91. Conserved Epidermal Factors Direct Bone Growth in Mouse Digit Tip Regeneration

PURPOSE: An important goal of regenerative medicine is to understand endogenous mechanisms of tissue regeneration in mammals. The ability to regenerate limbs and appendages exists in various forms in the natural world, including axolotl salamander limb, zebrafish fin, and mouse digit tip (MDT). In all examples of appendage regeneration, a first critical step is re-epithelialization of the amputation stump to form a specialized regeneration epidermis (RE). Despite the universal importance of the RE, there is much to learn about its cellular composition, signaling pathways, and inter-species conservation between organisms that display innate abilities in appendage regeneration. We asked whether conserved factors in the RE exist between evolutionarily divergent species and if these factors play critical roles in MDT regeneration. METHODS: Given the parallels between development and regeneration, we employed an approach of profiling regenerating zebrafish fin for factors potentially involved in mammalian limb development pathways. Single cell RNA sequencing was performed on sorted basal epidermal cells harvested from zebrafish fin during the peak of RE formation. Mouse conditional knockout lines were generated using the CreER/LoxP system. Analysis of MDT regeneration was performed using immunofluorescence, in situ hybridization, and micro-CT. Regenerating axolotl limbs were harvested at various time points corresponding to stages of RE formation. Discarded specimens from staged human below-knee amputations were collected to evaluate expression in human limb tissue. RESULTS: Single cell RNA sequencing of zebrafish fin RE highlighted a family of epidermal transcription factors also involved in mouse limb development. In situ hybridization and immunofluorescent staining suggested that homologues of these factors are present in the RE of mouse digit tip and axolotl limb. Conditional epidermal knockout in mice revealed an impairment of digit tip regeneration manifested by reductions in both RE formation and bone regrowth. Analysis of secreted growth factors demonstrated a marked reduction after conditional knockout, implying that they act downstream of epidermal transcription factors. Finally, analysis of amputated human leg skin suggests that these factors are involved in the post-injury response in humans as well. CONCLUSION: The presence of common factors of the RE may reflect evolutionary conservation in the epidermal orchestration of appendage regeneration amongst divergent species. Epidermal transcription factors may have a prominent role in directing bone growth during mouse digit tip regeneration, which could reflect a reengagement of developmental signaling pathways. Work is underway to identify the transcriptional targets of these genes as well as viral manipulation during MDT regeneration. With a heightened understanding of the role of the RE in appendage regeneration, future work may lead to novel therapies for patients suffering from limb loss, bone defects, and chronic wounds.