The formation of correct neuronal pathways during the early stages of embryogenesis involves a number of navigational strategies. Prominent among them is the extension of growth cones along particular axon tracts, the selection of which is likely to me mediated by a hierarchy of molecular guidance cues. This process may play a crucial role in the correct wiring of the nervous system. The object of the present proposal is to increase our knowledge of such molecules by identifying and dissecting the molecular guidance mechanisms of a well defined population of peripheral sensory neurons in leech which make highly specific pathway choices. The promise of doing this in this system is that several novel molecules - the interactions between which are likely to be involved in this process - have already been identified and that these molecules have some of the most restricted axonal tract distributions yet described. Thus, we will perform a molecular and functional analysis of such proteins which are expressed by small subsets of axons forming specific fascicles in the leech with special emphasis on the cloning of lan3-2/4-2 antigens. In addition, we will molecularly characterize 1) a newly discovered protein with partial thrombospondin homology, L(tsp), which may interact with the lan3-2 antigen, and 2) a 200 kD protein, L(p200) which interacts with calsensin, a small EF-hand calcium-binding protein expressed in a subset of peripheral neurons fasciculating in a single axon tract. In these experiments we will in particular test the hypothesis that the lan3-2 antigen may be functioning both as a growth promoting homophilic adhesion molecule and that it may participate in heterophilic interactions with L(tsp), which is expressed by CNS effects, in a way that helps guide that axons of peripheral sensory neurons to the CNS. We will do this by in vivo function blocking assays with antibody and recombinant protein fragments and by CNS ablation and translocation experiments in the developing embryo. From the proposed experiments we will gain a basic understanding of the structure and functional significance of these molecules, their possible hierarchial organization, and their developmental regulation of expression. As our previous results demonstrate, the relatively simple and accessible system of the leech embryo provides a unique opportunity for identifying molecules mediating axonal pathway formation and to correlate their structure with their function in vivo. Since it has been established that many important structural protein sequence motifs have been functionally conserved throughout evolution, these investigations should enhance our basic understanding of neuronal recognition and selective fasciculation and provide new insights into the underlying causes of aberrant neural connections and abnormal brain development.
