Functional analysis of the role of Cdc42 in mammalian skeletal development

哺乳动物骨骼的发育是一个复杂的、在时间和空间上有组织的过程，在这个过程中，间充质组织要么直接分化为骨(膜内成骨)，要么首先形成软骨中间体，然后被骨取代(软骨内成骨)。骨骼形态发生受到细胞-基质相互作用的强烈影响，并受到细胞外蛋白质、生长因子、激素、转录因子和细胞内信号分子的严格调控。广泛的基因突变会导致各种骨软骨发育不良，即以生长和骨骼异常为特征的遗传性疾病。1整合素是骨形成过程中调节细胞-基质相互作用的主要黏附受体，在调节细胞外基质组装、细胞存活、增殖、分化和细胞骨架组织的两种成骨过程中都起着至关重要的作用。然而，相对较少的人知道在体内指导这些整合素功能的细胞内信号通路。Rho GTP酶是一个细胞内分子家族，参与粘附性和非粘附性受体的信号传递。CDC42是Rho家族的原型成员，与骨骼发育有关，但其功能尚未在动物模型中进行研究。为了阐明CDC42在骨骼形成中的作用，我们使用Cre-loxP系统有条件地灭活了小鼠成骨细胞和软骨细胞的间充质前体细胞中的CDC42基因。我们的初步数据显示，突变小鼠存在严重的骨骼缺陷，包括软骨和骨组织。本申请旨在1)详细描述这些缺陷的特征；2)确定影响骨骼形成的依赖于CDC42的信号通路；3)并提供一个复杂的机制观点：在骨形成过程中，CDC42如何整合细胞外和/或细胞内的信号。该项目涉及组织学、超微结构和免疫组织化学分析以及生化、细胞和基因表达方法。

Development of the mammalian skeleton is a complex, temporally and spatially organized process during which mesenchymal tissues either directly differentiate into bone (intramembranous ossification) or first form a cartilaginous intermediate which is subsequently replaced by bone (endochondral ossification). Skeletal morphogenesis is strongly influenced by cell-matrix interactions and tightly regulated via extracellular proteins, growth factors, hormones, transcription factors and intracellular signalling molecules. Mutations in a broad range of genes result in various osteochondrodysplasias, genetic diseases charactherized by growth and skeletal abnormalities. ß1 integrins, the major adhesion receptors that mediate cell-matrix interactions during skeletogenesis, have been shown to be crucial for both types of ossification processes regulating extracellular matrix assembly, cell survival, proliferation, differentiation and cytoskeletal organization. However, relativelly less is known about the intracellular signalling pathways directing these integrin functions in vivo. The Rho GTPases constitute a family of intracellular molecules involved in signalling from adhesive as well as nonadhesive receptors. Cdc42, a prototype member of the Rho family, is implicated in skeletal development but its function has not been studied in animal models. In order to elucidate the role of Cdc42 during skeletogenesis we have conditionally inactivated the mouse Cdc42 gene in mesenchymal precursor cells of osteoblasts and chondrocytes using the Cre-loxP system. Our preliminary data revealed severe skeletal defects of the mutant mice including both cartilaginous and bony tissues. Present application aims 1) to charactherize these defects in detail; 2) to identify Cdc42-dependent signaling pathways that affect skeletogenesis; 3) and to provide a complex mechanistic view how Cdc42 integrates extra- and/or intracellular signals during bone formation. The project involves histological, ultrastructural and immunohistochemical analyses as well as biochemical, cellular and gene expression approaches.