Epigenetic regulation of the regenerating axolotl forelimb's proximodistal axis by retinoic acid - PROJECT SUMMARY The axolotl salamander is capable of perfect, scar-free regeneration throughout its body. In order to regenerate a complex biological structure such as a limb, cells at the site of injury must correctly re-establish axial patterns such that only the missing tissues regenerate. Retinoic acid (RA) is a pleiotropic morphogen that has long been studied for its roles in development and regeneration. Intracellular RA can, broadly, be secreted to neighboring cells, catabolized in the cytoplasm by cytochrome P450 family 26 (CYP26) proteins, or imported to the nucleus. In the nucleus, RA binds to its family of receptors (RARs) at retinoic acid response elements (RAREs) located in the promoters of RA-responsive genes. Upon RA binding to RARs, local chromatin remodels to modify transcription of the primary target. Inhibiting RA breakdown by CYP26 reprograms regenerating distal cells to a proximal state in a dose-dependent manner, suggesting a gradient of RA concentration along the proximodistal axis maintained by differential RA catabolism. Despite decades of study, few primary targets of RA signaling have been confirmed, and the exact genetic mechanisms by which RA establishes a proximodistal axis remain under studied. In addition, connective tissue fibroblasts have been found to be key drivers of axial re- establishment in regeneration, but their ability to store and confer positional “memory” upon injury is not well understood. Previous work has also demonstrated that fibroblasts are specifically responsive to supplemental RA. From these foundational studies, I hypothesize that differential chromatin compaction promoted by RA signaling in connective tissue fibroblasts establishes the regenerating limb’s proximodistal axis. The proposed project intends to elucidate the epigenetic regulation by RA signaling in fibroblasts and its spatiotemporal progression during regeneration through two specific aims. Aim 1 is to use single-nuclear multiomic sequencing to generate fibroblast-specific candidate genes with putative RAREs and test their responsiveness to RA. Aim 2 is to functionally test candidates through assaying the regulatory activity of RA-responsive genes and functional ablation of normal RA-driven transcriptional regulation. The proposed experiments will provide deeper insight into the molecular role of RA in connective tissue fibroblasts that generates proximodistal positional identity during axolotl limb regeneration. The findings from these experiments will enhance our understanding of the regulatory conditions behind complex tissue regeneration which promote successful regeneration in some species, such as the axolotl, but not in humans. These studies will potentially generate novel targets for regenerative therapies in humans, whose regenerative capacity is largely limited to the distal-most digit tip.
