There is an urgent clinical need to develop new therapeutics to promote bone regeneration. A critical aspect of the bone healing process begins with the expansion of periosteal progenitors that occurs immediately after injury and then the differentiation of these progenitors to bone forming osteoblasts and chondrocytes, yet mechanisms that control skeletal progenitor/stem cell activation, expansion, and differentiation in response to injury are poorly described. Our project will study the role of the R-spondin (ligand) – Lgr (receptor) signaling axis in regulating these progenitors and bone regeneration. R-spondins (roof plate specific spondin) are a family of four secreted matricellular proteins (Rspo1-4) that bind to Leucine-rich repeat-containing G-protein coupled receptors 4/5/6 (Lgrs). Rspo-Lgr interaction potentiate canonical Wnt pathway by preventing the turnover of Wnt Frizzled receptors, and hence determines canonical Wnt signaling levels. While canonical Wnt signaling is known to play an important role in bone regeneration, very little research has explored positive modulators of Wnt signaling. In particular, the requirement of Rspo-Lgr in the context of fracture healing has never been examined due to lack of appropriate models. Our primary goal is to define the requirement of Rspo2/3 and Lgr6 in mesenchymal progenitors in response to bone injury. We have defined three specific aims to address this goal. In Aim1, we will use single and compound Rspo2 and Rspo3 floxed mice crossed with an alphaSMACreERT2 mouse to disrupt the Rspo2/3 genes in mesenchymal progenitors at the time of fracture. Bone healing will be assessed using microCT, histology, molecular analysis, and mechanical testing. Alterations in canonical Wnt signaling and osteogenic potential of Rspo2/3 deficient progenitors will be assessed. In Aim 2, Lgr6 knockout mice will be investigated for their bone healing properties using parameters similar to Aim 1. In Aim 3, Rspo2 will be delivered to bone injury sites and the impact on BMP and Wnt signaling, progenitor activation and differentiation, and bone healing assessed. Completion of this project will identify the requirement of Rspo2/3-Lgr6 interaction in fracture healing and will provide new therapeutic directions for enhancing bone healing.