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 functional role of FGF signaling 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. Two classes of receptors are necessary for FGF signaling, high affinity tyrosine kinase FGF receptors (FGFRs) and low affinity heparan sulfate proteoglycans (HSPGs). We will tes the involvement of the high affinity FGFRs in targeting decisions by altering the activity of the native receptors. To achieve this, we will 1) introduce dominant negative, constitutively activated and inducible forms of the FGFR into retinal ganglion cells in vivo by way of lipofection; and 2) add synthetic peptides that block bFGF-FGFR interactions to the developing pathway. Second, the possible role of low affinity HSPGs will be examined following our observation that a distinct bFGF-binding heparan sulfate (bFGF-HS) causes retinal axons to bypass the tectum. Because of the similarity of the mistargeting phenotypes induced by exogenous HS and bFGF, together with the fact that HS is a required co-factor for FGF/FGFR interactions, the proposal addresses whether the action of HS is mediated via the FGF signaling pathway. Third, the biological role of chondroitin sulfate (CS) will be characterized following our observations that CS disrupts axonal pathfinding. 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. 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.
