The Interactive Fly

Genes involved in tissue and organ development

Malpighian Tubules

  • Embryonic origin, functional domains and physiology of Malpighian tubules
  • Ecdysone regulates morphogenesis and function of malpighian tubules in Drosophila melanogaster through EcR-B2 isoform
  • Tip cells act as dynamic cellular anchors in the morphogenesis of looped renal tubules in Drosophila
  • The adult Drosophila malpighian tubules are maintained by multipotent stem cells
  • Drosophila serotonin receptors stimulate fluid secretion in the Malpighian tubules
  • White participates in vesicular transepithelial transport of cGMP
  • Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis
  • Two inwardly rectifying potassium channels, Irk1 and Irk2, play redundant roles in Drosophila renal tubule function
  • Early gene Broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster
  • KANK deficiency leads to podocyte dysfunction and nephrotic syndrome
  • Expression of polyQ aggregates in Malpighian tubules leads to degeneration in Drosophila melanogaster
  • Segment-specific Ca transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages
  • The corticotropin-releasing factor-like diuretic hormone 44 (DH) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster
  • Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila
  • The orphan pentameric ligand-gated ion channel pHCl-2 is gated by pH and regulates fluid secretion in Drosophila Malpighian tubules
  • Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes
  • Drosophila imaginal disc growth factor 2 is a trophic factor involved in energy balance, detoxification, and innate immunity
  • Identification of multiple functional receptors for tyramine on an insect secretory epithelium
  • Cold tolerance of Drosophila species is tightly linked to epithelial K+ transport capacity of the Malpighian tubules and rectal pads
  • CRISPR-induced null alleles show that Frost protects Drosophila melanogaster reproduction after cold exposure
  • Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster

    Genes expressed in Malpighian tubule development

    Embryonic origin, functional domains and physiology of Malpighian tubules

    The tubules arise during embryogenesis as four protuberances extending from the proctodeum. As this tissue is considered ectodermal, Malpighian tubules are classified as an ectodermally derived secretory tissue. The protuberances grow, first by cell proliferation and then by extensive rearrangement of the cells, to produce elongated blind end tubes composed of a single-cell-layered epithelium (Hoch, 1994).

    The development of Malpighian tubules reveals an essential role for the gene Krüppel. In each Malpighian tubule, one cell is singled out, the tip cell, whose function during embryogenesis is to promote cell division in its neighbours. The tip cell arises by division of a tip mother cell, which is selected from a cluster of equivalent cells, each expressing Krüppel in each tubule primordium. Each cluster is delineated by the expression of proneural genes; the selection of a single cell from each group involves lateral inhibition, mediated by the neurogenic genes. achaete is responsible for tip cell allocation, but Kr acts as the selector gene, responsible for tip cell fate. The tip cell directs the growth of the Malpighian tubules and organizes the mitotic response and migration of the other cells forming each tubule (Hoch, 1994). Therefore Krüppel is responsible for cell fate in the Malpighian tubules, a function quite distinct from Krüppel's role as a gap gene.

    Drosophila possesses two pair of Malpighian tubules. The right pair of tubules project forward from their point of insertion within the hindgut and lie at the anterior end of the abdomen, and the left pair extend backwards so that their tips become attached to the posterior part of the hindgut. Each tubule pair unites to form a common ureter, which enters the intestine between the midgut and hindgut. The two anterior Malpighian tubules are classically described as comprising a distal initial segment and a proximal main segment, joined by a narrow transitional segment; the two posterior tubules, in contrast, were thought to consist solely of a main segment. Contemporary studies, using enhancer trap lines, which place reporter genes under the control of tissue specific enhancers, confirm this viewpoint and thus the nomenclature "initial," "transitional," and "main" segments has been adopted to described these genetically deduced domains (Sözen, 1997).

    Enhancer trap studies reveal an unexpected complexity in Malpighian tubules in terms of both regions and cell types. Enhancer trap lines that delineate the initial and transitional segments of anterior tubules, reveal previously undescribed analogous domains in posterior tubules. It is also possible to subdivide the main segments. While the transitional-main segment boundary has been established in accordance with classical studies, an additional domain is found marking the lower third of the tubule and the ureter. This latter region, in turn, can be resolved into three subregions: a lower tubule and an upper and lower ureter (Sözen, 1997).

    Previous studies have described just two tubule cell types: principal (type I) and secondary or stellate (type II). Both can be further subdivided. Principal cells, for example, comprise at least two distinct subpopulations. Thus there appear to be differences in otherwise indistinguishable cells with respect to enhancer trap expression patterns and presumably with respect to function as well. Type II cells are distributed evenly throughout the initial, transitional and main segments of posterior tubules and within the main segment of anterior tubules. None of the enhancer trap markers mark cells in the lower tubule or ureter, suggesting that the mechanism by which type II cells are specified respects the newly defined lower tubule boundary. Several lines mark a "tiny" cell type found in lower tubules and posterior midgut but do not mark the same genetic domain as stellate cells. Possibly these previously undescribed cells are counterparts of the myoendocrine cells recently described in Formica. These cells may monitor the fluid collected in the ureter and secrete neurohormones basally into the hemolymph to regulate muscle contractility or ion transport (Sözen, 1997).

    Do discrete physiological properties map to the genetic domains that have been identified? With respect to a number of different transport processes, this is indeed the case. The obvious functional property of the tubule is to secrete urine. It has been reported that the initial segment of Drosophila anterior tubule does not secrete detectable fluid, that the lower third of the tubule is reabsorptive, and that only the main segment is responsible for fluid production. High levels of proton-pumping V-ATPases energize apical plasm membranes of several epithelia, including Malpighian tubules. The B-subunit of Drosophila V-ATPase is expressed in the initial and transitional segments and is much weaker in the reabsorptive main segment. The main segment consists entirely of the large, principal cell. This provides the first evidence that cation transport into the lumen of Malpighian tubules may be a unique property of principal, rather than type II, cells. A putative aquaporin has been cloned in Drosophila: this channel is found in stellate cell basolateral membranes. Given that stellate cells are found in secretory but not reabsorptive tubule regions, they may well play an essential role in fluid secretion. Another function ascribed to Malpighian tubules is the secretion of organic metabolites. This function is confined to the main segment. It is clear that the staining pattern for alkaline phosphatase precisely matches the lower tubule boundary, and is associated with the reabsorptive, rather than the secretory, domain of the tubule (Sözen, 1997).

    Secretion by Malpighian tubules is under hormonal control. CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates Malpighian tubule fluid secretion via cGMP, which in turn stimulates the nitric oxide signaling pathway. Liquid chromatography analysis of adult Drosophila reveals the presence of a CAP2b-like peptide, that coelutes with Manduca sexta CAP2b and synthetic CAP2b and that has CAP2b-like effects on the M. sexta heart. CAP2b stimulation elevates tubule cGMP levels but not those of cAMP. Both CAP2b and cGMP increase the transepithelial potential difference, suggesting that stimulation of vacuolar ATP action underlies the corresponding increases in fluid secretion (Davies, 1995).

    Calcium mobilization in identified cell types within an intact renal epithelium (the Drosophila Malpighian tubule) was studied by GAL4-directed expression of an aequorin transgene. Aequorin is a Ca2+ sensitive liminescent protein isolated from the coelenterate Aequorea victoria. It is a complex of apoaequorin, a 21 kDA polypeptide, and coelenterazine, a hydrophobic luminophore. Aequorin is used for monitoring Ca2+ changes. CAP2b, causes a rapid, dose-dependent rise in cytosolic calcium in only a single, genetically-defined, set of 77 principal cells in the main (secretory) segment of the tubule. In the absence of external calcium, the CAP2b-induced calcium response is abolished. In Ca2+-free medium, the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, elevates [Ca2+]i only in the smaller stellate cells, suggesting that principal cells do not contain a thapsigargin-sensitive intracellular pool. Assays for epithelial function confirm that calcium entry is essential for CAP2b to induce a physiological response in the whole organ. The data suggest a role for calcium signaling in the modulation of the nitric oxide signaling pathway in this epithelium. CAP2b must act to increase fluid secretion rates solely by an initial rise of [CA2+]i in principal cells. CAP2b stimulates tubule Nitric oxide synthase activity. It is probable that the CAP2b induced rise in [CA2+]i is sufficient to trigger the activation of Drosophila calcium sensitive Nitric oxide synthase. The maximal CAP2b concentrations employed elevate principal cell calcium levels from 87 to 255 nM, a value close to the EC50 of Drosophila NOS. This implies that Drosophila Nos is responsive over the range of the CAP2b concentrations employed. This may account for the observation that thapsigargin treatment results in increased basal cGMP levels that are not further increased on CAP2b stimulation. Thus the data provide strong evidence for a calcium-mediated link between CAP2b and NOS/cGMP activation of fluid secretion. The GAL4-targeting system allows general application to studies of cell-signaling and pharmacology that does not rely on invasive or cytotoxic techniques (Rosay, 1997).

    Other hormones likely to be involved in Malpighian tubule function are the leucokinins. Leucokinins are a group of widespread insect hormones. In tubules, their major action is to raise chloride permeability through stellate cells by binding to receptors on the basolateral membrane, and so ultimately to enhance fluid secretion (Julian Dow, personal communication).

    Ecdysone regulates morphogenesis and function of malpighian tubules in Drosophila melanogaster through EcR-B2 isoform

    Malpighian tubules are the osmoregulatory and detoxifying organs of Drosophila and its proper development is critical for the survival of the organism. They are made up of two major cell types, the ectodermal principal cells and mesodermal stellate cells. The principal and stellate cells are structurally and physiologically distinct from each other, but coordinate together for production of isotonic fluid. Proper integration of these cells during the course of development is an important pre-requisite for the proper functioning of the tubules. This study conclusively determined an essential role of ecdysone hormone in the development and function of Malpighian tubules. Disruption of ecdysone signaling interferes with the organization of principal and stellate cells resulting in malformed tubules and early larval lethality. Abnormalities include reduction in the number of cells and the clustering of cells rather than their arrangement in characteristic wild type pattern. Organization of F-actin and beta-tubulin also show aberrant distribution pattern. Malformed tubules show reduced uric acid deposition and altered expression of Na+/K+-ATPase pump. B2 isoform of Ecdysone receptor is critical for the development of Malpighian tubules and is expressed from early stages of its development (Gautam, 2014).

