Caractérisation de deux neuropeptides chez Drosophila melanogaster : la Leucokinine et l'IFamide

Nous avons isolé et identifié deux neuropeptides de la mouche du vinaigre, Drosophila melanogaster, la leucokinine et l'IFamide. La leucokinine, NSVVLGKKQRFHSWGamide, agit comme une hormone diurétique à une concentration seuil de 0,1 nM en utilisant le calcium comme second messager. L'IFamide, AYRKPPFNGSIFamide, est produit par quatre cellules dans le cerveau et nous avons démontré que des mâles dépourvus de ces quatre neurones sont bisexuels. Nous avons construit un gène artificiel codant un précurseur contenant plusieurs copies de neuropeptides et créé des mouches qui surexpriment ces neuropeptides artificiels.We have isolated and identified two neuropeptides from the fruitfly, Drosophila melanogaster , leucokinin and IFamide. Drosophila leucokinin, NSVVLGKKQRFHSWGamide, acts as a diuretic hormone with a threshold concentration of 0,1 nM and uses calcium as a second messenger. IFamide, AYRKPPFNGSIFamide, is produced by four neurons in the brain and males lacking these four peptidergic neurons are bisexuals. We have constructed an artificial precursor gene encoding several copies of neuropeptides and have made flies that over express the artificial neuropeptide gene

A novel role of Drosophila cytochrome P450-4e3 in permethrin insecticide tolerance

The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H2O2 and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H2O2 levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism

Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis

Epithelia frequently segregate transport processes to specific cell types, presumably for improved efficiency and control. The molecular players underlying this functional specialization are of particular interest. In Drosophila, the renal (Malpighian) tubule displays the highest per-cell transport rates known and has two main secretory cell types, principal and stellate. Electrogenic cation transport is known to reside in the principal cells, whereas stellate cells control the anion conductance, but by an as-yet-undefined route. Here, we resolve this issue by showing that a plasma membrane chloride channel, encoded by ClC-a, is exclusively expressed in the stellate cell and is required for Drosophila kinin-mediated induction of diuresis and chloride shunt conductance, evidenced by chloride ion movement through the stellate cells, leading to depolarization of the transepithelial potential. By contrast, ClC-a knockdown had no impact on resting secretion levels. Knockdown of a second CLC gene showing highly abundant expression in adult Malpighian tubules, ClC-c, did not impact depolarization of transepithelial potential after kinin stimulation. Therefore, the diuretic action of kinin in Drosophila can be explained by an increase in ClC-a–mediated chloride conductance, over and above a resting fluid transport level that relies on other (ClC-a–independent) mechanisms or routes. This key segregation of cation and anion transport could explain the extraordinary fluid transport rates displayed by some epithelia

Novel roles for GATAe in growth, maintenance and proliferation of cell populations in the Drosophila renal tubule

The GATA family of transcription factors is implicated in numerous developmental and physiological processes in metazoans. In Drosophila melanogaster, five different GATA factor genes (pannier, serpent, grain, GATAd and GATAe) have been reported as essential in the development and identity of multiple tissues, including the midgut, heart and brain. Here, we present a novel role for GATAe in the function and homeostasis of the Drosophila renal (Malpighian) tubule. We demonstrate that reduced levels of GATAe gene expression in tubule principal cells induce uncontrolled cell proliferation, resulting in tumorous growth with associated altered expression of apoptotic and carcinogenic key genes. Furthermore, we uncover the involvement of GATAe in the maintenance of stellate cells and migration of renal and nephritic stem cells into the tubule. Our findings of GATAe as a potential master regulator in the events of growth control and cell survival required for the maintenance of the Drosophila renal tubule could provide new insights into the molecular pathways involved in the formation and maintenance of a functional tissue and kidney disease

Tracing the evolutionary origins of insect renal function

Knowledge on neuropeptide receptor systems is integral to understanding animal physiology. Yet, obtaining general insight into neuropeptide signalling in a clade as biodiverse as the insects is problematic. Here we apply fluorescent analogues of three key insect neuropeptides to map renal tissue architecture across systematically chosen representatives of the major insect Orders, to provide an unprecedented overview of insect renal function and control. In endopterygote insects, such as Drosophila, two distinct transporting cell types receive separate neuropeptide signals, whereas in the ancestral exopterygotes, a single, general cell type mediates all signals. Intriguingly, the largest insect Order Coleoptera (beetles) has evolved a unique approach, in which only a small fraction of cells are targets for neuropeptide action. In addition to demonstrating a universal utility of this technology, our results reveal not only a generality of signalling by the evolutionarily ancient neuropeptide families but also a clear functional separation of the types of cells that mediate the signal

Targeted renal knockdown of Na+/H+ exchanger regulatory factor Sip1 produces uric acid nephrolithiasis in Drosophila

Nephrolithiasis is one of the most common kidney diseases with poorly understood pathophysiology, but experimental study has been hindered by lack of experimentally tractable models. Drosophilamelanogaster is a useful model organism for renal diseases because of genetic and functional similarities of Malpighian (renal) tubules with the human kidney. Here, we demonstrate the function of Sip1 (SRY-interacting protein 1) gene, an orthologue of human NHERF1 in Drosophila MTs, and its impact on nephrolithiasis. Abundant birefringent calculi were observed in Sip1 mutant flies, and the phenotype was also observed in renal stellate cell-specific RNAi Sip1 knockdowns in otherwise normal flies, confirming a renal aetiology. This phenotype was abolished in rosy flies (which model human xanthinuria) and by the xanthine oxidase inhibitor allopurinol, suggesting that the calculi were of uric acid. This was confirmed by direct assay for urate. Stones rapidly dissolved when the tubule was bathed in alkaline media, suggesting that Sip1 knockdown was acidifying the tubule. SIP1 was shown to co-locate with Na+/H+ exchanger NHE2, and with moesin, in stellate cells; and so a model was developed in which Sip1 normally regulates NHE2 activity and thus luminal pH. Drosophila renal tubule thus offers a useful model for urate nephrolithiasis

