Analysis of the mechanisms of mammalian palate and limb morphogenesis

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Cleft palate, the most frequent congenital craniofacial birth defect in humans, is caused by genetic and/or environmental perturbations in the multi-step process of palate development. Mutations in the TGF-β3 gene are associated with non-syndromic cleft palate in humans. However, little is known about why cleft palate is variable in its phenotype and penetrance and why mutation analysis using specific gene-deficient mice does not necessarily predict phenotypic consequences.In this study, we developed a novel suspension culture method of fetal mouse palatal shelves. By using the 'single palatal shelf in suspension culture' , we evaluated the differentiation of palatal medial edge epithelial (MEE) cells and its roles in palatal fusion. In addition, we established TGF-β3-defficient mice in various strains (C57BL/6J, 129/Sv, FVB/N, and ICR) as model systems for analyzing phenotypic variability of genetic cleft palate and the synergism of the master and modifier genes that regulate mammalian palatogenesis. In vivo analysis indicated that TGF-β3-null mutation causes severe cleft palate in inbred strains but partial cleft palate in outbred strains. In vitro analysis suggested that phenotypic variation of cleft palate in TGFβ3-defficient mice is closely associated with the difference in MEE cell differentiation between inbred and outbred mouse strains. However, such severe cleft palate in TGF-β3-null mice in inbred strains was partially rescued with a DNA methyltransferase inhibitor and produced milder phenotypes similar to those observed in outbred strains. Our results strongly suggest that TGFβ3-associated or -interacting imprinting genes may act as modifier genes and bring about phenotypic variation of mammalian non-syndromic cleft palate.