    Tip cells act as dynamic cellular anchors in the morphogenesis of looped renal tubules in Drosophila

    Tissue morphogenesis involves both the sculpting of tissue shape and the positioning of tissues relative to one another in the body. Using the renal tubules of Drosophila, this study shows that a specific distal tubule cell regulates both tissue architecture and position in the body cavity. Focusing on the anterior tubules, it was demonstrated that tip cells make transient contacts with alary muscles at abdominal segment boundaries, moving progressively forward as convergent extension movements lengthen the tubule (see Tip-Cell-Dependent Anchorage of Anterior Tubules to Alary Muscles). Tip cell anchorage antagonizes forward-directed, TGF-beta-guided tubule elongation, thereby ensuring the looped morphology characteristic of renal tubules from worms to humans. Distinctive tip cell exploratory behavior, adhesion, and basement membrane clearing underlie target recognition and dynamic interactions. Defects in these features obliterate tip cell anchorage, producing misshapen and misplaced tubules with impaired physiological function (Weavers, 2013; see Graphical Abstract).

    As the embryonic renal tubules assume their mature shape they interact with other tissues, responding to Dpp guidance cues as they take up their characteristic positions in the body cavity (Bunt, 2010). This study shows that, in addition, a single cell at the distal end of each renal tubule makes specific transitory, and finally long-term, contacts with target tissues. These cells express a distinctive pattern of genes and show characteristic exploratory activity, which is crucial for the stereotypical looped shape and position of the tubules in the body cavity. In turn, these features have profound consequences for the efficacy of fluid homeostasis in the whole animal (Weavers, 2013).

    It is suggested that the elongation and forward extension of the tubules result from the combined effects of cell rearrangements that lengthen the tubule and the response of kink region cells to regional Dpp guidance cues. The evidence indicates that the tip cells act as anchors, through their interactions with alary muscles, so that tubules are tethered at both ends, the proximal end being attached through ureters to the hindgut. These attachments perform two functions: they stabilize the looped architecture, maintaining the kink close to the tubule midpoint, and they limit forward and ventral movement to ensure the stereotypical tubule arrangement in the body cavity (Weavers, 2013).

    If tip cell contact with the alary muscles is lost, the kink 'unravels,' shifting distalward, and the tubule as a whole extends too far into the anterior, with the distal region lying more ventrally close to the Dpp-expressing gastric caeca. Confirming the existence of a forward tractive force responsible for tip cell detachment are the distortion of transient alary muscle targets before the tip cell detaches and the characteristic 'recoil' seen when the tip cell is ablated. Evidence that this results from the response to guidance cues is the failure of tip cells to detach from their first alary muscle contact (A5/A6) in the absence of the midgut Dpp guidance cue and the more anterior location of the kink region (close to the gastric caeca) in tubules where the tip cell stalk is greatly extended, for example when the activity of RhoA is repressed. The critical nature of the balance between these forward and restraining influences is also revealed when adhesion between the tip cells and alary muscles is increased by manipulating tip cell number or adhesive strength. In each case, the tip cells remain attached to alary muscles posterior to their normal final contacts, and this results in more posterior positioning of the whole tubule. Together, these results strongly suggest that tip cells detach because the forward movement of tubules overcomes the adhesive strength of their early transient contacts (Weavers, 2013).

    The final tip cell/alary muscle target is highly reproducible, suggesting recognition through segmental identity, the A3/A4 target being the first encountered by the tip cell that expresses Ubx. However, altering Ubx expression in alary muscles has no effect on the final tip cell contact. Instead, it appears that tip cells adhere to each alary muscle they contact, and the final target depends on the balance between forward tubule movement and the strength of tip cell/target adhesion. Consistent with this view, when all the normal muscle targets are ablated, tip cells can make stable contacts with the A2/A3 alary muscle (Weavers, 2013).

    From the time that they are specified, tip cells show distinctive patterns of gene expression, morphology, and behavior critical to their ability to make alary muscle contacts; they form dynamic filopodia, which explore the alary muscle surface, remain denuded of the BM that envelops the rest of the tubule, and express cell-adhesion proteins, including Neuromusculin (Nrm) and integrins. Protrusive activity depends on the rapid turnover of actin, mediated by regulators such as Rac GTPase and the actin-capping protein Enabled, which are active in tip cells (Weavers, 2013).

    BM deposition basally around the tip cells severely inhibits protrusive behavior, and tip cells therefore employ multiple mechanisms to ensure that they remain denuded. These include the absence of expression of factors that promote BM deposition and stabilization (hemocyte attractants, BM components, or receptors), the removal by transcytosis of any BM that is deposited, and expression of MMP1, which is able to cleave matrix components. MMP1 is expressed late during tubule elongation and the protein is localized apically in tip cells, suggesting that its function might be to degrade transcytosed BM proteins (Weavers, 2013).

    Protrusive exploratory behavior results in adhesive contacts made possible through tip cell expression of Nrm and integrins. Nrm is a homophilic cell-adhesion molecule of the Ig-domain superfamily. The binding partner for tip cell Nrm is unclear, as alary muscles do not express it. However, driving nrm expression in alary muscles induces strong adhesion, resulting in tip cells remaining bound to their first target in A5/A6. It is possible that Nrm in tip cells normally makes heterophilic associations with Ig domain-containing proteins such as Dumbfounded (Kirre), which is expressed in alary muscles and is sufficient, when overexpressed, to induce more posterior target adhesio (Weavers, 2013).

    Tip cells express integrins, and complexes accumulate as each target contact is made, but initially they do not lead to long-term adhesion. It is suggested that the strength of adhesion increases with successive contacts, either through increased expression of integrins and their associated factors or by regulated adhesive complex turnover, as shown in other tissues. Once the final tip cell contact is made, BM accumulates around the tip cellalary muscle surface, increasing the concentration of integrin ligands at the junction. The accompanying decline in the protrusive activity of the tip cell could also result from integrin-mediated adhesion, which is known to reduce levels of the actin-capping protein Enabled. This sequence of events parallels the mechanism by which elongated myotubes and tendon cells establish their myotendinous junctions (Weavers, 2013).

    Once the anterior tubule tip cells make their final alary muscle contact, they remain attached throughout development into adult life. Such interaction of excretory tubule tips with muscles is a common feature of renal systems in insects, either with alary muscles or with fine striated muscles that spiral along the tubule. Muscle contacts increase tubule movement, maximizing the effectiveness of excretion, by increasing hemolymph sampling and enhancing tubule flow. Similar contacts are found outside the arthropods; the flame cells that cap planarian protonephridial tubes develop prominent filopodia and interact with nearby muscle fibers, providing anchorage, thought to be important during branching morphogenesis in this system (Weavers, 2013).

    Tip cells or groups of cells at the distal tips of outgrowing epithelial tubes act as organizers in tubular systems, from the migrating Dictyostelium slug to the branching epithelial scaffolds of human organs. As in fly renal tubules, these distinctive cells regulate cell division and guided tubule extension, and in mammalian systems they control branching morphogenesis (Weavers, 2013).

    However, in distinct contrast to the role of tip cells in the morphogenesis of these systems, the tip cells of the anterior renal tubules play no role in leading outgrowth. Instead, they act to counteract outgrowth, and importantly this leads to the development of a looped tubular structure both by tethering the distal tips of tubules close to their proximal junction with the ureter and by maintaining the tightness of the tubule kink region. Looped tubular structures are relatively uncommon; a tubule tree as in the lung, pancreas, or liver or an anastomosing network as in the vascular system is more frequently seen. However, a striking example of looped tubules is found in the mammalian kidney, where the distal and proximal convoluted tubules together with the loop of Henle connect the tubule tip (at the glomerulus) to the collecting duct (close to the site of urine outflow). Looping of both the nephron and its vascular supply creates a countercurrent system that maximizes the efficiency of ion and fluid homeostasis. Such exchange systems also occur in insects with specialized diets or those living in dry conditions. Countercurrent exchange has not been demonstrated in Drosophila melanogaster tubules, where it is more likely that the looped tubule structure is important for effective hemolymph sampling (Weavers, 2013).

    In the development of the mammalian nephron, as in fly renal tubules, both the site of connection to the ureter and the tubule tip, the renal corpuscle, are established early in organ development so that tubule extension, by both cell proliferation and rearrangements, occurs between these fixed points. It will be interesting to discover whether similar tissue interactions stabilize the position of the developing glomerulus, and so play a prominent role in maintaining the looped structure as kidney tubules extend, resulting in the final intricate and regular array of nephrons apparent in the mature mammalian kidney (Weavers, 2013).

    The adult Drosophila malpighian tubules are maintained by multipotent stem cells

    All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration after ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue. In Drosophila, the Malpighian tubules are thought to be very stable and no stem cells have been identified. This study has identified multipotent stem cells in the region of lower tubules and ureters of the Malpighian tubules. Using lineage tracing and molecular marker labeling, it was demonstrated that several differentiated cells in the Malpighian tubules arise from the stem cells and an autocrine JAK-STAT signaling regulates the stem cells' self-renewal. Identifying adult kidney stem cells in Drosophila may provide important clues for understanding mammalian kidney repair and regeneration during injury (Singh, 2008).

    The regenerating renal cells may come from one of the three possible sources, based on previous studies. First, the circulating blood contains bone marrow-derived stem cells able to differentiate into non-haematopoietic cells, such as cells of the kidney. Second, the differentiated glomerular and tubular cells may also be able to dedifferentiate into stem-like cells to repair the damaged tissues. Third, large numbers of slowly cycling cells have recently been identified in the mouse renal papilla region; these cells may be adult kidney stem cells and may participate in renal regeneration after ischemic injury. Further, the ureter and the renal collecting ducts were formed from the epithelium originating from the ureteric bud, and the nephrons and glomeruli were formed from the metanephric mesoderm-derived portion during kidney development. Two distinguished stem cell types have been proposed as responsible for repairing the renal collecting tubules and the nephrons. This study identified a type of pluripotent stem cells (RNSCs) in the Drosophila renal organ. The stem cells are able to generate all cell types of the adult fly MTs. In the region of lower tubules and ureters, autocrine JAK-STAT signaling regulates the stem cell self-renewal. Weak JAK-STAT signaling may convert an RNSC into a renalblast (RB), which will differentiate into an RC in the region of lower tubules and ureters, and a type I or type II cell in the upper tubules. These data indicate that only one type of stem cell may be responsible for repair and regeneration of the whole damaged tissues in mammalian kidney (Singh, 2008).

    The Drosophila RNSCs represent a unique model to study the molecular mechanisms that regulate stem cell or cancer stem cell behavior. In most of the stem cell systems that has been well characterized to date, stem cells always reside in a specialized microenvironment, called a niche. A niche is a subset of neighboring stromal cells and has a fixed anatomical location. The stromal cells often secrete growth factors to regulate stem cell behavior. The stem cell niche plays an essential role in maintaining stem cells, and stem cells will lose stem cell status once they are detached from the niche. The niche often provides the balanced (proliferation-inhibiting and proliferation-stimulating) signals that keep the stem cells dividing slowly. The inhibitory signals keep the stem cell quiescent most of the time while the stimulating signals promote stem cell division, to replenish lost differentiated cells. Maintaining the balance between proliferation-inhibiting and proliferation-stimulating signals is the key to maintaining tissue homeostasis (Singh, 2008).

