Din Oversees Mesenchymal Stem Cell Homeostasis in Mouse Incisors

The murine incisor presents an excellent model for investigating stem cell homeostasis due to its regenerative capacity and continuous growth throughout the lifetime. Proper homeostasis of the dental epithelial stem cells (ESCs) and mesenchymal stem cells (MSCs) is pivotal for the continuous growth, tissue turnover and injury healing in murine incisors. By employing a newly developed knockout mouse model, we revealed that a predicted gene, Din ( 4930453N24Rik ), plays pivotal roles in the homeostasis of MSCs in murine incisors. Din -deficient incisors exhibited arrested growth after eruption, and severely compromised healing/regeneration ability following injury. Although Din showed expression in multiple cell types in murine incisors, including both dental epithelium- and dental mesenchyme-derived naïve and differentiated cells, lineage-specific knockout of Din from epithelium, cranial neural crest, Col1a1 -expressing cells, and Gli1 + MSCs indicated that Din is essential for the dental MSCs in murine incisors but dispensable for the dental ESCs and differentiated ameloblasts and odontoblasts. Single-cell RNA sequencing (scRNA-seq) analysis revealed a decline in Din expression levels along the MSC differentiation trajectory, with highest levels in MSCs and transit amplifying cells (TACs), followed by low levels in pulp fibroblasts and odontoblasts. Din -deficient MSCs exhibited receded stemness, reduced motility, accelerated aging, and compromised osteogenesis potential whilst enhanced adipogenesis potential. Our transcriptomic, proteomic, and GLISA assays collectively suggest that Din may oversee multiple aspects of MSC homeostasis in murine incisors through Rho GTPases.