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Motor neuron function depends on neurotransmitter release from synaptic vesicles (SVs). Here we show that the UNC-4 homeoprotein and its transcriptional corepressor protein UNC-37 regulate SV protein levels in specific Caenorhabditis elegans motor neurons. UNC-4 is expressed in four classes (DA, VA, VC, and SAB) of cholinergic motor neurons. Antibody staining reveals that five different vesicular proteins (UNC-17, choline acetyltransferase, Synaptotagmin, Synaptobrevin, and RAB-3) are substantially reduced in unc-4 and unc-37 mutants in these cells; nonvesicular neuronal proteins (Syntaxin, UNC-18, and UNC-11) are not affected, however. Ultrastructural analysis of VA motor neurons in the mutant unc-4(e120) confirms that SV number in the presynaptic zone is reduced ( approximately 40%) whereas axonal diameter and synaptic morphology are not visibly altered. Because the UNC-4-UNC-37 complex has been shown to mediate transcriptional repression, we propose that these effects are performed via an intermediate gene. Our results are consistent with a model in which this unc-4 target gene ("gene-x") functions at a post-transcriptional level as a negative regulator of SV biogenesis or stability. Experiments with a temperature-sensitive unc-4 mutant show that the adult level of SV proteins strictly depends on unc-4 function during a critical period of motor neuron differentiation. unc-4 activity during this sensitive larval stage is also required for the creation of proper synaptic inputs to VA motor neurons. The temporal correlation of these events may mean that a common unc-4-dependent mechanism controls both the specificity of synaptic inputs as well as the strength of synaptic outputs for these motor neurons.

The Caenorhabditis elegans UNC-13 protein and its mammalian homologues are important for normal neurotransmitter release. We have identified a set of transcripts from the unc-13 locus in C. elegans resulting from alternative splicing and apparent alternative promoters. These transcripts encode proteins that are identical in their C-terminal regions but that vary in their N-terminal regions. The most abundant protein form is localized to most or all synapses. We have analyzed the sequence alterations, immunostaining patterns, and behavioral phenotypes of 31 independent unc-13 alleles. Many of these mutations are transcript-specific; their phenotypes suggest that the different UNC-13 forms have different cellular functions. We have also isolated a deletion allele that is predicted to disrupt all UNC-13 protein products; animals homozygous for this null allele are able to complete embryogenesis and hatch, but they die as paralyzed first-stage larvae. Transgenic expression of the entire gene rescues the behavior of mutants fully; transgenic overexpression of one of the transcripts can partially compensate for the genetic loss of another. This finding suggests some degree of functional overlap of the different protein products.

Mutations in the unc-17 gene of the nematode Caenorhabditis elegans produce deficits in neuromuscular function. This gene was cloned and complementary DNAs were sequenced. On the basis of sequence similarity to mammalian vesicular transporters of biogenic amines and of localization to synaptic vesicles of cholinergic neurons in C. elegans, unc-17 likely encodes the vesicular transporter of acetylcholine. Mutations that eliminated all unc-17 gene function were lethal, suggesting that the acetylcholine transporter is essential. Molecular analysis of unc-17 mutations will allow the correlation of specific parts of the gene (and the protein) with observed functional defects. The mutants will also be useful for the isolation of extragenic suppressors, which could identify genes encoding proteins that interact with UNC-17.