The Interactive Fly

Genes involved in tissue and organ development

Malpighian Tubules


Embryonic origin, functional domains and physiology of Malpighian tubules

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

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).

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).

References

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

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

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


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