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）： 颅缝是许多严重颅面畸形的主要原因。 异常 这个项目的长期目标是了解如何 颅缝发育并抵抗骨质闭塞，直到神经颅 成长完成了。 此更新应用程序的重点是功能 成纤维细胞生长因子（FGF）信号系统在发育和 缝合融合。 这些假设是，某些FGFs从 骨基质（FGF 2）、缝细胞和硬脑膜（除FGF 2外 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（FGFR）1、2和3， 它们以特定的重叠模式介导细胞信号， 缝线形态发生所需的活动--增殖， 分化或凋亡。 FGFR 1和FGFR 2，负调节 骨骼和纤维组织的生长。 FGFRs还可以 在缝合重建过程中发出凋亡信号。 的细胞应答 对FGF的依赖性可以作为FGF的可用浓度的函数而变化， 存在的FGF的类型和表达的FGFR的库。 由于FGF过量，可能会发生缝线闭塞 成骨细胞中的分化信号传导或增殖细胞的丢失， 缝合干细胞 检验假设的具体目标包括： 缝发育和融合过程中FGF和FGFr的表达模式（目的 1）表征体外缝线形成和融合中的FGF信号传导 (aim 2）和体内（目的3），并研究FGF/FGFR信号通路 在初级缝和颅骨细胞中（目的4）。 该研究将采用 大鼠，在胎龄19-21天（F19-F21）期间形成缝线。 方法学包括免疫组织化学定位、原位 杂交和PT/PCR分析了来自 非融合（冠状），融合（后额内），和实验- 诱导融合缝合。 体外缝线开发将用于测试 合适的FGF替代硬脑膜预防 核聚变 FGF和FGF的FGF抑制剂（中和抗体 和反义寡核苷酸）和酪氨酸的特异性抑制剂 FGFR，SU 5402的激酶活性将在体外和体内使用， 通过子宫外手术在F19胎仔中植入珠粒递送， N1新生儿阻塞或导致缝线融合。 在每种情况下， 骨和缝线的生长和闭塞将通过 组织形态计量学 FGFR和标记物的细胞分布模式 增殖、成骨分化和凋亡的过程将是 由co-localization决定。 最后，分离缝线和成骨细胞 细胞将被用于测试适当的FGF（S）在一系列的 影响增殖和分化的浓度， 在酪氨酸激酶信号通路中的潜在中介作用， 将缝合细胞与成纤维细胞中鉴定的那些进行比较。