Gene functions of the Ambystoma altamirani skin microbiome vary across space and time but potential antifungal genes are widespread and prevalent.

Amphibian skin microbiomes can play a critical role in host survival against emerging diseases by protecting their host against pathogens. While a plethora of biotic and abiotic factors have been shown to influence the taxonomic diversity of amphibian skin microbiomes it remains unclear whether functional genomic diversity varies in response to temporal and environmental factors. Here we applied a metagenomic approach to evaluate whether seasonality, distinct elevations/sites, and pathogen presence influenced the functional genomic diversity of the A. altamirani skin microbiome. We obtained a gene catalogue of 92 107 nonredundant annotated genes and a set of 50 unique metagenome assembled genomes (MAGs). Our analysis showed that genes linked to general and potential antifungal traits significantly differed across seasons and sampling locations at different elevations. Moreover, we found that the functional genomic diversity of A. altamirani skin microbiome differed between B. dendrobatidis infected and not infected axolotls only during winter, suggesting an interaction between seasonality and pathogen infection. In addition, we identified the presence of genes and biosynthetic gene clusters (BGCs) linked to potential antifungal functions such as biofilm formation, quorum sensing, secretion systems, secondary metabolite biosynthesis, and chitin degradation. Interestingly genes linked to these potential antifungal traits were mainly identified in Burkholderiales and Chitinophagales MAGs. Overall, our results identified functional traits linked to potential antifungal functions in the A. altamirani skin microbiome regardless of variation in the functional diversity across seasons, elevations/sites, and pathogen presence. Our findings suggest that potential antifungal traits found in Burkholderiales and Chitinophagales taxa could be related to the capacity of A. altamirani to survive in the presence of Bd, although further experimental analyses are required to test this hypothesis.

Microhabitat types promote the genetic structure of a micro-endemic and critically endangered mole salamander (Ambystoma leorae) of Central Mexico.

The reduced immigration and emigration rates resulting from the lack of landscape connectivity of patches and the hospitality of the intervening matrix could favor the loss of alleles through genetic drift and an increased chance of inbreeding. In order for isolated populations to maintain sufficient levels of genetic diversity and adapt to environmental changes, one important conservation goal must be to preserve or reestablish connectivity among patches in a fragmented landscape. We studied the last known population of Ambystoma leorae, an endemic and critically threatened species. The aims of this study were: (1) to assess the demographic parameters of A. leorae and to distinguish and characterize the microhabitats in the river, (2) to determine the number of existing genetic groups or demes of A. leorae and to describe possible relationships between microhabitats types and demes, (3) to determine gene flow between demes, and (4) to search for geographic locations of genetic discontinuities that limit gene flow between demes. We found three types of microhabitats and three genetically differentiated subpopulations with a significant level of genetic structure. In addition, we found slight genetic barriers. Our results suggest that mole salamander's species are very sensitive to microhabitat features and relatively narrow obstacles in their path. The estimates of bidirectional gene flow are consistent with the pattern of a stepping stone model between demes, where migration occurs between adjacent demes, but there is low gene flow between distant demes. We can also conclude that there is a positive correlation between microhabitats and genetic structure in this population.

Early gene expression during natural spinal cord regeneration in the salamander Ambystoma mexicanum.

In contrast to mammals, salamanders have a remarkable ability to regenerate their spinal cord and recover full movement and function after tail amputation. To identify genes that may be associated with this greater regenerative ability, we designed an oligonucleotide microarray and profiled early gene expression during natural spinal cord regeneration in Ambystoma mexicanum. We sampled tissue at five early time points after tail amputation and identified genes that registered significant changes in mRNA abundance during the first 7 days of regeneration. A list of 1036 statistically significant genes was identified. Additional statistical and fold change criteria were applied to identify a smaller list of 360 genes that were used to describe predominant expression patterns and gene functions. Our results show that a diverse injury response is activated in concert with extracellular matrix remodeling mechanisms during the early acute phase of natural spinal cord regeneration. We also report gene expression similarities and differences between our study and studies that have profiled gene expression after spinal cord injury in rat. Our study illustrates the utility of a salamander model for identifying genes and gene functions that may enhance regenerative ability in mammals.

