Worms defecate by executing three sets of muscle contractions every 45-50 seconds when they are feeding. A calcium spike in the posterior intestine immediately precedes and probably initiates the first of these muscle contractions (1), called the posterior body-wall muscle contraction (pBoc). Approximately 3 seconds after the pBoc, an anterior body-wall muscle contraction (aBoc) pushes the posterior pharynx backward into the anterior intestine. Immediately following the aBoc, a contraction of the enteric muscles in the region of the anus expels intestinal contents (Exp). We have been working to characterize
aex-2 , mutations in which cause defects in both the aBoc and Exp (Aex) steps of the defecation motor program. We have cloned
aex-2 and shown that it encodes a 7-transmembrane domain protein with homology to the G protein-coupled receptor family. However, the
aex-2 sequence does not fall into any specific small molecule receptor subfamily, suggesting that it mediates a novel signaling pathway in C. elegans . This signaling pathway may be peptidergic, since mutations in two enzymes known to process small signaling peptides also cause an Aex phenotype.
aex-5 encodes a proprotein convertase, which cleave proproteins into small signaling peptides (2), and
egl-21 encodes a carboxypeptidase E (3), which process peptides once they have been cleaved.
aex-2 may encode a receptor for one or more peptides processed by these two gene products. An
aex-2::gfp fusion expresses GFP in the enteric muscles, NSM, AWB, and several other cells in the head of the worm, including possibly AVL, a neuron responsible for stimulating the aBoc. AEX-2 may be receiving a signal in AVL that in turn activates the anterior body-wall muscles to execute the aBoc. AEX-2 may also be receiving a signal in the enteric muscles, inducing them to execute the expulsion. An unknown signal or signals from the intestine initiates the process of defecation, but how that signal is transmitted to activate the three motor steps is unknown. In an effort to identify other proteins functioning in this pathway, we are screening for suppressors of the
aex-2 expulsion defect. We have screened over 10,000 haploid genomes and have identified more than ten alleles that suppress
aex-2(
sa3) , a hypomorphic allele that has a missense mutation in the sixth transmembrane domain. While no mutation confers complete suppression, many have interesting phenotypes, including three mutants of a novel class that occasionally execute more than one expulsion per cycle. None of these mutations suppresses the aBoc phenoytype of
aex-2 , suggesting that while the aBoc and Exp phenotypes are caused by one mutation, they are mechanistically separable. We are currently mapping and further characterizing these new alleles. 1. Dal Santo, P, et al., 1999. Cell 98: 757-67. 2. Thacker, C. and A.M. Rose, 2000. Bioessays 22: 1-9. 3. Carey, T. and J.M. Kaplan West Coast Worm Meeting Abstract, 2000.