Regulation of GABAergic Neuron Differentiation

描述（由申请人提供）：γ-氨基丁酸（GABA）是 哺乳动物中枢神经系统中主要的抑制性神经递质。 虽然γ-氨基丁酸能神经元对神经系统的正常发育至关重要， 和功能，对它们的形成和分化知之甚少。 拟议实验的长期目标是了解 GABA能分化的调控机制。拟议的工作将 剖析GABA能所需基因的协调调节， 表型。这些研究主要集中在胚胎表达和调控方面 哺乳动物谷氨酸脱羧酶基因Gad 1和Gad 2， 编码囊泡GABA转运蛋白（VGAT，也称为囊泡GABA转运蛋白）的蛋白质。 抑制性氨基酸转运蛋白或VIAAT）。Gad基因编码GABA 而VGAT/VIAAT基因编码转运蛋白， 将GABA包装成突触囊泡。它们的协同激活， 调节是正常GABA能分化和功能所必需的。的 实验分为三个具体目标。上 Gad 1、Gad 2和VGAT/VIAAT的表达模式正在被进一步研究。 通过小鼠胚胎中的原位杂交分析来定义。这很有启发性 这些基因在早期神经分化过程中的共表达。在 第二个目标是研究人员开发的一种新型细胞培养系统， 实验室用于测试特定转录因子的活性， 激活神经祖细胞中的Gad和VGAT/VIAAT基因。这 培养系统是基于小鼠胚胎干细胞的分化 含有Gad 1、Gad 2或VGAT/VIAAT基因的标记的“敲入”等位基因。的 所测试的因子在发育中的中脑中表达， 间脑和端脑，并参与GABA能发育。的 干细胞实验测量这些蛋白质对内源性 Gad和VGAT/VIAAT控制序列。在第三个目标中， 通过PI和转录因子开发的Gad 1-lacZ“敲入”小鼠品系 敲除小鼠用于测试这些蛋白质在Gad 1调节中的作用， vivo.在目标2中检查的相同转录调节因子正在进行测试， 目标3的基因杂交这种体外和体内结合的方法 允许细胞系统中特定因子的活性与 它在完整胚胎中的功能。拟议的实验将导致新的 GABA能神经元分化的协调调节 在胎儿发育期间。

DESCRIPTION (provided by applicant): Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. Although GABAergic neurons are essential for proper nervous system development and function, very little is known about their formation and differentiation. The long-term goal of the proposed experiments is to gain an understanding of the mechanisms regulating GABAergic differentiation. The proposed work will dissect the coordinate regulation of the genes required for the GABAergic phenotype. These studies are focused on the embryonic expression and regulation of the mammalian glutamate decarboxylase genes, Gad1 and Gad2, and the gene encoding the vesicular GABA transporter (VGAT, also known as the vesicular inhibitory amino acid transporter or VIAAT). The Gad genes encode the GABA biosynthetic enzymes while the VGAT/VIAAT gene encodes the transporter that packages GABA into synaptic vesicles. Their coordinate activation and regulation is necessary for normal GABAergic differentiation and function. The proposed experiments are organized into three Specific Aims. In the first Specific Aim, the expression patterns of Gad1, Gad2 and VGAT/VIAAT are being defined by in situ hybridization analysis in mouse embryos. This is revealing the co-expression of these genes during early neural differentiation. In the second Aim, a novel cell culture system developed in the investigators laboratory is used to test the activity of specific transcription factors in activating the Gad and VGAT/VIAAT genes in neural progenitor cells. This culture system is based on differentiation of mouse embryonic stem cells containing tagged "knockin" alleles of the Gad1, Gad2 or VGAT/VIAAT genes. The factors being tested are expressed in the developing mesencephalon, diencephalon and telencephalon and are involved in GABAergic development. The stem cell experiments measure the activity of these proteins on the endogenous Gad and VGAT/VIAAT control sequences. In the third Aim, genetic crosses between a Gad1-lacZ "knockin" mouse strain developed by the PI and transcription factor knockout mice are used to test the role of these proteins in Gad1 regulation in vivo. The same transcriptional regulators examined in Aim 2 are being tested in the genetic crosses of Aim 3. This combined in vitro and in vivo approach allows the activity of a particular factor in the cell system to be linked with its function in the intact embryo. The proposed experiments will lead to new insights into the co-ordinate regulation of GABAergic neuron differentiation during fetal development.