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

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.

Creation of chimeric mutant axolotls: a model to study early embryonic heart development in Mexican axolotls.

The Mexican axolotl (Ambystoma mexicanum) provides an excellent model for studying heart development since it carries a cardiac lethal mutation in gene c that results in failure of contraction of mutant embryonic myocardium. In cardiac mutant axolotls (c/c) the hearts do not beat, apparently because of an absence of organized myofibrils. To date, there has been no way to analyze the genotypes of embryos from heterozygous spawnings (+/c x +/c) until stage 35 when the normal (+/c or +/+) embryos first begin to have beating hearts; mutant (c/c) embryos fail to develop normal heartbeats. In the present study, we created chimeric axolotls by using microsurgical techniques. The general approach was to transect tailbud embryos and join the anterior and posterior halves of two different individuals. The chimeric axolotl is composed of a normal head and heart region (+/+), permitting survival and a mutant body containing mutant gonads (c/c) that permits the production of c/c mutant offspring: 100% c/c offspring were obtained by mating c/c chimeras (c/c x c/c). The mutant phenotypes were confirmed by the absence of beating hearts and death at stage 41 in 100% of the embryos. Examination of the mutant hearts with electron microscopy and comfocal microscopy after immunofluorescent staining for tropomyosin showed identical images to those described previously in naturally-occurring c/c mutant axolotls (i.e., lacking organized sarcomeric myofibrils). These "c/c chimeric" axolotls provide a useful and unique way to investigate early embryonic heart development in cardiac mutant Mexican axolotls.

Isolation, amino acid sequence determination and binding properties of two fatty-acid-binding proteins from axolotl (Ambistoma mexicanum) liver. Evolutionary relationship.

Up until now, the primary structure of fatty-acid-binding proteins (FABPs) from the livers of four mammalian (rat, human, cow and pig) and three nonmammalian (chicken, catfish and iguana) species has been determined. Based on amino acid sequence comparisons, it has been suggested that mammalian and nonmammalian liver FABPs may be paralogous proteins that originated by gene duplication, rather than as a consequence of mutations of the same gene. In this paper we report the isolation and amino acid sequence determination of two FABPs from axolotl (Ambistoma mexicanum) liver. One of them is similar to mammalian liver FABPs (L-FABPs) and the other to chicken, catfish and iguana liver FABPs (Lb-FABPs). The finding of both L-FABP and Lb-FABP in a single species, as reported here, indicates that they are paralogous proteins. The time of divergence of these two liver FABP types is estimated to be of approximately 694 million years ago. The ligand-binding properties of axolotl liver FABPs were studied by means of parinaric-acid-binding and parinaric-acid-displacement assays. L-FABP binds two fatty acids per molecule but Lb-FABP displays a fatty-acid-conformation-dependent binding stoichiometry; L-FABP shows a higher affinity for fatty acids, especially oleic acid, while Lb-FABP has a higher affinity for other hydrophobic ligands, especially retinoic acid. In addition, the tissue-expression pattern is different, L-FABP is present in liver and intestinal mucosa while the expression of Lb-FABP is restricted to liver. Data indicate distinct functional properties of both liver FABP types.

Structure and developmental expression of Ikaros in the Mexican axolotl.

The Ikaros family of transcription factors plays an essential role in hematopoiesis. We report here the structure of cDNA clones encoding two Ikaros isoforms, Ikl and Ik2, in the Mexican axolotl. The Ik1 cDNA sequence is very similar to that of the rainbow trout, chicken, and mammalian Ik1 sequences. However, a 96 base pair region which encodes the first N-terminal zing finger (F1) is lacking from axolotl Ik1, both in clones from a cDNA library and clones isolated from direct polymerase chain reaction products. A region corresponding to exon 3 is completely absent from the axolotl Ik2 sequence and thus the Ik1 and Ik2 isoforms possess the same number of zinc finger motifs. The structure of these five CC-HH motifs is very well conserved in the axolotl, including the structural deviations from its amino acid consensus composition which are identical in all species analyzed to date. The axolotl Ik1 3' untranslated region sequence is very long (2538 bp) and contains two UA-rich motifs known as instability determinants and which could play a role in mRNA translational efficiency. Ikaros transcripts are first detected in the ventral blood island of stage 36 embryos, about 24 h before the first heartbeats (late tailbud stage), and then in the major lymphopoietic organs of the developing larvae. In situ hybridization demonstrates that Ikaros transcripts are abundant at the periphery of the thymus lobes, in the presumptive site of early thymocyte differentiation.

