RESUMEN
Macrophages are an ancient blood cell lineage critical for homeostasis and defence against pathogens. Although their numbers were long thought to be sustained solely by haematopoietic organs, it has recently become clear that their proliferation, or self-renewal, also plays a major role. In the Drosophila larva, macrophages undergo a phase of rapid self-renewal, making this an attractive model for elucidating the signals and regulatory mechanisms involved. However, a central self-renewal pathway has not been identified in this system. Here, we show that the PDGF- and VEGF-receptor related (Pvr) pathway fulfils this role. Our data show that two of the three known Pvr ligands, PDGF- and VEGF-related factor 2 (Pvf2) and Pvf3, are major determinants of overall macrophage numbers, yet they each act in a temporally independent manner and via distinct mechanisms. While Pvf3 is needed prior to the self-renewal period, we find that Pvf2 is critical specifically for expanding the larval macrophage population. We further show that Pvf2 is a potent macrophage mitogen that is kept at limiting quantities by its transient expression in a remarkably small number of blood cells. Together, these data support a novel mechanism for the regulation of macrophage self-renewal rates by the dynamic transcriptional control of Pvf2. Given the strong parallels that exist between Drosophila and vertebrate macrophage systems, it is likely that a similar self-renewal control mechanism is at play across animal phyla.
Asunto(s)
Proteínas de Drosophila , Factor A de Crecimiento Endotelial Vascular , Animales , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Larva/genética , Larva/metabolismo , Macrófagos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial VascularRESUMEN
BACKGROUND: Congenital heart diseases are the major cause of death in newborns, but the genetic etiology of this developmental disorder is not fully known. The conventional approach to identify the disease-causing genes focuses on screening genes that display heart-specific expression during development. However, this approach would have discounted genes that are expressed widely in other tissues but may play critical roles in heart development. RESULTS: We report an efficient pipeline of genome-wide gene discovery based on the identification of a cardiac-specific cis-regulatory element signature that points to candidate genes involved in heart development and congenital heart disease. With this pipeline, we retrieve 76% of the known cardiac developmental genes and predict 35 novel genes that previously had no known connectivity to heart development. Functional validation of these novel cardiac genes by RNAi-mediated knockdown of the conserved orthologs in Drosophila cardiac tissue reveals that disrupting the activity of 71% of these genes leads to adult mortality. Among these genes, RpL14, RpS24, and Rpn8 are associated with heart phenotypes. CONCLUSIONS: Our pipeline has enabled the discovery of novel genes with roles in heart development. This workflow, which relies on screening for non-coding cis-regulatory signatures, is amenable for identifying developmental and disease genes for an organ without constraining to genes that are expressed exclusively in the organ of interest.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Cardiopatías Congénitas/genética , Corazón/crecimiento & desarrollo , Animales , Biología Computacional , Drosophila/genética , Drosophila/fisiología , Pruebas Genéticas , Genoma , Genómica , Interferencia de ARN , Elementos Reguladores de la Transcripción , Proteínas Ribosómicas/genéticaRESUMEN
In insects, many critical olfactory behaviours are mediated by the large odorant receptor (Or) gene family, which determines the response properties of different classes of olfactory receptor neurons (ORNs). While ORN responses are generally conserved within and between Drosophila species, variant alleles of the D. melanogaster Or22 locus have previously been shown to alter the response profile of an ORN class called ab3A. These alleles show potential clinal variation, suggesting that selection is acting at this locus. Here, we investigated if the changes seen in ab3A responses lead to changes in olfactory-related behaviours. We show that variation at the Or22 locus and in the ab3A neurons are not fully compensated for by other ORNs and lead to overall changes in antennal odorant detection. We further show that this correlates with differences in odorant preference behaviour and with differences in oviposition site preference, with flies that have the chimaeric short allele strongly preferring to oviposit on banana. These findings indicate that variation at the Or22 locus leads to changes in olfactory-driven behaviours, and add support to the idea that the ab3A neurons are of especial importance to the ecology of Drosophila flies.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Odorantes/análisis , Neuronas Receptoras Olfatorias/fisiología , Oviposición , Receptores Odorantes/metabolismo , Animales , Proteínas de Drosophila/genética , Femenino , Masculino , Neuronas Receptoras Olfatorias/citología , Receptores Odorantes/genéticaRESUMEN
In both mammals and insects, steroid hormones play a major role in directing the animal's progression through developmental stages. To maximize fitness outcomes, steroid hormone production is regulated by the environmental conditions experienced by the animal. In insects, the steroid hormone ecdysone mediates transitions between developmental stages and is regulated in response to environmental factors such as nutrition. These environmental signals are communicated to the ecdysone-producing gland via the action of neuropeptide and peptide hormone signalling pathways. While some of these pathways have been well characterized, there is evidence to suggest more signalling pathways than has previously been thought function to control ecdysone production, potentially in response to a greater range of environmental conditions. Here, we review the neuropeptide and peptide hormone signalling pathways known to regulate the production of ecdysone in the model genetic insect Drosophila melanogaster, as well as what is known regarding the environmental signals that trigger these pathways. Areas for future research are highlighted that can further contribute to our overall understanding of the complex orchestration of environmental, physiological and developmental cues that together produce a functioning adult organism.
