EXON-SPECIFIC FIBRONECTIN ISOFORMS AND CHONDROGENESIS

纤维连接蛋白是组织细胞外基质中的一种连接分子，其结构在胚胎肢体发育中的软骨形成（间充质分化为软骨）过程中发生变化。 纤连蛋白异构体的变化是由mRNA选择性剪接事件引起的，涉及纤连蛋白基因中至少三个外显子（IIIB、IIIA和V）。 在软骨形成过程中，纤维连接蛋白mRNA的剪接方式由软骨前间质的B+A+V+变为软骨的B+A-V+。由外显子IIIA编码的区域在软骨形成过程中发生的凝聚事件后从间充质纤维连接蛋白中消失。 这种结构上的变化反过来又可能导致功能上的变化。 我们最近的研究表明，除了特异性的外显子IIIA编码的区域的单克隆抗体高密度四肢间充质微团文化抑制软骨形成，可能是通过干扰细胞凝聚的形成和/或维持。 我们计划测试的假设，一个特定的纤连蛋白亚型导致mRNA选择性剪接是必要的软骨发育的不同阶段。 前4个目标将集中在间充质纤维连接蛋白的结构，特别是外显子IIIA编码的区域，是否需要在软骨形成过程中发生的间充质凝聚事件的某些方面。目的1中的实验将研究在存在和不存在外显子特异性抗体和肽的情况下，间充质细胞与细胞粘附测定中的各种纤连蛋白亚型的相互作用。 目的2将使用迁移测定和聚集测定来确定间充质纤连蛋白是否支持参与软骨形成凝聚的间充质细胞迁移和/或聚集事件。 目的3将评估纤连蛋白异构体对间充质软骨形成的影响，特别是与细胞密度的依赖性和可能的功能串扰与N-钙粘蛋白介导的细胞-细胞相互作用。 目的4将通过表征与TGF-β 1的软骨调节活性的功能关系来检查纤连蛋白亚型对间充质软骨形成的特异性作用的调节。 最终目标（目标5）将开始通过表征纤连蛋白与分化的软骨细胞的相互作用及其对软骨细胞表型维持的影响来研究软骨纤连蛋白（B+A+V+）中外显子IIIA缺失的生物学意义。从这些实验中得到的信息将进一步我们了解的功能，纤连蛋白在软骨形成的不同阶段，并提供有用的见解的目的选择性剪接事件的纤连蛋白基因，在软骨发育过程中表现出的组织特异性的方式。

The structure of fibronectin, a connecting molecule in the extracellular matrices of tissues, changes during chondrogenesis, the differentiation of mesenchyme into cartilage, in the developing embryonic limb. Fibronectin isoform-changes result from mRNA alternative splicing events involving at least three exons in the fibronectin gene (IIIB, IIIA, and V). During chondrogenesis, the fibronectin mRNA splicing patterns change from B+A+V+ in precartilage mesenchyme to B+A-V+ in cartilage. The region encoded by exon IIIA disappears from mesenchymal fibronectin just after the condensation event that occurs during chondrogenesis. This structural change may in turn dictate a change in function. Our most recent studies show that addition of a monoclonal antibody specific for the region encoded by exon IIIA to high-density limb mesenchymal micromass cultures inhibits chondrogenesis, possibly by interfering with the formation and/or maintenance of cellular condensations. We plan to test the hypothesis that a specific fibronectin isoform resulting from mRNA alternative splicing is necessary for different stages of cartilage development. The first 4 aims will focus on whether the structure of mesenchymal fibronectin, specifically the region encoded by exon IIIA, is required for some aspect of the mesenchymal condensation event that occurs during chondrogenesis. Experiments in Aim 1 will investigate the interactions of mesenchymal cells with various fibronectin isoforms in cell adhesion assays in the presence and absence of exon-specific antibodies and peptides. Aim 2 will determine whether mesenchymal fibronectin supports mesenchymal cell migration and/or aggregation events involved in chondrogenic condensation using migration assays and aggregation assays. Aim 3 will assess the effect of fibronectin isoforms on mesenchymal condrogenesis, particularly related to the dependence on cell density and possible functional cross-talk with N-cadherin mediated cell-cell interactions. Aim 4 will examine the regulation of fibronectin isoform-specific effects on mesenchymal chondrogenesis by characterizing the functional relationship with the chondro- regulatory activities of TGF-beta1. The final aim (Aim 5) will begin to investigate the biological significance of the loss of exon IIIA in cartilage fibronectin (B+A+V+) by characterizing fibronectin interactions with differentiated chondrocytes and their effects on the maintenance of the chondrocyte phenotype. The information resulting from these experiments will further our understanding of the function of fibronectin at different stages of chondrogenesis and provide useful insights into the purpose for alternative splicing events in the fibronectin gene that are exhibited in a tissue-specific manner during cartilage development.