Mechanisms of Organ-Level Regeneration in Mammalian Skin

Abstract

Mammalian injury responses are typically restricted to tissue-specific repair. Skin wound healing is a prime example: after full-thickness wounding, although the epidermis is repaired, other cell types from diverse lineages fail to reform, including hair follicles, melanocytes, arrector pili muscles, complex innervations, lymphatic vessels, and adipocytes. While previous studies have shown that late gestational embryonic skin wounds can heal without excessive collagen deposits, it is unclear whether skin at this stage can regenerate across lineages and what mechanisms underlie this ability. Here, I present studies that define the developmental window of organ-level regeneration in the skin, uncover the mechanisms that block regeneration, and demonstrate strategies that overcome the barrier. First, I delineate the responses to wounding in late gestational embryonic skin and postnatal skin. Using newly optimized staining and imaging procedures, I demonstrate that skin wounded at embryonic day (E)16.5 regenerates diverse, interconnected cell types. Through a physiological challenge, I show that these regenerated cell types are functional. Regenerative ability decreases progressively, with a complete loss of multi-lineage regeneration by postnatal day (P)5. Beyond scarring, wounding at P5 also causes hyperinnervation, epidermal hyper-thickening, and excessive myeloid cell infiltration. By comparing how skin responds to wounding at E16.5 vs P5, we now have the opportunity to identify and test mechanisms that control organ-level regeneration in the same model system. Second, I identify key genes that inhibit regeneration. With single-cell RNA sequencing, I identified a postnatal wound-specific fibroblast (PWF) population that is absent in unwounded skin and after embryonic wounding. This population is enriched in secreted factors and is the only wound-specific population to emerge. I then outline my viral-based screening approach that enabled rapid in vivo testing of secreted factor candidates that inhibit regeneration. From the screen, I discovered that three PWF-enriched genes — Timp1, Cxcl12, and Ccl7 — inhibit organ-level regeneration when overexpressed in E16.5 wounds. Third, I demonstrate that the regeneration block can be removed. I show that overexpression of PWF-enriched factors not only blocks the reformation of diverse cell types but also drives hyperinnervation, and that hyperinnervation alone is sufficient to prevent regeneration in E16.5 wounds. With cell type-specific knockout approaches, I further show that PWFs directly drive hyperinnervation. By reducing neuronal activity — either by blocking synaptic vesicle release or by temporally ablating peripheral neurons — I demonstrate that multi-lineage regeneration can be restored in P5 wounds. These strategies can also promote regeneration in adult wounds, further highlighting therapeutic potential. Together, these studies suggest that the intrinsic ability to regenerate at the organ level may not be irreversibly lost, but rather blocked. By comparing wounding responses in E16.5 vs P5 skin, I identify fibroblast-driven hyperinnervation as a key barrier. This regeneration block can be removed either genetically, chemically, or with a viral vector approach, thereby unlocking latent regenerative potential in non-regenerating skin. These efforts may pave the way for treatments that promote regeneration of functional skin, and they also provide a framework for identifying and targeting similar regeneration blocks in other organ systems.