The mammalian nervous system is comprised of a vast repertoire of neuronal types. Its reasonable to assume that this repertoire is generated by a complex network of genetic control. However, to date very little is known about this network. We propose to analyze a very fundamental developmental choice made by neurons: whether to be excitatory or inhibitory. The decision to be inhibitory usually involves the selective expression of the genes for glutamic acid decarboxylase (GAD). We propose to analyze the mechanisms that lead to the expression of the GAD67 gene in the correct neurons of the mammalian brain. This should give insight into the larger problem of how the genome can guide the development of so many separate neuronal types. Preliminary data strongly suggests that there are several transcriptional start sites for the GAD67 gene. Segments of DNA surroundin the transcriptional start sites will be cloned and transfected into cell lines and used to create lines of transgenic mice. In this way we will determine the minimal unit of DNA necessary to give cell type specific expression of the GAD67 gene. Once this is determined, deletion analysis will be performed to locate regulatory sites in and adjacent to the GAD67 gene. Knowledge of the location and sequence of these sites will make it possible to systematically search for trans-acting factors that control the expression of the GAD gene. P19 mouse teratocarcinoma cells can be induced to differentiate into neuronal and glial like cells by treatment with retinoic acid. After induction levels of GAD67 RNA transcripts rise dramatically. However the functional properties of the cells which are expressing GAD are not known. The ability of these cells to make functiona GAD enzyme and GABAergic presynaptic terminals will be investigated. Since there are 2 genes coding for GAD it will be important to know the relative contribution of each one. As a step in that direction we will create mice in which both alleles of the GAD67 gene are interrupted. We will then determine the effect of eliminating the functional GAD67 gene on development. Taken together these studies will provide basic information o mechanisms that lead to the emergence of a major subset of brain neurons. The work will have relevance to human diseases that are caused by or involv disturbances of normal brain development and gene expression. Recently GAD has been shown to be a dominant autoantigen in juvenile diabetes and the stiff man syndrome. This work will provide additional basic data about thi important disease-related set of genes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS012867-19
Application #
2262486
Study Section
Neurology C Study Section (NEUC)
Project Start
1979-04-01
Project End
1996-07-31
Budget Start
1994-08-01
Budget End
1996-07-31
Support Year
19
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Washington University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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Bain, G; Ray, W J; Yao, M et al. (1996) Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture. Biochem Biophys Res Commun 223:691-4
Yao, M; Bain, G; Gottlieb, D I (1995) Neuronal differentiation of P19 embryonal carcinoma cells in defined media. J Neurosci Res 41:792-804
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Bain, G; Ray, W J; Yao, M et al. (1994) From embryonal carcinoma cells to neurons: the P19 pathway. Bioessays 16:343-8
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Turetsky, D M; Huettner, J E; Gottlieb, D I et al. (1993) Glutamate receptor-mediated currents and toxicity in embryonal carcinoma cells. J Neurobiol 24:1157-69
Morales, M J; Gottlieb, D I (1993) A polymerase chain reaction-based method for detection and quantification of reporter gene expression in transient transfection assays. Anal Biochem 210:188-94
Bain, G; Ramkumar, T P; Cheng, J M et al. (1993) Expression of the genes coding for glutamic acid decarboxylase in pluripotent cell lines. Brain Res Mol Brain Res 17:23-30
Ray, W J; Gottlieb, D I (1993) Expression of ionotropic glutamate receptor genes by P19 embryonal carcinoma cells. Biochem Biophys Res Commun 197:1475-82

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