The C. elegans AWC olfactory neurons are a bilaterally symmetric pair of similar neurons. However, the promoter of the candidate odorant receptor
str-2 drives asymmetric GFP expression in only one of the two AWC neurons. Cell-cell communication specifies asymmetric AWC cell fates through a novel pathway that includes a voltage-gated calcium channel (
unc-2,
unc-36) and CaMKII (
unc-43), which in turn activate a MAP kinase cascade initiated by NSY-1, the C. elegans ortholog of the mammalian
p38/jnk-regulatory MAPKKK ASK1. A screen for mutant worms that express
str-2 ::GFP in both AWC cells (2 AWC ON ) yielded n euronal sy mmetry ( nsy ) genes that map to novel loci.
nsy-2 is defined by a single allele,
ky388. Genetic epistasis analysis suggests that
nsy-2 acts downstream of the CaMKII
unc-43 and upstream of the MAPKKK
nsy-1 . Temperature-shift assays with the temperature-sensitive
nsy-2(
ky388 ) allele suggest that the onset of the requirement for
nsy-2 activity in
str-2 asymmetry occurs in late embryogenesis, a few hours after the initiation of axon outgrowth. This result is consistent with a role for
nsy-2 in the initial communication between the AWC neurons.
nsy-2 was cloned by rescue with one cosmid, which contains only one full-length open reading frame. This open reading frame has also been called
tir-1, with five predicted splice forms. Our sequence analysis of cDNA clones corresponding to the gene revealed three novel splice forms with alternative 5' ends. In the
nsy-2 (
ky388) allele, a C->T mutation, which results in a premature stop codon, was found in an early exon of one splice form. We refer to the specific
tir-1 splice form that is affected by
ky388 as
nsy-2 . Expression of NSY-2 in AWC under a specific promoter rescues the
str-2 expression defect in
nsy-2 mutant, suggesting that
nsy-2 acts in AWC to affect its cell fate. Overexpression of NSY-2 leads to a 2 AWC OFF phenotype opposite to the loss of function phenotype. Mosaic analysis with overexpression of NSY-2 indicates that NSY-2 acts cell autonomously to execute the AWC OFF cell fate.
nsy-2 encodes a cytoplasmic protein with a combination of Heat-Armadillo repeats, two SAM domains, and one TIR (Toll-interleukin-1 receptor) domain. A single NSY-2-like protein is present in C. elegans , Drosophila , mouse, and human. The mammalian ortholog is known as SARM, and its function is unknown. We hypothesize that NSY-2 acts as a scaffold between UNC-43 and the MAPK cascade. NSY-2, UNC-43, and NSY-1 could be co-immunoprecipitated when coexpressed in HEK 293 cells and COS cells, suggesting that NSY-2 physically associates with UNC-43 and NSY-1. NSY-2 displays a punctate pattern along the AWC axon that co-localizes with NSY-1 and a post-synaptic marker. We suggest that AWC lateral signaling is executed by NSY-2 activation of NSY-1/ASK1 at AWC synapses.