Humans generally heal skin injuries with a fibrous scar, which results in increased skin tightness, altered cosmesis, and morbidity when scars form over joints. Under specific and rare circumstances, humans and mammals exhibit spontaneous tissue regeneration, which heals tissue to their original architecture and function without a scar. Thus, the molecular machinery for tissue regeneration exists in mammals and understanding how to elicit regeneration will help us attain our long-term goal of regenerating injured human skin. Prior work from our group and others used ear hole closure to demonstrate that “regeneration-competent” mouse strains, including p21–/–, wildtype aged, and skin-specific CXCL12–/– mice, close ear holes to ~90% of the wounded size compared to control mice, which close holes to ~50%. Despite this improved healing, only 1% of regeneration-competent mice achieved complete ear hole closure. During the past funding cycle, we increased the frequency of complete hole closure, and most recently showed that topical imiquimod activates TRPA1+ neurons to drive complete ear hole closure in 20% of mice. This 20-fold improvement is substantial, and we continue to aim higher. In our preliminary data, we performed single-cell RNA-sequencing on wound- edge tissue from TRPA1+ activated and aged mice to identify signaling pathways driving tissue regeneration. Ligand-receptor interaction analysis identified a new receptor-mediated signaling pathway that promotes tissue regeneration. Unexpectedly, mice lacking this receptor exhibited the opposite phenotype; they demonstrated faster wound closure, decreased scar formation, and improved tissue regeneration in 3 different skin injury models. However, topical imiquimod treatment synergizes with receptor deficient mice to increase the frequency of complete ear hole closure to 90%. Taken together, we conclude that this newly identified receptor regulates both speed and quality of wound repair, which counters the current paradigm that the speed of wound closure and quality of wound healing are inversely related. Moreover, this pathway is independent from TRPA1+ neurons to promote regeneration. We hypothesize that this receptor pathway promotes scar formation and blocking this receptor permits tissue regeneration. In Aim 1, we elucidate the molecular mechanism of how this receptor promotes scar formation. We will use mouse genetics and our injury models to identify which cell types expressing this receptor are necessary and sufficient to drive scar formation. Next, we will use mouse genetics and pharmacologic inhibitors to identify the molecular mechanisms downstream of the receptor that promote scar formation. In Aim 2, a major gap in the wound healing field is the limited understanding of human wound healing due to sample availability. To fill this gap, we created the infrastructure to collect human skin in a dense time course after injury for single-cell RNA-sequencing. We expect increased recruitment ...