Project summary/Abstract MSCs play critical roles in tissue turnover and injury healing processes, making them highly valuable in the field of regenerative medicine. The murine teeth present an excellent model for investigating MSC homeostasis due to the rapid turnover and regenerative abilities in incisors and the limited injury healing capacity in molars that resembles human teeth. Understanding how MSCs homeostasis is regulated in murine teeth will provide valuable insights for applying MSCs to regenerative dentistry. MSC homeostasis involves the maintenance of stem cell niche, directed migration, re-entry into the cell cycle for proliferation, and guided differentiation, etc. By employing a newly developed knockout mouse model, we found that a predicted gene, Din ( 4930453N24Rik ), plays pivotal roles in governing MSC homeostasis in murine teeth by regulating Rho GTPases, RhoA and RAC1. The incisor growth in Din-KO mice arrested after eruption, and the injury healing process was significantly impaired in both incisors and molars. A series of lineage-specific knockouts revealed that Din is crucial for dental MSCs but dispensable for differentiated odontoblasts and dental epithelium-derived cells. Din-KO MSCs showed diminished stemness, reduced motility and sign of aging, along with compromised osteogenesis ability and enhanced adipogenesis potential. Transcriptomic, proteomic and computational analyses identified DIN as a novel regulator of the binary molecular switches, Rho GTPases. The interactions between DIN and GTPases likely occur through potential binding sites within a highly conserved NTF2 domain in DIN and several critical function domains in GTPases, which appear to further trigger downstream signaling, such as canonical WNT pathway. Based on these findings, we hypothesize that Din regulates MSC homeostasis in murine teeth during tissue turnover and injury healing by interacting with Rho GTPases and their downstream signaling pathways, including the canonical WNT signaling pathway. To test this hypothesis, we propose two Specific Aims: 1) To determine Din role and the cell fate of Din+ vs. Din- MSCs in different MSC subsets during tissue turnover and injury healing processes, and 2) To determine how Din regulates Rho GTPases RhoA and Rac1 and their downstream effectors. Successful completion of the proposed study will advance our understanding of the regulatory mechanism governing MSC homeostasis in murine teeth during tissue turnover and injury healing processes. The discovery of a novel GTPases regulator and the elucidation of its associated mechanism will have broad impact beyond the dental research field.