Project Summary Regenerative medicine techniques have become an important option for the many patients suffering from poorly healing musculoskeletal injuries. Benefits observed from stem cells therapies have, in part, been attributed to their paracrine actions that initiate appropriate cell-cell communication in the injured tissue. Despite great strides in understanding molecular controls of spatial cues during tissue patterning, a major knowledge gap exists regarding the role of timekeeping in coordinated tissue responses. Time-keeping genes involved in the molecular clock are principally organized to synchronize cells, especially in the day-night rhythms. However, the oscillatory nature of timekeeping genes may also allow them to contribute to the properties of multipotent cells that activate upon injury. Despite evidence that clock genes have been shown to play a role in synchronizing cell states during intestinal regeneration and contribute to the regenerative capacity of basal epithelium and cartilage, the role of the clock system across multiple cell types during whole limb regeneration is unknown. This proposal aims to uncover and exploit the relationship between clock genes and regeneration during development, with the goal of controlling the speed and capacity for tissue regeneration in the Xenopus laevis. Xenopus larvae are capable of tail regeneration and exhibit regenerative and regeneration-incompetent developmental stages, making them a useful model for interrogating the process of tissue repair. Xenopus also develop quickly and ex vivo, permitting easy monitoring of morphology and the outcomes of genetic manipulation. Although mammals have a more limited capacity for self-regeneration, understanding the role of timekeeping genes in Xenopus can provide important insights into how we may control tissue regeneration in humans. In Aim 1, biological time- keeping machinery will be characterized in cells of the regenerative and non-regenerative Xenopus tail using in vivo DNA reporters and single-cell transcriptomics. Single-cell transcriptomics will be used to define which cell type(s) act as drivers of time keeping processes to maintain the collective actions of greater than 40 cell types during tail regeneration. Aim 2 will determine how the clock gene system affects regenerative capacity and the speed of regrowth by assessing timekeeping control at both network and single-gene levels. Regenerative capacity will be evaluated when the clock gene network is amplified or damped using small molecule treatments. CRISPR/Cas9 clock gene overexpression and knock downs will be used to determine the role of five core time- keeping genes known to affect regeneration in other tissues. The effects of small molecules and clock gene knockouts on the cell synchronization landscape will be monitored using single-cell sequencing. This project will be the first to closely characterize the clock system in whole-limb regeneration and may lead to greater i...