This proposal aims to elucidate the molecular nature of growth cone guidance and target recognition in the developing visual projection of the vertebrate, Xenopus laevis. A multidisciplinary approach is proposed to address questions in three main areas. First, the role of a polypeptide growth factor, basic fibroblast growth factor (bFGF), will be examined based on our recent finding that bFGF, when added exogenously to living brains, causes the axons of retinal ganglion cells to by-pass, rather than innervate, their target the optic tectum. The effects of bFGF on retinal axon growth will be assayed in culture and in vivo with timelapse videomicroscopy and the normal expression pattern of bFGF, and the FGF receptor (FGFR), will be characterized with immunocytochemistry. The functional roles of bFGF and FGFR signaling will be tested by a) the introduction of dominant negative, constitutively activated and inducible forms of the FGFR into retinal ganglion cells in vivo; and b) the addition of synthetic peptides that block bFGF-FGFR interactions to the developing pathway. Second, the biological roles of two glycosaminoglycans, heparin and chondroitin sulfate (CS) will be characterized following our observations that heparin, like bFGF, causes retinal axons to by-pass the tectum whereas CS abolishes pathfinding. The similarity of the mistargeting phenotypes induced by exogenous heparin and bFGF, together with the fact that heparin/heparan sulfate is a required co-factor for FGF/FGFR interactions, suggests that the action of heparin is mediated via the bFGF or FGFR signaling pathways. Experiments are proposed to test this idea. The in vivo role of CS will be investigated with immunolocalization, enzymatic degradation and binding studies, and an in vitro substrate choice assay system will be used to address how CS might modulate growth cone steering. Third, the question of when the neuroepithelium first develops patterned cues that retinal axons use to navigate by will be addressed with transplants of embryonic brain tissue. Retinal ganglion cell axons will be challenged with increasingly younger pieces of optic tract and tectum to determine when pathway guidance and target recognition cues first arise. The experiments here focus primarily on in vivo development with the goal of identifying the molecular processes that are biologically relevant. The possibility that growth factors and glycosaminoglycans play fundamental roles in axon guidance and target recognition in the developing retinal projection has not been examined previously and our proposed studies promise to yield novel insights into this area.
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