Asunto(s)
Proteínas de Drosophila/metabolismo , Ecdisona/biosíntesis , Neuropéptidos/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Ecdisona/genética , Regulación del Desarrollo de la Expresión Génica , Neuropéptidos/genéticaRESUMEN
As juvenile animals grow, their behavior, physiology, and development need to be matched to environmental conditions to ensure they survive to adulthood. However, we know little about how behavior and physiology are integrated with development to achieve this outcome. Neuropeptides are prime candidates for achieving this due to their well-known signaling functions in controlling many aspects of behavior, physiology, and development in response to environmental cues. In the growing Drosophila larva, while several neuropeptides have been shown to regulate feeding behavior, and a handful to regulate growth, it is unclear if any of these play a global role in coordinating feeding behavior with developmental programs. Here, we demonstrate that Neuropeptide F Receptor (NPFR), best studied as a conserved regulator of feeding behavior from insects to mammals, also regulates development in Drosophila Knocking down NPFR in the prothoracic gland, which produces the steroid hormone ecdysone, generates developmental delay and an extended feeding period, resulting in increased body size. We show that these effects are due to decreased ecdysone production, as these animals have reduced expression of ecdysone biosynthesis genes and lower ecdysone titers. Moreover, these phenotypes can be rescued by feeding larvae food supplemented with ecdysone. Further, we show that NPFR negatively regulates the insulin signaling pathway in the prothoracic gland to achieve these effects. Taken together, our data demonstrate that NPFR signaling plays a key role in regulating animal development, and may, thus, play a global role in integrating feeding behavior and development in Drosophila.
Asunto(s)
Tamaño Corporal , Proteínas de Drosophila/genética , Estadios del Ciclo de Vida/genética , Receptores de Neuropéptido/genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Ecdisona/metabolismo , Conducta Alimentaria , Receptores de Neuropéptido/metabolismo , Transducción de SeñalRESUMEN
Blood cells, known as hemocytes in invertebrates, play important and conserved roles in immunity, wound healing and tissue remodelling. The control of hemocyte number is therefore critical to ensure these functions are not compromised, and studies using Drosophila melanogaster are proving useful for understanding how this occurs. Recently, the embryonic patterning gene, torso-like (tsl), was identified as being required both for normal hemocyte development and for providing immunity against certain pathogens. Here, we report that Tsl is required specifically during the larval phase of hematopoiesis, and that tsl mutant larvae likely have reduced hemocyte numbers due to a reduced larval growth rate and compromised insulin signaling. Consistent with this, we find that impairing insulin-mediated growth, either by nutrient deprivation or genetically, results in fewer hemocytes. This is likely the result of impaired insulin-like signaling in the hemocytes themselves, since modulation of Insulin-like Receptor (InR) activity specifically in hemocytes causes concomitant changes to their population size in developing larvae. Taken together, our work reveals the strong relationship that exists between body size and hemocyte number, and suggests that insulin-like signaling contributes to, but is not solely responsible for, keeping these tightly aligned during larval development.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Animales , Tamaño Corporal , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Hemocitos , Insulina , LarvaRESUMEN
