Developmental pathways in the nervous system

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DESCRIPTION (provided by applicant): The development of the mammalian nervous system requires the generation of a large number of different types of neurons, characterized by the specific expression of neurotransmitters and their receptors and by the formation of highly specific axonal connections. These developmental processes are regulated in part by transcription factors that bind to specific DNA sequences and activate or repress the expression of neural genes. We are currently studying Brn3a, a member of the POU subclass of homeodomain transcription factors. The expression pattern of Brn3a indicates a role in the development of specific neurons in the CNS and sensory system. Mice lacking Brn3a exhibit marked defects in sensory axon growth, undergo extensive neuronal death in the sensory ganglia in late gestation, and die at birth. The mechanisms by which these transcription factors regulate neuronal phenotype and survival, and the specific downstream "target genes" they control, are not well understood. In prior work, we characterized the DNA recognition properties of Brn3a, and developed a rapid screening method for locating functional Brn3a binding sites in large regions of genomic DNA, called "Complex Stability Screening." In recent studies we have used this method to identify autoregulatory sites within an enhancer region that regulates Brn3a expression in sensory neurons. We then demonstrated the in vivo role of these sites in Brn3a mutant mice. These studies, combined with the application of gene expression arrays and a wealth of information recently available from the mouse and human genome projects, provide the basis for a general approach to identifying the downstream targets of Brn3a. Specific Aims: 1) Use gene expression arrays ("genechips") to compare the patterns of gene expression in the embryonic sensory ganglia and midbrain of Brn3a mutant and wild-type mice. 2) Use the comparison of mouse and human genomic sequences and Complex Stability Screening of the genomic loci of potential regulatory targets to establish a direct transcriptional relationship with Brn3a. 3) Test the activity of transgenic reporters for selected Brn3a target genes in Brn3a mutant and wild type mice. 4) Test whether Bm3a functions generally as a positive or negative regulator of transcription by the transgenic expression of dominant positive and negative forms of this factor in sensory neurons, and over express selected Brn3a targets in transgenic mice to determine their developmental effects.