This is a proposal to investigate molecular genetic mechanisms that underlie normal and abnormal craniofacial development. We showed recently that a mutation in the homeobox of the human MSX2 gene (substitution of a proline for a histidine in position 7 of the homeodomain) is associated with a craniofacial genetic anomaly known as craniosynostosis, Boston type, an autosomal dominant disorder identified in a large kindred in the Northeastern United States. This disorder is characterized by the premature fusion of calvarial sutures which results in abnormal skull shape. We intend to examine further the relationship between the Msx2 gene, craniofacial developmental, and the etiology of craniosynotosis. To this end, we will pursue three specific aims: First, we will examine the temporal and spatial pattern of Msx2 expression during calvarial development and suture formation in the mouse. These studies will provide the basis for further analyses on the role of the Msx2 gene in craniosynotosis and in the normal development of the skull. Second, we will examine the effect of mutations in the Msx2 gene on the development of the mouse embryo, particularly the calvaria. That the Msx2 gene is expressed during inductive tissue interactions, that it becomes inactive upon differentiation, and that the dominant Pro7His mutation is associated with premature fusion of calvaria suggest that the Msx2 gene participates in cell signalling events that maintain a population of cells in an undifferentiated state. We will test this hypothesis using gene targeting and transgenic mouse technologies. Homologous recombination in embryonic stem (ES) cells is currently being utilized to create a null mutation in the mouse Msx2 gene, and will be used to introduce the Pro7His mutation into Msx2. Trans-dominant approaches, using pronuclear injection, are also underway to introduce the Pro7His mutation into the mouse Msx2 gene. Third, we will examine the effect of the Pro7His mutation on macromolecular interactions of the Msx2 protein. Computer modeling studies indicate that the Pro7His mutation should have little effect on the binding of the Msx2 protein to its target DNA sequence, and preliminary el shift analyses are consistent with this prediction. These findings, together with the fact that the Pro7His mutation is in a region of the homeodomain that has been implicated in protein-protein interactions, suggest that the Pro7His mutation does not significantly affect the affinity or specificity of DNA binding, but rather affects interactions between Msx2 and an accessory protein(s). We will test this hypothesis by (A) closely examining the interactions of wild type and mutant proteins with their binding sites, and (B) using the yeast two hybrid system to screen for proteins that interact with Msx2, and whose interaction is perturbed by the Pro7His mutation.
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