To persist when nutrient sources are limited, aerobic soil bacteria metabolize atmospheric hydrogen (H2). This process is the primary sink in the global H2 cycle and supports the productivity of microbes in oligotrophic environments. H2-metabolizing bacteria possess [NiFe] hydrogenases that oxidize H2 to subatmospheric concentrations. The soil saprophyte Mycobacterium smegmatis has two such [NiFe] hydrogenases, designated Huc and Hhy, that belong to different phylogenetic subgroups. Both Huc and Hhy are oxygen-tolerant, oxidize H2 to subatmospheric concentrations, and enhance bacterial survival during hypoxia and carbon limitation. Why does M. smegmatis require two hydrogenases with a seemingly similar function? In this work, we resolved this question by showing that Huc and Hhy are differentially expressed, localized, and integrated into the respiratory chain. Huc is active in late exponential and early stationary phases, supporting energy conservation during mixotrophic growth and transition into dormancy. In contrast, Hhy is most active during long-term persistence, providing energy for maintenance processes following carbon exhaustion. We also show that Huc and Hhy are obligately linked to the aerobic respiratory chain via the menaquinone pool and are differentially affected by respiratory uncouplers. Consistently, these two enzymes interacted differentially with the respiratory terminal oxidases. Huc exclusively donated electrons to, and possibly physically associated with, the proton-pumping cytochrome bcc-aa3 supercomplex. In contrast the more promiscuous Hhy also provided electrons to the cytochrome bd oxidase complex. These results indicate that, despite their similar characteristics, Huc and Hhy perform distinct functions during mycobacterial growth and survival.
Asunto(s)
Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium smegmatis/metabolismo , Oxidorreductasas/metabolismo , Aerobiosis , Mycobacterium smegmatis/enzimologíaRESUMEN
Axis specification is a fundamental developmental process. Despite this, the mechanisms by which it is controlled across insect taxa are strikingly different. An excellent example of this is terminal patterning, which in Diptera such as Drosophila melanogaster occurs via the localized activation of the receptor tyrosine kinase Torso. In Hymenoptera, however, the same process appears to be achieved via localized mRNA. How these mechanisms evolved and what they evolved from remains largely unexplored. Here, we show that torso-like, known for its role in Drosophila terminal patterning, is instead required for the integrity of the vitelline membrane in the hymenopteran wasp Nasonia vitripennis We find that other genes known to be involved in Drosophila terminal patterning, such as torso and Ptth, also do not function in Nasonia embryonic development. These findings extended to orthologues of Drosophila vitelline membrane proteins known to play a role in localizing Torso-like in Drosophila; in Nasonia these are instead required for dorso-ventral patterning, gastrulation and potentially terminal patterning. Our data underscore the importance of the vitelline membrane in insect development, and implies phenotypes caused by knockdown of torso-like must be interpreted in light of its function in the vitelline membrane. In addition, our data imply that the signalling components of the Drosophila terminal patterning systems were co-opted from roles in regulating moulting, and co-option into terminal patterning involved the evolution of a novel interaction with the vitelline membrane protein Torso-like.This article has an associated First Person interview with the first author of the paper.
