Mechanisms of Craniosynostosis in Bent Bone Dysplasia Syndrome

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PROJECT SUMMARY Craniosynostosis, or the premature fusion of one or more suture joints in the skull, is the second most common congenital craniofacial birth defect with a prevalence of around 1/2500 births. Mutations to Fibroblast growth factor receptor 2 (FGFR2) are common genetic causes behind craniosynostosis syndromes, including the FGFR2M391R and the FGFR2Y381D variants which cause Bent Bone Dysplasia Syndrome (BBDS). Our lab has demonstrated that these mutations affect osteoblast fate via relocalization of Fgfr2 from the plasma membrane to the nucleus in vitro, but lack of an animal model has prevented the connection of these genetic and molecular changes to specific symptoms of BBDS such as craniosynostosis. We have developed a mouse model harboring a Cre-inducible knock-in of the Fgfr2M391R allele. Activating this mutation in neural crest cells (NCCs) via the Wnt1-Cre driver leads to progressive postnatal fusion of the frontal, coronal, sagittal, and lambdoid sutures. Both the sagittal and lambdoid sutures consist entirely of mesoderm-derived bone, however activation of the Fgfr2M391R allele via the Mesp1-Cre driver does not induct pan-suture craniosynostosis. Genetic lineage tracing of the embryonic mouse calvaria shows that NCCs give rise to the frontal bones, as well as the majority of non- osteogenic dense connective tissue at the sutures. This suggests that these previously uncharacterized non- osteogenic sutural cells are regulated by Fgfr2 and mechanistically involved in suture development and maintenance, as well as the pathogenesis of craniosynostosis in BBDS. Using a combination of genetic lineage tracing and single genomics, I will determine how expression of the Fgfr2M391R variant alters cell identity and contributions of dense connective tissues in the postnatal sutures. Among these cells are a population of recently observed osteofibrous progenitor cells which have yet to be fully characterized but are likely crucial to maintenance of postnatal suture patency. I will better resolve this population of cells and identify the role of Fgfr2 in maintaining the balance between their differentiation into osteoblasts vs. fibroblasts. I will also expand upon the knowledge of the nuclear role of mutant Fgfr2M391R and how it alters gene regulation within sutural cells to induce craniosynostosis. Due to its previously established role in altering chromatin remodeling and gene expression, I predict that Fgfr2M391R alters chromatin accessibility at additional gene targets. These may include members of the p53 and Wnt signaling pathways, which I find to be differentially expressed in postnatal sutures of our Wnt1-Cre; Fgfr2M391R/+ mouse model and have a known role in calvaria development and craniosynostosis. Using single nuclei ATAC-sequencing, I will determine how the Fgfr2M391R variant alters chromatin accessibility, cross referencing this dataset with differentially expressed genes from our bulk and single cell RNA-seq datasets. This will identify the function of nuclear Fgfr2 and novel signaling crosstalk necessary for regulating osteofibrous cells in the calvarial sutures. Completion of this study will advance our understanding of the connection between cavarial dense connective tissues, suture development, and the pathogenesis of craniosynostosis.