Regulation of GABAergic Neuron Differentiation

描述(申请人提供)：γ-氨基丁酸(GABA)是 哺乳动物中枢神经系统中的主要抑制性神经递质。 尽管GABA能神经元对神经系统的正常发育是必不可少的 和功能，人们对它们的形成和分化知之甚少。 拟议中的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的表达和调控上。 哺乳动物谷氨酸脱羧酶基因Gad1和Gad2，以及该基因 编码囊泡型GABA转运蛋白(VGAT，也称为囊泡型 抑制性氨基酸转运体或VIAAT)。Gad基因编码GABA 生物合成酶，而VGAT/VIAAT基因编码转运蛋白 将GABA包装成突触小泡。它们的配位激活和 调节是正常的GABA能分化和功能所必需的。这个 拟议的实验被组织成三个具体目标。在第一个 针对Gad1、Gad2和VGAT/VIAAT的表达模式 通过小鼠胚胎的原位杂交分析来定义。这是发人深省的 这些基因在早期神经分化过程中的共表达。在 第二个目标，研究人员开发了一种新的细胞培养系统 实验室被用来检测特定转录因子的活性 激活神经前体细胞中的Gad和VGAT/VIAAT基因。这 小鼠胚胎干细胞分化培养体系的建立 包含标记的Gad1、Gad2或VGAT/VIAAT基因的“敲门”等位基因。这个 被测试的因子在发育中的中脑中表达， 间脑和端脑，并参与GABA能的发育。这个 干细胞实验测量了这些蛋白质对内源性 GAD和VGAT/VIAAT控制序列。在第三个目标中，基因杂交在 用PI和转录因子培育的Gad1-LacZ“敲门”小鼠品系 基因敲除小鼠被用来测试这些蛋白在Gad1调控中的作用 活着。在AIM 2中检测到的相同转录调控因子正在进行测试 Aim的遗传杂交3.体外和体内相结合的方法 允许细胞系统中的特定因子的活动与 它在完整胚胎中的功能。拟议的实验将导致新的 GABA能神经元分化的协同调控研究 在胎儿发育过程中。

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.