Pathway choices which are made by neuronal growth cones during development ultimately are responsible for establishing the specificity of synaptic connections within the embryonic nervous system. In vitro and in vivo studies in vertebrates have examined the cellular parameters governing axonal pathfinding. In addition, a number of adhesion molecules have been identified which appear to play a role in neuronal development. However, the expression patterns of these molecules, including N-CAM and N-cadherin, seen to be too general to account for the specificity observed during axonal pathfinding. Thus, the molecular mechanisms involved in growth cone guidance have for the most part remained obscure. A detailed examination of these mechanisms will provide a basis for understanding the development of the mammalian nervous system both before and after birth. Such studies are relevant to an eventual understanding of genetic diseases which affect the structure of the brain and motor and sensory systems. One relatively simple system which is amenable to analysis of the events involved in axonal guidance is the segmental ganglia of insects. These ganglia contain a small number of identified neurons early in development, and their growth cones have been shown to make a genetically programmed set of pathway choices. In addition, the basic cellular architecture is conserved between different insect species, allowing studies at both the cellular and molecular levels. Previous work in Drosophila has allowed the identification of a molecule termed fasciclin III. which is expressed on a subset of axonal bundles in the developing nervous system, and thus is a good candidate for a molecule involved in growth cone guidance. It has subsequently been shown that fasciclin III represents a novel class of adhesion protein which mediates cell aggregation through a homphilic mechanism. The studies proposed here are directed at determining the structural motifs which are important in mediating homotypic binding. This will be accomplished by isolating a fasciclin III homologue from a second insect species, and determining potentially important structural motifs by identifying sequences which are conserved between the two homologues. Alternatively, epitopes recognized by monoclonal antibodies which inhibit fasciclin III-mediated aggregation will be mapped to localize regions potentially involved in binding. The role of regions identified by either technique in mediating binding will be examined by testing the ability of peptides based on the defined regions to inhibit cellular aggregation. The role of fasciclin III in supporting neurite extension will also be examined in an in vitro system, and structural features important in mediating this role will be defined by similar techniques. The contribution of the cytoplasmic domain of fasciclin III to adhesion will be addressed by analyzing the effects of deletions of this domain on cellular aggregation and neurite outgrowth. Interactions with cytoskeletal elements will be of fasciclin III in signal transduction by a number of second messenger systems will also be examined, including the role of the cytoplasmic domain in mediating signal transduction. In the cases of other well defined homophilic adhesion molecules, heterotypic interactions have also been described. The possibility that fasciclin III is also involved in such interactions will be addressed by identifying putative heterotypic ligands based on their affinity for fasciclin III. Such putative counter-receptors will subsequently be characterized biochemically and functionally. Finally, additional molecules possessing the structural motifs defined by fasciclin III will be identified and characterized through a combination of biochemical and molecular genetic approaches.
| Strong, R K; Vaughn, D E; Bjorkman, P J et al. (1994) Expression and crystallization of a soluble form of Drosophila fasciclin III. J Mol Biol 241:483-7 |
