Honors Theses

Document Type

Thesis

Date of Completion

Spring 4-30-2026

Academic Year

2025-2026

Department

Biology

Academic Major

Biochemistry and Molecular Biology

Faculty Advisor

Trixie Pittman, Ph.D.

Abstract

Cleft palate is a common craniofacial birth defect that arises when the molecular and morphogenetic events guiding secondary palate formation lose coordination during a narrow developmental window. In mice, successful palatogenesis requires the paired palatal shelves to grow vertically, elevate above the tongue, and fuse at the midline; disruption of any of these steps can result in clefting. Two important regulators of this process are canonical Wnt signaling and the transcription factor Pax9, both of which contribute to normal palatal mesenchymal growth and patterning during early development. This paper first examines whether altered Dkk1/Wnt signaling contributes to the Pax9-null palate phenotype. Evidence from mouse studies shows that Pax9-null embryos fail to complete palatal shelf growth and elevation, producing a persistent cleft phenotype. When Dkk1 is reduced specifically in neural crest-derived mesenchyme, shelf growth, elevation, and midline contact are restored in most embryos, demonstrating that the Pax9-null phenotype is at least partially reversible. In a complementary experiment, overexpression of Dkk1 during the critical window of palatogenesis is sufficient to recreate a cleft palate even when Pax9 is still present. Together, these rescue and phenocopy experiments show that altered Dkk1/Wnt signaling contributes substantially to the Pax9-null palate phenotype and support a model in which Pax9 helps maintain the Wnt signaling environment required for normal palatal growth by limiting excessive Dkk1-mediated inhibition. This paper then uses single-cell RNA sequencing analysis of the E13.5 anterior palate to examine whether Pax9 expression and canonical Wnt target-gene activity overlap within mesenchymal cells before palatal shelf elevation. Using the Ozekin et al. dataset, the four mesenchymal clusters were grouped into a single mesenchymal population, and Pax9 expression was compared with a canonical Wnt module score based on Axin2, Lef1, and Sp5. Scatterplot analysis showed no strong global correlation between Pax9 expression and canonical Wnt activity across the entire mesenchyme, with a Spearman correlation of ρ = 0.0535. However, mapping highly co-active cells back onto the mesenchymal UMAP revealed that Pax9-Wnt overlap was not broadly distributed but instead appeared in localized pockets of transcriptional space. These results suggest that Pax9 and canonical Wnt signaling are coordinated within specific mesenchymal niches rather than uniformly across the developing palate, providing a more cell-resolved framework for understanding how these pathways interact during early palatogenesis.

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