FMRFamide

DEVELOPMENTAL BIOLOGY

In situ hybridization techniques were used to describe the cellular distribution of transcripts from a Drosophila gene that encodes multiple FMRFamide-related neuropeptides. Exon-specific oligonucleotide probes were used to assay transcription in both embryonic and larval stages. A pattern of hybridization signals is found that is restricted to the central nervous system and, within that tissue, is cell-specific. The pattern included 36 distinct signals distributed throughout both the brain and segmental nerve cord (ventral ganglion). These observations suggest that the cell-specific pattern of FMRFamide-like neuropeptide expression in the Drosophila CNS is due to the restricted expression of specific gene transcripts. With few exceptions, all previously identified FMRFamide-immunoreactive neurons in Drosophila larvae express FMRFamide gene transcripts. The 36 hybridization regions of the CNS could be divided into three categories, based on their signal intensities (strong, moderate, and weak). The differences in intensity are reproducible and suggest that steady-state levels of specific neuropeptide RNA differ among individual neurons. The two exon-specific probes produce patterns that are indistinguishable both in pattern and in intensity. This supports the previous conclusion that the one detectable FMRFamide transcript contains both exons. A single identifiable signal is detected during embryogenesis (beginning at stage 16), but the mature complement of signals is not fully established until the final larval stages (Schneider, 1991).

Protein expression of the FMRFamide neuropeptide gene in Drosophila has been mapped with polyclonal antisera against three small peptides whose sequences were derived from the Drosophila proFMRFamide precursor. One antiserum is affinity-purified and extensively characterized. The enriched antibodies label 15-21 bilaterally symmetric pairs of neurons in a pattern that correspond very closely to the pattern of in situ hybridization that was determined previously. The other antisera produce complementary results. These findings suggest that the antisera specifically label cells that express the FMRFamide gene. Strong staining was observed in identified larval interneurons and neuroendocrine cells, and moderate to weak staining in neurons of unknown function. The adult pattern of expression includes both larval neurons whose immunoreactivity persists through metamorphosis and adult-specific neurons. During metamorphosis, transient staining is observed in a small number of neurons and in specific neuropil regions that included the central body, the protocerebral bridge, and the optic ganglia. Based on these morphological features, it is suggested that the FMRFamide-like neuropeptides in Drosophila play a number of functional roles, perhaps affecting both physiological and developmental phenomena. Such roles include general modulation throughout all post-embryonic stages, via the blood, and also more stage- and region-specific modulation within the CNS (Schneider, 1993a).

DPKQDFMRFamide is one of five different FMRFamide-containing peptides encoded in the Drosophila FMRFamide gene. To study the cellular expression of DPKQDFMRFamide, antisera was generated 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 is 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 (Nichols, 1995a).

The expression of SDNFMRFamide, one of five different FMRFamide-containing peptides encoded by the Drosophila melanogaster FMRFamide gene, has been determined. To study expression, antisera to the N-terminus of SDNFMRFamide was generated 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 (Nichols, 1995b).

The Drosophila FMRFamide gene encodes multiple FMRFamide-related peptides. These peptides are expressed by neurosecretory cells and may be released into the blood to act as neurohormones. The effects of eight of these peptides were examined on nerve-stimulated contraction (twitch tension) of Drosophila larval body-wall muscles. Seven of the peptides strongly enhance twitch tension, and one of the peptides is inactive. Their targets are distributed widely throughout the somatic musculature. The effects of one peptide, DPKQDFMRFamide, are unchanged after the onset of metamorphosis. The seven active peptides show similar dose-response curves. Each has a threshold concentration near 1 nM, and the EC50 for each peptide is approximately 40 nM. At concentrations of <0.1 microM, the responses to each of the seven excitatory peptides follows a time course that matches the fluctuations of the peptide concentration in the bath. At higher concentrations, twitch tension remains elevated for 5-10 min or more after wash-out of the peptide. When the peptides are presented as mixtures predicted by their stoichiometric ratios in the dFMRFamide propeptide, the effects are additive, and there are no detectable higher-order interactions among them. One peptide was tested and found to enhance synaptic transmission. At 0.1 microM, DPKQDFMRFamide increases the amplitude of the excitatory junctional current to 151% of baseline within 3 min. Together, these results indicate that the products of the Drosophila FMRFamide gene function as neurohormones to modulate the strength of contraction at the larval neuromuscular junction. In this role these seven peptides appear to be functionally redundant (Hewes, 1998).

Adult

Changes in the pattern of specific neuropeptide gene expression were studied during the metamorphosis of the Drosophila nervous system. Prior to metamorphosis, the Drosophila FMRFamide gene is expressed exclusively within the central nervous system in a stereotyped pattern that comprises roughly 60 neurons. The FMRFamide gene is continuously expressed throughout all stages examined: at each of 15 stages of adult development and through at least the first 10 days of adult life. There are no differences between the results observed with 2 different exon-specific hybridization probes, thus indicating little if any alternative splicing during postembryonic development. Despite many changes in the positions of individual hybridization signals due to the large-scale reorganization of the nervous system, the continuous pattern of gene expression through adult development permits many adult signals to be identified as larval signals. It is concluded that the adult pattern of FMRFamide gene expression is largely derived from persistent larval neurons. Adult-specific hybridization signals in the brain and ventral ganglion are also detected and these correspond to many of the approximately 40 adult-specific FMRFamide-immunoreactive neurons. One specific larval signal is lost during adult development and the intensities of other signals fluctuate in reproducible manners. These stereotyped differences in hybridization signal intensity resemble similar observations made in larval stages and support the hypothesis that the steady-state levels of FMRFamide transcripts are differentially regulated among the diverse neurons that express the gene (O'Brien, 1991).

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