Despite the abundance and diversity of peptide neurotransmitters, their role in neuronal function and development is not well understood. We are particularly interested in the role of neuropeptides in the ASH sensory circuit. Based on laser ablations experiments, the ASH sensory neurons are primarily responsible for detecting nose touch, high osmolarity and volatile repellents. Nose touch elicts a different type of synaptic output than other stimuli detected by ASH. Specifically, neuropeptide neurotransmitters may be released in response to high osmolarity or volatile repellents (CGC 2309). To date, the only neuropeptides characterized in C. elegans are the FMRFamides (FaRPs) encoded by
flp-1 and related genes (C. Li, et. al. WBG 11(2):43). However, FaRP immunoreactivity is not detected in ASH (CGC 1500) suggesting that novel neuropeptides are utilized in this circuit. We have searched public databases for additional C. elegans neuropeptides (based on the nearly complete genomic sequence). Our strategy is based on the structure of previously charactetized neuropeptide preproproteins. They contain a signal peptide and interrupted multiple repeats of related neuropeptides. The active neuropeptides are released from the proprotein by endoproteolytic cleavage at dibasic (or occasionally monobasic) amino acids. During the last year, we have identified 31 putative neuropeptide genes, 17 of which encode FaRPs. However, the other 14 genes encode putative neuropeptides which can be classified as either similar to previously characterized neuropeptides (Nathoo and Hart, WBG 15(1):43) or completely novel. The first step in our characterization of these novel neuropeptide genes is analysis of the cellular expression pattern using GFP reporter constructs. To date, we have analyzed genes distantly related to Aplysia myomodulin (T24D8.3-5) and to Aplysia buccalin (C01C4.1). In adults expressing the C01C4.1::GFP construct, GFP is detected in neurons including ASI, ASE, AWC, PHB, and in the posterior and anterior intestinal cells. For the T24D8.3-5::GFP construct, the expression pattern is more complex; it includes many neurons and secretory cells near the vulva. No GFP expression is detected in ASH with either construct. We are now generating similar GFP reporter constructs for the other 12 putative neuropeptide genes. Our long range goals include: identifying the ASH neuropeptide(s), characterizing these and other novel neuropeptides, determining mutant phenotypes for these genes, and assessing the role of these putative neuropeptides in function and development.