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.
