RESUMO
Transporting epithelia provide a protective barrier against pathogenic insults while allowing the controlled exchange of ions, solutes and water with the external environment. In invertebrates, these functions depend on formation and maintenance of 'tight' septate junctions (SJs). However, the mechanism by which SJs affect transport competence and tissue homeostasis, and how these are modulated by ageing, remain incompletely understood. Here, we demonstrate that the Drosophila renal (Malpighian) tubules undergo an age-dependent decline in secretory capacity, which correlates with mislocalisation of SJ proteins and progressive degeneration in cellular morphology and tissue homeostasis. Acute loss of the SJ protein Snakeskin in adult tubules induced progressive changes in cellular and tissue architecture, including altered expression and localisation of junctional proteins with concomitant loss of cell polarity and barrier integrity, demonstrating that compromised junctional integrity is sufficient to replicate these ageing-related phenotypes. Taken together, our work demonstrates a crucial link between epithelial barrier integrity, tubule transport competence, renal homeostasis and organismal viability, as well as providing novel insights into the mechanisms underpinning ageing and renal disease.
RESUMO
Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 genes of the major intrinsic protein family are expressed at a very high level in the fly renal tissue: the aquaporins (AQPs) Drip and Prip and the aquaglyceroporins Eglp2 and Eglp4 As predicted from their structure, and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip, and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes result in impaired hormone-induced fluid secretion. The Drosophila tubule has 2 main secretory cell types: active cation-transporting principal cells, wherein the aquaglyceroporins localize to opposite plasma membranes, and small stellate cells, the site of the chloride shunt conductance, with these AQPs localizing to opposite plasma membranes. This suggests a model in which osmotically obliged water flows through the stellate cells. Consistent with this model, fluorescently labeled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects in regulating their internal environments.
Assuntos
Transporte Biológico/fisiologia , Drosophila melanogaster/fisiologia , Túbulos Renais/metabolismo , Água/metabolismo , Animais , Aquagliceroporinas/genética , Aquagliceroporinas/metabolismo , Aquaporinas/genética , Aquaporinas/metabolismo , Permeabilidade da Membrana Celular , Cloretos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Túbulos Renais/citologia , Masculino , Túbulos de Malpighi/metabolismo , Modelos Animais , Oócitos/metabolismo , Osmorregulação , XenopusRESUMO
The insect renal (Malpighian) tubule has long been a model system for the study of fluid secretion and its neurohormonal control, as well as studies on ion transport mechanisms. To extend these studies beyond the boundaries of classical physiology, a molecular genetic approach together with the 'omics technologies is required. To achieve this in any vertebrate transporting epithelium remains a daunting task, as the genetic tools available are still relatively unsophisticated. Drosophila melanogaster, however, is an outstanding model organism for molecular genetics. Here we describe a technique for fluid secretion assays in the D. melanogaster equivalent of the kidney nephron. The development of this first physiological assay for a Drosophila epithelium, allowing combined approaches of integrative physiology and functional genomics, has now provided biologists with an entirely new model system, the Drosophila Malpighian tubule, which is utilized in multiple fields as diverse as kidney disease research and development of new modes of pest insect control.
Assuntos
Rim/citologia , Rim/metabolismo , Túbulos de Malpighi/citologia , Animais , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Nefropatias/metabolismo , Túbulos de Malpighi/metabolismoRESUMO
BACKGROUND: Neuropeptides are central to the regulation of physiological and behavioural processes in insects, directly impacting cold and desiccation survival. However, little is known about the control mechanisms governing these responses in Drosophila suzukii. The close phylogenetic relationship of D. suzukii with Drosophila melanogaster allows, through genomic and functional studies, an insight into the mechanisms directing stress tolerance in D. suzukii. RESULTS: Capability (Capa), leucokinin (LK), diuretic hormone 44 (DH44 ) and DH31 neuropeptides demonstrated a high level of conservation between D. suzukii and D. melanogaster with respect to peptide sequences, neuronal expression, receptor localisation, and diuretic function in the Malpighian tubules. Despite D. suzukii's ability to populate cold environments, it proved sensitive to both cold and desiccation. Furthermore, in D. suzukii, Capa acts as a desiccation- and cold stress-responsive gene, while DH44 gene expression is increased only after desiccation exposure, and the LK gene after nonlethal cold stress recovery. CONCLUSION: This study provides a comparative investigation into stress tolerance mediation by neuroendocrine signalling in two Drosophila species, providing evidence that similar signalling pathways control fluid secretion in the Malpighian tubules. Identifying processes governing specific environmental stresses affecting D. suzukii could lead to the development of targeted integrated management strategies to control insect pest populations. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Assuntos