    Drosophila RNSCs are controlled differently. This study has demonstrated that the JAK-STAT signaling regulates the stem cell self-renewal. Both the ligand Upd and the receptor Dome are expressed in the RNSCs and the autocrine JAK-STAT signaling regulates the stem cell self-renewal; thus, the self-sufficient stem cells control their self-renewal or differentiation and do not need to constrained to a fixed niche. However, the RNSCs are still confined to the region of lower tubules and ureters even in the Upd overexpressed flies, suggesting that some other factors besides the JAK-STAT signaling may restrict the RNSCs to the region of the lower tubules and ureters (Singh, 2008).

    Recent studies also suggest that tumors may arise from small populations of so-called cancer stem cells (CSCs). The CSCs probably have arisen from mutations that dysregulate normal stem cell self-renewal. For example, mutations that block the proliferation-inhibiting signals or promote the proliferation-stimulating signals can convert the normal stem cells into CSCs. This study demonstrates that amplifying the JAK-STAT signaling by overexpressing its ligand Upd stimulates the RNSCs to proliferate and also to differentiate into RC, which results in tumorous overgrowth in the MT. Therefore, the Drosophila RNSC system may also be a valuable in vivo system in which to study CSC regulation (Singh, 2008).

    The RNSCs are located in the region of the lower tubules and ureter of the MTs, while ISCs are located at the posterior midgut. The MTs' ureters connect to the posterior midgut. The two types of stem cells are at close anatomical locations in the adult fly digestion system and also share some properties. For example, both of them are small nuclear cells, Arm-positive, and express esg. However, RNSCs and ISCs produce distinctly different progenies. ISCs produce progenies that include either Su(H)GBE-lacZ- or Pros-positive cells, which are not among the progenies of RNSCs because Su(H)GBE-lacZ and Pros are not expressed in the MTs. RNSCs produce progenies that include Cut- or TSH-positive cells, which are not among the progenies of ISCs because Cut and TSH are not expressed in the posterior midgut. One possibility for this difference is that, although RNSCs and ISCs originate from the same stem cell pool, their particular environments restrict their differentiation patterns. Future experiments, such as transferring RNSCs to the posterior midgut and vice versa, should be able to test this model (Singh, 2008).

    The JAK-STAT signaling regulates self-renewal of the male germline, the male somatic, female escort stem cells in fly. The signaling also regulates self-renewal and maintenance of mammalian embryonic stem cells. This study reports that the JAK-STAT signaling regulates self-renewal of RNSCs. The JAK-STAT signaling may be a general stem cell signaling and also regulate stem cell self-renewal in other, un-characterized stem cell systems (Singh, 2008).

    esg has been used as a marker of both male germline stem cells. This study has demonstrated that the esg-Gal4. UAS-GFP transgene is specifically expressed in RNSCs. The function of the esg gene is to maintain cells as diploid in Drosophila imaginal cells. Stem cells may have to be diploid, and esg may be a general stem cell factor. Identifying a stem cell signaling pathway (such as the JAK-STAT signal transduction pathway) and a stem cell factor (such as esg) will provide useful tools for identifying stem cells in other systems and for understanding stem cell regulation in general (Singh, 2008).

    Drosophila serotonin receptors stimulate fluid secretion in the Malpighian tubules

    Every living cell must detect, and respond appropriately to, external signals. Thus, the functions of intracellular second messengers, such as guanosine 3'5'-cyclic monophosphate (cGMP), adenosine 3'5'-cyclic monophosphate (cAMP), and intracellular calcium, are intensively studied. However, artifact-free manipulation of these messengers is problematic, and simple pharmacology may not allow selective intervention in distinct cell types in a real, complex tissue. A method has been devised by which second messenger levels can be manipulated in cells of choice using the GAL4/UAS system. By placing different receptors (rat atrial natriuretic peptide [ANP] receptor and Drosophila serotonin receptors [5HTDro7 and 5HTDro1A]) under UAS control, they can be targeted to arbitrary defined populations of cells in any tissue of the fly, and second messenger levels can be manipulated simply by adding the natural ligand. The potential of the system is illustrated in the Drosophila renal (Malpighian) tubule, where each receptor has been shown to stimulate fluid secretion, to act through its cognate second messenger, and to be blocked by appropriate pharmacological antagonists. The results have uncovered a new role for cGMP signaling in tubules and also demonstrate the utility of the tubule as a possible in vivo test bed for novel receptors, ligands, or agonists/antagonists (Kerr, 2004).

    Ectopic expression of the rat ANP receptor (GC-A) in tubules was achieved under control of both principal cell and stellate cell GAL4 drivers and a heat-shock (hs) promoter. Expression of the GC-A transgene was confirmed by RT-PCR. Expression of GC-A in tubules confers sensitivity to ANP, with resultant production of cGMP. Measurement of cAMP levels in tubules that express GC-A in principal cells (using the c42 GAL4 driver), stellate cells (using the c742 driver), or ubiquitously, show that cGMP levels are stimulated neither by GC-A receptor, nor by ANP, alone. ANP raises cGMP in a dose-dependent manner in c42-GC-A and c724-GC-A tubules, with a nearly 4-fold maximal increase in cGMP levels in c42-GC-A tubules and a 2-fold cGMP increase in c724-GC-A tubules. Since stellate cells make up only a small fraction of the tubule volume, the apparently lower fold increase in stellate cell cGMP probably reflects a larger absolute rise in cGMP in these cells. The EC50 for ANP in both principal and stellate cells is similar, 10-8 M (Kerr, 2004).

    Similar increases in fluid transport are observed upon stimulation with ANP, when GC-A is expressed either in only principal cells or in stellate cells. Although cGMP signaling has been shown in principal cells, a diuretic role for cGMP in stellate cells had not been demonstrated before. The effects of cGMP in principal and stellate cells are additive; when GC-A is expressed ubiquitously under hs control, maximal secretion rates are higher than when it is targeted to either cell alone (Kerr, 2004).

    cGMP signals in principal cells may activate CNG-type calcium channels, resulting in calcium increase and fluid transport. Targeted expression of the calcium reporter, aequorin, was used to measure changes in intracellular calcium ([Ca2+]i) in GC-A tubules. In tubule principal cells, a biphasic elevation of [Ca2+]i is observed upon ANP stimulation, followed by a sustained secondary rise in [Ca2+]i. By contrast, no change in stellate cell [Ca2+]i was observed upon ANP challenge. Thus, major cellular targets for cGMP in principal cells are CNG channels. Stellate cells, however, must contain uncharacterized cGMP-activated targets that modulate fluid transport (Kerr, 2004).

    ANP-mediated cGMP signaling and increased fluid transport are a result of specific ligand-receptor interactions, since the ANP antagonist, anantin, abolishes ANP stimulation of both cGMP and fluid transport in hs-GC-A tubules in a dose-dependent manner (Kerr, 2004).

    Pilot experiments have shown that Drosophila tubules are insensitive to 5HT; there is thus scope to modulate second messengers by ectopic expression of the cognate GPCRs. The Drosophila 5HT7Dro receptor raises cAMP levels in cultured cells by activating adenylate cyclase. When 5HT7Dro is expressed in either principal or stellate cells using the appropriate GAL4 drivers, 5HT induces dose-dependent production of cAMP. EC50 for 5HT in both lines was 10-7 M. Similarly, the heat shock construct elicits elevation of [cAMP] upon 5HT treatment. Controls (non-heat-shocked hs-5HT7Dro and heat-shocked wild-type or parental lines) show no response to 5HT. Furthermore, there is no detectable impact on cGMP levels when 5HT7Dro is driven either in principal cells, stellate cells, or ubiquitously (Kerr, 2004).

    As would be expected from the literature, increased [cAMP] in principal cells stimulates fluid transport. However, as for cGMP, a previously undocumented diuretic role of cAMP in stellate cells was uncovered, and the maximal rates of 5HT-induced fluid secretion observed with c42-5HT7Dro and c724-5HT7Dro tubules appear to be additive: the sum of maximal rates is approximately equal to the maximal 5HT-induced rate observed in heat-shocked hs-5HT7Dro tubules. It was thus hypothesized that, if stellate cells have the machinery to respond functionally to cAMP and cGMP, then they are also likely to have the machinery to produce the signals (Kerr, 2004).

    As with GC-A, the possibility that [Ca2+]i may be altered upon activation of 5HT7Dro was investigated. In principal cells of 5HT7Dro/c42-aeq tubules, 5HT stimulation results in a biphasic elevation of [Ca2+]i, similar to that seen in GC-A/c42-aeq tubules. However, no [Ca2+]i rise was observed in 5HT-stimulated 5HT7Dro/c710-aeq tubules. These results are consistent with both cAMP and cGMP acting on a CNG channel that is expressed only in principal, and not in stellate, cells (Kerr, 2004).

    It proved possible to reproduce the known pharmacology of this 5HT receptor. The antagonist (+)-butaclamol almost completely attenuated 5HT-stimulated production of cAMP in a dose-dependent manner, with an IC50 of 2.5 × 10-8 M. This agrees precisely with values obtained for the receptor in cell lines. A maximal dose of (+)-butaclamol (10-5 M) also reduced 5HT-stimulated fluid transport in hs-5HT7Dro tubules (Kerr, 2004).

    Another Drosophila 5HT receptor, 5HT1ADro, is known to mobilize intracellular calcium. Expression of 5HT1ADro in either principal or stellate cells results in 5HT-induced calcium responses. As would be expected from the actions of capa and leucokinin (ligands known to act through [Ca2+]i), 5HT also stimulated fluid transport, when 5HT1ADro was expressed in either principal cells or stellate cells, and no effect was seen without a GAL4 driver. Although a comprehensive dose-response curve was not performed, 5HT-stimulated fluid transport was inhibited by yohimbine. The effective concentration for yohimbine in tubules (10-5 M) is comparable with the Ki (18 μM) obtained for this receptor in cell lines (Kerr, 2004).

    The 5HT1ADro receptor has been previously shown to inhibit adenylate cyclase in vitro. Accordingly, cAMP and cGMP were measured in 5HT-stimulated c42-5HT1ADro and c724-5HT1ADro tubules. Activation of the 5HT1ADro receptor in tubules does not affect cyclic nucleotide levels, and thus the stimulatory effects of 5HT on fluid transport are solely due to stimulation of increased [Ca2+]i (Kerr, 2004).

