Premature fusion of the cranial sutures is the primary cause of many severe craniofacial abnormalities. The long term goal of this project is to understand how cranial sutures develop and resist osseous obliteration until neurocranial growth is complete. This renewal application focuses on the function of the fibroblast growth factor (FGF) signaling system in the development and fusion of sutures. The hypotheses are that certain FGFs released from bone matrix (FGF2), the suture cells, and the dura mater (other that FGF2 or 7) diffuse to the extracellular matrix of the developing suture. There the cells express fibroblast growth factor receptors (FGFRs) 1,2 and 3 in specific, overlapping patterns where they mediate signals for cellular activities required for suture morphogenesis-- proliferation, differentiation, or apoptosis. FGFR1 and FGFR2, negatively regulate growth of the bones and fibrous tissues, respectively. FGFRs may also signal apoptosis during remodeling of the suture. The cellular response to the FGFs may vary as a function of the available concentration of FGF, the types of FGFs present, and the repertoire of the FGFR(s) expressed. Obliteration of the suture may occur as a result of excess FGF differentiative signalling in the osteoblasts or loss of the proliferative suture stem cells.
Specific aims to test the hypotheses include to may FGF and FGFr expression pattern during suture development and fusion (aim 1), characterize FGF signaling in formation and fusion of sutures in vitro (aim 2) and in vivo (aim 3), and investigate FGF/FGFR signalling pathways in primary suture and calvarial cells (aim 4). The study will employ the rat, in which the sutures are formed during fetal days 19-21 (F19-F21). Methodology includes immunohistochemical localization, in situ hybridization and PT/PCR analysis of mRNA of dissected tissues from nonfusing (coronal), fusing (posterior intrafrontal), and experimentally- induced fusing sutures. Suture development in vitro will be used to test the ability of appropriate FGFs to substitute for dura in preventing fusion. FGFs inhibitors of the FGFs and FGRFs (neutralizing antibodies and antisense oligonucleotides) and a specific inhibitor of tyrosine kinase activity of FGFRs, SU5402, will be employed in vitro and in vivo, delivered by bead implantation in F19 fetuses by ex utero surgery and to N1 neonates to block or cause suture fusion. In each case the extent of bone and suture growth and obliteration will be determined by histomorphometry. The cellular distribution pattern of FGFRs and markers of proliferation, osteogenic differentiation, and apoptosis will be determined by co-localization. Finally, isolated suture and osteoblastic cells will be used to test the appropriate FGF(s) over a range of concentrations for influence on proliferation and differentiation and potential intermediated in the tyrosine kinase signalling pathway in suture cells will be compared to those identified in fibroblasts.
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