Differentiation of osteoblast lineage cells that form dermal bones and sutures of the anterior cranial vault in development and disease

Abstract

FGFR-related craniosynostosis syndromes, such as Apert syndrome, are characterized in part by dysmorphology of the anterior cranial vault bones and early closure of the coronal suture. Understanding the cellular processes during early formation of the anterior cranial vault bones and the coronal suture is key to the development of therapies for these syndromes. Runt-related transcription factor 2 (RUNX2) and Osterix (OSX) play critical roles in the regulation of osteoblast differentiation and function and are first expressed at different stages of osteoblast lineage cell (OLC) development. RUNX2 is known to act upstream of OSX and is expressed during an earlier stage of OLC differentiation, but relatively little is known about the behavior and developmental sequence of OLCs that contribute to the initial formation of cranial dermal bones and the sutures between them. To address this issue, we made the R2Tom transgenic mouse line by introducing the tdTomato reporter gene joined with an enhancer fragment of the human RUNX2 gene and the Hsp68 minimal promoter sequence that labels developmentally early OLCs. Crossing this novel R2Tom mouse line with the Osx-GFP line produced R2Tom;Osx-GFP mice, in which expression of RUNX2 and Osx is detected with different fluorescent reporters. The R2Tom;Osx-GFP mouse line was then crossed with Fgfr2+/P253R;Emx1-Cre (referred to below as Apert) mice that show phenotypes similar to Apert syndrome patients to produce Apert;R2Tom;Osx-GFP mice and “unaffected” littermates that carry the fluorescent marker but do not express the mutant Fgfr2 gene. Using large field, high-resolution fluorescent microscopy (ranging between 12.5x and 112x) and light sheet microscopy, we captured expression of RUNX2 and Osx across the surface of whole mouse embryos and at the cellular level. R2Tom;Osx-GFP and Apert;R2Tom;Osx-GFP mice were developmentally staged to provide precise information about the developmental sequence of expression of RUNX2 and Osx and 3D physical organization of early OLCs during the cellular assembly of what will be the frontal and parietal bones and initiation of the coronal suture in mouse embryos between E11.5 to E14.5. Our preliminary data on Apert;R2Tom;Osx-GFP mice at E12.5 and E13.5 reveal differences in the timing and pattern of the developmental sequence of RUNX2 and Osx expression in the anterior cranial vault of mice carrying the Fgfr2+/P253R mutation relative to their unaffected litter mates. These results provide our first view of OLC differentiation in the anterior cranial vault of Fgfr2+/P253R Apert mice prior to the formation of anterior cranial vault bones and the coronal suture and suggest that differences in the timing of OLC patterning play an import role in dysmorphologies of the anterior cranial vault.