The proper functioning of the vertebrate brain depends on the formation of specific connections between different, often distant, neurons. This specificity is generated during embryogenesis when axons first navigate to their synaptic targets. Axonal navigation involves specific interactions between the advancing tips of axons, the growth cones, and their microenvironment.
The aim of this proposal is to elucidate the cellular and molecular basis of these interactions in the vertebrate visual system in vivo. We have recently developed a technique for transfecting retinal cell precursors in the living embryo (Xenopus) with foreign DNA. In the first series of experiments, we propose to use this method to alter the expression of adhesive components on the surfaces of growth cones of single retinal ganglion cells (RGCs) by transfecting these cells with different forms of N-CAM, N-cadherin and integrin cDNAs. The axons of transfected RGCs will be assayed for abnormal phenotypes by immunolocalizing a coexpressed luciferase reporter gene. This type of approach will permit for the first time, manipulations of gene expression in selected cells in vivo without altering their environment. As such, it will provide a unique test for the role of adhesive interactions in axonal navigation in vivo. In a second series of experiments, the microenvironment of normal RGC growth cones will be manipulated to determine the cellular and molecular characteristics of the substrate that are important for navigation. Embryological transplantation experiments are proposed that will challenge normal optic axons to grow through: 1) pieces of optic tract neuroepithelium with genetically altered adhesivity to test the role of this property in axonal guidance, and 2) progressively less mature pieces of presumptive optic tract tissue to determine when critical guidance cues first arise in the neuroepithelium.
We aim to characterize the nature of these cues with a combination of molecular, immunological and transplant experiments.
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