. Within Yosemite, we found significant genetic substructure both at the level of major drainages and among breeding ponds. Kings Canyon samples show a different pattern, with substantial variation among breeding sites, but no substructure among drainages. Across the range of B. canorus as well as among Yosemite ponds, we found an isolation-by-distance pattern suggestive of a stepping stone model of migration. However, in Kings Canyon we found no hint of such a pattern, suggesting that movement patterns of toads may be quite different in these nearby parklands. Our data imply that management for B. canorus should focus at the individual pond level, and effective management may necessitate reintroductions if local extirpations occur. A brief review of other pond-breeding anurans suggests that highly structured populations are often the case, and thus that our results for B. canorus may be general for other species of frogs and toads.

Genetic analysis of steel and the PG-M/versican-encoding gene AxPG as candidates for the white (d) pigmentation mutant in the salamander Ambystoma mexicanum.

Vertebrate non-retinal pigment cells are derived from neural crest (NC) cells, and several mutations have been identified in the Mexican axolotl Ambystoma mexicanum (Ambystomatidae) that affect the development of these cell lineages. In "white" (d) mutant axolotls, premigratory NC cells differentiate as pigment cells, yet fail to disperse, survive, or both, and this leads to a nearly complete absence of pigment cells in the skin. Previous studies revealed that d affects pigment cell development non-autonomously, and have reported differences between white and wild-type axolotls in the structure and composition of the extracellular matrix through which NC and pigment cells migrate. Here we test the correspondence of d and two candidate genes: steel and AxPG. In amniotes, Steel encodes the cytokine Steel factor (mast cell growth factor; stem cell factor; kit ligand), which is expressed along the migratory pathways of melanocyte precursors and is required by these cells for their migration and survival; mammalian Steel mutants resemble white mutant axolotls in having a deficit or complete absence of pigment cells. In contrast, AxPG encodes a PG-M/versican-like proteoglycan that may promote the migration of A. mexicanum pigment cells, and AxPG expression is reduced in white mutant axolotls. We cloned a salamander orthologue of steel and used a partial genetic linkage map of Ambystoma to determine the genomic locations of steel, AxPG, and d. We show that the three genes map to different linkage groups, excluding steel and AxPG as candidates for d.

Adaptive evolution via a major gene effect: paedomorphosis in the Mexican axolotl.

Although adaptive evolution is thought to depend primarily on mutations of small effect, major gene effects may underlie many of the important differences observed among species in nature. The Mexican axolotl (Ambystoma mexicanum) has a derived mode of development that is characterized by metamorphic failure (paedomorphosis), an adaptation for an entirely aquatic life cycle. By using an interspecific crossing design and genetic linkage analysis, a major quantitative trait locus for expression of metamorphosis was identified in a local map of amplified fragment length polymorphisms. These data are consistent with a major gene hypothesis for the evolution of paedomorphosis in A. mexicanum.

What insights into the developmental traits of urodeles does the study of interspecific hybrids provide?

Natural and artificial hybrids represent an important source of material for developmental and evolutionary studies of urodeles. We review the available literature on hybrid salamanders, emphasizing the unique developmental insights that these organisms provide. Of particular interest is the application of new molecular tools to identify DNA markers for traditional characters in developmental research, and we discuss our own results using Bulk Segregant Analysis to identify RAPD markers for the white phenotype in the axolotl. We pay particular attention to the inferences that can be drawn from the many disparate crosses between ambystomatid salamanders that vary in their metamorphic response. These crossing experiments suggest that 1) metamorphosis is dominant to paedomorphosis, 2) that different ambystomatids use different genetic mechanisms to block metamorphosis and become sexually mature, larval paedomorphs, and 3) metamorphosis may be controlled by a few genetic loci. As increasingly sophisticated molecular approaches are applied to these and other hybrid crossing schemes, it should be possible to understand the mechanistic basis of a wide variety of developmental characters that differentiate urodele species.

Esophageal dysfunction and chest pain in patients with mitral valve prolapse: a prospective study utilizing provocative testing during esophageal manometry.

PURPOSE: The cause of chest discomfort in patients with mitral valve prolapse (MVP) remains unknown. Our aim was to determine prospectively the incidence of esophageal disorders and abnormal responses to edrophonium chloride and esophageal acid infusions in patients with MVP and troublesome non-ischemic chest pain. PATIENTS AND METHODS: After coronary artery disease was excluded, 20 patients with MVP and chest pain underwent esophageal manometry and provocative testing with edrophonium chloride and acid infusion. Seven patients with MVP but without chest pain served as control subjects; they also underwent esophageal manometry with provocative testing. RESULTS: Esophageal manometry revealed esophageal disorders in 16 patients: diffuse esophageal spasm in 14 patients, nutcracker esophagus in one, and hypotensive lower esophageal sphincter in one. Esophageal motility was normal in four patients. Injection of edrophonium and acid infusion tests evoked typical chest discomfort in three of 18 and five of 19 patients, respectively. In six of seven control subjects with MVP but with no chest discomfort, esophageal motility was normal and provocative testing did not produce chest discomfort (p less than 0.05 versus results in patients). CONCLUSION: Esophageal disorders were common and may account for chest discomfort in certain patients with MVP and persistent chest pain syndromes.