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 FGFR2 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 (Fgfr2C342Y) 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), Fgfr2C342Y/+ heterozygous (HET) and Fgfr2C342Y/C342Y homozygous (HOM) mouse embryos from embryonic day (E)12.5-E17.5 stages. Gene expression (Sox9, Shh, Fgf10 and Runx2) was studied by in situ 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 Sox9 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 Sox9 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 Fgfr2C342Y/C342Y mouse embryo paves the way towards better understanding the clinical attributes of human Crouzon–Pfeiffer syndrome. FGFR2 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.

Predicting calvarial growth in normal and craniosynostotic mice using a computational approach

“…; Peskett et al. ). Most importantly, (iv) they provided the reference data required for validation of the FE modelling approach.…”

“…; Peskett et al. ). This line is particularly interesting, as it exhibits robust phenotypic abnormalities with features recapitulating clinical abnormalities observed in patients.…”

“…; Peskett et al. ). Thus, Crouzon Fgfr2 C342Y/+ mutant type (MT) and WT mice provide an invaluable tool with which to understand the biomechanics of craniosynostotic and normal skull growth during postnatal development.…”

“…In contrast, human ICV reaches 65% of the adult volume by 1 year, with minimal further growth after 10 years (Dekaban, 1977;Sperber, 1989). The Crouzon mouse model (Fgfr2 C342Y/+ ) has been extensively studied and has become well-established for investigating craniosynostosis (Eswarakumar et al 2004;Perlyn et al 2006;Liu et al 2013;Mart ınez-Abad ıas et al 2013;Peskett et al 2017). This line is particularly interesting, as it exhibits robust phenotypic abnormalities with features recapitulating clinical abnormalities observed in patients.…”

Mouse Models of Syndromic Craniosynostosis

“…In addition to introducing GOF mutations, increasing gene dosage also allows the identification of novel phenotypes in animal models. For example, a detailed analysis of Fgfr2c C342Y/C342Y homozygotes identified exencephaly, overt cleft of the secondary palate, and a series of segmentation defects along the axial skeleton [Peskett et al, 2017].…”