The basic architecture of a nervous system is largely determined by the pathway choices made by neuronal growth cones during embryonic development. Although the behavior of mammalian growth cones has been extensively studied in tissue culture, little is known about the molecular mechanisms by which specific pathway choices are determined in vivo. Knowledge about these mechanisms would help to explain how the mammalian nervous system develops before and after birth. This is relevant to the eventual understanding of genetic diseases that affect the structure of the brain and of sensory and motor systems. The development of the insect segmental ganglia is a good experimental system in which to isolate and study pathfinding events in vivo, because these ganglia are composed of a very small number of neurons, each of which makes a unique, genetically programmed set of pathway choices. Furthermore, the basic ganglion architecture is conserved between species suitable for cell biology studies, such as the grasshopper, and species with well-developed genetics, such as Drosophila. A variety of data suggest that individual axons or axon bundles in insect segmental ganglia are differentially labeled by surface recognition molecules that are used for growth cone guidance. The cell surface proteins known as fasciclins are good candidates for such recognition molecules. The gene encoding one of these proteins, fasciclin I, has been isolated in both grasshopper and Drosophila, and mutation in the Drosophila gene has been identified. Although this mutation alone does not cause a visible alteration of the embryonic nervous system, embryos bearing both the fasciclin I mutation and a mutation in the Drosophila homolog of the c-abl oncogene have a lethal phenotype in which the nervous system is disrupted. The nature of the description, however, is not understood. In the experiments described in this proposal, the role of fasciclin I in the development of the embryonic nervous system will be studied by analyzing in detail the phenotypes of embryos lacking both fasciclin I and abl. This will be done by examining the structure of the nervous system in double mutant embryos using light-level immunohistochemistry and electron microscopic reconstruction. Double mutant combinations will also be made with other mutations in genes encoding proteins expressed in the nervous system, and their phenotypes similarly analyzed. Potential cell-adhesion activities of fasciclin I will be studied by expressing the molecule on the surface of tissue culture cells and studying the aggregation behavior of the transformed cells. In second part of the proposal, a genetic and a biochemical method to identify other potential neuronal recognition molecules are described. In the genetic approach, new molecules that may be involved in the pathway of action of fasciclin I will be identified by screening for mutations in other genes that can be mutated to an abl -dependent lethal phenotype. The biochemical approach utilizes a monoclonal antibody against a carbohydrate moiety shared by many insect neuronal surface proteins. Two of these proteins are fasciclins; the others are presently unidentified and could include other neuronal recognition protein species will then be generated and used to isolate cDNA clones encoding them.
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