The long-range goal of the proposed research is to understand the roles of the extracellular matrix (ECM) proteins fibronectin (FN) and laminin (LAM) in peripheral nervous system (PNS) development. ECM molecules regulate cell shape, differentiation, and movement during embryogenesis. In the PNS, ECM molecules appear to be involved in neural crest migration and in development and regeneration of neuromuscular junctions. Growth cones of nerve fibers migrating from the neural tube or peripheral ganglia traverse substrate pathways that are also likely to contain adhesive molecules. The adhesive glycoproteins FN and LAM and good candidates for mediating particular stages of PNS cell migration and differentiation, due to their locations in embryos and interactions with PNS cells in vitro. First, what is the relevance of the cell binding domains of FN and LAM to neuronal interaction with the molecules? Two domains of FN mediate neuron-substratum interaction in a cell type-specific manner. Functional regions of LAM are less well-defined, but also appear to mediate cell type-specific behavior. In vitro studies will probe 1) roles of adhesion in cell interaction with specific domains, 2) behavior of living cells as they interact with each domain, and 3) cytoskeletal correlates of these interactions. Key tools in a variety of inhibition assays will be proteolytic FN and LAM fragZ01MH02395 and domain specific reagents that selectively interfere with cell-substratum interactions. Second, how are FN and LAM (and specific regions of each) used by cells in the developing PNS? Approaches will be 1) immunochemistry at the light and electron microscopic levels, to reveal temporal and spatial associations between nerve fibers and ECM molecules, 2) analysis of neuronal interaction with complex matrices in vitro, and 3) perturbation of neuron-ECM interactions in vivo. Third, how do defined regions of FN and LAM contribute to migration and differentiation of neural crest cells? Crest cells interact with FN and LAM in vitro and are exposed to both molecules in vivo. Tissue culture studies will address 1) mechanisms of crest cell migration and 2) differentiation of crest subpopulations in response to these molecules. With our background in cell-ECM interactions and the tools now available to us, we will be able to evaluate precisely the roles of FN and LAM in PNS development.
