FORMATION OF NEURAL PATHWAYS IN THE EMBRYO

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This application seeks to explore the underlying mechanisms of axonal pathfinding and target recognition in the retinal projection of the embryonic Xenopus CNS, a system which is very amenable to such studies because of a recently developed in vivo time-lapse system, modified for the perfusion of drugs. This proposal, a continuation of a long term study of this system, has three major goals. The first is to understand the role of growth cone filopodia in axonal guidance. Retinal axons treated with cytochalasin, an actin depolymerizer, do not pathfind properly in the brain. In this proposal, the filopodial activity of individual axons treated with cytochalasin at various times and doses, will be correlated with their pathfinding behavior. Second, it has become increasingly clear that growth cones in culture can to respond particular cues by growing, stopping, turning or collapsing. Signal transduction cascades of various sorts must be used to translate these signals into growth cone action Which messenger systems are used for which purpose in the developing brain, however, has not yet been approached. One goal of this proposal, therefore is to investigate the role of particular second messenger systems in axon guidance and target recognition in the intact embryonic brain. A screening procedure based on the effects of particular membrane permeant agents has already been used to implicate some of the signaling pathways involved, particularly a G- protein cascade, and to suggest that other pathways, like the calcium pathway may be uninvolved or redundant. In addition, it has been demonstrated that growth filopodia are necessary for pathfinding in the brain, suggesting that this may be the sites of signal transduction. Experiments are proposed to pursue these suggestions with rigorous experiments that distinguish alternate interpretations, and to extend the pathway molecularly. Third, retinal axons dX to the tectum using local positional cues in the neuroepithelium. Evidence suggests that certain positional information genes, such as homeobox genes and paired-box genes, are involved in establishing topographic identity during early vertebrate neurogenesis. Recently, it has been shown that at least some of these genes can directly regulate tahe expression of particular cell adhesion molecules, making it likely that they therefore also affect axonal navigation in the CNS. Advances in gene introduction technology in vivo have made it possible to misexpress genes tin the embryonic Xenopus nervous system. In this proposal, Xenopus homeobox- containing genes, including engrailed, distal-less, Pax-6, and ganglion cells or in the neuroepithelial cells through which the growing retinal axons normally navigate. The misexpressing embyros will be examined for defects in pathfinding and target recognition.