Study on the mechanisms by which cleft palate shows its phenotypic polymorphism in mice

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Cleft palate is the most frequent congenital craniofacial birth defect in humans. The cause of human cleft palate is still unclear but has been assumed to be multi-factorial. However, recent studies using transgenic mice tend to conclude that cleft palate results from mutation of a certain gene. In addition, very 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 its phenotypic consequences.In this study, we transferred the Tgfb3-deficient allele into various mouse strains, such as C57BL/6J, 129/Sv, FVB/N, SJL/J, and ICR by backcrossing over 8 generations, and actually analyzed the variance of the cleft palate phenotypes in the Tgfb3-null fetuses (n>50 of each strain). We thereby found that all the Tgfb3-null fetuses show only compete cleft palate in C57BL/6J, but they show only incomplete cleft type in ICR. In 129/Sv, FVB/N, and SJL/J mouse strains, the cleft palate showed both of complete and incomplete cleft types. In addition, the complete cleft palate in C57BL/6J was changed into incomplete cleft palate by re-backcrossing with ICR, indicating that the cleft palate phenotype is reversible between complete and incomplete cleft types and is dependent on the genetic background. Furthermore, the severe (complete) cleft palate in the Tgfb3-null mice of C57BL/6J strain was partially rescued in culture with 5-Aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, and resulted in the incomplete cleft palate equivalent to that observed in ICR strain. Our results strongly suggest that the phenotypic modifier of the lack of Tgfb3-induced cleft palate is the imprinting gene(s) and its specific methylation pattern is inheritable in inbred strains. Thus, the phenotype of cleft palate can be determined by synergy of genetic mutation and epigenetic modifier(s).