RESUMEN
Insect odorant receptor (Or) genes determine the responses of sensory neurons that mediate critical behaviors. The Drosophila melanogaster Or22 locus represents an interesting example of molecular evolution, with high levels of sequence divergence and copy number variation between D. melanogaster and other Drosophila species, and a corresponding high level of variability in the responses of the neuron it controls, ab3A. However, the link between Or22 molecular and functional diversity has not been established. Here, we show that several naturally occurring Or22 variants generate major shifts in neuronal response properties. We determine the molecular changes that underpin these response shifts, one of which represents a chimeric gene variant previously suggested to be under natural selection. In addition, we show that several alternative molecular genetic mechanisms have evolved for ensuring that where there is more than one gene copy at this locus, only one functional receptor is generated. Our data thus provide a causal link between the striking levels of phenotypic neuronal response variation found in natural populations of D. melanogaster and genetic variation at the Or22 locus. Since neuronal responses govern animal behavior, we predict that Or22 may be a key player in underlying one or more olfactory-driven behaviors of significant adaptive importance.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Evolución Molecular , Neuronas Receptoras Olfatorias/fisiología , Receptores Odorantes/genética , Animales , Femenino , Variación Genética , Proteínas de Insectos/genética , Masculino , FenotipoRESUMEN
Many of the links between diet and cancer are controversial and over simplified. To date, human epidemiological studies consistently reveal that patients who suffer diet-related obesity and/or type II diabetes have an increased risk of cancer, suffer more aggressive cancers, and respond poorly to current therapies. However, the underlying molecular mechanisms that increase cancer risk and decrease the response to cancer therapies in these patients remain largely unknown. Here, we review studies in mouse cancer models in which either dietary or genetic manipulation has been used to model obesity and/or type II diabetes. These studies demonstrate an emerging role for the conserved insulin and insulin-like growth factor signaling pathways as links between diet and cancer progression. However, these models are time consuming to develop and expensive to maintain. As the world faces an epidemic of obesity and type II diabetes we argue that the development of novel animal models is urgently required. We make the case for Drosophila as providing an unparalleled opportunity to combine dietary manipulation with models of human metabolic disease and cancer. Thus, combining diet and cancer models in Drosophila can rapidly and significantly advance our understanding of the conserved molecular mechanisms that link diet and diet-related metabolic disorders to poor cancer patient prognosis.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Dieta , Neoplasias/genética , Obesidad/genética , Animales , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Drosophila , Humanos , Ratones , Neoplasias/complicaciones , Neoplasias/patología , Obesidad/complicaciones , Obesidad/patologíaRESUMEN
In Drosophila, key developmental transitions are governed by the steroid hormone ecdysone. A number of neuropeptide-activated signaling pathways control ecdysone production in response to environmental signals, including the insulin signaling pathway, which regulates ecdysone production in response to nutrition. Here, we find that the Membrane Attack Complex/Perforin-like protein Torso-like, best characterized for its role in activating the Torso receptor tyrosine kinase in early embryo patterning, also regulates the insulin signaling pathway in Drosophila We previously reported that the small body size and developmental delay phenotypes of torso-like null mutants resemble those observed when insulin signaling is reduced. Here we report that, in addition to growth defects, torso-like mutants also display metabolic and nutritional plasticity phenotypes characteristic of mutants with impaired insulin signaling. We further find that in the absence of torso-like, the expression of insulin-like peptides is increased, as is their accumulation in insulin-producing cells. Finally, we show that Torso-like is a component of the hemolymph and that it is required in the prothoracic gland to control developmental timing and body size. Taken together, our data suggest that the secretion of Torso-like from the prothoracic gland influences the activity of insulin signaling throughout the body in Drosophila.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Hemolinfa , Insulina/metabolismo , Transducción de Señal , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Expresión Génica , Estudios de Asociación Genética , Genotipo , Mutación , FenotipoRESUMEN
