Gas signaling regulates Wnt and Hedgehog-responsive cells during postnatal tooth development

Objectives: Dental stem cell differentiation is perhaps regulated by Wnt and Hedgehog (Hh) signaling pathways, but mechanisms are unclear. Our lab recently demonstrated that a G-protein subunit (Gas) may regulate Wnt/Hh signaling to facilitate stem cell differentiation. We therefore aim to identify potential Wnt- and Hh-responsive dental stem cells and determine whether Gas balances these pathways to encourage tooth growth. Methods: We generated mouse models to simultaneously track and alter Gas signaling in Wnt- and Hh-responsive cells. Dams were injected with tamoxifen to activate tracking and Gas mutations in pups. Juveniles were sacrificed for histological examination and FACS. RNAseq was performed on sorted Wnt- and Hh-responsive cells to identify changes in gene expression. Results: Wnt and Hh-responsive cells were located in dentin, dental pulp, incisor alveoulus, mandible, and bone directly beneath the root. Groups with activated Gas signaling exhibited thicker dentin layers, fibrous tissue formation instead of bone near the tooth root, shortened incisors, and stunted mandible growth. The percentage of Wnt- and Hh-responsive incisor cells was significantly higher (p < 0.0001) compared to those in the periosteum and bone marrow. These percentages were altered in Wnt-responsive cells with abnormal Gas signaling. RNAseq of Wnt-responsive dental cells revealed that the following signaling pathways were altered with abnormal Gas signaling: Wnt, Hippo, cAMP, calcium, stem cell pluripotency, and osteogenic differentiation. Conclusion: Our data suggest that Wnt- and Hh-responsive cells are present throughout the developing jaw and give rise to tooth-forming progeny. Interestingly, the incisor contains a higher percentage of these cells compared to bone marrow, which is the preferred source of stem cells for regeneration. More importantly, we show that Gas signaling plays a role in tooth development and identified additional pathways and specific genes involved in this process. Overall, we made strides to distinguish therapeutic targets for enhanced implant fixation and tooth regeneration.