GROWTH CONE GUIDANCE--INTERACTIONS AND SURFACE MOLECULES

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We have identified and begun the molecular and functional characterization of two new cell surface molecules involved in neurogenesis. Lazarillo and Conulin were found by making mAbs specific to the developing nervous system and then using those mAbs as biochemical reagents to purify and characterize each antigen. We have cloned the gene encoding Lazarillo and we are proposing to clone the gene encoding Conulin in this application. Lazarillo represents a new class of molecule, a lipocalin, involved in neuronal path findings and suggests a new molecular signaling pathway. We are proposing experiments to characterize how Lazarillo regulates growth cone and filopodial behavior to guide neurons along a specific pathway. This will entail developing a "molecular knockout" anti-sense technology to specifically inhibit the expression of Lazarillo in identified neurons. We will test the hypothesis that Lazarillo mediates its regulation of growth cone behavior via a signal transduction pathway that includes a small lipophilic ligand and a protein ligand. Structural and functional experiments are proposed to identify both the small lipophilic ligand that binds in the lipocalin hydrophobic pocket and the protein ligand that binds to the putative protein protein interaction domain suggested by our modeling studies. We predict that homologues of Lazarillo exist in other animals, including vertebrates, and function in a similar way during the development of their nervous systems. We have proposed a straightforward PCR based approach to identify these homologues and will use sequence information and in situ hybridization to verify the homology. Conulin's unique spacial and temporal localization to a subset of neuronal growth cones makes it a good candidate for a novel molecular function. Experiments to identify Conulin include immunoaffinity and biochemical purification, and expression cloning. We hypothesize that Conulin functions in neuronal path finding as growth cones switch from one axonal fascicle to another in the central ganglionic neuropil. We will test this hypothesis by observing the behavior of identified growth cones that have had conulin removed from their surfaces. It is clear that many of the basic molecular mechanisms in developmental processes have been highly conserved during the evaluation of nervous systems as diverse as nematode, fly and mouse. We propose that Lazarillo and Conulin will lead us to new molecular mechanisms used by all developing nervous systems.