It has been shown that the enzyme glutamic acid decarboxylase (GAD; EC 4.1.1.15), which catalyzes the conversion of L-glutamate to y-aminobutyric acid in the central nervous system of vertebrates, can be first detected in rodents at late embryonic stages. In contrast, we have found that the gene coding for the 67-kDa form of GAD is already transcriptionally active at embryonic day E10.5 in the mouse. In addition to the 3.5-kb adult-type mRNA, we have detected two 2-kb embryonic messages that contain alternatively spliced exons of 80 (1-80) and 86 (1-86) bp, respectively. The overlapping stop-start codon TGATG, found in the embryonic exons, converts the monocistronic adult-type transcript into a bicistronic one, coding for a 25-kDa leader peptide and a 44-kDa enzymatically active truncated GAD. A second stop codon at the 3' end of the 86-bp exon abolishes the expression of truncated GAD. The products of the two embryonic mRNAs were identified in a rabbit reticulocyte in vitro translation system, COS cells, and mouse embryos. The two GAD embryonic forms represent distinct functional domains and display characteristic developmental patterns, consistent with a possible role in the formation of the y-aminobutyric acid-ergic inhibitory synapses.

Glutamic acid decarboxylase (GAD) is the biosynthetic enzyme for gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates. In addition to the adult CNS, GABA and GAD also have been detected in embryos, although their precise localization and specific functions in embryonic development have not been elucidated. In this paper, the authors studied the cellular distribution of two GAD isoforms, GAD65 and GAD67, in midgestation mouse embryos by in situ hybridization histochemistry. With few exceptions, it was found that GAD65 and GAD67 mRNAs are localized in overlapping cellular domains of the embryonic CNS that later develop into regions with a strong GABAergic contribution. The GAD-expressing cells are situated in the differentiating zone of the embryonic day 10.5 (E10.5) through E11.5 CNS and in the subventricular zone and the mantle zone of the E12.5 CNS, which suggests that they are committed neuronal precursors. By using a specific serum for GABA, a similar pattern of distribution was obtained, indicating that GAD mRNAs are translated efficiently into enzymatically active GAD, which produces embryonic GABA. The expression domains of GAD overlap with those of genes that are known to be involved in the patterning of the embryonic CNS. The two GAD mRNAs also are detected outside of the embryonic CNS in various cell types, mainly those of placodal and neural crest origin. This pattern of expression is consistent with the notion that GAD and its product, GABA, play a signaling role during development.

In addition to being the major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) is thought to play a morphogenetic role in embryonic development. During the last decade, considerable progress has been made in elucidating the molecular mechanisms involved in GABA synthesis and biological action. The present review is an attempt to summarise recent results on the ontogeny of the different components of embryonic GABA signalling with an emphasis on the synthesis of GABA by different molecular forms of glutamic acid decarboxylase (GAD).

A series of overlapping clones coding for L-glutamic acid decarboxylase was purified from a mouse brain cDNA library, the longest of which contains a 1869 bp open reading frame and 913 bp of non-coding sequence. By comparison with the corresponding sequences from the mouse genome, it was determined that the first methionine in the longest cDNA represents the initiation codon. Expression of this cDNA in eukaryotic cells produces a 62 kd protein that is recognized by antiserum against rat GAD and which displays GAD activity commensurate with the amount of protein produced. Antibodies raised against the purified product of this cDNA recognize a 62 kd protein from mouse brain on immunoblots, specifically stain GABA-ergic neurons in brain sections, and are capable of immunoprecipitating most GAD activity from mouse brain extracts. These results provide the first definitive identification of a cDNA coding for the larger of two forms of GAD in mouse brain, and suggest that the two forms are closely related.