Project Summary Tissue integrity is maintained by specialized adult stem cells that replenish old and damaged cells. During aging and in disease states, stem cell function is compromised leading to progressive degeneration of the structure and function of vital organs. This is particularly apparent in the skeleton, which becomes increasingly fragile and prone to fractures as we age. To uncover and counteract the mechanisms of skeletal degeneration and enhance bone repair, we must first define skeletal stem and progenitor cell (SSPC) behavior in vivo and determine the essential regulators of cellular function. This is a technically challenging feat as, in contrast to other organs where stem cells reside within a defined physical location and are readily identified by gene and protein expression, cells with progenitor activity can be found throughout bone and there are no specific markers to identify these in situ. As a result, we still know remarkably little about SSPCs, including whether a multipotent stem cell exists in vivo or if bone is maintained by a pool of lineage-restricted progenitors. Additionally, it is unclear whether progenitors from different locations are functionally equivalent. Recently, we identified several distinct skeletal populations with stem cell characteristics that demonstrate distinct dynamic responses to injury. We hypothesize that these correspond to SSPC subtypes from different anatomical regions of bone and that they play unique roles in skeletal regeneration in homeostasis and injury repair. We will leverage cutting-edge technologies to investigate this hypothesis. In the first part of this proposal, we will perform parallel in vivo clonal functional and transcriptional analysis to identify cells with progenitor activity in an unbiased manner, interrogate the differentiation potential of SSPCs in defined bone regions and distinguish gene expression signatures associated with precise cellular behaviors. These experiments will, for the first time, establish the phenotype and function of individual SSPCs in their native environment and across distinct local niches, providing a holistic overview of the SSPC landscape to enable the identification of novel regulators of stem cell activity and markers to enable the isolation of therapeutically useful cell populations. In the second part of this proposal, we will chart the dynamic response and contribution of individual progenitors to the repair of various types of bone injury. These data will reveal the key cell populations driving discrete stages of bone healing and their transcriptional response to injury, including the signaling pathway signatures enriched in progenitors. Together this study will dissect the complex web of skeletal stem cell states to facilitate the development of targeted strategies to improve bone health and fracture healing.