FGFR2 is an Upstream Regulator of MSX2 Gene Expression in Mouse Calvarial Bone Cells

Activating mutations of FgfR2 and Msx2 cause craniosynostosis syndromes. Whereas haploinsufficiency and the knockout of Msx2 cause calvarial bone ossification defects in human and mice. Interestingly, the transgenic mice expressing bone-targeted dominant negative (DN) mutation of FgfR2 display calvarial bone defects similar to the knockout of Msx2. Furthermore both FgfR2 and Msx2 are expressed in osteogenic precursor cells and inhibit late osteogenic differentiation, suggesting that FgfR2 and Msx2 belong to a same genetic pathway in the regulation of osteoblast differentiation. Objectives: The purpose was to determine whether FgfR2 is an upstream regulator of the Msx2 gene, forming a linear regulatory pathway for cranial bone and suture formation. Methods: First, calvarial bone cells from postnatal day 10 mice were treated with FGF9, a cognate ligand for FgfR2, for 48 hours and Northern analysis determined the effect of FGF9 on Msx2 gene expression. Second, the expression level of the Msx2 gene in calvarial bone cells was compared between transgenic mice expressing DN or ligand-dependent activating (P253R) mutations of FgfR2 and their wild-type littermates. Calvarial cells from transgenic mice were also treated with FGF9 and examined for the effect of FgfR2 mutations on FGF9-induced Msx2 gene expression. Results: FGF9 treatment of wild-type cells significantly increases Msx2 gene expression. Similarly, analyses of transgenic mouse bone cells reveal that the Msx2 gene expression is elevated in cells expressing P253R mutation of FgfR2, which was further increased by FGF9 treatment. In contrast, Msx2 gene expression was lower in cells expressing DN mutation compared to wild-type littermates, and was not affected by FGF9 treatment. Conclusions: Based on these results, we concluded that FgfR2 is an upstream regulator of Msx2 gene. We also propose that some of FgfR2's functions in calvarial bone cells may be mediated by Msx2. (Supported by NICHD HD40282 and grants from AAOF, BK21)