Identifying Epigenetic Mechanisms that Connect Limb Development and Regeneration with the Genomic Landscape

This project proposes to characterize mechanisms associated with limb development and regeneration in a primary animal model of tissue regeneration research. The Mexican axolotl (Ambystoma mexicanum) is widely regarded to be the best vertebrate model for studies of limb regeneration. With recent expansion of genetic and genomic resources, the Mexican axolotl is poised to make significant contributions to stem cell biology and regenerative medicine. The logical next step is to build upon systems level knowledge of how transcription is regulated during axolotl limb regeneration to identify the upstream, epigenetic mechanisms that deploy endogenous programs of regeneration. In this application, we propose to address the following question: Are epigenetic mechanisms that orchestrate regeneration unique to salamanders, or are they shared with other tetrapods including humans? As tetrapods, salamanders and humans develop their homologous limbs in the same way; signaling pathways are deployed to activate progenitor cells and regulate patterns of cellular proliferation and differentiation that ultimately form tissues. It is not known if conserved mechanisms of tetrapod limb development are re-deployed during salamander regeneration. Two research objectives are proposed to address this question. First, limb development and limb regeneration enhancers will be identified throughout the axolotl genome using ATAC-Seq (Assay for Transposase-Accessible Chromatin). By computationally and functionally associating enhancers to genes, the relative conservation of enhancer use between limb development and regeneration will be determined. Second, transgenic methods will be we developed and implemented to validate axolotl enhancer functions. An understanding of epigenetic mechanisms that allow the highly regenerative salamander to reform whole organs will improve approaches in stem cell biology and lead to the development of therapies for inducing tissue repair and regeneration in humans.