Patterning of the Drosophila embryonic termini by the Torso (Tor) receptor pathway has long served as a valuable paradigm for understanding how receptor tyrosine kinase signaling is controlled. However, the mechanisms that underpin the control of Tor signaling remain to be fully understood. In particular, it is unclear how the Perforin-like protein Torso-like (Tsl) localizes Tor activity to the embryonic termini. To shed light on this, together with other aspects of Tor pathway function, we conducted a genome-wide screen to identify new pathway components that operate downstream of Tsl. Using a set of molecularly defined chromosomal deficiencies, we screened for suppressors of ligand-dependent Tor signaling induced by unrestricted Tsl expression. This approach yielded 59 genomic suppressor regions, 11 of which we mapped to the causative gene, and a further 29 that were mapped to <15 genes. Of the identified genes, six represent previously unknown regulators of embryonic Tor signaling. These include twins (tws), which encodes an integral subunit of the protein phosphatase 2A complex, and α-tubulin at 84B (αTub84B), a major constituent of the microtubule network, suggesting that these may play an important part in terminal patterning. Together, these data comprise a valuable resource for the discovery of new Tor pathway components. Many of these may also be required for other roles of Tor in development, such as in the larval prothoracic gland where Tor signaling controls the initiation of metamorphosis.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genoma de los Insectos , Estudio de Asociación del Genoma Completo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Alelos , Animales , Tipificación del Cuerpo/genética , Cruzamientos Genéticos , Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Mutación , Fenotipo , Tubulina (Proteína)/metabolismoRESUMEN
Zinc absorption in animals is thought to be regulated in a local, cell autonomous manner with intestinal cells responding to dietary zinc content. The Drosophila zinc transporter Zip88E shows strong sequence similarity to Zips 42C.1, 42C.2 and 89B as well as mammalian Zips 1, 2 and 3, suggesting that it may act in concert with the apically-localised Drosophila zinc uptake transporters to facilitate dietary zinc absorption by importing ions into the midgut enterocytes. However, the functional characterisation of Zip88E presented here indicates that Zip88E may instead play a role in detecting and responding to zinc toxicity. Larvae homozygous for a null Zip88E allele are viable yet display heightened sensitivity to elevated levels of dietary zinc. This decreased zinc tolerance is accompanied by an overall decrease in Metallothionein B transcription throughout the larval midgut. A Zip88E reporter gene is expressed only in the salivary glands, a handful of enteroendocrine cells at the boundary between the anterior and middle midgut regions, and in two parallel strips of sensory cell projections connecting to the larval ventral ganglion. Zip88E expression solely in this restricted subset of cells is sufficient to rescue the Zip88E mutant phenotype. Together, our data suggest that Zip88E may be functioning in a small subset of cells to detect excessive zinc levels and induce a systemic response to reduce dietary zinc absorption and hence protect against toxicity.
Asunto(s)
Proteínas Portadoras/fisiología , Drosophila/genética , Inactivación Metabólica/genética , Zinc/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Portadoras/genética , Dieta , Drosophila/embriología , Embrión no Mamífero , Ojo/embriología , Anomalías del Ojo/genética , Absorción Intestinal/genética , Zinc/toxicidadRESUMEN
The rapid and orderly folding of epithelial tissue during developmental processes such as gastrulation requires the precise coordination of changes in cell shape. Here, we report that the perforin-like protein Torso-like (Tsl), the key extracellular determinant for Drosophila embryonic terminal patterning, also functions to control epithelial morphogenesis. We find that tsl null mutants display a ventral cuticular hole phenotype that is independent of the loss of terminal structures, and arises as a consequence of mesoderm invagination defects. We show that the holes are caused by uncoordinated constriction of ventral cell apices, resulting in the formation of an incomplete ventral furrow. Consistent with these data, we find that loss of tsl is sensitive to gene dosage of RhoGEF2, a critical mediator of Rho1-dependent ventral cell shape changes during furrow formation, suggesting that Tsl may act in this pathway. In addition, loss of tsl strongly suppressed the effects of ectopic expression of Folded Gastrulation (Fog), a secreted protein that promotes apical constriction. Taken together, our data suggest that Tsl controls Rho1-mediated apical constriction via Fog. Therefore, we propose that Tsl regulates extracellular Fog activity to synchronize cell shape changes and coordinate ventral morphogenesis in Drosophila Identifying the Tsl-mediated event that is common to both terminal patterning and morphogenesis will be valuable for our understanding of the extracellular control of developmental signaling by perforin-like proteins.
Asunto(s)
Proteínas de Drosophila/genética , Gastrulación , Morfogénesis , Animales , Proteínas de Ciclo Celular , Drosophila/embriología , Drosophila/genética , Proteínas de Drosophila/metabolismo , Epitelio/embriología , Mutación , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The Membrane Attack Complex Perforin-like/Cholesterol-Dependent Cytolysin (MACPF) superfamily is an ancient and biologically diverse group of proteins that are best known for pore-forming roles in mammalian immunity and bacterial pathogenesis. Intriguingly, however, some eukaryotic proteins which contain the MACPF domain that defines this family do not act in attack or defence, and instead have distinct developmental functions. It remains unclear whether these proteins function via pore formation or have a different mechanism of action. Of these, by far the best characterised is Torso-like (Tsl), the only MACPF member that has been identified in the fruit fly, Drosophila melanogaster. While it has long been known to have a role in embryonic patterning, recent studies have shown that Tsl in fact has multiple roles in development. As such, it presents an excellent opportunity to investigate how the MACPF domain functions in a developmental context. Here, we review what is known about Tsl in Drosophila and other insects, and discuss the potential molecular mechanism by which Tsl and thus other developmental MACPF proteins may function.
