2004 West Coast Worm Meeting abstract 63
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
HHMI, Department of Anatomy, UCSF
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 AWCON) yielded neuronal symmetry (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 AWCOFF 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 AWCOFF 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.