Investigating the roles of Wnt/beta-catenin signaling in craniofacial development

Craniofacial malformations such as oral clefts and dental abnormalities are common congenital diseases, which arise from the disruption of various steps of normal craniofacial development. Previous studies have established important roles of several core signaling pathways, including Shh, Bmp and Fgf signaling, in secondary palate and early tooth development. However, very little is known about the role of canonical Wnt signaling in palatogenesis. Moreover, whereas canonical Wnt signaling has been shown to play critical roles in the developing dental epithelium, whether it is also required in the tooth mesenchyme for activation of the odontogenic potential is not known.;In this thesis, I showed that canonical Wnt signaling was not activated in most palatal mesenchymal cells by analysis of canonical Wnt signaling reporter mice. To test the requirement of inactivation/inhibition of canonical Wnt signaling in normal palatogenesis, I utilized the Cre/Lox system to conditionally stabilize beta-catein in palatal mesenchymal cells. Persistent activation of canonical Wnt signaling in palatal mesenchymal cells led to cleft palate. Detailed histological analysis indicated that elevation of palatal shelves did not occur in the beta-catenin gain-of-function mutants. To investigate the cellular mechanisms underlying the cleft palate phenotype, I performed BrdU labeling and TUNEL assays to detect alterations in cell proliferation and cell death, and found that cell proliferation was reduced in both palatal epithelium and mesenchyme in mutants compared to those of control littermates at E13.5, while cell death was similar in control and mutant littermates. To investigate which downstream gene(s)/pathway(s) were affected, I analyzed the expression of genes important for secondary palate development by in-situ hybridization, and revealed that the expression of Lef1, Bmp4, Bmp2, and Osr2 was increased, while the expression of Shox2 was totally abolished in palatal mesenchyme of mutant embryos. In conclusion, our data indicate canonical Wnt signaling is actively inhibited in palatal mesenchymal cells during normal palatogenesis.;I also investigated the role of mesenchymal Wnt/beta-catenin in early tooth development. I found that tissue-specific inactivation of beta-catenin, a central component of the canonical Wnt signaling pathway, in the developing early tooth mesenchyme, caused tooth developmental arrest at the bud stage in mice. I further showed that mesenchymal beta-catenin function was required for expression of Lef1 and Fgf3 in the developing tooth mesenchyme and for induction of primary enamel knot in the developing tooth epithelium. Moreover, I found that constitutive stabilization of beta-catenin in the developing palatal mesenchyme induced ectopic tooth initiation from the palatal epithelium. Together, these results revealed that Wnt ligands expressed in the presumptive dental epithelium signal directly to the developing tooth mesenchyme to activate mesenchymal odontogenic program during tooth initiation.