Skin protects our body against the environment, and its ability to repair upon injury is directly connected to both disease and survival. Failure to properly repair injured tissue can result in chronic wounds, which are associated with severe complications and even death. Multiple cell types, like epithelial cells and fibroblasts, must coordinate their behaviors to achieve injury repair. These processes have so far mainly been studied in genetically homogeneous mouse models. However, healthy skin contains many clones harboring somatic mutations, including oncogenic Ras mutations which are the leading cause of Squamous Cell Carcinoma. How genetically diverse epithelial cells affect injury repair is unknown. The goal of this proposal is to unravel how cell behaviors are properly orchestrated on the single-cell and tissue-scale level during repair. The critical barrier to addressing these fundamental questions lies in the inability to study these dynamic processes in an intact mammal. To this end, my laboratory has established an in vivo strategy to directly visualize and manipulate epithelial cells and fibroblasts in the skin of live mice. We have previously used this strategy to define the roles of epithelial cells and fibroblasts in homeostasis, and the complex spatiotemporal organization of epithelial cell behaviors during repair. We hypothesize that epithelial cells and fibroblasts use flexible behaviors to enable proper wound healing of genetically mosaic skin. We will first define how a mosaic epithelium responds to injury (Aim 1). Our preliminary results show that a mosaic Hras mutant skin epithelium heals at a normal rate and does not induce tumors, but that WT and Hras mutant cells exhibit different behaviors during repair. We hypothesize that the behavioral flexibility of WT cells confers the ability to contain their Hras mutant neighbors and ultimately achieve normal injury repair. To test this, we will define the epithelial cell behaviors and evaluate the roles of signaling pathways during injury repair of an Hras-mosaic skin epithelium. We will then examine the roles of fibroblasts in contact with epithelial cells during injury repair (Aim 2). We hypothesize that direct communication between fibroblasts in the upper dermis and epithelial cells in the epidermis coordinates cell behaviors and repair. To test this, we will define the behaviors and functions of fibroblasts in the presence of WT epithelial cells and an Hras-mosaic epithelium. We will achieve both these aims by combining intravital microscopy with genetic and pharmacological manipulations of distinct repair behaviors or resident cell types in vivo. The proposed experiments will allow us to dissect the coordination and functional significance of distinct cell activities, populations, and interactions during repair using an integrated approach of cutting-edge imaging technology, genetic manipulation, cell biology, and single cell sequencing. Given that many aspects of injur...