The overall goal of the proposed research is to understand the mechanism of neuronal migration in the vertebrate brain. Two broad goals are to discover the genes required for the migration of CNS neurons on glial substrates and to develop an experimental approach to imaging large cohorts of migrating neurons, in situ, in real time. In prior cycles of this grant, the investigator discovered a gene astrotactin (Astn), that functions as a neuronal ligand for granule cell migration along glial guides. The predicted protein structure of Astn has an extra cellular region containing two potential glycosylation sites, and regions of homology to adhesion molecules of the fibronectin III family and the EGF receptor.
The first aim of the proposed research is to carry out a quantitative analysis of a targeted deletion of Astn, examining the size of primary structures, and the development of the two principal cerebellar neurons, the granule cell and the Purkinje cell. In preliminary studies, they discovered an Astn2 and are searching for other family members. In another group of experiments, they will use biochemical methods to isolate the glial receptor for ASTN. Finally, they will extend their long-standing interest in migration to the in vivo setting, using newly developed computer-based methods to follow the migration of large cohorts of granule cells, labeled with retroviral transfer of the GFP marker or with GFP-Astn """"""""knock-in"""""""" mice. The new imaging experiments should provide the first information on the movements of granule cells in vivo, using 3D time-lapse recordings of large cohorts of cells. Together these experiments will generate information on the neuron-glial ligand astrotactin, and its role in migration. As the discovery of a three more astrotactin genes demonstrates that Astn is part of a gene family, they will examine the redundancy in function of these genes. The discovery of the glial receptor will provide fundamental information on mechanisms of glial differentiation, information critical to both brain development and brain trauma. Finally, being able to view the movements of granule cells in real time in situ will inform about the relationship between axon (parallel fiber) extension and the locomotion of the granule neuron down the Bergmann glial fiber.
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