    Although receptor guanylate cyclases (like GC-A) are self-sufficient, the strategy of modulating second messengers by ectopic expression of GPCRs depends on the expression of cognate G proteins in the target cell type. A priori, it was expected that most G proteins would be widely expressed, but this was confirmed in tubules by RT-PCR with primers against all known G protein α, β, and γ subunits. As predicted, all the Gα subunit genes found in Drosophila are expressed in tubules. Tubules also express the full complement of genes encoding the Gγ subunit; the only G protein subunit that does not appear to be expressed in tubules is Gβ76C (Kerr, 2004).

    Although a particular target cell type might not express cognate G proteins, an RT-PCR strategy compared with measurement of second messengers should be informative. As well as the apoaequorin calcium reporter, others are now available, and successful cAMP and cGMP measurements in the tiny (160-cell) tubule suggest that radioimmunoassay is sufficiently sensitive for most Drosophila tissues of interest (Kerr, 2004).

    Obviously, the target cell must not normally express receptors for, or respond to, the ligand of choice. In the case of Drosophila, this condition is satisfied; there is no atrial natriuretic peptide-like sequence encoded within the Drosophila genome. For 5HT receptors or other GPCRs, more caution must be exercised; nonetheless, standard experimental controls would quickly identify any problems. In the case of 5HTDro1A, expression has only been documented in some cells of the embryonic nervous system; 5HTDro7 expression has been found in cells of the embryonic ventral midline, and in adult head, but not body. It is thus likely that these constructs will be useful in most Drosophila nonnervous tissues (Kerr, 2004).

    This study demonstrates successful ectopic expression of vertebrate and Drosophila receptors in Malpighian tubules and quantifies the effects of such expression on signal transduction pathways and physiological output. There are clear results from this approach: (1) the interactions between 3 second messenger pathways were studied in unprecedented detail, in two cell types, in an organotypic context; (2) the results validated all that was known about signaling through known neuropeptides in the tubule; (3) a diuretic role for cAMP and cGMP in stellate cells was demonstrated, inviting the intriguing question as to which extracellular ligands normally activate these pathways; (4) this conserved pharmacology of a vertebrate receptor (rat GC-A) expressed in a model organism illustrates another possibility for functional genomics, i.e., that novel genes could be characterized relatively cheaply and easily by ectopic expression in a model organism where detailed organotypic-phenotypic analysis is possible. The Drosophila Malpighian tubule is an ideal such 'test bed' for genes where organotypic analysis may be important for normal function (Kerr, 2004).

    The utility of this experimental system is more general, allowing sensitive and specific intervention in second messenger signaling in any Drosophila tissue for which there exists a GAL4 driver. Combined with the ready availability of real-time calcium reporters in Drosophila and the possibility of measuring cAMP and cGMP similarly, using transgenic FRET reporters, this simple model organism now has an impressive genetic toolbox for cell signaling studies (Kerr, 2004).

    For more information about Malpighian tubule function, see The Drosophila melanogaster Malpighian tubule WWW page, maintained by Julian Dow at the University of Glasgow. In addition, the Dow laboratory maintains a sensitive map illustrating the physiology of Malpighian tubule secretory cells. Functioning of the V-ATPase is described at Julian Dow's V-ATPase site.

    White participates in vesicular transepithelial transport of cGMP

    Guanosine 3'-5' cyclic monophosphate (cGMP) and adenosine 3'-5' cyclic monophosphate (cAMP) are important regulators of cell and tissue function. However, cGMP and cAMP transport have received relatively limited attention, especially in model organisms where such studies can be conducted in vivo. The Drosophila Malpighian (renal) tubule transports cGMP and cAMP and utilises these as signalling molecules. This study shows via substrate competition and drug inhibition studies that cAMP transport - but not cGMP transport - requires the presence of di- or tri-carboxylates; and that transport of both cyclic nucleotides occurs via ATP binding cassette sub-family G2 (ABCG2), but not via ABC sub-family C (ABCC), transporters. In Drosophila, the white (w) gene is known for the classic eye colour mutation. However, gene expression data show that of all adult tissues, w is most highly expressed in Malpighian tubules. Furthermore, as White is a member of the ABCG2 transporter class, it is a potential candidate for a tubule cGMP transporter. Assay of cGMP transport in w- (mutant) tubules shows that w is required for cGMP transport but not cAMP transport. Targeted over-expression of w in w- tubule principal cells significantly increases cGMP transport compared with that in w- controls. Conversely, treatment of wild-type tubules with cGMP increases w mRNA expression levels, implying that cGMP is a physiologically relevant substrate for White. Immunocytochemical localisation reveals that White is expressed in intracellular vesicles in tubule principal cells, suggesting that White participates in vesicular transepithelial transport of cGMP (Evans, 2008).

    The first Drosophila mutation to be identified was white (w), and it was instrumental to Morgan's description of genes and chromosomes. Despite over 3000 publications on w since then, few have investigated the biological function of White protein, tending instead to concentrate on the genetics of w. The most prominent phenotype of w mutants is the pronounced lack of eye colour. However, eye colour phenotypes can also reflect defects in Malpighian tubule structure and function, because several eye pigment precursors [notably the transport of compounds in the xanthommatin biosynthetic pathway are stored and processed in the larval tubule before being released into the pupal haemocoel for uptake by the developing adult eye. The Malpighian tubules of w- Drosophila are clear or whitish in appearance due to the absence of tryptophan metabolites and pteridines, unlike wild-type tubules, which are yellow in appearance (Evans, 2008 and references therein).

    Insect Malpighian tubules are critical for survival and play essential roles in osmoregulation, homeostasis and immune function (Dow, 2005). Excess fluid and solutes are transported and excreted largely across these blind-ended tubules, with selective re-absorption occurring in the rectum. Drosophila tubules provide a unique model for studying transport in a live polarised epithelial tissue, with added benefits of the availability of genetic tools. cGMP and cAMP signalling has been studied in the Drosophila Malpighian tubule for over 10 years, but the transport mechanisms of these cyclic nucleotides have received rather less attention. Nitric oxide (NO)-cGMP signalling was first identified to stimulate fluid transport, with more recent work implicating NO signalling in the immune response (McGettigan, 2005). Furthermore, NO-cGMP signalling can be induced by activation of the capa receptor in Dipteran tubules from several species (Pollock, 2004); and so is an important feature of tubule function in insect vectors of disease. Exogenous cAMP also stimulates fluid transport, with fluid transport also being stimulated by corticotropin-releasing factor (CRF)-like peptide (Cabrero, 2002) and calcitonin-like peptide (Evans, 2008 and references therein).

    cGMP and cAMP are transported into the Malpighian tubules; cGMP transport across the tubule (efflux) is modulated by cGMP-dependent phosphodiesterases (cG-PDEs). In mammalian systems, cyclic nucleotide transport has been attributed to a number of ATP binding cassette (ABC) transporters and solute carriers (members of the SLC22 family). These transporters have been widely studied in mammals but equivalent transporters have not previously been identified in dipteran insects (Evans, 2008).

    White is a member of the ABC transporter subfamily G, with greatest sequence similarity to human ABCG2. Investigations of the role of White in Malpighian tubules were carried out ~30 years ago, and tentatively identified location of and transport substrates for White. The location of White in the tubules was thought to be either the basolateral membrane or the pigment storage vesicle membranes. Potential substrates for White included tryptophan, kynurenine, 3-hydroxykynurenine, guanine and riboflavin (Evans, 2008 and references therein).

    This study demonstrates that cGMP transported by the tubule occurs via ABCG2 transporters, and that White is required for cGMP but not cAMP, transport across the Malpighian tubule. Localisation of White to intracellular vesicles in the principal cells in the main, fluid-transporting segment of the tubule, indicates a possible storage-excretion mechanism of cGMP transport via vesicles (Evans, 2008).

    Uptake and excretion of cGMP by Drosophila Malpighian tubules has been documented (Riegel, 1998). This study shows that the transport of cGMP is sensitive to the electrical gradient, suggesting that a secondary active transport mechanism is involved. Secreted fluid is capable of stimulating cGMP-dependent protein kinase (cGK) activity, suggesting that as in mammals, cGMP can be transported across the tubule membrane in unaltered form in Drosophila. Although primary active transport could not be shown unambiguously, the ABCG transport ATPase, White, is nonetheless necessary for significant cGMP flux; it must thus, at least, facilitate the diffusion of cGMP. In tubules, White is expressed throughout the cytoplasm in large vesicles of the main segment. These vesicles are probably the pigment vesicles which were suspected to be the location of White in a previous study (Sullivan, 1980). Other sub-families of ABC transporters also show intracellular localisation: ABCC4 transporters localise to the dense granules of platelets, and not at the plasma membrane. Thus, it seems that sub-families of the ABC transporter family, in both vertebrates and invertebrates, can be localised to vesicular structures in the cytoplasm of the cell. The first identified ABCG transporter in Leishmania, LiABCG4, has been shown to localise to the plasma membrane and to post-Golgi secretory vesicles when overexpressed in yeast (Castanys-Munoz, 2007). However, in the parasite, LiABCG4 is mainly localised to the plasma membrane, with some localisation in flagellar pockets; suggesting that the localisation in secretory vesicles in yeast may be due to over-expression of the ABCG4 transporter in the yeast system. Although the vesicular localisation of White in tubule cells may be due to targeted over-expression of w+, localisation of White in non-transgenic tubules (Sullivan, 1980) suggests that vesicular localisation for White is not associated with expression artifacts in vivo (Evans, 2008).

    The primarily vesicular localisation of White may also indicate trafficking of cGMP across the Malpighian tubules in vesicles, a novel mechanism of transepithelial cGMP transport that would not compromise the integrity of intracellular cGMP signalling pathways. This would explain why fluid transport assays on tubules from w loss-of-function mutants show similar rates of cGMP-induced fluid transport (Davies, 1995; Dow, 1994b) to wild-type tubules. The current model is thus that cGMP is transported into the cell by a basolateral plasma membrane cGMP transporter that still remains to be discovered; once in the cell, it can act to stimulate fluid transport. However, White sequesters cGMP into vesicles, contributing [perhaps together with the action of DmPDE6 (Day, 2006) to its clearance from the cell; and these vesicles are excreted from the apical surface of the cell, presumably as part of a general purpose organic solute clearance mechanism. In the absence of White, cGMP is still transported into cells but due to reduced uptake into intracellular vesicles, is transported into the lumen at a much reduced rate. It will be interesting in due course to try to identify the plasma membrane transporter for cGMP. Importantly, this multi-stage transport model explains why it was not possible to demonstrate accumulation of cGMP beyond Nernst-predicted ratios, despite the involvement of a transport ATPase in the process (Evans, 2008).

