Analysis of the Fgfr2C342Y mouse model shows condensation defects due to misregulation of Sox9 expression in prechondrocytic mesenchyme

Abstract

Syndromic craniosynostosis caused by mutations in <i>FGFR2</i> is characterised by developmental pathology in both endochondral and membranous skeletogenesis. Detailed phenotypic characterisation of features in the membranous calvarium, the endochondral cranial base and other structures in the axial and appendicular skeleton has not been performed at embryonic stages. We investigated bone development in the Crouzon mouse model (<i>Fgfr2</i>^C342Y) at pre- and post-ossification stages to improve understanding of the underlying pathogenesis. Phenotypic analysis was performed by whole-mount skeletal staining (Alcian Blue/Alizarin Red) and histological staining of sections of CD1 wild-type (WT), <i>Fgfr2</i>^C342Y/+ heterozygous (HET) and <i>Fgfr2</i>^C342Y/C342Y homozygous (HOM) mouse embryos from embryonic day (E)12.5-E17.5 stages. Gene expression (<i>Sox9</i>, <i>Shh</i>, <i>Fgf10</i> and <i>Runx2</i>) was studied by <i>in situ</i> hybridisation and protein expression (COL2A1) by immunohistochemistry. Our analysis has identified severely decreased osteogenesis in parts of the craniofacial skeleton together with increased chondrogenesis in parts of the endochondral and cartilaginous skeleton in HOM embryos. The <i>Sox9</i> expression domain in tracheal and basi-cranial chondrocytic precursors at E13.5 in HOM embryos is increased and expanded, correlating with the phenotypic observations which suggest FGFR2 signalling regulates <i>Sox9</i> expression. Combined with abnormal staining of type II collagen in pre-chondrocytic mesenchyme, this is indicative of a mesenchymal condensation defect. An expanded spectrum of phenotypic features observed in the <i>Fgfr2</i>^C342Y/C342Y mouse embryo paves the way towards better understanding the clinical attributes of human Crouzon-Pfeiffer syndrome. <i>FGFR2</i> mutation results in impaired skeletogenesis; however, our findings suggest that many phenotypic aberrations stem from a primary failure of pre-chondrogenic/osteogenic mesenchymal condensation and link FGFR2 to SOX9, a principal regulator of skeletogenesis.