Cloning of a complementary DNA encoding an Ambystoma mexicanum metallothionein, AmMT, and expression of the gene during early development.

We have used a polymerase chain reaction strategy to isolate a metallothionein (MT) cDNA from the amphibian Ambystoma mexicanum (axolotl). This cDNA is 875-bp long and encodes a 60 amino acid protein, AmMT, typical for family 1 MTs. It contains 20 cysteine (Cys) residues that can be aligned with those of other vertebrate MTs. The overall structure of the protein is unique among vertebrates in having only two amino acid residues before the first Cys at the amino-terminal end. Northern analyses showed that AmMT is expressed throughout embryogenesis, giving rise to three mRNA species of 650, 750, and 1,600 nucleotides (nt). The 750 and 1,600 nt transcripts appear to result from differential use of polyadenylation signals, whereas the 650 nt RNA could arise from deadenylation of the 750-nt transcript. Both the 750- and 1,600-nt RNAs were presented in embryos before the mid-blastula transition (MBT). After the MBT, the 750-nt RNA was replaced by the 650-nt RNA which was gradually degraded to undetectable levels in post-neurulation embryos. Levels of the 1,600-nt transcript increased at gastrulation and reach a maximum in Stage 30 embryos. In adult animals, levels of the 750-nt RNA were high in liver and testes, and very low in lung, gut, skin, and oviducts, whereas levels of the 1,600-nt transcript were similar and moderately elevated in all tissues examined. In contrast, in Xenopus laevis, Northern analysis did not detect XIMT-A mRNA in embryos before late neurulation (Stage 24). XIMT-A mRNA levels then increased sharply in Stage 36 hatched embryos at levels similar to those found in adult livers. These results show that AmMT presents a unique expression pattern among metazoans being transcribed as two transcripts differing in the length of their 3' untranslated regions, the levels of which vary during embryogenesis and in adult tissues.

Structure and diversity of the heavy chain VDJ junctions in the developing Mexican axolotl.

The immune capacity of young and adult axolotls (Ambystoma mexicanum) was evaluated by examining the combinatorial and junctional diversity of the VH chain. A large number of VDJ rearrangements isolated from 2.5-, 3.5-, 10-, and 24-month-old animals were sequenced. Six JH segments were identified with the canonical structure of all known vertebrate JHs, including the conserved Trp103-Gly104-X-Gly106 motif. Four core DH-like sequences were used by most (80%) of the VDJ junctions. These G-rich sequences had structures reminiscent of the TCRB DB sequences, and were equally used in their three reading frames. About 25% of the Igh, VDJ junctions from 3.5-month-old axolotls were out of frame, but most rearrangements were in frame at 10 and 24 months, suggesting that there is active selection of the productively rearranged Igh chains in the developing animals. There was no significant difference between the size of CDR3 in young (3.5 months) and subadult (10 months) axolotls (mean: 8.5 amino acids). However, the CDR3 loop was 1 amino acid longer in 2-year-old adult animals (mean: 9.5 residues). Several pairs of identical VDJ/CDR3 sequences were shared between 3.5-month-old individually analyzed axolotls, or between groups of axolotl of different ages. These identical rearrangements might be provided by the selection of some B-cell clones important for species survival, although the probability that different 3.5-month-old axolotl larvae would produce identical junctions seems very low, considering their limited number of B cells (less than 10(5)). The high frequency of tyrosine residues and the paucity of charged residues in the axolotl CDR3 loops may explain the polyreactivity of natural antibodies, and also clarify why it is so difficult to raise specific antibodies against soluble antigens.

Differential expression of a novel isoform of alpha-tropomyosin in cardiac and skeletal muscle of the Mexican axolotl (Ambystoma mexicanum).