    Although the w gene has almost exclusively been researched in association with its role as an eye colour marker, recent microarray data of adult fly tissues (Chintapalli, 2007) has shown that w is most highly expressed in Malpighian tubule, a tissue enriched for organic anion transporters. Previous studies have shown that White can act as a heteromeric transporter: with Scarlet, it is a tryptophan transporter, responsible for brown eye colour; with Brown, it transports guanine, the precursor of the red pigment in eye. Mutation of key residues such as glycine 589 in the fifth transmembrane helix of White, significantly reduces guanine transport by White-Brown heterodimers, suggesting the importance of G589 in heterodimerisation and in guanine transport. Interestingly, mutation of amino acid 553 in TM5 of ABCG2 (a well-conserved residue corresponding to G589 in White) disrupts function and trafficking of ABCG2, implying conservation of dimerisation function of these residues across evolution (Evans, 2008 and references therein).

    Even if White does play a key role in cGMP transport, organic solute transporters are heavily represented in the tubule transcriptome, and so there are other potential candidate transporters. In mammals, transporters of the ABCC class, notably MRP4 (ABCC4) and MRP5 (ABCC5) have been shown to transport cyclic nucleotides out of the cell. Treatment of Drosophila tubules with glibenclamide, a broad-spectrum inhibitor of ABC transporters, results in inhibition of both cAMP and cGMP transport. However, utilising either known inhibitors or competing substrates for ABBC transporters shows that such transporters are not involved in cAMP/cGMP transport by Malpighian tubules. Interestingly, tubules express five of the seven Drosophila homologues of mammalian ABCC transporters (Day, 2006); with one gene, CG9270, being expressed only in tubules of the adult fly. It would be interesting to screen the product of this gene for ABCC transporter function, and to determine its substrate specificity. In any case, the lack of effect of ABCC-specific drugs on the tubule could indicate evolutionary divergence between mammalian and Drosophila ABCC transporters, or may simply reflect inaccessibility of these transporters to specific drugs if the transporters are localised to the apical membrane (Evans, 2008).

    This study has shown that cGMP transport by the tubule is specific and possesses distinct properties from cAMP transport. Previous investigations have suggested that there may be some overlapping function of cyclic nucleotide transporters. The different conclusions reached by these different studies may be a reflection of the concentration of each cyclic nucleotide used - the competing cyclic nucleotide was greatly in excess in the Riegel study. This study shows that cAMP transport requires the presence of di- or tri-carboxylates; but that cGMP transport is unaffected by these compounds. cAMP transport probably requires an OAT-like transporter at the basolateral membrane, whereas cGMP is transported via a different mechanism. Thus in the tubules, the mechanisms of transport of cGMP and cAMP are largely independent and specific. This could reflect the importance of these signalling molecules in the tubules. Alternatively, it could reflect the transport mechanisms necessary in a tissue for which there is a requirement for an established potential gradient to enable solute uptake and excretion (Evans, 2008).

    Overall, though, the results suggest that the extraordinary abundance of White in the adult Malpighian tubule may reflect a novel role that continues beyond the need to handle visual pigment precursors in the larva and pupa. White is thus a more versatile transport protein that previously suspected (Evans, 2008).

    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. This study resolved 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 (see Leucokinin) 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 (Cabrero, 2014).

    Epithelia provide essential barrier and vectorial transport capabilities intrinsic to the success of higher organisms. Depending on their roles and specializations, epithelia may be termed tight or leaky, based on their electrical conductance, which in turn depends on the patency of their paracellular spaces. Tight epithelia are thought to have a highly restricted paracellular route because of prominent tight junctions (septate junctions in insects), so constraining transepithelial fluxes to a transcellular route. Leaky epithelia, traditionally associated with high flux rates, lack such prominent tight junctions (Cabrero, 2014).

    Insect renal (Malpighian) tubules move fluid at the highest rates observed in biology, and thus would seem to be ideal candidates for leaky epithelia. However, insect tubule cells are typically large and multinucleate or polyploid (large cell diameters minimize the junctional circumference per unit transporting membrane), and the cells are also surrounded by prominent septate junctions. Nonetheless, it has been argued that, in tubules of the dengue fever mosquito Aedes aegypti, the rapid neurohormonally controlled chloride shunt conductance is paracellular, caused by remodeling of the septate junctions with switch-like speed under the influence of diuretic peptides of the kinin family (Cabrero, 2014).

    Drosophila melanogaster is a member of one of the largest insect orders, the Diptera, and these tubules are distinguished by a prominent secondary cell type, the stellate cell. The powerful transgenic toolbox available for Drosophila allows cell-specific contributions to tissue-level function to be probed; by expressing the luminescent calcium reporter apoaequorin transgenically in specific cell types, it was possible to show that kinin signals specifically through intracellular calcium in only the stellate cells-and not the principal cells-consistent with the observed expression of the kinin receptor in just stellate cells. The biogenic amine tyramine also acts similarly; that is, it stimulates fluid secretion by activating a chloride shunt conductance by raising intracellular calcium levels in the stellate cells. Thus-at least in Drosophila-the stellate cell plays a crucial role in transducing the diuretic kinin signal into a rapid increase in chloride conductance (Cabrero, 2014).

    In practice, both transcellular and paracellular routes of chloride conductance are likely to contribute to both resting and kinin-stimulated fluid secretion, but the relative importance of the two routes is not known. This study used Drosophila transgenics-combined with physiology, electrophysiology, and imaging-to show that a CLC chloride channel encoded by ClCa/ CG31116 is a basolateral and apical plasma membrane chloride channel, uniquely localized to stellate cells, that is essential for neuropeptide-stimulated, but not resting, levels of secretion. Furthermore, by generating Drosophila that were transgenic for a membrane-targeted fluorescent chloride reporter, this study demonstrated that tubule stellate cells displayed a characteristic intracellular chloride signature upon kinin stimulation. Therefore, this issue has bee resolved in Drosophila; kinin stimulates chloride flux by a transcellular route that is confined to the stellate cells (Cabrero, 2014).

    Only two of the three CLC genes encoded by the D. melanogaster genome show highly abundant expression in adult Malpighian tubule. By contrast, ClC-b shows low-level expression consistent with a predicted role as a housekeeping endomembrane channel. Drosophila ClC-b (CG8594) is a homolog of human CLCN7 (CLC-7), an endosomal and lysosomal Cl- channel that is thought to act as a H+/2Cl- exchanger that provides the major chloride conductance of lysosomes. Based on these distributions- and the functions of their respective homologs in humans-this study focused attention on the functional roles of ClC-a and -c (Cabrero, 2014).

    Drosophila ClC-a (CG31116) is a homolog of human CLCN2 (CLC-2), a plasma membrane inwardly rectifying chloride channel functionally significant in airway epithelia and the central nervous system. In epithelia, CLCN2 is apical and has been suggested to play a role in Cl- efflux (Cabrero, 2014).

    The GAL4/UAS binary expression system allows the knockdown of specific transcripts in specific cells within an otherwise normal organism, under control of the appropriate GAL4 driver lines. The Vienna UAS-dsRNA line ClC-akk101247 produced a significant knockdown (>70%) in overall tubule expression of ClC-a when driven in stellate cells, but no knockdown when driven in principal cells, suggesting that ClC-a is expressed uniquely in the stellate cells (Cabrero, 2014).

    To determine whether ClC-a knockdown in stellate cells affected tubule physiology, tubule secretion was measured in control and ClC-a knockdown tubules. ClC-a RNAi knockdown in stellate cells had no effect on basal fluid secretion levels, but abolished the stimulation of fluid secretion normally induced by Drosophila kinin, a neuropeptide that activates the tubule chloride shunt pathway in Drosophila and other insects. By contrast, knockdown of ClC-a in principal cells had no effect on either resting or stimulated secretion. Therefore, ClC-a in stellate cells is essential for kinin-stimulated, but not resting, fluid secretion (Cabrero, 2014).

    ClC-a knockdown might have an impact indirectly on fluid secretion, rather than directly on chloride conductance. The electrophysiological signature of kinin action is a rapid abolition of by a completely different route-for example, a paracellular conductance (Cabrero, 2014).

    Stellate-cell-specific ClC-a knockdown also produced an inflated abdomen phenotype in adult flies, implying defective osmoregulation. To confirm that the abdominal bloating was due to increased haemolymph water content, wet-dry weight measurements were performed. Wet weight measurements revealed that ClC-a knockdown male and female adults are significantly heavier compared with control flies, whereas dry weight measurements are equivalent. Interestingly, the same distended abdomen phenotype was observed upon knockdown of teashirt, a transcription factor that regulates stellate cell differentiation and renal physiology in Drosophila (Cabrero, 2014).

    If kinin is indeed activating a stellate-cell-specific chloride shunt conductance, it may be possible to detect transient changes in intracellular chloride on kinin stimulation. To monitor stellate cell intracellular concentration of Cl- ([Cl-]i), flies were made transgenic for a genetically encoded fluorescent combined Cl-/pH - biosensor (ClopHensor), under UAS control, allowing its cell-specific expression. The fluorescence of the PalmPalm-ClopHensor confirms that this transgenic sensor is typically localized to plasma and intracellular membranes, as seen in other systems (Cabrero, 2014).

    First, the ratiometric changes were measured in RCl (F458/F545) of the PalmPalm-ClopHensor, in response to application of 75 mM KCl to either single stellate cells or individual Malpighian tubules, using confocal microscopy or spectrophotometer recording, respectively, to confirm the chloride sensitivity of the system. Kinin stimulation in the tubule caused a strong increase of [Cl-]i, likely due to chloride ion passage through the basolateral plasma membrane. After peaking, [Cl-]i rapidly stabilized to values similar to normal, possibly due to partial desensitization of the kinin response and redistribution of chloride ions through the ClC-a chloride channel in the stellate cells or a transient mismatch between activation of basal and apical conductances. These changes were dramatically reduced in reporter lines in which ClC-a was specifically knocked down in only in tubule stellate cells, confirming that the changes seen were due to ClC-a chloride channel activation by kinin (Cabrero, 2014).

    Real-time PCR data show that ClC-a expression is primarily in the stellate cells, because only knockdown in stellate cells significantly impinges on overall tubule expression level. Although ClC-a is a homolog of a plasmamembrane chloride channel, and its knockdown leads to specific effects on transepithelial transport, it is conceivable that it could be an intracellular channel that in some way impinges indirectly on transepithelial chloride flux. Accordingly, it is important to establish where in the stellate cell it resides (Cabrero, 2014).

