EXON-SPECIFIC FIBRONECTIN ISOFORMS AND CHONDROGENESIS

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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.