Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain, and dysfunction of GABAergic neurons can have profound pathological implications. In C. elegans, based on promoter fusion analysis, 26 neurons are known to express conserved GABAergic terminal differentiation genes, such as the enzyme producing GABA (GAD/unc-25), the GABA-specific vesicular transporter (VGAT/unc-47) and the protein that targets VGAT to the synaptic membrane (a LAMP-like protein/unc-46). 25 of these worm GABAergic neurons are motoneurons and are involved in (i) head movements (4 RMEs), (ii) defecation (AVL and DVB), (iii) locomotion (19 D-type). Only one neuron, RIS, is an interneuron, like the dominant type of GABA neurons in vertebrates.Using fosmid recombinering technology, we studied the expression of
unc-47 and found that the VGAT is expressed strongly in 28 other neurons and weakly in at least 30 other, just in the head. The current identification of all the neurons where
unc-47 is expressed is under progress. In parallel, since the immunostaining may not be sensitive enough, we are generating an
unc-25 CRISPR line in order to determine if these cells expressed GABA or if
unc-47 is having another role not determined yet. In the course of improving the technique of anti-GABA immunostaining, we have found an additional neuron on the dorsal side of the larvae head. In parallel, we have drawn a more precise picture of how the differentiation of the 26 classic GABAergic neurons is controlled. As published it is known that UNC-30 is required for expression of GABA terminal differentiation genes in the D-type neurons. It is currently unknown what the terminal selectors for the other GABAergic neurons are. Through a candidate gene approach, we confirmed with anti-GABA immunostaining that LIM-6 -a LIM homeobox transcription factor- controls RIS, AVL and DVB fate. In addition, we generated a null allele of
nhr-67 -a Tailless/TLX ortholog- and also using anti-GABA immunostaining and marker analysis, we concluded that NHR-67 is involved in RIS, AVL and RMEs terminal differentiation, suggesting that NHR-67 and LIM-6 act together to control RIS and AVL GABAergic fate. We also confirmed that CEH-10 is involved in RME terminal differentiation. Moreover, after an EMS mutagenenesis screen, we found that TAB-1, a homeodomain transcription factor, is involved in RMEL/R GABAergic fate, suggesting that NHR-67, CEH-10 and TAB-1 work together to control RME fate.Together, these results will give us an updated picture of GABA neuron identification and developement.