Neuropsychiatric development disorders involving the neocortex have a molecular basis that is virtually unknown. The long-term goal of this work is to identify key molecules used in normal development that will serve as a foundation for understanding these disorders. This proposal uses a novel strategy based upon my ability to generate stable cell lines with neuronal phenotypes from neuroblasts that are destined for specific layers during cortical neurogenesis. Although the characterization of these lines is incomplete, the preliminary data indicate the feasibility of generating cell lines based on cortical layer and demonstrate the advantages that these lines offer for studying the genes that underlie neocortical development. A most basic developmental question about the formation of layers in the neocortex is whether the adult neurons composing a layer are genetically committed prior to positioning within that layer, or are initially plastic, molded by subsequent environmental interactions within the neocortex to form the layer. Towards answering this question, I will test the hypothesis that new cell lines, derived from neuroblasts destined for a cortical layer, express novel genes that specifically identify young neurons destined for the same layer in vivo. Neuronal lines will be generated with a novel procedure based on targeting neuronal progenitor cells with retroviruses, resulting in the cellular expression of oncogene combinations. Using this procedure I've produced over 35 stable cell lines from the murine neocortex. Preliminary data on two of these lines have shown that they are phenotypically stable by neurofilament and neuron specific enolase expression and are clonal. A key advantage of a cell line is that mRNAs, that are easily isolated in large quantities, are a vastly less complex population compared with intact brain. This will allow the detection of even single-copy mRNAs and I will use this advantage to generate unbiased nucleotide probes against mRNAs selected by subtractive techniques; these probes have no inherent bias for molecular motifs that might identify a neuron in vivo. The complementary approach will bias probes for a class of molecules shown to be important for mammalian muscle and Drosophila nervous system differentiation, the helix-loop-helix transcription factors. Unbiased and biased probes will be screened on developing neocortical tissue by northern blots and in situ hybridization. Probes labelling discreet neocortical populations of neurons will be characterized by cDNA and genomic cloning of the genes followed by sequence analyses.
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