S. Randal Voss

Skin Regeneration in Adult Axolotls: A Blueprint for Scar-Free Healing in Vertebrates

While considerable progress has been made towards understanding the complex processes and pathways that regulate human wound healing, regenerative medicine has been unable to develop therapies that coax the natural wound environment to heal scar-free. The inability to induce perfect skin regeneration stems partly from our limited understanding of how scar-free healing occurs in a natural setting. Here we have investigated the wound repair process in adult axolotls and demonstrate that they are capable of perfectly repairing full thickness excisional wounds made on the flank. In the context of mammalian wound repair, our findings reveal a substantial reduction in hemostasis, reduced neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM) during scar-free healing. Additionally, we test the hypothesis that metamorphosis leads to scarring and instead show that terrestrial axolotls also heal scar-free, albeit at a slower rate. Analysis of newly forming dermal ECM suggests that low levels of fibronectin and high levels of tenascin-C promote regeneration in lieu of scarring. Lastly, a genetic analysis during wound healing comparing epidermis between aquatic and terrestrial axolotls suggests that matrix metalloproteinases may regulate the fibrotic response. Our findings outline a blueprint to understand the cellular and molecular mechanisms coordinating scar-free healing that will be useful towards elucidating new regenerative therapies targeting fibrosis and wound repair.

HYBRIDIZATION BETWEEN A RARE, NATIVE TIGER SALAMANDER (AMBYSTOMA CALIFORNIENSE) AND ITS INTRODUCED CONGENER

Exotic species threaten native biodiversity through predation, competition, and habitat alteration, but also by hybridizing with native species. A lack of reproductive isolation between exotic and native species can lead to genetic swamping, loss of native genetic diversity, and, in rare or endangered species, extirpation or extinction. We examined hybridization between a declining native salamander, the California tiger salamander, Am- bystoma californiense, and an introduced congener, A. tigrinum. Ambystoma californiense is restricted to central California where A. tigrinum has been deliberately introduced as fish bait. In the Salinas Valley, we sampled salamanders from four artificial ponds and two natural vernal pools. Based on mitochondrial DNA and two nuclear loci, we found that hybrids were present in all six ponds, and that these hybrids were viable and fertile. No potentially pure A. californiense were present in three of the six ponds, and only one pond had more than 8% possibly pure native animals. Despite a relatively ancient split and wide genetic divergence between these taxa, they are interbreeding and threatening the genetic purity of the native species. Our data also suggest that the extent of the genetic mixing depends on the breeding habitat. There is little evidence of barriers to gene exchange in the four artificial breeding ponds. However in the two vernal pools, we found significantly fewer larvae with hybrid genotypes and significantly more with pure parental genotypes than expected. Linkage disequilibria revealed positive associations between native alleles and genotypes, and neg- ative associations between native and introduced alleles and genotypes in these two ponds. Despite rampant hybridization, these data provide evidence of some constraints on hy- bridization in the native breeding habitats. Our results suggest that habitat characteristics of native species should be exploited in management strategies to limit hybridization with exotics.

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.

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

BackgroundEstablishing 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.ResultsApproximately 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.ConclusionsOur 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.

Multiple nuclear gene sequences identify phylogenetic species boundaries in the rapidly radiating clade of Mexican ambystomatid salamanders

Delimiting the boundaries of species involved in radiations is critical to understanding the tempo and mode of lineage formation. Single locus gene trees may or may not reflect the underlying pattern of population divergence and lineage formation, yet they constitute the vast majority of the empirical data in species radiations. In this study we make use of an expressed sequence tag (EST) database to perform nuclear (nDNA) and mitochondrial (mtDNA) genealogical tests of species boundaries in Ambystoma ordinarium, a member of an adaptive radiation of metamorphic and paedomorphic salamanders (the Ambystoma tigrinum complex) that have diversified across terrestrial and aquatic environments. Gene tree comparisons demonstrate extensive nonmonophyly in the mtDNA genealogy of A. ordinarium, while seven of eight independent nuclear loci resolve the species as monophyletic or nearly so, and diagnose it as a well‐resolved genealogical species. A differential introgression hypothesis is supported by the observation that western A. ordinarium localities contain mtDNA haplotypes that are identical or minimally diverged from haplotypes sampled from a nearby paedomorphic species, Ambystoma dumerilii, while most nDNA trees place these species in distant phylogenetic positions. These results provide a strong example of how historical introgression can lead to radical differences between gene trees and species histories, even among currently allopatric species with divergent life history adaptations and morphologies. They also demonstrate how EST‐based nuclear resources can be used to more fully resolve the phylogenetic history of species radiations.

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.

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

BackgroundThe 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.ResultsAnnotation 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!ConclusionThis 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.

Sal-Site: Integrating new and existing ambystomatid salamander research and informational resources

Salamanders of the genus Ambystoma are a unique model organism system because they enable natural history and biomedical research in the laboratory or field. We developed Sal-Site to integrate new and existing ambystomatid salamander research resources in support of this model system. Sal-Site hosts six important resources: 1) Salamander Genome Project: an information-based web-site describing progress in genome resource development, 2) Ambystoma EST Database: a database of manually edited and analyzed contigs assembled from ESTs that were collected from A. tigrinum tigrinum and A. mexicanum, 3) Ambystoma Gene Collection: a database containing full-length protein-coding sequences, 4) Ambystoma Map and Marker Collection: an image and database resource that shows the location of mapped markers on linkage groups, provides information about markers, and provides integrating links to Ambystoma EST Database and Ambystoma Gene Collection databases, 5) Ambystoma Genetic Stock Center: a website and collection of databases that describe an NSF funded salamander rearing facility that generates and distributes biological materials to researchers and educators throughout the world, and 6) Ambystoma Research Coordination Network: a web-site detailing current research projects and activities involving an international group of researchers. Sal-Site is accessible at http://www.ambystoma.org.

Species boundaries, phylogeography and conservation genetics of the red-legged frog ( Rana aurora/draytonii ) complex

The red‐legged frog, Rana aurora, has been recognized as both a single, polytypic species and as two distinct species since its original description 150 years ago. It is currently recognized as one species with two geographically contiguous subspecies, aurora and draytonii; the latter is protected under the US Endangered Species Act. We present the results of a survey of 50 populations of red‐legged frogs from across their range plus four outgroup species for variation in a phylogenetically informative, ∼400 base pairs (bp) fragment of the mitochondrial cytochrome b gene. Our mtDNA analysis points to several major results. (1) In accord with several other lines of independent evidence, aurora and draytonii are each diagnosably distinct, evolutionary lineages; the mtDNA data indicate that they do not constitute a monophyletic group, but rather that aurora and R. cascadae from the Pacific northwest are sister taxa; (2) the range of the draytonii mtDNA clade extends about 100 km further north in coastal California than was previously suspected, and corresponds closely with the range limits or phylogeographical breaks of several codistributed taxa; (3) a narrow zone of overlap exists in southern Mendocino County between aurora and draytonii haplotypes, rather than a broad intergradation zone; and (4) the critically endangered population of draytonii in Riverside County, CA forms a distinct clade with frogs from Baja California, Mexico. The currently available evidence favours recognition of aurora and draytonii as separate species with a narrow zone of overlap in northern California.

Relationship between stream order and length of larval period in the Salamander Eurycea wilderae

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