I. Lim

Cellular expression of the Drosophila melanogaster FMRFamide neuropeptide gene product DPKQDFMRFamide. Evidence for differential processing of the FMRFamide polypeptide precursor.

DPKQDFMRFamide is one of five different FMRFamide-containing peptides encoded in the Drosophila FMRFamide gene. To study the cellular expression of DPKQDFMRFamide, we have generated antisera to DPKQD, the N-terminal sequence of the peptide, to avoid crossreactivity with other -FMRFamide-containing peptides. The antisera were purified and the specificity characterized. DPKQDFMRFamide immunoreactive material is first observed in the embryonic central nervous system (CNS) in one cell of the subesophageal ganglion and one cell in each of the three thoracic ganglia. This pattern of expression is observed in larval, pupal, and adult neural tissue, albeit with increased signal intensity. In larva, pupa, and adult, additional cells in the superior protocerebrum, a thoracic ganglion, and an abdominal ganglion express DPKQDFMRFamide immunoreactive material. Immunoreactivity is observed in a cell in the lateral protocerebrum of pupa and adult and cells in the optic lobe of adult. No immunoreactive material was observed in gut tissue. DPKQDFMRFamide antisera stain a subset of cells previously identified by in situ hybridization and immunocytochemistry to express the FMRFamide transcript and polypeptide precursor. These data suggest that the Drosophila FMRFamide polypeptide precursor undergoes differential processing to produce DPKQDFMRFamide immunoreactive material in a limited number of cells expressing the FMRFamide precursor.DPKQDFMRFamide is one of five different FMRFamide-containing peptides encoded in theDrosophila FMRFamide gene. To study the cellular expression of DPKQDFMRFamide, we have generated antisera to DPKQD, the N-terminal sequence of the peptide, to avoid crossreactivity with other-FMRFamide-containing peptides. The antisera were purified and the specificity characterized.DPKQDFMRFamide immunoreactive material is first observed in the embryonic central nervous system (CNS) in one cell of the subesophageal ganglion and one cell in each of the three thoracic ganglia. This pattern of expression is observed in larval, pupal, and adult neural tissue, albeit with increased signal intensity. In larva, pupa, and adult, additional cells in the superior protocerebrum, a thoracic ganglion, and an abdominal ganglion express DPKQDFMRFamide immunoreactive material. Immunoreactivity is observed in a cell in the lateral protocerebrum of pupa and adult and cells in the optic lobe of adult. No immunoreactive material was observed in gut tissue.DPKQDFMRFamide antisera stain a subset of cells previously identified byin situ hybridization and immunocytochemistry to express the FMRFamide transcript and polypeptide precursor. These data suggest that theDrosophila FMRFamide polypeptide precursor undergoes differential processing to produce DPKQDFMRFamide immunoreactive material in a limited number of cells expressing the FMRFamide precursor.

Multiple Antigenic Peptides Designed to Structurally Related Drosophila Peptides

We have isolated TDVDHVFLRFamide (DMS), FDDYGHMRFamide (DSK), and DPKQDFMRFamide from Drosophila melanogaster. These peptides, structurally related by a common C-terminus -XRFamide, where X = L or M, are encoded by three different genes. To determine cellular expression, we have generated antisera to multiple antigenic peptides and performed double-label immunofluorescence using antisera raised in the same species host animal. Our results indicate that DMS and DSK immunoreactive materials have unique, non-overlapping expression patterns, while DMS and DPKQDFMRFamide immunoreactive materials colocalize in two superior protocerebrum neurons, and DSK and DPKQDFMRFamide immunoreactive materials colocalize in one superior protocerebrum neuron, one subesophageal ganglion neuron, and three thoracic ganglia neurons.

Spatial and temporal analysis of the Drosophila FMRFamide neuropeptide gene product SDNFMRFamide: Evidence for a restricted expression pattern

The expression of SDNFMRFamide, one of five different FMRFamide-containing peptides encoded by the Drosophila melanogaster FMRFamide gene, has been determined. To study expression, we generated antisera to the N-terminus of SDNFMRFamide to avoid crossreactivity with FMRFamide-containing peptides. The antisera were purified and the specificity characterized. SDNFMRFamide immunoreactive material is present in the central nervous system throughout development. Immunoreactivity is first observed in embryonic neural tissue in a cluster of cells in the subesophageal ganglion and immunoreactive fibers projecting from these cells to the brain and ventral ganglion. This pattern of expression is also observed in neural tissue dissected from larva, pupa, and adult. Double-labelling experiments indicate that cells recognized by SDNFM-antisera are also stained with FMRFamide antisera. Based on position, SDNFMRFamide immunoreactive material is expressed in a limited number of cells that contain the FMRFamide polypeptide precursor. This finding suggests that the Drosophila FMRFamide precursor undergoes differential post-translational processing.

