PROJECT SUMMARY Orthopedic injuries represent one of the most significant health and economic burdens in our nation. Bone fragility with age due to diseases such as osteoporosis increases the risk for fracture, while at the same time leading to delayed bone healing resulting in a higher risk for non-union. With a growing aging population, the demand for therapeutics to combat age-related bone loss is one of the pillars of the American Orthopedic Association’s “Own-the-Bone” initiative. Current treatments mainly focus on preventing further bone loss rather than increasing bone mass in a substantial manner. Skeletal stem and progenitor cells (SSPCs) are essential for proper bone mass and bone healing. SSPCs are known to decline in frequency and function with age. Therefore, SSPCs represent the most direct target to increase bone mass. Previous studies and our own data suggest that Notch signaling plays a pivotal role in SSPC homeostasis and self-renewal. We recently identified a conditional Notch knockout mouse model which exhibits a striking phenotype of increasing bone formation in all skeletal elements in aging mice. This mouse model, the Ncstnf/f;LepR-cre mouse, is deficient in Nicastrin (Ncstn), an essential component of the Notch signal transduction machinery of all 4 Notch receptors in LepR+ SSPCs. This model has exciting therapeutic potential to combat age-related bone loss. Based on this cKO model and our preliminary data I hypothesize that Notch signaling is essential in maintenance of the skeletogenic stem cell pool during adulthood and aging. In order to investigate the molecular mechanism by which Notch signal inhibition leads to increased trabecular bone mass with age, we performed bulk RNA sequencing on LepR+ SSPCs from middle-aged WT and cKO mice. This unbiased sequencing approach identified Ebf3 as a significantly downregulated gene in cKO cells and therefore potential downstream mediator of Notch signaling in promoting SSPC differentiation into osteoblasts. Previous work has linked Ebf3 to the inhibition of osteogenesis, however, the upstream regulators of Ebf3 are not yet known. Also, while others have looked into the role of Notch in SSPCs, the downstream mechanisms by which Notch is controlling SSPC function are still largely unknown. Therefore, this proposal aims to i) define the precise role of Notch signaling in SSPCs during aging/adulthood on proliferation, self-renewal and differentiation and ii) uncover the molecular mechanism by which Notch signaling is having the effects on proliferation, self-renewal and differentiation. These aims will provide novel specific mechanistic insights into the mechanism by which a Notch-Ebf3 signaling axis maintains skeletal stem cells in their primitive state throughout the lifespan of an animal.