Scope: Developing Regeneration Resources for a Model Amphibian

This Multi-PD/PI application seeks to develop resources that will better enable studies of a highly regenerative amphibian, the Mexican axolotl (Ambystoma mexicanum). There is need to probe axolotl regeneration more deeply, with the same powerful approaches that have proven so effective in genetic model organisms. Specific Aim 1 will accomplish the first chemical genetic screen of axolotl regeneration using an embryonic tail regeneration assay. Using pre-feeding axolotl embryos that are efficiently reared in micro-titer plates, approximately 10,000 soluble chemicals from commercial, clinical-stage, and novel natural products libraries will be tested for impact on tail regeneration. Preliminary data show that the chemical screen and tail regeneration assay are likely to identify new molecules that impact regeneration. Positive hits from this screen will be reported to the community and seven chemicals, including inhibitors of Wnt, Tgfâ, and Fgf signaling, will be investigated further under Specific Aim 2, using histological and transcriptional approaches. In particular, assays will test for chemical effects on formation of the wound epidermis, which acts as an early signaling center in the recruitment of progenitor/stem cells. Also, assays will be used to assess cellular de-differentiation and proliferation, two processes that are associated with endogenous regeneration. Genes found to be expressed differently between control and chemically treated embryos will be prioritized for knock out using the CRISPR/Cas9 method. For each gene target, two gRNA pairs will be designed and injected into single cell axolotl embryos. Embryos will be reared to assess viability, and then administered tail amputations to confirm CRISPR gene editing and test for regeneration competence. Embryos associated with CRISPRs that block or cause abnormal regeneration, will be prioritized for founding stable transgenic lines. Specific Aim 3 will develop genetic constructs with cell specific promoters and fluorescent protein reporters, targeting signaling pathways and novel genes that are activated during tail regeneration. The expression patterns of constructs will be characterized in control and regenerating axolotl embryos. Embryos carrying the most robust and precise expression constructs will be reared to maturity to found stable transgenic lines. The chemical and genetic hits, and biological information arising from this model will be shared through a community website (Sal- Site). The proposed transgenics will be distributed by the Ambystoma Genetic Stock Center. Overall, this project integrates expertise across chemical screening, pharmacology, histology, transcription, transgenesis, and vertebrate biology to discover reagents and develop tools that are needed to enhance the axolotl for stem cell biology and regeneration.