Selective elimination/RNAi silencing of FMRF-related peptides and their receptors decreases the locomotor activity in Drosophila melanogaster

Highlights

• RNAi knockdown of FaRP genes decreased startle-induced locomotor activity of flies.

• Three Gal4 drivers were constructed with 5′ regulatory sequences of the Fmrf gene.

• The new drivers express Gal4 in partial FMRF neuronal pattern and some ectopic cells.

• Cell ablation targeted by the new drivers can also influence adult locomotor activity.

Abstract

Five neuropeptide genes are classified in the FMRF-related (FaRP) group: the Fmrf, dromyosuppressin (Dms), drosulfakinin (Dsk), neuropeptide F (npf) and short neuropeptide F (sNPF) genes coding for 8, 1, 2, 1 and 4 peptides, respectively. In order to compare their effects on the locomotor activity of Drosophila adults, we made RNAi knockdown of the peptides and their specific receptor genes. In addition, we constructed Gal4 drivers with three distinct parts of the Fmrf gene’s 5′ regulatory sequence (RS8-Gal4, RS11-Gal4, RS17-Gal4), and used them to ablate FMRF-positive neurons inducing apoptosis by expressing the reaper (rpr) gene. We examined the locomotor activity of flies by measuring the mean velocity of movement (MVM) following repeated air-puffs. Locomotor activity was decreased by RNAi knockdown induced in the CNS by the elav-Gal4 driver. According to the MVM curve profiles, RNAi knockdown most effectively decreased the velocity when the DmsR-1 and DmsR-2 genes were silenced together (DmsR-1-RNAi/elav-Gal4; DmsR-2-RNAi/+). Similar effect was observed in Dsk-RNAi/ elav-Gal4; DskR-2-RNAi/+, while moderate effects were found in three other combinations (Fmrf-RNAi/elav-Gal4; FR-RNAi/+, Dms-RNAi/ elav-Gal4;DmsR-2-RNAi/+, CCKLR-17D1-RNAi/elav-Gal4; CCKLR-17D3-RNAi/+), and weak effect in DmsR-2-RNAi/elav-Gal4; DmsR-1-RNAi/+. Male and female flies were not different in this respect. In the cell ablation experiment, the MVM profiles of the female flies were different from the controls when the UAS-rpr transgene was driven by RS8-Gal4 or RS17-Gal4. The RS11-Gal4 and Fmrf-Gal4 drivers were ineffective. In the males only the RS17-Gal4 showed a weak effect. RNAi silencing of the FaRP and FaRP-receptor genes effectively decreased the startle-induced locomotor activity of flies. Ablation of FMRF-positive neurons by the RS8-Gal4 and/or RS17-Gal4 drivers also decreased the flies’ activity.

Introduction

Specific neurons in the central nervous system (CNS) produce and secrete peptides and biogenic amines. Some of these neuropeptides reach the target organ through the circulation and act as neurohormones while others remain within the CNS and modify the activity of other neurons as neuromodulators. Neuropeptides constitute a large group of signaling molecules. They influence many important processes of life in all Metazoans, like neurotransmission, nociception, feeding and metabolism, energy and osmotic homeostasis, learning and memory, etc. Insects as well use a plethora of neuropeptides to control and modulate physiological, developmental and behavioral events. The fruitfly Drosophila melanogaster, the genetically best known organism among the higher Eukaryotes, offers an excellent model system for studying the action mechanism of neuropeptides.

The availability of Drosophila genome sequence (Adams et al., 2000), has given a large impetus to the neuropeptide field as well: within a few years, many of the putative peptide and receptor genes have been identified in the fruitfly (Hewes and Taghert, 2007, Nässel and Winther, 2010. Bioinformatic approach predicted 119 peptide precursor genes in the Drosophila genome. Out of them, at least 42 genes may code for “prepropeptide” proteins containing one or more copies of identical and/or closely related peptide sequences flanked by proteolytic cutting sites. The peptidomic analysis has verified 46 neuropeptides which are derived from 19 of these precursor genes (Clynen et al., 2010).

Almost all the receptors which bind peptide ligands belong to the G protein-coupled receptors (GPCRs). GPCRs represent one of the largest protein family, their genes comprise 1.5% of the Drosophila genome (Brody and Cravchik, 2000). Currently about 160 GPCRs are known in Drosophila, of which 44 are known to bind peptide ligands (Hewes and Taghert, 2007, Hauser et al., 2006a, Hauser et al., 2006b, Nässel and Winther, 2010). However, much less is known about the signaling pathways beyond the neuropeptide ligands and the cognate receptors.

