Genetic and Signaling Mechanisms of Secondary Palate Development and Clefts
- Reynolds, Kurt Sean
- Advisor(s): Zhou, Chengji
Abstract
Orofacial clefts are the most common birth defect worldwide. One key component of craniofacial development is the formation of the palate separating the oral and nasal cavities, which is a complex process that requires extensive coordination of many factors and signaling pathways that if disrupted may result in cleft palate. I employed single-cell RNA sequencing to describe the transcriptome of the palate and identify the various cell populations that comprise the developing secondary palatal shelves, to gain a deeper insight into their regulatory mechanisms. Through these efforts, I identified key cell populations that contribute to the medial edge epithelium, which facilitates fusion between the paired palatal shelves. I further described novel marker genes of the MEE and found that the Tgf-β receptors I and II are necessary for their specification. Wnt5a is an essential palatogenetic regulator necessary in both palate epithelium and mesenchyme. I show that Wnt5a antagonizes proliferation at the anterior palate, but in contrast with prior reports, does not promote proliferation at the posterior. I then worked to describe several of the key Wnt5a effectors that are dysregulated due to its loss of function, both at the transcriptional level using single-cell data, and at the protein level through immunostaining and immunoblot techniques. Finally, I demonstrated that mesenchymal Wnt5a contributes to activation of Rho family GTPases in the developing palate, and that their ectopic activation can partially rescue the Wnt5a mutant phenotype in culture. I also show that epithelial β-catenin is not required for secondary palate development in multiple models. Contrary to prior reports, the MEE is appropriately specified, and the palate completely fuses in its absence.