Temperatura Baixa , Dessecação , Proteínas de Drosophila/genética , Drosophila/fisiologia , Túbulos de Malpighi/fisiopatologia , Neuropeptídeos/genética , Animais , Drosophila/genética , Proteínas de Drosophila/metabolismo , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Transdução de Sinais/genética , TermotolerânciaRESUMO
Multicellular organisms rely on cell adhesion molecules to coordinate cell-cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most abundantly expressed in the adult renal (Malpighian) tubule rather than in neuronal tissues. The role Fas2 serves in this epithelium is unknown. Here we show that Fas2 is essential to brush border maintenance in renal tubules of Drosophila. Fas2 is dynamically expressed during tubule morphogenesis, localizing to the brush border whenever the tissue is transport competent. Genetic manipulations of Fas2 expression levels impact on both microvilli length and organization, which in turn dramatically affect stimulated rates of fluid secretion by the tissue. Consequently, we demonstrate a radically different role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Drosophila melanogaster/metabolismo , Túbulos Renais/metabolismo , Microvilosidades/metabolismo , Animais , Transporte Biológico , Moléculas de Adesão Celular Neuronais/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Túbulos Renais/embriologia , Microtúbulos/metabolismoRESUMO
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. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH44) and Drosophila melanogaster kinin (Drome-kinin, DK) 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. Drome-kinin receptor, encoded by the leucokinin receptor gene, 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, we demonstrate a novel role 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.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Hormônios de Inseto/metabolismo , Neuropeptídeos/metabolismo , Animais , Animais Geneticamente Modificados , Desidratação , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Técnicas de Silenciamento de Genes , Hormônios de Inseto/genética , Túbulos de Malpighi/metabolismo , Neuropeptídeos/genética , Transdução de Sinais , Inanição/metabolismo , Estresse Fisiológico/genéticaRESUMO
BACKGROUND: After mating, Drosophila females undergo a remarkable phenotypic switch resulting in decreased sexual receptivity and increased egg laying. Transfer of male sex peptide (SP) during copulation mediates these postmating responses via sensory neurons that coexpress the sex-determination gene fruitless (fru) and the proprioceptive neuronal marker pickpocket (ppk) in the female reproductive system. Little is known about the neuronal pathways involved in relaying SP-sensory information to central circuits and how these inputs are processed to direct female-specific changes that occur in response to mating. RESULTS: We demonstrate an essential role played by neurons expressing the sex-determination gene doublesex (dsx) in regulating the female postmating response. We uncovered shared circuitry between dsx and a subset of the previously described SP-responsive fru(+)/ppk(+)-expressing neurons in the reproductive system. In addition, we identified sexually dimorphic dsx circuitry within the abdominal ganglion (Abg) critical for mediating postmating responses. Some of these dsx neurons target posterior regions of the brain while others project onto the uterus. CONCLUSIONS: We propose that dsx-specified circuitry is required to induce female postmating behavioral responses, from sensing SP to conveying this signal to higher-order circuits for processing and through to the generation of postmating behavioral and physiological outputs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Peptídeos/metabolismo , Células Receptoras Sensoriais/metabolismo , Comportamento Sexual Animal/fisiologia , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Membrana Celular/metabolismo , Copulação , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Feminino , Cistos Glanglionares/metabolismo , Regulação da Expressão Gênica , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Receptores de Peptídeos , Diferenciação Sexual/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Útero/citologia , Útero/metabolismoRESUMO
Doublesex proteins, which are part of the structurally and functionally conserved Dmrt gene family, are important for sex determination throughout the animal kingdom. We inserted Gal4 into the doublesex (dsx) locus of Drosophila melanogaster, allowing us to visualize and manipulate cells expressing dsx in various tissues. In the nervous system, we detected differences between the sexes in dsx-positive neuronal numbers, axonal projections and synaptic density. We found that dsx was required for the development of male-specific neurons that coexpressed fruitless (fru), a regulator of male sexual behavior. We propose that dsx and fru act together to form the neuronal framework necessary for male sexual behavior. We found that disrupting dsx neuronal function had profound effects on male sexual behavior. Furthermore, our results suggest that dsx-positive neurons are involved in pre- to post-copulatory female reproductive behaviors.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Diferenciação Sexual/genética , Comportamento Sexual Animal/fisiologia , Animais , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Feminino , MasculinoRESUMO
In a study in this issue, Clyne and Miesenböck (2008) apply an ingenious optogenetic technology to activate neurons that generate male-specific courtship song in flies. This work sheds new light on the neural circuitry underlying sexually dimorphic behaviors in Drosophila.
