Gonadotropin-releasing hormone mediates central nervous system control of the endocrine hierarchy which controls reproductive function. The complex pathways which govern transcription of this important gene in the brain remain to be elucidated. We will focus on regulation of GnRH gene expression by neuromodulators and on the mechanisms which determine the expression of individual releasing hormone genes in tightly specified patterns to exclusive populations of neurons in the hypothalamus. We have isolated immortal cell lines of the GnRH neurons by targeting oncogene expression in transgenic mice. These cell lines provide a unique opportunity to study regulation of an idual CNS neuroendocrine hormone gene in an isolated clonal population of renewable neurons under controlled culture conditions. Our first goal is to construct a work in which to understand the control of GNRH by an integrated set of neuromodulatory influences. We plan a systematic analysis of the responses to known modulatory agents in the GnRH neuronal cen line, GT1. Our second goal is to unravel the genetic control mechanisms which specify GnRH expression to an exclusive population of neurons in the hypothalamus. Using transfection of the GnRH gene into GT1 cells in culture, we have localized a neuron-specific enhancer upstream of the GnRH gene. We plan to delineate the individual elements important for neuronal expression, to isolate the specific DNA-binding proteins involved, and to investigate their mechanisms of action. Our Preliminary Studies demonstrate the feasibility of using targeted oncogenesis to transform specific dffferentiated neurons which can be established in culture with maintenance of highly differentiated phenotypes and set the stage for the third major direction proposed, to immortalize different unique neurons of the brain. We plan to target gene expression to corticotropin-releasing hormone neurons, and ultimately to other distinct populations of neurons in the CNS, with the ultimate goal of establishing immortal cell lines of several individual neuronal cell lineages in the brain.
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