Background The role of fibroblast growth factor and receptor (FGF/FGFR) signaling in bone development is well studied, partly because mutations in FGFRs cause human diseases of achondroplasia and FGFR-related craniosynostosis syndromes including Crouzon syndrome. The FGFR2c C342Y mutation is a frequent cause of Crouzon syndrome, characterized by premature cranial vault suture closure, midfacial deficiency and neurocranial dysmorphology. Here, using newborn Fgfr2cC342Y/+ Crouzon syndrome mice, we tested whether the phenotypic effects of this mutation go beyond the skeletal tissues of the skull, altering the development of other non-skeletal head tissues including the brain, the eyes, the nasopharynx and the inner ears. Results Quantitative analysis of 3D multimodal imaging (high resolution micro computed tomography and magnetic resonance microscopic images) revealed local differences in skull morphology and coronal suture patency between Fgfr2cC342Y/+ mice and unaffected littermates, as well as changes in brain shape but not brain size, significant reductions in nasopharyngeal and eye volumes, and no difference in inner ear volume in Fgfr2cC342Y/+ mice. Conclusion These findings provide an expanded catalogue of clinical phenotypes in Crouzon syndrome caused by aberrant FGF/FGFR signaling and evidence of the broad role for FGF/FGFR signaling in development and evolution of the vertebrate head.

SUMMARYApert syndrome is a congenital disorder characterized by severe skull malformations and caused by one of two missense mutations, S252W and P253R, on fibroblast growth factor receptor 2 (FGFR2). The molecular bases underlying differential Apert syndrome phenotypes are still poorly understood and it is unclear why cleft palate is more frequent in patients carrying the S252W mutation. Taking advantage of Apert syndrome mouse models, we performed a novel combination of morphometric, histological and immunohistochemical analyses to precisely quantify distinct palatal phenotypes in Fgfr2+/S252W and Fgfr2+/P253R mice. We localized regions of differentially altered FGF signaling and assessed local cell patterns to establish a baseline for understanding the differential effects of these two Fgfr2 mutations. Palatal suture scoring and comparative 3D shape analysis from high resolution μCT images of 120 newborn mouse skulls showed that Fgfr2+/S252W mice display relatively more severe palate dysmorphologies, with contracted and more separated palatal shelves, a greater tendency to fuse the maxillary-palatine sutures and aberrant development of the inter-premaxillary suture. These palatal defects are associated with suture-specific patterns of abnormal cellular proliferation, differentiation and apoptosis. The posterior region of the developing palate emerges as a potential target for therapeutic strategies in clinical management of cleft palate in Apert syndrome patients.

Background Connective tissue growth factor (CTGF/CCN2) is a matricellular protein that is highly expressed during bone development. Mice with global CTGF ablation (knockout, KO) have multiple skeletal dysmorphisms and perinatal lethality. A quantitative analysis of the bone phenotype has not been conducted. Results We demonstrated skeletal site-specific changes in growth plate organization, bone microarchitecture, and shape and gene expression levels in CTGF KO compared with wild-type mice. Growth plate malformations included reduced proliferation zone and increased hypertrophic zone lengths. Appendicular skeletal sites demonstrated decreased metaphyseal trabecular bone, while having increased mid-diaphyseal bone and osteogenic expression markers. Axial skeletal analysis showed decreased bone in caudal vertebral bodies, mandibles, and parietal bones in CTGF KO mice, with decreased expression of osteogenic markers. Analysis of skull phenotypes demonstrated global and regional differences in CTGF KO skull shape resulting from allometric (size-based) and nonallometric shape changes. Localized differences in skull morphology included increased skull width and decreased skull length. Dysregulation of the transforming growth factor-β-CTGF axis coupled with unique morphologic traits provides a potential mechanistic explanation for the skull phenotype. Conclusions We present novel data on a skeletal phenotype in CTGF KO mice, in which ablation of CTGF causes site-specific aberrations in bone formation.

COVER PHOTOGRAPH: Multimodal imaging and segmentation used to visualize skull (yellow), brain (pink), inner ear (green), nasopharynx (green) and globe of the eye (blue) in Fgfr2cC342Y/+Crouzon syndrome mice (top) and normal littermates (bottom). From Martínez‐Abadías et al., Developmental Dynamics 242:80–94, 2013.

Our goal was to quantify the maturation of individual craniofacial bones and determine how the Fgfr2 P253R mutation modifies cranial bone maturation. Bone volumes and relative bone density histograms were measured from computed tomography images of Fgfr2+/P253R Apert craniosynostosis syndrome model mice and unaffected littermates at E15.5, E16.5, E17.5, P0, and P2. This data was used to quantify ontogenetic change in individual bone density and to test for differences in relative density between the two genotypes. Individual bones increase in volume and density at different rates across embryonic and perinatal development. While many bones of the Fgfr2+/P253R mice display larger volumes than those of littermates by P0, they generally display smaller volumes at P2. Relative bone density of the frontal and some facial/palatal bones is reduced for Fgfr2+/P253R mice at P2, but not before. These differences suggest a postnatal shift in the effect of the mutation on bone growth and maturation. Some of the most significant differences in craniofacial maturation between the two genotypes occur within the facial skeleton, identified as a region of significant dysmorphology in these mice and in humans carrying mutations for FGFR‐related craniosynostosis syndromes. Work supported in part by grants from the NSF to CJP (BCS‐1061554); the NIDCR/NIH and ARRA to JTR (R01DE018500; 3R01DE018500–02S1; R01DE022988).Grant Funding Source: NIDCR/NIH; NSF