The overall goal of this research is to understand the molecular mechanism of glial-guided neuronal migration and its genetic regulation in the developing mammalian brain. To analyze the molecular mechanism of migration, the proposed research will use an in vitro model system, developed in our laboratory, to define the role of cell adhesion ligands in the locomotion of the cerebellar granule neuron along the glial guide. We will assay the effects of antibodies gainst the neuron-glia ligand astrotactin, and compare these with effects of antibodies against the neuron-neuron ligands L1, N-CAM, N-cadherin and TAG-1, and of the receptor for fibronectin and laminin, integrin. Migrating cells will be imaged with video-enhanced differential interference contrast microscopy in microcultures to examine the frequency, rate of movement or cytology of migrating granule neurons. In companion studies, we will develop methods to image the dynamics of migration of dye-labeled granule neurons in tissue slices of cerebellum, and assess the effects of antibodies against cell adhesion ligands on migration in situ. To provide information on the predicted protein structure of astrotactin, including its overall structure, interaction with the membrane and homology with other cell adhesion ligands, affinity- purified anti-astrotactin antibodies will be used to clone cDNAs for the 100KD protein component of the astrotactin activity. We will use our biological assays to demonstrate that putative astrotactin clones encode functional epitopes of the astrotactin activity. Astrotactin clones will be analyzed at the molecular level and the function of astrotactin cDNAs will be xamined by expression of identified clones in PC12 neurons and weaver granule cells. To analyze the genetic regulation of neuronal migration, we will continue our studies on neurological mutant mice with defects in glial-guided migration, weaver and meander tail. Meander tail is an exciting new mutation with a severe disruption of cellular organization restricted restricted to the anterior lobe of the cerebellum. In vitro recombination experiments will be used to define the cellular site of action of the meander tail gene. The dynamics of granule cell migration in tissue slices of meander tail cerebellum will then be compared with results from weaver. To analyze whether cell-cell interactions with neurons or axons provide the """"""""stop signals"""""""" for glial-guided neuronal migration, we will examine whether cerebellar granule neurons terminate migration upon encountering lanes of neurons or axons in and in vitro model system.
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