Abstract: How skeletal stem cells are activated to expand, migrate to the injury site, and become osteoblast cells to restore damaged bone are central questions in skeletal regeneration, which is key for the maintenance of a functional skeletal system. Craniofacial bones mainly form through intramembranous ossification with limited bone marrow space and skeletal stem cells that reside in the suture, a special fibrous joint that connects calvarial bones of the skull together, have been identified as a major cell population that are required for craniofacial bone homeostasis and injury repair. Numerous studies in both human and mice have highlighted the importance of stem cell niche, a microenvironment where stem cells reside and also regulate stem cell behaviors. As the craniofacial skeleton encases the brain and protects it, calvarial bone defects in the skull, if failed to heal with bony tissue and consequently fibrous non-unions occur, are associated with high morbidity and mortality. Therefore, reconstruction and regeneration of calvarial defects, in particular critical-size defects, continuously poses as an unmet therapeutic challenge. The G protein stimulatory α-subunit (Gas), encoded by GNAS gene, transduces signals from G protein coupled receptors (GPCRs) and has emerged as a critical regulator of osteoblast differentiation by inhibiting Hedgehog (Hh) signaling. Studies in both mouse models and human genetic diseases supports the critical roles of both Gas and Hh signaling in regulating skeletal stem cells in multiple contexts. Having showed that activation of Hh signaling by loss of Gas is a common pathway that critically regulates intramembranous ossification in calvarial bone formation as in heterotopic ossification (HO), we have also made novel findings in unpublished preliminary studies in an calvarial bone injury model that directed suture stem cells (SuSCs) migration to the injury site correlates with upregulated expression of chemokine (C-X-C motif) ligand 12 (Cxcl12) and Sonic Hedgehog (Shh), to a less extent Indian Hedgehog (Ihh), in SuSC niche. Further, inhibition of the Cxcl12 cognate receptor Cxcr4, which is coupled to Gai that counteracts Gas signaling, severely impaired calvarial bone regeneration. Loss of Gas enhanced Shh expression, which induced osteoblast differentiation. We therefore hypothesize that Cxcl12 and Shh are critical niche factors that are induced by calvarial injury and coordinately promote SuSC migration, expansion and osteoblast differentiation, all of which are essential for calvarial bone injury repair. This hypothesis will be tested in three specific aims: 1) To better define SuSCs and determine the roles of Cxcl12 and Gai/Gas signaling in directing SuSC migration to the injury site during calvarial bone regeneration; 2) To determine the roles of Gai/Gas and Shh signaling in SuSC expansion and osteoblast differentiation during calvarial bone regeneration; 3) To determine the interaction of Cxcl12 and Sh...