The defecation process in C.elegans is a rhythmic motor program that consists of three sequential muscle contractions: posterior muscle contraction (pBoc), anterior muscle contraction (aBoc) and enteric muscle contraction, which leads to the expulsion (Exp) of the gut contents. Previous studies have shown that the Exp step is regulated by a putative neuropeptide signaling pathway from the intestine that involves the activation of the GPCR
aex-2 expressed in a pair of GABAergic motor neurons(Mahoney, Luo et al. 2008). However, the neuropeptide has not been identified. Here we report that
nlp-40, which encodes a conserved neuropeptide-like protein in nematodes, is the missing neuropeptide signal from the intestine that controls the Exp step. The
vj3 allele of
nlp-40, identified in our lab, and
tm4085 allele from the knockout consortium are both putative null alleles, since both of them delete sequences in the putative mature NLP-40 peptide (Husson, Clynen et al. 2005).
nlp-40 mutants lack about 96% of Exp steps, although neither the cycle length nor the pBoc step of
nlp-40 mutants is affected. We found that
nlp-40 is exclusively expressed in all cells of the intestine. The Exp defects can be fully rescued by transgenes expressing NLP-40::GFP fusion protein under the endogenous
nlp-40 promoter or a heterologous intestine-specific promoter. The NLP-40::GFP fusion protein localizes to discrete puncta on the basolateral surface of the intestine. In addition, the fluorescence is also detected in coelomocytes. Together, these data indicate that NLP-40 is stored in dense core vesicles and is released from the intestine. Several results suggest that NLP-40 may be the ligand of AEX-2. First, both
nlp-40 and
aex-2 null mutants reduce expulsion frequency by about 96%. Second, both
aex-2 and
nlp-40 mutants are recessive, but double heterozygotes have a~50% reduction in Exp frequency. Finally, ectopic expression of
nlp-40 can partially rescue the Exp defects of
nlp-40 mutants but not those of
nlp-40;
aex-2 double mutants. The NLP-40 pre-propeptide is predicted to be processed into at least three short peptides. We have identified a region of NLP-40 containing one of these peptides that is sufficient to rescue the Exp defects of
nlp-40 mutants. We are directly testing whether this peptide is the ligand of AEX-2 in vitro by testing whether a synthetic peptide can activate AEX-2 in HEK cells. We are also examining how NLP-40 release from the intestine is regulated and how its release controls the execution of Exp. Together, these results suggest that a neuropeptide signaling pathway initiated by the intestinal release of NLP-40 and activating AEX-2 regulates the execution of the rhythmic defecation motor program.