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.

When introduced or cultivated plants or animals hybridize with their native relatives, the spread of invasive genes into native populations might have biological, aesthetic, and legal implications. Models suggest that the rate of displacement of native by invasive alleles can be rapid and inevitable if they are favored by natural selection. We document the spread of a few introduced genes 90 km into a threatened native species (the California Tiger Salamander) in 60 years. Meanwhile, a majority of genetic markers (65 of 68) show little evidence of spread beyond the region where introductions occurred. Using computer simulations, we found that such a pattern is unlikely to emerge by chance among selectively neutral markers. Therefore, our results imply that natural selection has favored both the movement and fixation of these exceptional invasive alleles. The legal status of introgressed populations (native populations that are slightly genetically modified) is unresolved by the US Endangered Species Act. Our results illustrate that genetic and ecological factors need to be carefully weighed when considering different criteria for protection, because different rules could result in dramatically different geographic areas and numbers of individuals being protected.conservation | California Tiger Salamander | genetics | hybridization | Ambystoma

BackgroundMicroarray 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.ResultsConsiderable 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.ConclusionMany 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.

BackgroundFollowing 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.ResultsWe 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.ConclusionOur 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 neural and epidermal factors. Our findings indicate the general value of quantitative proteomic analysis in understanding the regeneration of complex structures.

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, Ambystoma 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 negative associations between native and introduced alleles and genotypes in these two ponds. Despite rampant hybridization, these data provide evidence of some constraints on hybridization 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.