ABSTRACT
Most bone develops either by intramembranous ossification where bone forms within a soft connective tissue, or by endochondral ossification by way of a cartilage anlagen or model. Bones of the skull can form endochondrally or intramembranously or represent a combination of the two types of ossification. Contrary to the classical definition of intramembranous ossification, we have previously described a tight temporo-spatial relationship between cranial cartilages and dermal bone formation and proposed a mechanistic relationship between chondrocranial cartilage and dermal bone. Here, we further investigate this relationship through an analysis of how cells organize to form cranial cartilages and dermal bone. Using Wnt1-Cre2 and Mesp1-Cre transgenic mice, we determine the derivation of cells that comprise cranial cartilages from either cranial neural crest (CNC) or paraxial mesoderm (PM). We confirm a previously determined CNC-PM boundary that runs through the hypophyseal fenestra in the cartilaginous braincase floor and identify four additional CNC-PM boundaries in the chondrocranial lateral wall, including a boundary that runs along the basal and apical ends of the hypochiasmatic cartilage. Based on the knowledge that as osteoblasts differentiate from CNC- and PM-derived mesenchyme, the differentiating cells express the transcription factor genes RUNX2 and osterix (OSX), we created a new transgenic mouse line called R2Tom. R2Tom mice carry a tdTomato reporter gene joined with an evolutionarily well-conserved enhancer sequence of RUNX2. R2Tom mice crossed with Osx-GFP mice yield R2Tom;Osx-GFP double transgenic mice in which various stages of osteoblasts and their precursors are detected with different fluorescent reporters. We use the R2Tom;Osx-GFP mice, new data on the cell derivation of cranial cartilages, histology, immunohistochemistry, and detailed morphological observations combined with data from other investigators to summarize the differentiation of cranial mesenchyme as it forms condensations that become chondrocranial cartilages and associated dermal bones of the lateral cranial wall. These data advance our previous findings of a tendency of cranial cartilage and dermal bone development to vary jointly in a coordinated manner, promoting a role for cranial cartilages in intramembranous bone formation.
ABSTRACT
The Fgfr2c C342Y/+ Crouzon syndrome mouse model carries a cysteine to tyrosine substitution at amino acid position 342 (Cys342Tyr; C342Y) in the fibroblast growth factor receptor 2 (Fgfr2) gene equivalent to a FGFR2 mutation commonly associated with Crouzon and Pfeiffer syndromes in humans. The Fgfr2c C342Y mutation results in constitutive activation of the receptor and is associated with upregulation of osteogenic differentiation. Fgfr2cC342Y/+ Crouzon syndrome mice show premature closure of the coronal suture and other craniofacial anomalies including malocclusion of teeth, most likely due to abnormal craniofacial form. Malformation of the mandible can precipitate a plethora of complications including disrupting development of the upper jaw and palate, impediment of the airway, and alteration of occlusion necessary for proper mastication. The current paradigm of mandibular development assumes that Meckel's cartilage (MC) serves as a support or model for mandibular bone formation and as a template for the later forming mandible. If valid, this implies a functional relationship between MC and the forming mandible, so mandibular dysmorphogenesis might be discerned in MC affecting the relationship between MC and mandibular bone. Here we investigate the relationship of MC to mandible development from the early mineralization of the mandible (E13.5) through the initiation of MC degradation at E17.7 using Fgfr2c C342Y/+ Crouzon syndrome embryos and their unaffected littermates (Fgfr2c +/+ ). Differences between genotypes in both MC and mandibular bone are subtle, however MC of Fgfr2c C342Y/+ embryos is generally longer relative to unaffected littermates at E15.5 with specific aspects remaining relatively large at E17.5. In contrast, mandibular bone is smaller overall in Fgfr2c C342Y/+ embryos relative to their unaffected littermates at E15.5 with the posterior aspect remaining relatively small at E17.5. At a cellular level, differences are identified between genotypes early (E13.5) followed by reduced proliferation in MC (E15.5) and in the forming mandible (E17.5) in Fgfr2c C342Y/+ embryos. Activation of the ERK pathways is reduced in the perichondrium of MC in Fgfr2c C342Y/+ embryos and increased in bone related cells at E15.5. These data reveal that the Fgfr2c C342Y mutation differentially affects cells by type, location, and developmental age indicating a complex set of changes in the cells that make up the lower jaw.