Structure, Function, and Expression of Drosophila melanogaster FMRFamide-related Peptides

Abstract: In 1977, Price and Greenberg 1 identified the tetrapeptide FMRFamide as a cardioexcitatory molecule from mollusc. Subsequent to this discovery, FMRFamide‐related peptides (FaRPs) have been identified in both invertebrates and vertebrates. 2, 3 Peptides in the FaRP family contain a common RFamide C‐terminus and act as modulators and messengers of neural and gastrointestinal functions. 2, 3 Like other organisms, Drosophila melanogaster contains several genes 4–6 that encode for numerous FaRPs. 7, 8 Elucidating the processing and activities of multiple FaRPs encoded in a single precursor is critical to establishing their roles in physiology. In this manuscript, we describe the distribution of FMRFamide immunoreactive materials in the Drosophila central nervous system and gut, and correlate it with the expression of specific FaRPs and their activities. The unique distributions 9–13 and biological activities 14, 15 of Drosophila FaRPs suggest that the precursors are highly processed and the structurally related peptides are not functionally redundant. The complete distribution of FaRPs in the central nervous system and gut as detected by FMRFamide antisera is not accounted for by the sum of the individual expression patterns of the known Drosophila peptides. Thus, these data suggest that one or more Drosophila FaRPs or structurally related peptides remain to be discovered.

Regulation of Drosophila FMRFamide neuropeptide gene expression

Physiologically important peptides are often encoded in precursors that contain several gene products; thus, regulation of expression of polypeptide proteins is crucial to transduction pathways. Differential processing of precursors by cell- or tissue-specific proteolytic enzymes can yield messengers with diverse distributions and dissimilar activities. FMRFamide-related peptides (FaRPs) are present throughout the animal kingdom and affect both neural and gastrointestinal functions. Organisms have several genes encoding numerous FaRPs with a common C-terminal structure but different N-terminal amino acid extensions. We have isolated SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide contained in the Drosophila FMRFamide gene. To investigate the regulation of expression of FMRFamide peptides, we generated antisera to distinguish among the three neuropeptides. We have previously reported the distribution of SDNFMRFamide and DPKQDFMRFamide. In this article, we describe TPAEDFMRFamide expression. TPAEDFMRFamide antisera stain cells in embryonic, larval, pupal, and adult thoracic and abdominal ganglia. In addition, TPAEDFMRFamide-immunoreactive material is present in a lateral protocerebrum cell in adult. Thus, TPAEDFMRFamide antisera staining of neural tissue is different from SDNFMRFamide or DPKQDFMRFamide. In addition, TPAEDFMRFamide antisera stain larval, pupal, and adult gut, while SDNFMRFamide and DPKQDFMRFamide do not. TPAEDFMRFamide immunoreactivity is present in cells stained by FMRFamide antisera. Taken together, these data support the conclusion that TPAEDFMRFamide is differentially processed from the FMRFamide polypeptide protein precursor and may act in both neural and gastrointestinal tissue.

Neuropeptide precursor processing detected by triple immunolabeling

Abstract Peptides that play critical physiological roles are often encoded in precursors that contain several gene products. Differential processing of a polypeptide precursor by cell-specific proteolytic enzymes can yield multiple messengers with diverse distributions and functions. We have isolated SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide from Drosophila melanogaster. The peptides are encoded in the FMRFamide gene and have a common C-terminal FMRFamide but different N-terminal extensions. In order to investigate the regulation of expression of FMRFamide peptides, we generated antisera to distinguish between the structurally related neuropeptides. We established a triple-label immunofluorescence protocol using antisera raised in the same host species and mapped the neural distribution of SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide. Each peptide has a unique, nonoverlapping cellular expression pattern, suggesting that the precursor is differentially processed. Thus, our data indicate that D. melanogaster contains cell-specific proteolytic enzymes to cleave a polypeptide protein precursor, resulting in unique expression patterns of neuropeptides.

Antisera to multiple antigenic peptides detect neuropeptide processing

Peptides act as critical messengers of essential physiological function. Frequently, several peptides are encoded in the same precursor and, often, there is structure relatedness among the gene products. The complexity of protein precursors and presence of homologous peptides raises issues about regulation of gene expression and function of structurally-related peptides. We have determined the cellular location of DPKQDFMRFamide and SDNFMRFamide encoded in the Drosophila FMRFamide gene. We raised antisera that distinguish between the two peptides and conducted double label immunostaining utilizing antisera raised in the same host species. We found that DPKQDFMRFamide and SDNFMRFamide are present in distinct distribution patterns. We also established that the peptides are present in cells stained by FMRFamide antisera. Thus, our data are consistent with the conclusion that Drosophila contains cell-specific proteolytic processing enzymes capable of posttranslationally cleaving a polypeptide protein precursor to yield unique expression patterns of neuropeptides that may have diverse activities.

Dromyosuppressin and drosulfakinin, two structurally related Drosophila neuropeptides, are uniquely expressed in the adult central nervous system.

Drosophila myosuppressin (TDVDHVFLRFamide; DMS) and sulfakinin (FDDYGHMRFamide; DSK) have similar C-terminal structures. To determine the neuronal expression patterns of these structurally related peptides, we have generated DMS- and DSK-specific antisera to multiple antigenic peptides and performed double-label immunochemistry with antisera raised on different animals of the same species host animal. Our data indicate that DMS and DSK staining patterns in the adult central nervous system are unique and nonoverlapping.