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UBC Theses and Dissertations

Bone morphogenetic protein signaling molecules in facial morphogenesis and dysmorphogenesis Ashique, M. Amir


Facial morphogenesis is a dynamic multi-step process involving the formation of the neural crest cell derived facial prominences, their coordinated outgrowth, fusion and differentiation to generate the final facial form. Defects in each of these processes are associated with facial dysmorphogenesis. The identification of genes that are involved in cleft lip is the first aim of this thesis. PCR based differential display was used to compare gene expression profiles in cleft and non-cleft chicken embryos. The second manner to identify candidate genes involved in orofacial clefting was by examining expression patterns of previously cloned genes at the specific time and place where lip fusion occurs. This approach identified Bone morphogenetic proteins (BMPs) as potential genes that are involved with the outgrowth and fusion of facial prominences. Little was known about where BMP receptors and BMP antagonists are expressed in the face or the function of endogenous BMPs in the face. The expression of BMP ligands (Bmp-2, Bmp-4 and Bmp- 7) was correlated with the expression of BMP receptors (BmpR-IA, BmpR-IB and BmpR-II) and noggin, a BMP antagonist, in order to predict where endogenous BMP signaling occurs in the face. Bmp-2, Bmp-4 and Bmp-7 are expressed in the zone of fusion between the frontonasal mass and maxillary prominences. Noggin expression is restricted to the frontonasal mass epithelium and is downregulated at the corners of the frontonasal mass just prior to fusion. Later in development, Bmps are expressed in the perichondrium of cartilage whereas noggin is expressed within differentiating cartilage. The BmpR-IA and BmpR-II are expressed ubiquitously whereas BmpR-IB is expressed in a subset of tissues. This expression data was used to design studies that examined BMP function during primary palate closure and facial cartilage morphogenesis. Noggin protein was applied to regions with high BMP expression in the zone of fusion and subsequently induced clefts. These experiments show for the first time that endogenous BMPs regulate two aspects of lip closure; outgrowth of facial prominences and thinning of the frontonasal epithelium prior to fusion. We also examined the well-established retinoic acid model for cleft lip to see whether BMPs were mediating this defect. Ectopic retinoic acid increases Bmp-2 and Bmp-4 expression prior to the induction of cleft lip in chickens. Mimicking this result by applying BMP-2 to the face also induced clefts. These results are related to non-syndromic human orofacial clefting and demonstrate the utility of studying cleft lip in non-mammalian model systems. Finally, experiments were carried out that would directly test the function of the BMP receptors in craniofacial development. Whereas expression of dominant-negative (dn) and constitutively active (ca) BMP type I receptors seldom affected early facial morphology, there were significant effects on chondrogenesis. These mutant forms of the type IA and IB receptors regulated the size and shape of cartilage elements. The dn form of the BMPRIB inhibited chondrogenesis, whereas ca-BMPRIA and ca-BMPRIB increased cartilage formation. The embryos injected with active viruses also lacked feather germs over much of the head and about 50% of specimens did not form an egg tooth. It appears that type IA and type IB receptors play similar roles in regulating cartilage and feather formation in the skull. Overall, these findings identify BMP signalling molecules as critical regulators of facial morphogenesis and dysmorphogenesis.

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