    A specific antibody was raised against ClC-a, and it was validated by Western blotting. Immunocytochemistry showed specific labeling of only stellate cells within the tubule, and confocal microscopy revealed that the location was on the plasma membrane. By overexpressing in the stellate cell a known apical membrane aquaporin channel, Drip, labeled with enhanced YFP (eYFP), it was possible to show that ClC-a both colocalizes apically with DRIP, and prominently labels the basal membrane. So although additional chloride transport routes cannot be excluded, ClC-a is present in both apical and basal plasma membranes and is both necessary and sufficient for kinin response. Therefore, a plasma membrane ClC-a chloride channel, uniquely in the stellate cells, is essential for the action of kinin to stimulate fluid production by increasing the chloride conductance, so collapsing the transepithelial membrane potential (Cabrero, 2014).

    It was also possible to confirm by immunocytochemistry the efficiency of the knockdown of ClC-a expression at the protein level. Only down-regulation of ClC-a in the stellate cell was able to replicate the effect of blocking the antibody with the antigenic peptide (Cabrero, 2014).

    ClC-a is not the only chloride channel enriched in the tubule; ClC-c (CG5284), a homolog of human CLCN3 (CLC-3) known to play both plasma membrane and epithelial roles, is also tubule-enriched. In the plasma membrane of epithelia, it is implicated in cell swelling. It is also strongly expressed in the nervous system, where it is found in endosomal compartments and synaptic vesicles; mice deficient for CLCN3 show gross neurological defects, such as blindness and loss of the hippocampus. Accordingly, the equivalent experiments were performed on a knockdown of ClC-c. By contrast with ClC-a, knockdown of ClC-c in principal cells knocked down overall tubule Clc-c expression levels, whereas knockdown in stellate cells produced compensatory up-regulation of whole tubule expression, suggesting that ClC-c is expressed in both cell types and does not play a stellate-specific role. Similarly, fluid secretion was only partially reduced in stellate-specific ClC-c knockdown. Significantly, electrophysiological analysis showed that knockdown of ClC-c in either principal or stellate cells did not have an impact on kinin-induced depolarization of the transepithelial potential, implying that ClC-c is not involved in the chloride shunt pathway, and so any impact on fluid secretion is through some other mechanism. Consistent with the quantitative PCR (q-PCR) data, a specific antibody against ClC-c, stained both principal and stellate cells uniformly. Therefore, it is concluded that, although ClC-c is important for tubule function, it is not a direct participant in transepithelial chloride ion transport (Cabrero, 2014).

    The relative contributions of transcellular and paracellular flux and of specialized cell types in the integrated performance of an epithelium is a fundamental and important issue, in which experimental techniques have been limiting. By selecting the Drosophila as an experimental subject, powerful cell-type–specific transgenics provide valuable methodologies to address such questions. Previously studies used cell-type–specific calcium reporters to show that the kinin-induced calcium signal is exclusively expressed in stellate cells, implying that they alone mediate the tissue response to kinin; this study directly show that kinin actions on chloride flux- evidenced independently by increase in fluid secretion, increase in intracellular chloride, and decrease in transepithelial potential- all depend obligatorily on the presence of the plasma membrane chloride channel ClC-a. These results confirm the proposed spatial segregation of cation and anion transport in Drosophila into two distinct cell types and suggest that the paracellular pathway is more likely to account for basal, rather than kinin-stimulated, fluid secretion (Cabrero, 2014).

    Although these transgenic technologies are not readily applied to other species, the model developed in Drosophila is likely to have more general application. In both the dengue vector Aedes aegypti, and the malaria vector Anopheles gambiae, kinin receptors have been localized to the stellate cells. Indeed, stellate cells are considered general to the Diptera. Excitingly, differentiated characters of the stellate role are under control of teashirt in Drosophila, and orthologs of teashirt have been found to be localized to previously undocumented secondary cells in highly diverged insect groups such as Orthoptera and Coleoptera. It may well be that the uniquely high secretory rates of many insect renal tubules can be explained by the model shown in this study (Cabrero, 2014).

    Two inwardly rectifying potassium channels, Irk1 and Irk2, play redundant roles in Drosophila renal tubule function

    Inwardly rectifying potassium channels play essential roles in renal physiology across phyla. Barium-sensitive K+ conductances are found on the basolateral membrane of a variety of insect Malpighian (renal) tubules, including Drosophila melanogaster. This study found that barium decreases the lumen-positive transepithelial potential difference in isolated perfused Drosophila tubules, and decreases fluid secretion and transepithelial K+ flux. In those insect species in which it has been studied, transcripts from multiple genes encoding inwardly rectifying K+ channels are expressed in the renal (Malpighian) tubule. In Drosophila melanogaster, this includes transcripts of the Irk1, Irk2 and Irk3 genes. The role of each of these gene products in renal tubule function is unknown. Simultaneous knockdown of Irk1 and Irk2 in the principal cell of the fly tubule decreases transepithelial K+ flux, with no additive effect of Irk3 knockdown, and decreases barium sensitivity of transepithelial K+ flux by approximately 50%. Knockdown of any of the three inwardly rectifying K+ channels individually has no effect, nor does knocking down Irk3 simultaneously with Irk1 or Irk2. Irk1/Irk2 principal cell double knockdown tubules remain sensitive to the kaliuretic effect of cAMP. Inhibition of the Na+/K+-ATPase with ouabain and Irk1/Irk2 double knockdown have additive effects on K+ flux, and 75% of transepithelial K+ transport is due to Irk1/Irk2 or ouabain-sensitive pathways. In conclusion, Irk1 and Irk2 play redundant roles in transepithelial ion transport in the Drosophila melanogaster renal tubule, and are additive to Na+/K+-ATPase-dependent pathways (Wu, 2015).

    Early gene Broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster

    In insects, humoral response to injury is accomplished by the production of antimicrobial peptides (AMPs) which are secreted in the hemolymph to eliminate the pathogen. Drosophila Malpighian tubules (MTs), however, are unique immune organs that show constitutive expression of AMPs even in unchallenged conditions and the onset of immune response is developmental stage dependent. Earlier reports have shown ecdysone positively regulates immune response after pathogenic challenge however, a robust response requires prior potentiation by the hormone. This study provides evidence to show that MTs do not require prior potentiation with ecdysone hormone for expression of AMPs and they respond to ecdysone very fast even without immune challenge, although the different AMPs Diptericin, Cecropin, Attacin, Drosocin show differential expression in response to ecdysone. Early gene Broad complex (BR-C) could be regulating the IMD pathway by activating Relish and physically interacting with it to activate AMPs expression. BR-C depletion from Malpighian tubules renders the flies susceptible to infection. It was also shown that in MTs ecdysone signaling is transduced by EcR-B1 and B2. In the absence of ecdysone signaling the IMD pathway associated genes are down-regulated and activation and translocation of transcription factor Relish is also affected (Verma, 2015).

    KANK deficiency leads to podocyte dysfunction and nephrotic syndrome

    Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of progressive renal function decline and affects millions of people. This study identified recessive mutations in kidney ankyrin repeat-containing protein 1 (KANK1), KANK2, and KANK4 in individuals with nephrotic syndrome. In an independent functional genetic screen of Drosophila cardiac nephrocytes, which are equivalents of mammalian podocytes, it was determined that the Drosophila KANK homolog (dKank) is essential for nephrocyte function. RNAi-mediated knockdown of dKank in nephrocytes disrupted slit diaphragm filtration structures and lacuna channel structures. In rats, KANK1, KANK2, and KANK4 all localized to podocytes in glomeruli, and KANK1 partially colocalized with synaptopodin. Knockdown of kank2 in zebrafish recapitulated a nephrotic syndrome phenotype, resulting in proteinuria and podocyte foot process effacement. In rat glomeruli and cultured human podocytes, KANK2 interacted with ARHGDIA, a known regulator of RHO GTPases in podocytes that is dysfunctional in some types of nephrotic syndrome. Knockdown of KANK2 in cultured podocytes increased active GTP-bound RHOA and decreased migration. Together, these data suggest that KANK family genes play evolutionarily conserved roles in podocyte function, likely through regulating RHO GTPase signaling (Gee, 2002).

    Expression of polyQ aggregates in Malpighian tubules leads to degeneration in Drosophila melanogaster

    Polyglutamine (polyQ) disorders are caused by expanded CAG (Glutamine) repeats in neurons in the brain. The expanded repeats are also expressed in the non-neuronal cells, however, their contribution to disease pathogenesis is not very well studied. This study expressed a stretch of 127 Glutamine repeats in Malpighian tubules (MTs) of Drosophila melanogaster as these tissues do not undergo ecdysone induced histolysis during larval to pupal transition at metamorphosis. Progressive degeneration, which is the hallmark of neurodegeneration was also observed in MTs. The mutant protein forms inclusion bodies in the nucleus resulting in expansion of the nucleus and affect chromatin organization which appear loose and open, eventually resulting in DNA fragmentation and blebbing. A virtual absence of tubule lumen was observed followed by functional abnormalities. As development progressed, severe abnormalities affecting pupal epithelial morphogenesis processes were observed resulting in complete lethality. Distribution of heterogeneous RNA binding protein (hnRNP), HRB87F, Wnt/wingless and JNK signaling and expression of Relish was also found to be affected. Expression of multi-drug resistance genes following polyQ expression was up regulated. The study gives an insight into the effects of polyQ aggregates in non-neuronal tissues (Yadav, 2015).

    Segment-specific Ca transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages

    Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca2+ transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of Drosophila. Basolateral Ca2+ transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca2+ fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca2+ regulation. Basolateral Ca2+ transport across distal tubules of Drosophila varied throughout the life cycle; Ca2+ was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults 168h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca2+ uptake and Ca2+ release across the basolateral surface of the distal segment of the same tubule, indicating that Ca2+ transport is bidirectional. Ca2+ uptake across the distal segment of tubules of young adults and Ca2+ release across the distal segment of tubules of older adults was also suggestive of reversible Ca2+ storage. These results suggest that the distal tubules of Drosophila are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca2+ sequestration during periods of high dietary calcium intake and passive Ca2+ release during periods of calcium deficiency (Browne, 2016).

    The corticotropin-releasing factor-like diuretic hormone 44 (DH) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster

    Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. This study demonstrates roles for CRF-like diuretic hormone 44 (DH44) and Leukokinin (Lk) in desiccation and starvation tolerance. Gene expression and labelled DH44 ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH44 in neurons of the pars intercerebralis and DH44 receptor (DH44-R2) in Malpighian tubule principal cells, indicate that suppression of DH44 signalling improves desiccation tolerance of the intact fly. Leucokinin receptor (Lkr), is expressed in DH44 neurons as well as in stellate cells of the Malpighian tubules. Lkr knockdown in DH44-expressing neurons reduces Malpighian tubule-specific Lkr, suggesting interactions between DH44 and LK signalling pathways. Finally, although a role for DK in desiccation tolerance was not defined, a novel role was demonstrated for Malpighian tubule cell-specific Lkr in starvation tolerance. Starvation increases gene expression of epithelial LKR. Also, Malpighian tubule stellate cell-specific knockdown of LKR significantly reduced starvation tolerance, demonstrating a role for neuropeptide signalling during starvation stress (Cannell, 2016).

    Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila

    Multiple neuropeptides are known to regulate water and ion balance in Drosophila melanogaster. Several of these peptides also have other functions in physiology and behavior. Examples are corticotropin-releasing factor-like diuretic hormone (diuretic hormone 44; DH44) and leucokinin (LK), both of which induce fluid secretion by Malpighian tubules (MTs), but also regulate stress responses, feeding, circadian activity and other behaviors. This study investigated the functional relations between the LK and DH44 signaling systems. DH44 and LK peptides are only colocalized in a set of abdominal neurosecretory cells (ABLKs). Targeted knockdown of each of these peptides in ABLKs leads to increased resistance to desiccation, starvation and ionic stress. Food ingestion is diminished by knockdown of DH44, but not LK, and water retention is increased by LK knockdown only. Thus, the two colocalized peptides display similar systemic actions, but differ with respect to regulation of feeding and body water retention. It was also demonstrated that DH44 and LK have additive effects on fluid secretion by MTs. It is likely that the colocalized peptides are coreleased from ABLKs into the circulation and act on the tubules where they target different cell types and signaling systems to regulate diuresis and stress tolerance. Additional targets seem to be specific for each of the two peptides and subserve regulation of feeding and water retention. These data suggest that the ABLKs and hormonal actions are sufficient for many of the known DH44 and LK functions, and that the remaining neurons in the CNS play other functional roles (Zandawala, 2017).

    This study reveals that a portion of the LK-expressing neurosecretory cells (ABLKs) in abdominal ganglia co-express DH44, similar to earlier findings in the moth Manduca sexta, the locust Locusta migratoria, and blood-sucking bug Rhodnius prolixus. Colocalization of these peptides in multiple insect orders, including basal orders, suggests that this colocalization and the subsequent functional interaction between these signaling systems evolved early on during insect evolution. Since ABLKs are the sole neurons producing both peptides in Drosophila, it was possible to use GAL4 lines to knock down each of the two peptides in these cells only and thereby isolate the contribution of the ABLKs to the physiology. This enabled establishing that these neuroendocrine cells are sufficient for many of the functions assigned to DH44 and LK and therefore these functions are hormonally mediated. In contrast, earlier studies were based upon altering peptide levels or activity in entire populations of DH44 and LK neurons. Also, this study showed that the LK-GAL4 driver includes a set of ectopic brain cells (ipc-1) that do not express LK, but another peptide ITP. The ipc-1 neurons produce sNPF and tachykinin in addition to ITP and have been found to regulate stress responses. This means that in earlier studies, where the LK-GAL4 line was used to inactivate or activate neurons, additional phenotypes are likely to have arisen. Using the current approach, only ABLK neurons were targetted, it was found that both DH44-RNAi and Lk-RNAi in these cells increase resistance to desiccation, starvation, and ionic stress. This suggests that diminishing the release of these two peptides from ABLKs is sufficient for this phenotype to occur. However, food intake is not affected by LK knockdown in ABLKs, whereas DH44 knockdown diminishes feeding, and conversely the knockdown of LK in ABLKs results in increased body water content that is not seen after DH44-RNAi. Thus, the two colocalized peptides appear to display similar systemic actions, but differ with respect to feeding and water retention. When knocking down LK in all LK neurons, a very similar set of effects was obtained to those when only the ABLKs were targeted, indicating that in the assays that were performed in this study the other two sets of LK neurons (LHLK and SELK) played a minimal role (Zandawala, 2017).

    Interestingly, knockdown of DH44 in ABLKs increases resistance to desiccation and decreases feeding, but these effects were not seen when DH44 was diminished in all DH44 neurons. This is consistent with previous work where inactivation or activation of DH44 neurons had no effect on food intake. Perhaps, the effects seen following ABLK manipulations could be compensated by action of the six DH44-expressing MNCs in the brain. Similarly, reduction of LK in ABLKs causes a slight increase in time of recovery from chill coma, but this is not noted after global knockdown of LK. This minor difference could possibly be attributed to the strength of the two GAL4 driver lines used and, thus, the efficiency of LK knockdown in ABLKs (Zandawala, 2017).

    This study also demonstrated that DH44 and LK have additive effects on fluid secretion in MTs. It is likely that these two colocalized peptides are released together and act on the MTs where they target different cell types, receptors, signaling systems, and effectors to regulate fluid secretion. The action of these peptides on the MTs may also in part be responsible for the regulation of stress responses seen in the assays, as shown earlier for CAPA peptide and DH44. It is, however, not clear whether the altered food intake and water retention after DH44 and LK knockdown, respectively, are direct actions on target tissues or indirect effects caused by altered water and ion regulation in the fly (Zandawala, 2017).

    Not only do the ABLKs produce two diuretic hormones, but they also seem to be under tight neuronal and hormonal control. Receptors for several neurotransmitters and peptides have been identified on these cells in adults: the serotonin receptor 5-HT1B, LK receptor (LkR), and the insulin receptor dInR. Knockdown of the 5-HT1B receptor in ABLK neurons diminished LK expression, increased desiccation resistance, and diminished food intake, but manipulations of dInR expression in these cells generated no changes in physiology in the tests performed. In larvae, all ABLKs colocalize LK and DH44, and several receptors have been detected in addition to 5-HT1B and dInR [29], namely RYamide receptor, SIFamide receptor, and the ecdysis-triggering hormone (ETH) receptor ETHR-A. However, the expression of these receptors on adult ABLKs has so far not been investigated. Interestingly, the functions of ABLKs in larvae, studied so far, seem to be primarily related to regulating muscle activity and ecdysis motor patterns. The 5-HT-1B receptor on ABLKs was shown to modulate locomotor turning behavior, whereas ETH-mediated activation of ETHR-A on ABLKs initiates the pre-ecdysis motor activity. In this context, it is worth noting that during metamorphosis six to eight novel ABLKs differentiate anteriorly in the abdominal ganglia, and these are the ones that display the strongest expression of DH44. In adult flies, the ABLKs are neurosecretory cells with restricted arborizations in the CNS, but widespread axon terminations along the abdominal body wall and in the lateral heart nerves, whereas in larvae the same cells send axons that terminate on segmental abdominal muscles, muscle 8. It is not yet known whether larval ABLKs are involved in the regulation of diuresis and other related physiological functions in vivo, but certainly larval functions in locomotion and ecdysis behavior are specific to that developmental stage. Thus, it seems that there is a developmental switch of function in this set of peptidergic neuroendocrine cells (Zandawala, 2017).

    In summary, this study shows that a set of abdominal neuroendocrine cells, ABLKs, co-expressing DH44 and LK, are sufficient for regulation of resistance to desiccation, starvation and ionic stress, as well as modulating feeding and water content in the body. These ABLKs represent a subset of neurons that express DH44 and LK, and the functions of the remaining neurons are yet to be determined (Zandawala, 2017).

    The orphan pentameric ligand-gated ion channel pHCl-2 is gated by pH and regulates fluid secretion in Drosophila Malpighian tubules

    Pentameric ligand-gated ion channels (pLGICs) constitute a large protein superfamily in metazoa whose role as neurotransmitter receptors mediating rapid, ionotropic synaptic transmission has been extensively studied. Although the vast majority of pLGICs appear to be neurotransmitter receptors, the identification of pLGICs in non-neuronal tissues and homologous pLGIC-like proteins in prokaryotes points to biological functions, possibly ancestral, that are independent of neuronal signaling. This study reports the molecular and physiological characterization of a highly divergent, orphan pLGIC subunit, pHCl-2 (CG11340), in Drosophila melanogaster. pHCl-2 forms a channel that is insensitive to a wide array of neurotransmitters, but is instead gated by changes in extracellular pH. pHCl-2 is expressed in the Malpighian tubules, which are non-innervated renal-type secretory tissues. pHCl-2 is localized to the apical membrane of the epithelial principal cells of the tubules, and loss of pHCl-2 reduces urine production during diuresis. These data implicate pHCl-2 as an important source of chloride conductance required for proper urine production, highlighting a novel role for pLGICs in epithelial tissues regulating fluid secretion and osmotic homeostasis (Feingold, 2016).

    Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes

    The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. This study analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. These results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease (Fu, 2017).

    Drosophila imaginal disc growth factor 2 is a trophic factor involved in energy balance, detoxification, and innate immunity

    Drosophila imaginal disc growth factor 2 (IDGF2) is a member of chitinase-like protein family (CLPs) able to induce the proliferation of imaginal disc cells in vitro. This study characterized physiological concentrations and expression of IDGF2 in vivo as well as its impact on the viability and transcriptional profile of Drosophila cells in vitro. IDGF2 was shown to be independent of insulin and protects cells from death caused by serum deprivation, toxicity of xenobiotics or high concentrations of extracellular adenosine (Ado) and deoxyadenosine (dAdo). Transcriptional profiling suggested that such cytoprotection is connected with the induction of genes involved in energy metabolism, detoxification and innate immunity. IDGF2 was hown to be an abundant haemolymph component, which is further induced by injury in larval stages. The highest IDGF2 accumulation was found at garland and pericardial nephrocytes supporting its role in organismal defence and detoxification. These findings provide evidence that IDGF2 is an important trophic factor promoting cellular and organismal survival (Broz, 2017).

    Identification of multiple functional receptors for tyramine on an insect secretory epithelium

    The biogenic amine tyramine (TA) regulates many aspects of invertebrate physiology and development. Although three TA receptor subtypes have been identified (TAR1-3), specific receptors have not been linked to physiological responses in native tissue. In the Malpighian (renal) tubule of Drosophila melanogaster, TA activates a transepithelial chloride conductance, resulting in diuresis and depolarization of the transepithelial potential. In the current work, mutation or RNAi-mediated knockdown in the stellate cells of the tubule of TAR2 (tyrR, CG7431) resulted in a dramatic reduction, but not elimination, of the TA-mediated depolarization. Mutation or knockdown of TAR3 (tyrRII, CG16766) had no effect. However, deletion of both genes, or knockdown of TAR3 on a TAR2 mutant background, eliminated the TA responses. Thus while TAR2 is responsible for the majority of the TA sensitivity of the tubule, TAR3 also contributes to the response. Knockdown or mutation of TAR2 also eliminated the response of tubules to the related amine octopamine (OA), indicating that OA can activate TAR2. This finding contrasts to reports that heterologously expressed TAR2 is highly selective for TA over OA. This is the first report of TA receptor function in a native tissue and indicates unexpected complexity in the physiology of the Malpighian tubule (Zhang, 2017).