The Drosophila Fmrf gene codes for a prepropeptide which contains six peptide sequences sharing the characteristic FMRF amino acid sequence at the C-terminus, and two other peptides with more divergent C-terminal sequences (Wegener and Gorbashov, 2008, Nässel and Winther, 2010). In addition to FMRF, neuropeptides derived from four other genes have the RF motif at their C-terminal end. Traditionally, these five neuropeptide genes are classified as the FMRF-related group (FaRP) comprising the Fmrf (CG2346), dromyosuppressin (Dms, CG6440), drosulfakinin (Dsk, CG18090), neuropeptide F (npf, CG10342) and short neuropeptide F (sNPF, CG13968) genes (Nässel and Winther, 2010, and Flybase (http://flybase.org). The Dms and npf genes code for one peptide each, while sNPF and Dsk code for five and three peptides, respectively. The DSK1 and DSK2 peptides have been verified biochemically, DSK0 is putative because it has not been confirmed as an expressed peptide. Interestingly, Palmer et al. (2007) recently found that the DSK0 peptide induced crop contractions. All these peptides have a conserved C-terminus with the terminal RF-amide sequence, and a divergent N terminus which could be responsible for the specific biological effects.

The FaRP peptides have characteristic myoactivities, but they are not related functionally: their target organs, effective thresholds, time-course and expression patterns are different (Nichols et al., 2002). Drosophila FMRF peptides generally stimulate muscle contraction in the heart, crop, foregut and body wall (Nichols, 2003. DMS is a myoinhibitory peptide decreasing e.g. the movement of the heart and adult foregut/crop (Kaminski et al., 2002). DSK peptides stimulate the contractions of the heart and crop (Nichols et al., 2002, Nichols et al., 2009), and increase larval locomotion activity and induces escape response (Chen et al., 2012). DSKs and their CCKLR receptors are required for the growth of the neuromuscular junctions (Chen and Ganetzky, 2012).

All the FaRPs are expressed in the CNS in neurons arranged in bilaterally symmetric patterns, which are characteristic for each neuropeptide (Santos et al., 2007, Park et al., 2008, Nässel and Winther, 2010). The FMRF peptides are expressed in the Drosophila CNS in 17 various cell types, in about 60 neurons (Schneider et al., 1991, Schneider et al., 1993). Even peptides which are derived from the same FMRF prepropeptide protein by differential processing can show different tissue-specific expression: the SDNFMRF, DPKQDFMRF and TPAEDFMRF peptides are expressed in similar patterns in the thoraco-abdominal ganglion, but TPAEDFMRF is additionally expressed in the adult lateral protocerebrum and the gut (Nichols et al., 1999). DMS is expressed in the CNS but could be detected by immuno-staining in the crop and gut (“brain-gut peptide”, McCormick and Nichols, 1993, Merte and Nichols, 2002 DSK is also expressed in the CNS (Nichols and Lim, 1996). DSK is expressed in some of the DILP (Drosophila insulin-like peptide)-producing neurons which have axonal connection to neurohemal areas in the corpora cardiaca, aorta and the anterior intestine. DILP and DSK together control the feeding behavior as hormonal satiety signals (Söderberg et al., 2012). NPF and sNPF are expressed both in the CNS and the midgut (Veenstra, 2009). sNPF is also expressed in several thousand CNS neurons and consequently exerts a broad spectrum of modulatory effects (Nässel et al., 2008).

The determination of the neuronal cell fate and the neuropeptide expression patterns in the CNS are regulated by specific enhancers binding transcription factors in the genes’ regulatory regions (Benveniste and Taghert, 1999, Vanden Broeck, 2001). Specification of the identity of single neurons in the CNS is a multistep process which builds up a combinatorial code of the activated enhancers (Baumgardt et al., 2007). GPCR receptors of the neuropeptides and neurohormones are expressed on a broader scale in the target cells and organs Cazzamali and Grimmelikhuijzen, 2002, Duttlinger et al., 2002, Nichols, 2003, Bendena et al., 2012).

The experiments described here address the effects exerted by some of the FaRP peptides (FMRF, DMS, DSK) and receptors on the locomotor activity of Drosophila adults. For this purpose, we used two genetic approaches (i) RNAi silencing of FMRF-related peptides and their receptors: and (ii) construction of new Fmrf-Gal4 drivers which represent specific parts of the FMRF spatial expression in the CNS, and their application to remove the corresponding neural cells by reaper (rpr)-induced apoptosis. The results showed that these treatments decreased the locomotor activity of the flies in general.