Christine E. Holt - US grants

The axons of newly born neurons extend long distances before reaching their targets in the vertebrate brain. Pioneering retinal ganglion cell (RGC) axons in Xenopus laevis make this 450 Mum journey unerringly through a variety of cellular terrains. How are these early axons guided? The present proposal specifically addresses this question by investigating the interactions of the tips of RGC axons with their microenvironment as they pioneer the pathway. The experiments propose three main lines of investigation: 1) Light and electron microscopy of dye-filled RGC axons will be used to characterize the morphology and the microenvironment of pioneering growth cones at different stages of their advancement along the optic pathway. 2)\ Intracellular injection of Lucifer Yellow into RGCs will reveal whether their growth comes become dye-coupled to other cells in the pathway. If such coupling does occur, antibodies that specifically block gap junctional conductance will be used to test whether intercellular communication is necessary for axonal navigation. 3) To find out when the embryonic nervous system first becomes permissive to axonal pathfinding, eye primordia will be transplanted to progressively younger embryos. The earliest stage that directed axonal growth is supported will be determined and it will be asked whether specific guidance structures, identified ultrastructurally or immunologically, are associated with the onset of these permissive conditions. These experiments will provide a better understanding of the factors that influence the behavior of growing axons in vivo and help to define the cellular basis of axonal navigation in the embryonic CNS.

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.

Abstract not provided.