Alternative mRNA splicing is a fundamental process in eukaryotes that contributes to tissue-specific and developmentally regulated patterns of tropomyosin (TM) gene expression. Northern blot analyses suggest the presence of multiple transcripts of tropomyosin in skeletal and cardiac muscle of adult Mexican axolotls. We have cloned and sequenced two tropomyosin cDNAs designated ATmC-1 and ATmC-2 from axolotl heart tissue and one TM cDNA from skeletal muscle, designated ATmS-1. Nucleotide sequence analyses suggest that ATmC-1 and ATmC-2 are the products of the same alpha-TM gene produced via alternate splicing, whereas ATmC-1 and ATmS-1 are the identical isoforms generated from the alpha-gene. RT-PCR analysis using isoform-specific primer pairs and detector oligonucleotides suggests that ATmC-2 is expressed predominantly in adult axolotl hearts. ATmC-2 is a novel isoform, which unlike ATmC-1 and other known striated muscle isoforms expresses exon 2a instead of exon 2b.

Differential expression of C-protein isoforms in the developing heart of normal and cardiac lethal mutant axolotls (Ambystoma mexicanum).

Regulated assembly of contractile proteins into sarcomeric structures, such as A- and I-bands, is still currently being defined. The presence of distinct isoforms of several muscle proteins suggests a possible mechanism by which myocytes regulate assembly during myofibrillogenesis. Of several muscle isoforms located within the A-band, myosin binding proteins (MyBP) are reported to be involved in the regulation and stabilization of thick filaments during sarcomere assembly. The present confocal study characterizes the expression of one of these myosin binding proteins, C-protein (MyBP-C) in wild-type and cardiac lethal mutant embryos of the axolotl, Ambystoma mexicanum. C-protein isoforms are also detected in distinct temporal patterns in whole-mounted heart tubes and thoracic skeletal muscles. Confocal analysis of axolotl embryos shows both cardiac and skeletal muscles to regulate the expression of C-protein isoforms over a specific developmental window. Although the CPROAxslow isoform is present during the initial heartbeat stage, its expression is not retained in the adult heart. C-protein isoforms are simultaneously expressed in both cardiac and skeletal muscle during embryogenesis.

Identification and expression of a homologue of the murine HoxA5 gene in the Mexican axolotl (Ambystoma mexicanum).

An excellent model for studying heart development in vertebrates is the cardiac non-function lethal mutant (gene c) Mexican axolotl, Ambystoma mexicanum. In order to facilitate our analyses of the mutant system, we have undertaken a search for stage-specific molecular markers during embryonic development of the axolotl. We have concentrated on homeobox genes as suitable candidates for monitoring molecular changes during development. A 270-bp probe encoding a portion of the axolotl homeobox gene Ahox-1 was generated by PCR from a stage-18 axolotl embryonic cDNA library. 32P-labelled PCR-amplified Ahox-1 DNA was used as the probe for screening a lambda AM18 cDNA library using moderately stringent conditions. We isolated six clones and determined their partial nucleotide (nt) sequences. One of the clones, which has very high homology to human, mouse and rat Hox A5 (83 and 99% at the nt and amino-acid levels, respectively, in the homeodomain region), was analyzed further. RT-PCR analyses show that the level of expression of HoxA5 is very low at stage 11 of embryonic development (gastrula). The level of expression reaches maximum at stage 25 (tailbud) and then plateaus at stages 30 and 35 (heartbeat onset). Although the expression of Ahox-1 was also found to start at stage 11, it reaches a maximum level at stage 25 and declines at stage 35. We have also studied, using RT-PCR, the tissue-specific expression of HoxA5 and Ahox-1 in juvenile axolotl.

In vivo protein synthesis in developing hearts of normal and cardiac mutant axolotls (Ambystoma mexicanum).

