Mechanisms of Fetal Skin Regeneration - The ability to regenerate complex, multi-lineage organs remains one of the key challenges in regenerative medicine. Many mammalian organs, including the heart, digit tip, and skin, retain some ability to regenerate after damage during neonatal or fetal development, but the ability to regenerate decreases dramatically as the animal develops. Elucidating the mechanistic basis of this differential regenerative capacity is key to understanding the principles of regenerative control and laying the foundation for therapeutic reactivation of regeneration after injury. To this end, we propose to determine the genetic and molecular basis of scarless, multi-lineage regeneration after fetal skin injury. Healing of full-thickness injuries to postnatal skin is accompanied by fibrosis (scar formation), but injuries to fetal skin heal without scar formation, a phenomenon that has fascinated biologists for decades, but the mechanisms behind these differences remain poorly understood. We have overcome three technical hurdles and are now uniquely positioned to take on this challenge: (1) We have developed a robust surgical procedure to conduct wounding in fetal mice in utero. (2) We have optimized a cell isolation protocol to collect sufficient cells for single-cell RNA and ATAC analyses from wounded tissues. (3) We have established a strategy for rapid, in vivo genetic manipulation directly in wild-type mice by delivering targeted genetic cargo with viral vectors. Together, these approaches will enable us to identify and assay a relatively large number of genes in vivo for their involvement in either regeneration or scar formation. Furthermore, we have substantial preliminary data suggesting that injuries in fetal skin (E16.5) are healed with regeneration of diverse cell types from multiple lineages—hair follicles, innervations, blood and lymphatic vessels, and diverse dermal cell types—resulting in skin that is morphologically similar to unwounded skin. By contrast, these cell types fail to regenerate properly upon postnatal injury. We have also already identified one candidate factor—CXCL12, a postnatal wound-specific secreted factor, that inhibits regeneration when overexpressed in fetal skin. Building on this foundation, we propose to create comprehensive cellular and molecular atlases of fetal vs. postnatal wound healing processes using a single-cell multiomic approach (combining single-cell RNAseq and single-cell ATACseq) to assess both transcriptome and chromatin changes in the same single-cell. We will then determine if the reduction of CXCL12 in postnatal wounds promotes regeneration. We will also systematically test additional secreted factors that are unique to either fetal or postnatal wounds in their ability to drive or inhibit multilineage regeneration in the skin. Collectively, this work will provide critical insights into how regeneration ability is blocked or lost as an organ develops, open new research directions for studying organ- level regeneration in mammals, and guide new therapeutic strategies that promote regenerative healing under challenging conditions including burns and chronic non-healing wounds.
