Role of LGR+ connective tissue progenitors in TMJ development

PROJECT SUMMARY According to NIDCR, temporomandibular joint (TMJ) disorders affect up to 12% of the population and are a major priority for further research. The high incidence in children is unusual for a chronic pain condition, suggesting a significant developmental origin. TMJ development involves precise patterning of the mandibular bony processes and diverse connective tissues including the fibrous disc, stabilizing ligaments, and tendons that connect to jaw muscles. Skeletal and soft connective tissue structures of the TMJ derive from cranial neural crest cells, yet how cell fate decisions are balanced to generate the correct tissues with spatial precision remains unresolved. In a previously funded R21, PIs Gage Crump and Amy Merrill combined single-cell omics with zebrafish and mouse genetics to identify a new function of the nuclear receptor Nr5a2 in promoting connective tissue at the expense of skeletal fates at the developing TMJ. However, the identity of the stem/progenitor cells for TMJ connective tissues, and how they differ from skeletal stem cells, was unknown. Here, by analyzing new single-cell data of the developing mouse TMJ, we have identified the R-spondin receptors LGRs as markers of jaw connective tissue stem cells. Compared to roles in epithelial stem cell biology, potential roles of LGRs in mesenchymal tissues have been understudied. We propose that LGRs function to maintain the stem pool for sustained connective tissue generation at the TMJ by potentiating locally high Wnt signaling. In Aim 1, we will perform lineage tracing to test that Lgr4 and Lgr5 in mouse, and Lgr4 and Lgr6 in zebrafish, mark connective tissue stem cells at both embryonic and postnatal stages. We will also use targeted ablation to test the requirement of Lgr5+ cells for TMJ connective tissue development and postnatal growth. In Aim 2, we will perform real-time in vivo imaging in developing zebrafish to test that connective tissue progenitors transition from a high to low Wnt state as they differentiate into tendons and ligaments. We will then use conditional mouse genetics to test our model that sustaining Wnt signaling in Lgr5+ cells disrupts differentiation by locking cells in a stem cell state, and reciprocally blocking canonical Wnt signaling in Lgr5+ cells depletes stem cells and causes a failure to sustain connective tissue development. In Aim 3, we use mouse Lgr4 and Lgr5 floxed alleles provided by our collaborators at Novartis, and newly generated zebrafish lgr4 and lgr6 CRISPR mutants, to test that LGRs are redundantly required in Lgr5+ cells for high Wnt signaling and maintenance of connective tissue progenitors. We then test whether restoring Wnt signaling levels through genetic or pharmacological means can rescue connective tissue defects in LGR mutants. Strengths of the proposal include the PI team with complementary expertise in zebrafish and mouse models, unique single-cell datasets of the fish jaw and mouse TMJ, and sophisticated in vivo imaging and genetic tools in both species to enable mechanistic understanding of jaw connective tissue differentiation. Our work will reveal how alterations to Wnt and stem cell function impact the balance of skeletal and connective tissue formation, potentially predisposing to TMJ disorders.