Assuntos
Drosophila melanogaster/fisiologia , Comportamento Sexual Animal , Animais , Feminino , Luz , Masculino , Neurônios/fisiologia , Caracteres Sexuais , Asas de AnimaisRESUMO
Understanding how genes influence behavior, including sexuality, is one of biology's greatest challenges. Much of the recent progress in understanding how single genes can influence behavior has come from the study of innate behaviors in the fruit fly Drosophila melanogaster. In particular, the elaborate courtship ritual performed by the male fly has provided remarkable insights into how the neural circuitry underlying sexual behavior--which is largely innate in flies--is built into the nervous system during development, and how this circuitry functions in the adult. In this review we will discuss how genes of the sex determination pathway in Drosophila orchestrate the developmental events necessary for sex-specific behaviors and physiology, and the broader lessons this can teach us about the mechanisms underlying the development of sex-specific neural circuitry.
Assuntos
Drosophila melanogaster/genética , Processos de Determinação Sexual , Comportamento Sexual Animal/fisiologia , Animais , Evolução Biológica , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
BACKGROUND: How the central nervous system (CNS) develops to implement innate behaviors remains largely unknown. Drosophila male sexual behavior has long been used as a model to address this question. The male-specific products of fruitless (fru) are pivotal to the emergence of this behavior. These putative transcription factors, containing one of three alternative DNA binding domains, determine the neuronal substrates for sexual behavior in male CNS. RESULTS: We isolated the first fru coding mutation, resulting in complete loss of one isoform. At the neuronal level, this isoform alone controls differentiation of a male-specific muscle and its associated motorneuron. Conversely, a combination of isoforms is required for development of serotonergic neurons implicated in male copulatory behavior. Full development of these neurons requires the male-specific product of doublesex, a gene previously thought to act independently of fru. At the behavioral level, missing one isoform leads to diminished courtship behavior and infertility. We achieved the first rescue of a distinct fru behavioral phenotype, expressing a wild-type isoform in a defined subset of its normal expression pattern. CONCLUSION: This study exemplifies how complex behaviors can be controlled by a single locus through multiple isoforms regulating both developmental and physiological pathways in different neuronal substrates.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Fatores de Transcrição/fisiologia , Processamento Alternativo , Animais , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Feminino , Fertilidade , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Caracteres Sexuais , Comportamento Sexual Animal , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The fru4 allele of the sex determination gene fruitless is induced by insertion of a P[lacZ,ry+] enhancer trap element. This insert also acts to disrupt expression of the fru P1 promoter derived male-specific proteins, consequently impairing male courtship behavior. fru4 maps less than 2 kb upstream of the fru P3 promoter, whose function is essential for viability. We replaced this insert with a GAL4 element, P[GAL4,w+], recovering two lines with insertions in opposite orientations at the locus, one of which demonstrated fru-specific mutant phenotypes. Reporter expression of these lines recapitulated that of P3- and P4-derived proteins which, when correlated with a developmental and tissue specific survey of fru promoters' activities, uncovered a previously unsuspected complexity of fru regulation. These novel fru alleles provide the tools for manipulation of fru-expressing cells, allowing the consequent effects to be related back to specific fru functions and the regulatory units controlling these activities.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Marcação de Genes/métodos , Genes de Insetos , Proteínas do Tecido Nervoso/genética , Processos de Determinação Sexual , Fatores de Transcrição/genética , Alelos , Animais , Corte , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Imunofluorescência , Fatores de Transcrição Fushi Tarazu , Regulação da Expressão Gênica no Desenvolvimento , Homozigoto , Masculino , Microscopia Confocal , Mutação , Proteínas do Tecido Nervoso/biossíntese , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Transativadores/genética , Fatores de Transcrição/biossíntese , Transcrição GênicaRESUMO
Most insertional mutagenesis screens of Drosophila performed to date have not used target chromosomes that have been checked for their suitability for phenotypic screens for viable phenotypes. To address this, we have generated a selection of stocks carrying either isogenized second chromosomes or isogenized third chromosomes, in a genetic background derived from a Canton-S wild-type strain. We have tested these stocks for a range of behavioral and other viable phenotypes. As expected, most lines are statistically indistinguishable from Canton-S in most phenotypes tested. The lines generated are now being used as target chromosomes in mutagenesis screens, and the characterization reported here will facilitate their use in screens of these lines for behavioral and other viable phenotypes.