REGULATION OF CRANIALSUTURE MORPHOGENESIS

颅缝形态发生的调节

• 批准号: 6379745
• 负责人: Roy Clinton Ogle
• 金额: $ 30.4万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6379745
• 关键词: antisense nucleic acid; apoptosis; biological signal transduction; bone development; cell differentiation; cell growth regulation; congenital oral /facial /cranial defect; developmental neurobiology; dura mater; embryo /fetus; enzyme inhibitors; fibroblast growth factor; growth factor receptors; immunocytochemistry; in situ hybridization; laboratory rat; neutralizing antibody; newborn animals; osteoblasts; osteogenesis; periosteums; polymerase chain reaction; protein tyrosine kinase; receptor expression

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.

描述（改编自申请人的摘要）： 颅骨缝合线是许多严重颅面的主要原因 异常。 该项目的长期目标是了解 颅缝线会发育并抵抗骨闭塞直到神经骨骼 增长是完整的。 此更新应用的重点是 开发中的成纤维细胞生长因子（FGF）信号系统 缝合线的融合。 假设是某些FGF从 骨基质（FGF2），缝合细胞和硬脑膜（其他FGF2） 或7）扩散到发育中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 特定的重叠模式在其中介导信号的细胞信号 缝合形态发生所需的活动 - 增殖， 分化或凋亡。 FGFR1和FGFR2，负调节 骨骼和纤维组织的生长。 FGFR也可能 缝合线重塑期间的信号凋亡。 细胞反应 FGF可能会随着FGF的可用浓度而变化， FGF的类型以及FGFR（S）的曲目表示。 缝合线的闭塞可能是由于过量的FGF而发生的 成骨细胞中的分化信号传导或增殖的损失 缝合干细胞。 测试假设的具体目的包括至5月 FGF和FGFR表达模式在缝合缝合和融合期间（目的 1），表征FGF信号传导在体外形成和缝合线的融合中 （AIM 2）和体内（AIM 3），并研究FGF/FGFR信号通路 在原发性缝合线和钙细胞中（AIM 4）。 该研究将采用 大鼠，其中缝合线在胎儿天19-21（F19-F21）中形成。 方法包括原位免疫组织化学定位 解剖组织的mRNA的杂交和PT/PCR分析 不使用（冠状），融合（后额外）和实验 诱导的融合缝合线。 体外缝合缝线开发将用于测试 适当的FGF替代硬脑膜的能力 融合。 FGFS和FGRF的FGF抑制剂（中和抗体 和反义寡核苷酸）和酪氨酸的特定抑制剂 SU5402的FGFR的激酶活性将在体外和体内使用 通过子宫手术在F19胎儿中通过珠植入和 N1新生儿阻塞或引起缝合融合。 在每种情况下 骨骼和缝合线生长和闭塞将由 组织形态法。 FGFR和标记的细胞分布模式 增殖，成骨分化和凋亡将是 通过共定位确定。 最后，孤立的缝合线和成骨细胞 细胞将用于测试适当的FGF在一系列范围内 对增殖和分化的影响的浓度 潜在中介在酪氨酸激酶信号通路中 将缝合细胞与成纤维细胞中鉴定的细胞进行比较。