This proposal requests the continuation of R01-HD33442, part of the applicant's ongoing research program focused on how the diverse characteristics of vertebrate neurons are generated and maintained throughout life. Neuronal differentiation is controlled in part by the coordinated expression of DNA binding transcription factors in very specific times and places during neural development. The focus of the current study is the POU-domain factor Brn3a (Brn3.0, Pou4f1), expressed in primary sensory neurons at all axial levels and in selected neurons of the retina, diencephalon, brainstem and spinal cord. Targeted disruption of Brn3a in mice results in loss of neurons in the sensory ganglia and some CNS nuclei, followed by neonatal death. In the current award period we have identified a sensory-specific enhancer region of the Brn3a gene, and used reporter genes targeted by this enhancer to show marked defects in sensory axon growth in Brn3a knockout mice. We have also demonstrated that Brn3a acts as a negative regulator of its own expression, and that partial relief of this negative regulation in heterozygotes leads to nearly complete compensation for the defective allele. In further studies, we have used microarrays to perform a global assay of changes in gene expression in the sensory ganglia of Brn3a null mice, revealing the coordinated regulation of neurotransmitter systems, axonal components, and transcription factors by Brn3a.
The Specific Aims of this proposal are: 1) Use transgenic mice expressing the axonal tracer tauLacZ from the Brn3a locus to examine the CNS projections of Brn3a-expressing retinal ganglion cells, and determine whether the targeting of these axons to specific thalamic structures is dependent on early spontaneous depolarizations in the retina. 2) Use Brn3a/tauLacZ mice and microarray analysis of embryonic tissue in wild-type and mutant animals to determine the role of Brn3a in development and gene regulation in the habenula. 3) Use targeted insertion of a reporter gene into BAG clones derived from the mouse genome project to identify enhancer sequences directing Brn3a expression to the CNS. 4) Use locus-wide, real-time PCR-based chromatin immunoprecipitation assays, as well as database analysis and conventional protein-DNA interaction methodology, to define the regulatory inputs that direct Brn3a expression to primary sensory neurons throughout the neural axis. Together these aims will make a substantial contribution to understanding the basic mechanisms of neuronal development and advance our knowledge of the many neurological and behavioral illnesses which have a genetic or developmental component.
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