Asunto(s)
Colesterol/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Citotoxinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Perforina/metabolismo , Animales , Complejo de Ataque a Membrana del Sistema Complemento/genética , Citotoxinas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Perforina/genética , Transducción de Señal/genéticaRESUMEN
Early B cell factor 3 (EBF3) is a member of the highly evolutionarily conserved Collier/Olf/EBF (COE) family of transcription factors. Prior studies on invertebrate and vertebrate animals have shown that EBF3 homologs are essential for survival and that loss-of-function mutations are associated with a range of nervous system developmental defects, including perturbation of neuronal development and migration. Interestingly, aristaless-related homeobox (ARX), a homeobox-containing transcription factor critical for the regulation of nervous system development, transcriptionally represses EBF3 expression. However, human neurodevelopmental disorders related to EBF3 have not been reported. Here, we describe three individuals who are affected by global developmental delay, intellectual disability, and expressive speech disorder and carry de novo variants in EBF3. Associated features seen in these individuals include congenital hypotonia, structural CNS malformations, ataxia, and genitourinary abnormalities. The de novo variants affect a single conserved residue in a zinc finger motif crucial for DNA binding and are deleterious in a fly model. Our findings indicate that mutations in EBF3 cause a genetic neurodevelopmental syndrome and suggest that loss of EBF3 function might mediate a subset of neurologic phenotypes shared by ARX-related disorders, including intellectual disability, abnormal genitalia, and structural CNS malformations.
Asunto(s)
Mutación , Trastornos del Neurodesarrollo/genética , Factores de Transcripción/genética , Anomalías Múltiples/genética , Ataxia/genética , Sistema Nervioso Central/anomalías , Niño , Preescolar , Discapacidades del Desarrollo/genética , Femenino , Genitales/anomalías , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/genética , Masculino , Hipotonía Muscular/genética , Trastornos del Habla/genética , Síndrome , Dedos de Zinc/genéticaRESUMEN
Pore-forming members of the membrane attack complex/perforin-like (MACPF) protein superfamily perform well-characterized roles as mammalian immune effectors. For example, complement component 9 and perforin function to directly form pores in the membrane of Gram-negative pathogens or virally infected/transformed cells, respectively. In contrast, the only known MACPF protein in Drosophila melanogaster, Torso-like, plays crucial roles during development in embryo patterning and larval growth. Here, we report that in addition to these functions, Torso-like plays an important role in Drosophila immunity. However, in contrast to a hypothesized effector function in, for example, elimination of Gram-negative pathogens, we find that torso-like null mutants instead show increased susceptibility to certain Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis We further show that this deficit is due to a severely reduced number of circulating immune cells and, as a consequence, an impaired ability to phagocytose bacterial particles. Together these data suggest that Torso-like plays an important role in controlling the development of the Drosophila cellular immune system.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Larva/genética , Animales , Membrana Celular/genética , Membrana Celular/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Proteínas de Drosophila/inmunología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/inmunología , Enterococcus faecalis/inmunología , Enterococcus faecalis/patogenicidad , Sistema Inmunológico/crecimiento & desarrollo , Larva/crecimiento & desarrollo , Larva/inmunología , Perforina/inmunología , Fagocitosis/inmunología , Staphylococcus aureus/inmunología , Staphylococcus aureus/patogenicidadRESUMEN
Glutathione (GSH) is a tripeptide often considered to be the master antioxidant in cells. GSH plays an integral role in cellular redox regulation and is also known to have a role in mammalian copper homeostasis. In vitro evidence suggests that GSH is involved in copper uptake, sequestration and efflux. This study was undertaken to further investigate the roles that GSH plays in neuronal copper homeostasis in vivo, using the model organism Drosophila melanogaster. RNA interference-mediated knockdown of the Glutamate-cysteine ligase catalytic subunit gene (Gclc) that encodes the rate-limiting enzyme in GSH biosynthesis was utilised to genetically deplete GSH levels. When Gclc was knocked down in all neurons, this caused lethality, which was partially rescued by copper supplementation and was exacerbated by additional knockdown of the copper uptake transporter Ctr1A, or over-expression of the copper efflux transporter ATP7. Furthermore, when Gclc was knocked down in a subset of neuropeptide-producing cells, this resulted in adult progeny with unexpanded wings, a phenotype previously associated with copper dyshomeostasis. In these cells, Gclc suppression caused a decrease in axon branching, a phenotype further enhanced by ATP7 over-expression. Therefore, we conclude that GSH may play an important role in regulating neuronal copper levels and that reduction in GSH may lead to functional copper deficiency in neurons in vivo. We provide genetic evidence that glutathione (GSH) levels influence Cu content or distribution in vivo, in Drosophila neurons. GSH could be required for binding Cu imported by Ctr1A and distributing it to chaperones, such as Mtn, CCS and Atox1. Alternatively, GSH could modify the copper-binding and transport activities of Atox1 and the ATP7 efflux protein via glutathionylation of copper-binding cysteines.
