The function of a nervous system depends upon individual neurons and the synapses they make with each other. The molecules that define the wiring diagram are not known, however. Mutations in the
unc-4 gene disrupt movement and alter the pattern of synaptic input to one class of motor neurons in the C. elegans ventral nerve cord.
unc-4 mutant worms are unable to crawl backwards because the usual synapses to VA motor neurons are replaced with connections normally reserved for VB motor neurons (John White, pers. comm.). Our findings indicate that
unc-4 encodes a homeobox-containing transcription factor. Tcl insertions from the
unc-4 region were cloned and placed on the physical map. A partial homeobox motif,
ceh-4, was independently discovered in the nearby cosmid C07E2 (Burglin et al. Nature 341, 239243, 1989). We probed Southerns with C07E2 and identified a 1.8 kb Eco R1 fragment that is either altered or deleted in five different
unc-4 alleles. The 1.8 kb sequence includes two ORFs (198 bp & 110 bp) which together encode the first 46 amino acids of an apparent homeobox. The sequence of a PCR-derived cDNA clone shows that these presumptive exons are spliced with
ceh-4 which is located 0.8 kb away and encodes the carboxy terminal 14 amino acids of the homeodomain. At this point, we have identified over 600 bp of coding sequence in five exons. The
unc4 homeodomain is most similar (~55% identity) to the Drosophila prd family of homeobox sequences. We have not found significant homologies to coding regions outside the homeodomain. On northern blots, the 1.8 kb Eco R1 fragment hybridizes to a single, low abundance 1.2 kb poly A+ RNA that is absent from
unc-4 null alleles wd-1 and
e2308. Primer extension-PCR experiments indicate that
unc-4 RNA is most abundant in the embryo, L1 and early L2 stages. Temperature shifts with
unc-4(
e2322ts) (from M. Shen) have shown that the temperature sensitive period (tsp) begins in the late L1 and extends into the L3. The tsp is correlated with the birth of VA neuroblasts and the period in which VAs are wired into the ventral cord circuit (late L1). Notably,
unc-4 mutant embryos are not temperature sensitive. We conclude that ventral cord wiring is dependent upon
unc-4 protein function in early larvae but not in the embryo.
unc-4 may have a separate role in embryonic development that has not yet been detected. We would propose that in larval development the
unc-4 gene is selectively expressed in the VA motor neurons and therein controls transcription of one or more downstream genes which both define and sustain the VA pattern of synaptic input. We have raised antibodies to an
unc-4 fusion protein in order to test this hypothesis.