Specialized stellate cells offer a privileged route for rapid water flux in Drosophila renal tubule

Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 genes of the major intrinsic protein family are expressed at a very high level in the fly renal tissue: the aquaporins (AQPs) Drip and Prip and the aquaglyceroporins Eglp2 and Eglp4. As predicted from their structure, and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip, and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes result in impaired hormone-induced fluid secretion. The Drosophila tubule has 2 main secretory cell types: active cation-transporting principal cells, wherein the aquaglyceroporins localize to opposite plasma membranes, and small stellate cells, the site of the chloride shunt conductance, with these AQPs localizing to opposite plasma membranes. This suggests a model in which osmotically obliged water flows through the stellate cells. Consistent with this model, fluorescently labeled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects in regulating their internal environments

A nutrient-responsive hormonal circuit mediates an inter-tissue program regulating metabolic homeostasis in adult Drosophila

Animals maintain metabolic homeostasis by modulating the activity of specialized organs that adjust internal metabolism to external conditions. However, the hormonal signals coordinating these functions are incompletely characterized. Here we show that six neurosecretory cells in the Drosophila central nervous system respond to circulating nutrient levels by releasing Capa hormones, homologs of mammalian neuromedin U, which activate the Capa receptor (CapaR) in peripheral tissues to control energy homeostasis. Loss of Capa/CapaR signaling causes intestinal hypomotility and impaired nutrient absorption, which gradually deplete internal nutrient stores and reduce organismal lifespan. Conversely, increased Capa/CapaR activity increases fluid and waste excretion. Furthermore, Capa/CapaR inhibits the release of glucagon-like adipokinetic hormone from the corpora cardiaca, which restricts energy mobilization from adipose tissue to avoid harmful hyperglycemia. Our results suggest that the Capa/CapaR circuit occupies a central node in a homeostatic program that facilitates the digestion and absorption of nutrients and regulates systemic energy balance

Chloride Channels in Stellate Cells are Essential for Uniquely High Secretion Rates in Neuropeptide-stimulated \u3cem\u3eDrosophila\u3c/em\u3e Diuresis

Epithelia frequently segregate transport processes to specific cell types, presumably for improved efficiency and control. The molecular players underlying this functional specialization are of particular interest. In Drosophila, the renal (Malpighian) tubule displays the highest per-cell transport rates known and has two main secretory cell types, principal and stellate. Electrogenic cation transport is known to reside in the principal cells, whereas stellate cells control the anion conductance, but by an as-yet-undefined route. Here, we resolve this issue by showing that a plasma membrane chloride channel, encoded by ClC-a, is exclusively expressed in the stellate cell and is required for Drosophila kinin-mediated induction of diuresis and chloride shunt conductance, evidenced by chloride ion movement through the stellate cells, leading to depolarization of the transepithelial potential. By contrast, ClC-a knockdown had no impact on resting secretion levels. Knockdown of a second CLC gene showing highly abundant expression in adult Malpighian tubules, ClC-c, did not impact depolarization of transepithelial potential after kinin stimulation. Therefore, the diuretic action of kinin in Drosophila can be explained by an increase in ClC-a–mediated chloride conductance, over and above a resting fluid transport level that relies on other (ClC-a–independent) mechanisms or routes. This key segregation of cation and anion transport could explain the extraordinary fluid transport rates displayed by some epithelia

Insect capa neuropeptides impact desiccation and cold tolerance

This work was funded by grants from the UK Biotechnology and Biological Sciences Research Council (BB/G020620 and BB/L002647/1) (to S.-A.D., J.A.T.D., and S.T.); US Department of Agriculture/Department of Defense Deployed War Fighters Protection Grant Initiative (#6202-22000-029-00D) and US–Israel Binational Agricultural Research and Development Fund (BARD) (IS-4205-09C) (R.J.N.); and the National Science Foundation (IOS-0840772) (D.L.D.).The success of insects is linked to their impressive tolerance to environmental stress, but little is known about how such responses are mediated by the neuroendocrine system. Here we show that the capability (capa) neuropeptide gene is a desiccation- and cold stress-responsive gene in diverse dipteran species. Using targeted in vivo gene silencing, physiological manipulations, stress-tolerance assays, and rationally designed neuropeptide analogs, we demonstrate that the Drosophila melanogaster capa neuropeptide gene and its encoded peptides alter desiccation and cold tolerance. Knockdown of the capa gene increases desiccation tolerance but lengthens chill coma recovery time, and injection of capa peptide analogs can reverse both phenotypes. Immunohistochemical staining suggests that capa accumulates in the capa-expressing Va neurons during desiccation and nonlethal cold stress but is not released until recovery from each stress. Our results also suggest that regulation of cellular ion and water homeostasis mediated by capa peptide signaling in the insect Malpighian (renal) tubules is a key physiological mechanism during recovery from desiccation and cold stress. This work augments our understanding of how stress tolerance is mediated by neuroendocrine signaling and illustrates the use of rationally designed peptide analogs as agents for disrupting protective stress tolerance.Publisher PDFPeer reviewe