Over the past several years, our laboratory has been studying the genesis of astrocytes and oligodendrocytes during early CNS development. We have been and will continue to follow developmental fates and migration of progenitors from germinative zones of the postnatal rat CNS in vivo using replication deficient retroviral vectors and to chart the lineage and migration of immature cells and their differentiation into glia. We propose a series of integrated approaches to begin to answer the following questions. 1. What are the early stages of glial migration and differentiation? We will study early events in antigen acquisition and morphological changes during migration of forebrain subventricular zone (SVZ) cells into the cortex. We suggest that contact with blood vessels or pia is an early event in astrocyte development. We are developing an in vitro system in which SVZ cells aggregate in clusters, and migrate from clusters along glial """"""""cables"""""""". We will also visualize the migration and differentiation of immature cells in brain slices to study modes and directions of migration by infecting progenitors with a retrovirus encoding an endogenously fluorescent protein (GFP), using a beta-gal substrate in living cells, or marking SVZ cells with fluorescent dyes. We will begin to ask what are the signals that determine cell migration and fate during gliogenesis, using """"""""cable"""""""" cultures to give migrating SVZ cells choices between cables and either blood vessels or neurons (axons) to ask if such exposure will activate astrocyte or oligodendrocyte differentiation, respectively. We will introduce genes that may play roles in progenitor proliferation, migration, and differentiation into SVZ cells in vivo using retroviruses to see if expression will alter migration or developmental fate. 2. What is the extent of clonal dispersion and phenotypic heterogeneity of the progeny of postnatal SVZ cells? We will use a library of 100 different retroviruses coupled with polymerase chain reaction to recover and identify the specific virus in each cell in a single brain. 3. What are the patterns of gliogenesis in the cerebellum? We will examine the origins and migratory paths of glial progenitors in the cerebellum by retroviral labeling in vivo and after labeling in living, long-term cerebellar slices. 4. What is the nature of immature, proliferating cells in the adult CNS subcortical white matter and residual SVZ? What is (are) their normal fates? Do they continue to migrate through the CNS? If they do not normally differentiate into mature cells, do they have the potential to develop into neurons or glia? We will inject retroviruses into the SVZ and white matter of adult rats and characterizing the labeled cells in vivo and in vitro.
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