Molecular characterization of Drosophila melanogaster RFamide peptides.

Abstract

Although Drosophila melanogaster has served as a versatile experimental organism for many areas of research, little is known about the action of Drosophila neuropeptides due to the lack of peptide structure data, absence of whole organism assays, and the ambiguity of expression data. A peptide family of interest is the RFamide family due its participation in many physiological parameters and its presence in numerous invertebrates and vertebrates. Six RFamide peptides have been isolated from Drosophila (Nichols 1992a, b). In order to elucidate the physiological roles of these peptides, I determined the expression, processing, and activity(s) of Drosophila RFamide peptides. The peptides that I studied included DPKQDFMRFamide, SDNFMRFamide, and TPAEDFMRFamide, three peptides encoded in the Drosophila FMRFamide gene, and TDVDHVFLRFamide (DMS), encoded by the Drosophila myosuppressin (Dms) gene. To identify the peptide distribution, I raised peptide-specific antisera and determined expression in both neural and gastrointestinal tissue at all stages of development. Three antisera having different specificities recognizing DPKQDFMRFamide, SDNFMRFamide, or TPAEDFMRFamide were used to establish that the Drosophila FMRFamide polypeptide precursor is differentially posttranslationally processed to yield three unique peptide expression patterns. Based on the cellular expression patterns, it was possible to correlate the presence of tissue expression with activity, e.g., SDNFMRFamide is present in Drosophila heart tissue and affects heart beat, while the other two FMRFamide peptides are not expressed in this tissue and do not appear to affect heart beat. In addition, my work demonstrates that DMS is expressed in the heart, central nervous system, gastrointestinal tract, and reproductive tissue throughout development. These expression data are consistent with DMS activity. We have shown that Drosophila myosuppressin inhibits heart rate and, based on structure homology, it may inhibit gut motility and oviduct motility. In order to further study the Drosophila myosuppressin gene and peptide, I isolated and sequenced the Dms gene and established an in vitro heart rate assay. I also determined the effect of DMS on heart rate in ion channel mutants. Taken together, my studies demonstrate that the expression of Drosophila RFamides is highly regulated on a cell-specific level and that while they share a high degree of structure identity, the peptides have distinct activities. I also provide data that suggest RFamide peptides are important to the animal being present in both neural and gastrointestinal tissue throughout development.