Recessive mutant gene c in axolotls causes a failure of the hearts of affected embryos to function. The mutant hearts (c/c) lack organized sarcomeric myofibrils. The present study was undertaken to determine the overall pattern of in vivo protein synthesis and subsequent accumulation of the newly synthesized proteins for a 24-h period in normal (+/+ or +/c) and cardiac mutant (c/c) axolotl hearts at various stages of development. Additionally, selected cytoskeletal/myofibrillar proteins were analyzed in detail for their synthesis during heart development. For such analyses, the hearts were radiolabeled with 35S-methionine for 24 h and subjected to SDS-PAGE and autoradiography. Quantitative densitometric analyses of the bands show that, even though the overall protein pattern is similar in normal and mutant heart tissues, a general reduction in the synthesis of the proteins in mutant hearts is observed even at the earlier stages of development (stages 35-36 and 37-38). Synthesis and accumulation of most of the proteins is significantly inhibited in mutant hearts at later stages (stages 41-42). Tropomyosin synthesis in mutant hearts is at a level of only 72.6% of that in normal embryonic hearts at stage 35. The synthesis and the accumulation of the tropomyosin in mutant hearts decreases further with increasing age until the protein essentially stops being synthesized by stage 41.

Evolution of vertebrate IgM: complete amino acid sequence of the constant region of Ambystoma mexicanum mu chain deduced from cDNA sequence.

cDNA clones coding for the constant region of the Mexican axolotl (Ambystoma mexicanum) mu heavy immunoglobulin chain were selected from total spleen RNA, using a cDNA polymerase chain reaction technique. The specific 5'-end primer was an oligonucleotide homologous to the JH segment of Xenopus laevis mu chain. One of the clones, JHA/3, corresponded to the complete constant region of the axolotl mu chain, consisting of a 1362-nucleotide sequence coding for a polypeptide of 454 amino acids followed in 3' direction by a 179-nucleotide untranslated region and a polyA+ tail. The axolotl C mu is divided into four typical domains (C mu 1-C mu 4) and can be aligned with the Xenopus C mu with an overall identity of 56% at the nucleotide level. Percent identities were particularly high between C mu 1 (59%) and C mu 4 (71%). The C-terminal 20-amino acid segment which constitutes the secretory part of the mu chain is strongly homologous to the equivalent sequences of chondrichthyans and of other tetrapods, including a conserved N-linked oligosaccharide, the penultimate cysteine and the C-terminal lysine. The four C mu domains of 13 vertebrate species ranging from chondrichthyans to mammals were aligned and compared at the amino acid level. The significant number of mu-specific residues which are conserved into each of the four C mu domains argues for a continuous line of evolution of the vertebrate mu chain. This notion was confirmed by the ability to reconstitute a consistent vertebrate evolution tree based on the phylogenic parsimony analysis of the C mu 4 sequences.

Isolation and characterization of a developmentally regulated homeobox sequence in the Mexican axolotl Ambystoma mexicanum.

A homeobox-containing genomic DNA fragment was isolated from the Mexican axolotl. This clone was obtained from a partial genomic library enriched for sequences that cross-hybridized with the Drosophila Antp homeobox under low stringency hybridization conditions. DNA sequence analysis revealed that this sequence (Ahox1) was 66% homologous to the Antp homeobox sequence and was most closely related to the mouse Hox-1.6 (84% identity) and Drosophila lab (79% identity) homeobox sequences. Several cross-hybridizing fragments to Ahox1 were detected in both mouse and axolotl genomic DNA. This sequence was also shown to be conserved in other Ambystoma species. Northern blot analysis revealed that genes containing this sequence are developmentally regulated. Transcripts hybridizing to the Ahox1 homeobox probe were detected during the neurula and tail bud stages of development.

Analysis of the endocardium and cardiac jelly in truncal development in the cardiac lethal mutant axolotl Ambystoma mexicanum.

Recessive mutant gene c in axolotls results in a failure of the heart to function because of abnormal embryonic induction processes. The myocardium in this mutant lacks organized sarcomeric myofibrils. The present study was undertaken to determine if developmental abnormalities were evident in other areas of the heart besides the myocardium. A detailed comparative survey of the structure of developing normal and mutant hearts, including the endocardium, its cellular derivatives, and the extracellular matrix, known as cardiac jelly, showed that in the mutant there are fewer than the normal number of endocardial cells lining the heart lumen, the number of mesenchyme cells is reduced, and the cardiac jelly area is greatly enlarged in the posterior part of the truncus adjacent to the ventricle.