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Comparative Transcriptional Profiling of the Axolotl Limb Identifies a Tripartite Regeneration-Specific Gene Program

ABSTRACT: Identifying the genetic program that induces limb regeneration in salamanders is an important resource for regenerative medicine, which currently lacks tools to promote regeneration of functional body structures. The genetic network underlying limb regeneration has been elusive due to the complexity of the injury response that occurs concomitant to blastema formation. Here we performed parallel expression profile time courses of non-regenerative lateral wounds versus amputated limbs in axolotl. We show that limb regeneration occurs in three distinguishable phases--early wound healing followed by a transition phase leading to establishment of the limb development program. By focusing on the transition phase, we identified 93 strictly regeneration-associated genes involved in oxidative stress response, chromatin modification, epithelial development and limb development. The specific expression of the genes was confirmed by in situ hybridization. Regeneration-specific expression databases are critical resources for understanding how regeneration-relevant phenotypes can be induced from adult cells

ORGANISM(S): Ambystoma mexicanum

PROVIDER: GSE36451 | GEO | 2013/01/01

SECONDARY ACCESSION(S): PRJNA153543

REPOSITORIES: GEO

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Project description: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, a mass of progenitor cells that forms missing limb structures. An outstanding question is how molecular 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 highly similar between the three injury types, presumably because a common wound-healing program is employed across anatomical locations. However, we identified transcripts that were enriched in the limb compared to the flank and are associated with vertebrate limb development. Many of these genes were activated before blastema outgrowth and in situ hybridization showed that some of these genes were 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 that are associated with myelination of peripheral nerves, epidermal maintenance, and cell proliferation, suggesting that denervation affects myelinating Schwann cells, 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. We used microarray analysis to determine the gene expression changes that take place during limb regeneration, flank wound healing, and an denervated amputated limb. Epidermal tissue and cells adhered to the epidermis were collected as samples. Two harvested samples was pooled for each animal. Four biological replicates were collected from uninjured epidermis (D0) and at 1, 3, and 7 days post injury.

Project description:Variability of regenerative potential among animals has long perplexed biologists. Based on their amazing regenerative abilities, planarians have become important models for understanding the molecular basis of regeneration; however, planarian species with limited regenerative abilities are also found. Despite the importance of understanding the differences between closely related, regenerating and non-regenerating organisms, few studies have focused on the evolutionary loss of regeneration, and the molecular mechanisms leading to such regenerative loss remain obscure. Here we examine Procotyla fluviatilis, a planarian with restricted ability to replace missing tissues, utilizing next-generation sequencing to define the gene expression programs active in regeneration-permissive and regeneration-deficient tissues. We found that Wnt signaling is aberrantly activated in regeneration-deficient tissues. Remarkably, down-regulation of canonical Wnt signaling in regeneration-deficient regions restores regenerative abilities: blastemas form and new heads regenerate in tissues that normally never regenerate. This work reveals that manipulating a single signaling pathway can reverse the evolutionary loss of regenerative potential. RNA-seq experiments to identify gene expression changes following amputation in body regions with variable regenerative potential. Adult Procotyla fluviatilis were amputated at sites either anterior or posterior to the pharynx. After 24 hours post-amputation, tissues near the amputation site were excised and RNA was extracted. Similar tissues were excised from uncut control animals. Samples were processed for RNA-seq using Illumina procedures. We generated a de novo P. fluviatilis transcriptome and used RNA sequencing (RNA-seq) to characterize transcripts from excised tissue fragments in Reg+ and Reg- body regions 24 hours post-amputation. We performed parallel analyses on tissues excised from intact animals at identical body regions to account for regional differences in transcripts, thereby identifying changes resulting from amputation. Samples A1-A3 = Regeneration-proficient (Reg+) tissue excision 24 hours after amputation. Samples B1-B3 = Tissue excision from regeneration-proficient (Reg+) region but not amputated. Samples C1-C3 = Tissue excision from regeneration-deficient (Reg-) tissues 24 hours after amputation. Samples D1, D3-D4 = Tissue excision from regeneration-deficient (Reg-) region that was not amputated.

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Comparative Transcriptional Profiling of the Axolotl Limb Identifies a Tripartite Regeneration-Specific Gene Program

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