    Cold tolerance of Drosophila species is tightly linked to epithelial K+ transport capacity of the Malpighian tubules and rectal pads

    Insect chill tolerance is strongly associated with the ability to maintain ion and water homeostasis during cold exposure. Maintenance of K+ balance is particularly important due to its role in setting the cell membrane potential that is involved in many aspects of cellular function and viability. In most insects, K+ balance is maintained through secretion at the Malpighian tubules balancing reabsorption from the hindgut and passive leak arising from the gut lumen. This study examined K+ flux across the Malpighian tubules and the rectal pads in the hindgut in five Drosophila species that differ in cold tolerance. Chill tolerant species were found to be better at maintaining K+ secretion and supressing reabsorption during cold exposure. In contrast, chill susceptible species exhibited large reductions in secretion with no change, or a paradoxical increase, in K+ reabsorption. Using an assay to measure paracellular leak, chill susceptible species were found to experience a large increase in leak during cold exposure, which could explain the increased K+ reabsorption found in these species. The data therefore strongly support the hypothesis that cold tolerant Drosophila species are better at maintaining K+ homeostasis through an increased ability to maintain K+ secretion rates and through reduced leakage of K+ towards the hemolymph. These adaptations are manifested both at the Malpighian tubule and at the rectal pads in the hindgut and ensure that cold tolerant species experience less perturbation of K+ homeostasis during cold stress (Andersen, 2017).

    CRISPR-induced null alleles show that Frost protects Drosophila melanogaster reproduction after cold exposure

    The ability to survive and reproduce after cold exposure is important in all kingdoms of life. However, even in a sophisticated genetic model system like Drosophila melanogaster, few genes have been identified as functioning in cold tolerance. The accumulation of the Frost (Fst) gene transcript increases after cold exposure, making it a good candidate for a gene that has a role in cold tolerance. Despite extensive RNAi knockdown analysis, no role in cold tolerance has been assigned to Fst. CRISPR is an effective technique for completely knocking down genes, and is less likely to produce off-target effects than GAL4-UAS RNAi systems. CRISPR-mediated homologous recombination was used to generate Fst-null alleles, and these Fst alleles uncovered a requirement for FST protein in maintaining female fecundity following cold exposure. However, FST does not have a direct role in survival following cold exposure. FST mRNA accumulates in the Malpighian tubules, and the FST protein is a highly disordered protein with a putative signal peptide for export from the cell. Future work is needed to determine whether FST is exported from the Malpighian tubules and directly interacts with female reproductive tissues post-cold exposure, or whether it is required for other repair/recovery functions that indirectly alter energy allocation to reproduction (Newman, 2017).

    Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster

    At low temperatures, Drosophila, like most insects, lose the ability to regulate ion and water balance across the gut epithelia, which can lead to a lethal increase of [K(+)] in the hemolymph (hyperkalemia). Cold-acclimation, the physiological response to a prior low temperature exposure, can mitigate or entirely prevent these ion imbalances, but the physiological mechanisms that facilitate this process are not well understood. This study tested whether plasticity in the ionoregulatory physiology of the gut and Malpighian tubules of Drosophila may aid in preserving ion homeostasis in the cold. Upon adult emergence, D. melanogaster females were subjected to seven days at warm (25 ° C) or cold (10 ° C) acclimation conditions. The cold acclimated flies had a lower critical thermal minimum (CTmin), recovered from chill coma more quickly, and better maintained hemolymph K(+) balance in the cold. The improvements in chill tolerance coincided with increased Malpighian tubule fluid secretion and better maintenance of K(+) secretion rates in the cold, as well as reduced rectal K(+) reabsorption in cold-acclimated flies. To test whether modulation of ion-motive ATPases, the main drivers of epithelial transport in the alimentary canal, mediate these changes, the activities were measured of Na(+)-K(+)-ATPase and V-type H(+)-ATPase at the Malpighian tubules, midgut, and hindgut. Na(+)/K(+)-ATPase and V-type H(+)-ATPase activities were lower in the midgut and the Malpighian tubules of cold-acclimated flies, but unchanged in the hindgut of cold acclimated flies, and were not predictive of the observed alterations in K(+) transport. These results suggest that modification of Malpighian tubule and gut ion and water transport likely prevents cold-induced hyperkalemia in cold-acclimated flies and that this process is not directly related to the activities of the main drivers of ion transport in these organs, Na(+)/K(+)- and V-type H(+)-ATPases (Yerushalmi, 2018).


    Andersen, M. K., MacMillan, H. A., Donini, A. and Overgaard, J. (2017). Cold tolerance of Drosophila species is tightly linked to epithelial K+ transport capacity of the Malpighian tubules and rectal pads. J Exp Biol [Epub ahead of print]. PubMed ID: 28947500

    Browne, A. and O'Donnell, M. J. (2016). Segment-specific Ca transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages. J Insect Physiol 87: 1-11. PubMed ID: 26802560

    Broz, V., Kucerova, L., Rouhova, L., Fleischmannova, J., Strnad, H., Bryant, P. J. and Zurovec, M. (2017). Drosophila imaginal disc growth factor 2 is a trophic factor involved in energy balance, detoxification, and innate immunity. Sci Rep 7: 43273. PubMed ID: 28230183

    Bunt, S., Hooley, C., Hu, N., Scahill, C., Weavers, H. and Skaer, H. (2010). Hemocyte-secreted type IV collagen enhances BMP signaling to guide renal tubule morphogenesis in Drosophila. Dev Cell 19: 296-306. PubMed ID: 20708591

    Cabrero, P., Terhzaz, S., Romero, M. F., Davies, S. A., Blumenthal, E. M., Dow, J. A. (2014). Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. Proc Natl Acad Sci U S A. PubMed ID: 25228763

    Cannell, E., Dornan, A. J., Halberg, K. A., Terhzaz, S., Dow, J. A. and Davies, S. A. (2016). The corticotropin-releasing factor-like diuretic hormone 44 (DH) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster. Peptides [Epub ahead of print]. PubMed ID: 26896569

    Davies, S. A., et al. (1995). CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates tubule fluid secretion via cGMP. Am. J. Physiol. 269: R1321-1326. PubMed ID: 8594932

    Dow, J. A. and Davies, S. A. (2005). The Malpighian tubule: rapid insights from post-genomic biology. J. Insect Physiol. 52: 365-378. PubMed ID: 16310213

    Evans, J. M., Day, J. P., Cabrero, P., Dow, J. A. and Davies, S. A. (2008). A new role for a classical gene: white transports cyclic GMP. J. Exp. Biol. 211(Pt 6): 890-9. PubMed ID: 18310115

    Feingold, D., Starc, T., O'Donnell, M. J., Nilson, L. and Dent, J. A. (2016). The orphan pentameric ligand-gated ion channel pHCl-2 is gated by pH and regulates fluid secretion in Drosophila Malpighian tubules. J Exp Biol [Epub ahead of print]. PubMed ID: 27358471

    Fu, Y., Zhu, J. Y., Zhang, F., Richman, A., Zhao, Z. and Han, Z. (2017). Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes. Cell Tissue Res [Epub ahead of print]. PubMed ID: 28180992

    Gautam, N. K., Verma, P. and Tapadia, M. G. (2014). Ecdysone regulates morphogenesis and function of malpighian tubules in Drosophila melanogaster through EcR-B2 isoform. Dev Biol 398(2):163-76. PubMed ID: 25476260

    Gee, H. Y., et al. (2015). KANK deficiency leads to podocyte dysfunction and nephrotic syndrome. J Clin Invest 125: 2375-2384. PubMed ID: 25961457

    Hoch, M., Broadie, K., Jackle, H. and Skaer, H. (1994). Sequential fates in a single cell are established by the neurogenic cascade in the Malpighian tubules of Drosophila. Development 120: 3439-3450. PubMed ID: 7821213

    Kerr, M., Davies, S. A. and Dow, J. A. T. (2004). Cell-specific manipulation of second messengers: A toolbox for integrative physiology in Drosophila. Curr. Biol. 14: 1468-1474. PubMed ID: 15324663

    Newman, C. E., Toxopeus, J., Udaka, H., Ahn, S., Martynowicz, D. M., Graether, S. P., Sinclair, B. J. and Percival-Smith, A. (2017). CRISPR-induced null alleles show that Frost protects Drosophila melanogaster reproduction after cold exposure. J Exp Biol 220(Pt 18): 3344-3354. PubMed ID: 28705828

    Rosay, P., et al. (1997). Cell-type specific calcium signalling in a Drosophila epithelium. J. Cell Sci. 110: 1683-1692. PubMed ID: 9264456

    Sözen, M. A., et al. (1997). Functional domains are specified to single-cell resolution in a Drosophila epithelium. Proc. Natl. Acad. Sci. 94: 5207-5212. PubMed ID: 9144216

    Singh, S. R., Liu, W. and Hou, S. X. (2007). The adult Drosophila malpighian tubules are maintained by multipotent stem cells. Cell Stem Cell 1(2): 191-203. PubMed ID: 18371350

    Verma, P. and Tapadia, M. G. (2015). Early gene Broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster. Mol Immunol 66: 325-339. PubMed ID: 25931442

    Weavers, H. and Skaer, H. (2013). Tip cells act as dynamic cellular anchors in the morphogenesis of looped renal tubules in Drosophila. Dev Cell 27: 331-344. PubMed ID: 24229645

    Wu, Y., Baum, M., Huang, C. L. and Rodan, A. R. (2015). Two inwardly rectifying potassium channels, Irk1 and Irk2, play redundant roles in Drosophila renal tubule function. Am J Physiol Regul Integr Comp Physiol: ajpregu 00148 02015. PubMed ID: 26224687

    Yadav, S. and Tapadia, M.G. (2015). Expression of polyQ aggregates in Malpighian tubules leads to degeneration in Drosophila melanogaster. Dev Biol [Epub ahead of print]. PubMed ID: 26517966

    Yerushalmi, G. Y., Misyura, L., MacMillan, H. A. and Donini, A. (2018). Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster. J Exp Biol. PubMed ID: 29367271

    Zandawala, M., Marley, R., Davies, S. A. and Nassel, D. R. (2017). Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila. Cell Mol Life Sci 75(6):1099-1115. PubMed ID: 29043393

    Zhang, H. and Blumenthal, E. M. (2017). Identification of multiple functional receptors for tyramine on an insect secretory epithelium. Sci Rep 7(1): 168. PubMed ID: 28279025

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