Our lab is interested in the differentiation of the type D neurons, a set of 19 GABAergic motorneurons in the ventral cord of the worm. These neurons provide cross-inhibitory stimuli to body wall muscles and thus control coordinated sinusoidal locomotion. Laser ablation of the D neurons results in a "shrinker" phenotype in which affected worms hypercontract upon a gentle touch. Mutations in
unc-30, which encodes a homeodomain (HD) protein of the bicoid class and is expressed in the nuclei of the D neurons, cause a shrinker phenotype. The D neurons of
unc-30 mutant animals are defective in neurotransmitter production, axonal pathfinding, and synaptic partner choice (J.White, pers. comm.).
unc-30 may therefore control transcription of genes required for several aspects of D neuron function and development. We have identified two candidate transcriptional targets of
unc-30, both of which cause shrinker phenotypes when mutated.
unc-25 encodes the synthetic enzyme for GABA.
unc-47 encodes the vesicular transporter for GABA. Full-length
unc-25 and
unc-47 promoters drive GFP expression in all 26 GABAergic neurons in wild-type worms. We showed that expression of
unc-25-GFP and
unc-47-GFP constructs in the D neurons is abolished in
unc-30 null mutant animals but maintained in the other GABAergic neurons. We identified minimal
unc-25 and
unc-47 promoters required for expression in the D neurons. Sequence inspection revealed that these
unc-25 and
unc-47 minimal promoters both contain core consensus HD binding sequences recognized by bicoid-class HD proteins. We showed by site-directed mutagenesis that the core binding sites are absolutely required for expression in the D neurons in vivo. Moreover, in gel-shift assays the UNC-30 homeodomain binds to the minimal
unc-25 and
unc-47 promoters in a sequence-specific manner. Finally, footprint analysis of the minimal
unc-47 promoter shows that the UNC-30 homeodomain binds exclusively to the core HD site. Our results thus provide an example of coordinated control of two members of a neurotransmitter metabolic pathway by a single transcription factor.