unc-30 controls the terminal differentiation of the 19 type D GABAergic motor neurons in the ventral cord. Animals lacking type D neuron function are shrinkers, due to lack of cross-inhibition to the body wall muscles. In
unc-30 mutant animals, these neurons display multiple defects in axonal guidance and synaptic specificity (J. White, personal communication), and lack the neurotransmitter GABA1. Previously we showed that
unc-30 encodes a homeodomain transcription factor of a new class. The function of the UNC-30 protein is both necessary and sufficient for invoking D neuron specific characteristics such as GABA expression 2. To understand how
unc-30 controls these multiple aspects of D type GABAergic neuron differentiation, we have focused on identifying genes regulated by
unc-30.
unc-25, mutations in which confer a shrinker phenotype, is one such candidate target gene. In
unc-25 mutant animals all GABAergic neurons lack the neurotransmitter GABA 1. Previously we reported that
unc-25 encodes glutamic acid decarboxylase, the enzyme essential for producing GABA.
unc-25 is expressed in all GABAergic neurons at all larval and adult stages.
unc-25 expression in the type D neurons is abolished in
unc-30 mutant animals whereas its expression in other GABAergic neurons is unaffected. We have identified a promoter element of
unc-25 that is necessary for the regulation by
unc-30. This promoter contains two predicted high affinity DNA binding sites for homeodomain proteins. Mutating these two sites abolishes the expression of
unc-25 in the type D neurons, indicating that these sites are functionally important. We have further shown that in gel-mobility shift assays UNC-30 protein binds to the wild type
unc-25 promoter, but not the mutated form. We conclude that
unc-25 is most likely a direct target gene of
unc-30. To identify other genes that are controlled by
unc-30 and that may function in other aspects of the type D neuron differentiation, we have undertaken two approaches. One is to isolate dominant enhancers of a weak
unc-30 mutation. Several dominant enhancers have been isolated and are being characterized. The other is to use a cDNA representational difference analysis procedure 3 to identify transcripts whose expression depends on
unc-30. We have isolated at least three transcripts whose expressions are either reduced or abolished in
unc-30 mutant animals and none of which is
unc-25. We are also analyzing an intriguing phenomenon exhibited by a subset of the type D neurons. The 19 D neurons comprise 6 embryonically generated DD neurons and 13 postembryonically derived VD neurons. The six DD neurons reverse their synaptic connectivity as worms develop from L1 to older larvae 4. In collaboration with M. Nonet, we have used a VAMP-GFP marker that is specifically expressed in the type D neurons to label the presynaptic regions of these neurons, thus enabling us to observe directly the synaptic switch of the DDs in living worms. We will present preliminary analysis of the temporal control of this switch (Hallam and Jin, this meeting) and mutations that we isolated from a pilot screen for genes affecting this synaptic switch. 1. McIntire, S.L., Jorgensen, E. & Horvitz, H.R. Nature 364, 334-337 (1993). 2. Jin, Y., Hoskins, R. & Horvitz, H.R. Nature 372, 780-783 (1994). 3. Hubank, M. & Schatz, D.G. Nuc. Acids Res. 22, (1994). 4. White, J.G., Albertson, D.G. & Anness, M.A.R. Nature 271, 764-766 (1978).