REGULATION OF CRANIALSUTURE MORPHOGENESIS

描述(改编自申请人的摘要)：过早融合 颅缝是许多重型颅面骨折的主要原因。 异常现象。该项目的长期目标是了解如何 颅缝发展并抵抗骨性闭塞，直到脑颅 增长已经完成。本续费申请重点介绍了 成纤维细胞生长因子(成纤维细胞生长因子)信号系统在发育和发育中的作用 缝线融合术。假设是某些FGFs从 骨基质(FGF2)、缝合细胞和硬脑膜(FGF2除外 或7)扩散到正在发育的缝合线的细胞外基质。那里 细胞表达成纤维细胞生长因子受体(FGFRs)1、2和3 在特定的重叠模式中，它们为细胞传递信号 缝合形态发生所需的活动--增殖， 分化，或称细胞凋亡。FGFR1和FGFR2，负向调节 分别是骨骼和纤维组织的生长。FGFR还可以 在缝线重塑过程中发出细胞凋亡信号。细胞反应 对FGFs的影响可以随可用的成纤维细胞生长因子的浓度而变化， 目前存在的FGFs的类型，以及FGFR的曲目(S)表示。 由于过量的成纤维细胞生长因子，可能会发生缝合的堵塞。 成骨细胞中的分化信号或增殖性细胞的丢失 缝合干细胞。检验这些假说的具体目标包括 成纤维细胞生长因子和成纤维细胞生长因子受体在缝线发育和融合过程中的表达模式(目的 1)，研究了成纤维细胞生长因子在缝合线形成和融合过程中的信号特征 (目标2)和体内(目标3)，并研究成纤维细胞生长因子/成纤维细胞生长因子受体信号通路 在初级缝合和头盖骨细胞中(目标4)。这项研究将采用 大鼠，在19-21天(F19-F21)形成缝线。 方法学包括免疫组织化学定位、原位 大鼠解剖组织中mRNA的杂交和PT/PCR分析 不融合(冠状)，融合(额后部)，实验上- 诱导融合缝合。缝合线的体外开发将用于测试 合适的FGFs替代硬脑膜预防硬脑膜损伤的能力 核聚变。FGFs和FGFs(中和抗体)的抑制剂 和反义寡核苷酸)和酪氨酸的特异性抑制剂 FGFRs的激酶活性SU5402将在体外和体内应用， F19胎儿宫外手术植入珠子分娩 N1新生儿阻断或引起缝合融合。在每一种情况下， 骨和缝合的生长和闭塞将由以下因素决定 组织形态计量学。FGFRs及其标志物的细胞分布模式 增殖、成骨分化和细胞凋亡的 由共同本地化决定。最后，分离缝合和成骨细胞 细胞将用于测试适当的成纤维细胞生长因子(S)在一系列 影响增殖和分化的浓度和 酪氨酸激酶信号通路中的潜在介导物 缝合细胞将与成纤维细胞中鉴定的细胞进行比较。

DESCRIPTION (Adapted from the Applicant's Abstract): 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.