Asunto(s)
Cobre/deficiencia , Drosophila melanogaster/metabolismo , Glutatión/biosíntesis , Neuronas/patología , Animales , Axones/ultraestructura , Calcitonina/farmacología , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Cobre/uso terapéutico , Proteínas Transportadoras de Cobre , ATPasas Transportadoras de Cobre , Dieta , Proteínas de Drosophila/genética , Femenino , Técnicas de Silenciamiento del Gen , Glutamato-Cisteína Ligasa/genética , Larva , Neuronas/metabolismo , Neuropéptidos/biosíntesis , Fragmentos de Péptidos/farmacología , Interferencia de ARN , Alas de Animales/anomalíasRESUMEN
Dietary zinc is the principal source of zinc in eukaryotes, with its uptake and distribution controlled by a complex network of numerous membrane-spanning transport proteins. Dietary absorption is achieved by members of the SLC39A (ZIP) gene family, which encode proteins that are generally responsible for the movement of zinc into the cytosol. ZIP4 is thought to be the primary mammalian zinc uptake gene in the small intestine, with mutations in this gene causing the zinc deficiency disease Acrodermatitis enteropathica. In Drosophila, dual knockdown of the major dietary zinc uptake genes dZIP42C.1 (dZIP1) and dZIP42C.2 (dZIP2) results in a severe sensitivity to zinc-deficient media. However, the symptoms associated with ZIP4 loss can be reversed by zinc supplementation and dZIP42C.1 and 2 knockdown has minimal effect under normal dietary conditions, suggesting that additional pathways for zinc absorption exist in both mammals and flies. This study provides evidence that dZIP89B is an ideal candidate for this role in Drosophila, encoding a low-affinity zinc uptake transporter active in the posterior midgut. Flies lacking dZIP89B, while viable and apparently healthy, show indications of low midgut zinc levels, including reduced metallothionein B expression and compensatory up-regulation of dZIP42C.1 and 2. Furthermore dZIP89B mutants display a dramatic resistance to toxic dietary zinc levels which is abrogated by midgut-specific restoration of dZIP89B activity. We postulate that dZIP89B works in concert with the closely related dZIP42C.1 and 2 to ensure optimal zinc absorption under a range of dietary conditions.
Asunto(s)
Proteínas de Transporte de Catión/fisiología , Proteínas de Drosophila/fisiología , Zinc/metabolismo , Animales , Dieta , Regulación hacia Abajo , Drosophila melanogaster , Femenino , Tracto Gastrointestinal/metabolismo , Expresión Génica , Proteínas Fluorescentes Verdes/biosíntesis , Absorción Intestinal , Larva/metabolismo , Masculino , Metalotioneína/metabolismo , Especificidad de ÓrganosRESUMEN
Patterning of the Drosophila embryonic termini is achieved by localized activation of the Torso receptor by the growth factor Trunk. Governing this event is the perforin-like protein Torso-like, which is localized to the extracellular space at the embryo poles and has long been proposed to control localized proteolytic activation of Trunk. However, a protease involved in terminal patterning remains to be identified, and the role of Torso-like remains unknown. Here we find that Trunk is cleaved intracellularly by Furin proteases. We further show that Trunk is secreted, and that levels of extracellular Trunk are greatly reduced in torso-like null mutants. On the basis of these and previous findings, we suggest that Torso-like functions to mediate secretion of Trunk, thus providing the mechanism for spatially restricted activation of Torso. Our data represent an alternative mechanism for the spatial control of receptor signalling, and define a different role for perforin-like proteins in eukaryotes.