The goal of Project 5 is to analyze the molecular mechanisms which result in cell type and developmental stage specific expression of novel genes that are tightly regulated during cerebellar granule cell differentiation, with particular emphasis on those genes which require specific cell-cell interactions as signals for normal expression. Toward this goal, we will (1) Complete the characterization of a collection of cerebellar cDNAs which we have recently cloned from a stage specific granule cell cDNA library, screening for developmental stage and tissue specific expression. (2) Define the temporal and spatial patterns of expression of regulated clones by in situ hybridization of tissue from normal and neurologic mutant mice (including Lurcher (Lc), weaver (wv), and nervous (nr)) and (3) Isolate both full-length cDNAs and genomic clones of select tightly regulated novel cDNAs as tools for further analysis. To examine the transcriptional regulation of granule cell specific genes, we will carry out transient expression assays in cerebellar slice preparations and analyze critical constructs in transgenic mice. The transcription factors which interact with granule cell specific genes will be examined with particular emphasis on signal transduction pathways mediated by specific cell-cell interactions. Finally, we will examine the functions of selected gene products by (1) in-depth computer analysis of cDNA sequences, (2) immunocytochemical localization of polyclonal antisera against expressed gene products, (3) functional perturbation studies in vitro (see Project 4), and (4) molecular genetic perturbation studies in vivo.
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