Chondrogenesis involves the transition of mesenchymal cells to cartilage through a process of condensation, followed by dramatic changes in the composition of the extracellular matrices produced by the cells. The ECM glycoprotein fibronectin is present in both the mesenchymal cell and the differentiated cartilage, but the isoforms produced by the cells differ due to a change in alternative mRNA splicing. Mesenchymal fibronectin contains both alternative segments EIIIA and EIIIB, which are encoded by the eponymous exons, whereas cartilage FN lacks the EIIIA domain, due to skipping of exon EIIIA by the splicing apparatus. Evidence suggests that the mesenchymal isoform of FN plays a role in the process of chondrogenic condensation, and it has been hypothesized that the subsequent loss of EIIIA is necessary for chondrogenic differentiation. An understanding of the mechanisms that regulate alternative splicing of the exon encoding EIIIA should permit experimental manipulation of splicing and so establish the roles of the various FN isoforms.
Three aims are proposed to achieve these goals. First, cellular factors that interact with exon EIIIA will be identified by screening for sequence-specific RNA-protein interactions. The functional relevance of any such interactions will be established by co-transfection of cells with an exon EIIIA splicing reporter and an expression construct for each candidate RNA binding protein. Second, knowledge of the cis sequences and trans factors important for exon EIIIA recognition will be used to develop novel methods to perturb exon EIIIA splicing, by forcing exon inclusion or exclusion irrespective of cell type. Last, agents that perturb exon EIIIA splicing will be applied to a micromass model of chondrogenic condensation, to establish the roles of the FN isoforms in undifferentiated mesenchyme or differentiated chondrocytes. These studies will increase understanding of the early stages of cartilage formation, with implications for normal embryonic development as well as for bone replacement in adults.
