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.