Proper skull development is dependent upon complex epithelial-mesenchymal interactions between the developing flat bones of the calvaria, the mesenchymal blastema of the presumptive cranial sutures, and the underlying dura mater. Craniosynostosis, the premature fusion of calvarial bones at the sutures, is a common birth defect (1 in 3000 live births), with both genetic and environmental causes. This craniofacial anomaly represents a significant biomedical burden because the only option to correct severe craniosynostosis is surgical separation of the fused calvarial bones. It has been shown that a mutation in the homeodomain of MSX2 is associated with craniosynostosis, Boston type; affected individuals bear a proline to histidine substitution in position 7 (Pro7His) of the MSX2 homeobox. This human disorder has been recreated in mice by misregulation of either wildtype or Pro7His mutant Msx2 in transgenic mice. This mouse model for craniosynostosis, together with Msx2 homozygous null mutant mice, and normal mice, will be used to test the central hypothesis of this proposal; that Msx2 misregulation leads to craniosynostosis by disrupting epithelial-mesenchymal interactions in the developing calvaria. To test this hypothesis the following is proposed: To determine the effect of normal, misregulated, and absent Msx2 expression on the epithelial-mesenchymal interactions that regulate cranial suture formation. Calvarial rudiments and underlying dura mater will be used in heterotypic and homotypic tissue recombinations in vitro to determine the source of the aberrant epithelial-mesenchymal signal(s) responsible for craniosynostosis. A mutation in the fibroblast growth factor (FGF) receptor 2 gene has recently been shown to be associated with four human genetic disorders that also exhibit craniosynostosis: Crouzon, Jackson-Weiss, Pfeiffer and Apert syndromes. The effect of normal, misregulated, and absent Msx2 expression on the expression of fgfR2, the ligands of fgfR2, and molecular markers that identify osteogenic cell populations in vivo, and in heterotypic and homotypic tissue recombinations in vitro, will be determined. These studies will provide molecular characterizations of the craniosynostosis phenotype, and may provide insight into the epistatic relationships between Msx2, fgfR2, the ligands of fgfR2, and the osteogenic molecular markers. Msx2 has been implicated in preprogrammed cell death, or apoptosis, in other developmental processes such as neural crest migration and limb development; apoptosis in the developing calvaria may be the mechanism by which Msx2 misregulation leads to craniosynostosis. Preliminary data that Msx2 misregulation in cultured cells impairs cell growth and may lead to apoptosis are presented. To test the hypothesis that Msx2 misregulation leads to apoptosis in the developing calvaria, the effect of normal, misregulated, and absent Msx2 expression on cell growth and apoptosis in vivo, and in heterotypic and homotypic tissue recombinations in vitro will be determined. These studies should provide insight into the cellular and molecular genetic mechanisms that regulate normal as well as abnormal cranial suture development
