The Role of Din in Murine Bone and Tooth Development

Abstract

Bone- and tooth-related disorders are a major health burden in modern society. Identifying the cellular and molecular mechanisms of such disorders hold great potential in the development of novel therapeutic strategies. Recent genetic studies in mice have associated a peculiar tooth phenotype, “dense incisors (Din)” featuring overgrowth of dentin and arrested growth of incisors, with a predicted gene 4930453N24Rik (human ortholog C3orf38): Din. The spontaneous Din mutant mice showed smaller bodies and crania suggesting Din is a novel gene that has pivotal roles in bone and tooth development. LacZ-Knockin Din knockout (KO) allele and Din-flox mouse models were generated to study the cellular and molecular mechanisms associated with Din. The Din-KO mice completely recapitulated the phenotypes of spontaneous Din mutant mice. These phenotypes strongly suggested that Din may have critical roles in the homeostasis of osteogenic and odontogenic stem cells. Gene expression patterns, reverse genetics, bioinformatics analyses of in-house and open-access single RNA sequencing (scRNAseq) data, and double RNAScope colocalization experiments collectively revealed that Din functions in the skeletal and dental MSCs. Din was extensively expressed by multiple MSC subsets but only a portion of cells in each subset expressed Din. Din expression was indispensable for the homeostasis of MSCs. Thus, Din deficiency led to defective homeostasis of bone, suture, and tooth MSCs during postnatal growth and injury repair. Bioinformatic, transcriptomic, proteomic, and G-LISA assays were performed which revealed that Din plays a key role in governing the homeostasis of MSCs by interacting with small GTPases. Din was an immediate input regulator of small GTPases: RhoA and Rac1. Moreover, Din-KO mice had severely impaired osteogenesis and increased adipogenesis both in vivo and in vitro which highly suggested dysregulated Wnt signaling. The Wnt signaling changes were tested in Din-KO tissues through immunohistochemistry of Wnt signaling mediators: β-catenin, Lef1, Tcf1, and Cyclin D1. Also, a classical lithium chloride (LiCl) stimulation assay was performed on Din-KO BMSCs (in vitro) compared with their WT counterparts. Both analyses confirmed dysregulated Wnt signaling in Din-KO mice. Thus, we concluded that Din-GTPase interactions regulate Wnt signaling in the homeostasis of bone, suture, and tooth MSCs.