RESUMEN
The ribosome is a molecular machine essential for protein synthesis, which is composed of approximately 80 different ribosomal proteins (Rps). Studies in yeast and cell culture systems have revealed that the intracellular level of Rps is finely regulated by negative feedback mechanisms or ubiquitin-proteasome system, which prevents over- or under-abundance of Rps in the cell. However, in vivo evidence for the homeostatic regulation of intracellular Rp levels has been poor. Here, using Drosophila genetics, we show that intracellular Rp levels are regulated by proteasomal degradation of excess Rps that are not incorporated into the ribosome. By establishing an EGFP-fused Rp gene system that can monitor endogenously expressed Rp levels, we found that endogenously expressed EGFP-RpS20 or -RpL5 is eliminated from the cell when RpS20 or RpL5 is exogenously expressed. Notably, the level of endogenously expressed Hsp83, a housekeeping gene, was not affected by exogenous expression of Hsp83, suggesting that the strict negative regulation of excess protein is specific for intracellular Rps. Further analyses revealed that the maintenance of cellular Rp levels is not regulated at the transcriptional level but by proteasomal degradation of excess free Rps as a protein quality control mechanism. Our observations provide not only the in vivo evidence for the homeostatic regulation of Rp levels but also a novel genetic strategy to study in vivo regulation of intracellular Rp levels and its role in tissue homeostasis via cell competition.Key words: ribosomal protein, proteasomal degradation, Drosophila.
Asunto(s)
Drosophila , Proteínas Ribosómicas , Animales , Drosophila/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Biosíntesis de Proteínas , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Many organs of Drosophila show stereotypical left-right (LR) asymmetry; however, the underlying mechanisms remain elusive. Here, we have identified an evolutionarily conserved ubiquitin-binding protein, AWP1/Doctor No (Drn), as a factor required for LR asymmetry in the embryonic anterior gut. We found that drn is essential in the circular visceral muscle cells of the midgut for JAK/STAT signaling, which contributes to the first known cue for anterior gut lateralization via LR asymmetric nuclear rearrangement. Embryos homozygous for drn and lacking its maternal contribution showed phenotypes similar to those with depleted JAK/STAT signaling, suggesting that Drn is a general component of JAK/STAT signaling. Absence of Drn resulted in specific accumulation of Domeless (Dome), the receptor for ligands in the JAK/STAT signaling pathway, in intracellular compartments, including ubiquitylated cargos. Dome colocalized with Drn in wild-type Drosophila. These results suggest that Drn is required for the endocytic trafficking of Dome, which is a crucial step for activation of JAK/STAT signaling and the subsequent degradation of Dome. The roles of AWP1/Drn in activating JAK/STAT signaling and in LR asymmetric development may be conserved in various organisms.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Transducción de Señal/fisiología , Endocitosis/genética , Quinasas Janus/genética , Quinasas Janus/metabolismo , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismoRESUMEN
The function of the stem cell system is supported by a stereotypical shape of the niche structure. In Drosophila ovarian germarium, somatic cap cells form a dish-like niche structure that allows only two or three germ-line stem cells (GSCs) reside in the niche. Despite extensive studies on the mechanism of stem cell maintenance, the mechanisms of how the dish-like niche structure is shaped and how this structure contributes to the stem cell system have been elusive. Here, we show that a transmembrane protein Stranded at second (Sas) and its receptor Protein tyrosine phosphatase 10D (Ptp10D), effectors of axon guidance and cell competition via epidermal growth factor receptor (Egfr) inhibition, shape the dish-like niche structure by facilitating c-Jun N-terminal kinase (JNK)-mediated apoptosis. Loss of Sas or Ptp10D in gonadal apical cells, but not in GSCs or cap cells, during the pre-pupal stage results in abnormal shaping of the niche structure in the adult, which allows excessive, four to six GSCs reside in the niche. Mechanistically, loss of Sas-Ptp10D elevates Egfr signaling in the gonadal apical cells, thereby suppressing their naturally-occurring JNK-mediated apoptosis that is essential for the shaping of the dish-like niche structure by neighboring cap cells. Notably, the abnormal niche shape and resulting excessive GSCs lead to diminished egg production. Our data propose a concept that the stereotypical shaping of the niche structure optimizes the stem cell system, thereby maximizing the reproductive capacity.
Asunto(s)
Proteínas de Drosophila , Animales , Apoptosis/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células Germinativas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Nicho de Células Madre/genética , Proteínas Proto-Oncogénicas c-jun/metabolismoRESUMEN
Handicap theory explains that exaggeratedly developed sexual traits become handicaps but serve as honest signals of quality. Because very weak signals are less likely to provide benefits than to simply incur costs, it is interesting to elucidate how sexual traits are generated and developed during evolution. Many stalk-eyed fly species belonging to tribe Diopsini exhibit marked sexual dimorphism in their eye spans, and males with larger eye spans have larger bodies and reproductive capacities, which are more advantageous in terms of contests between males and acceptance for mating by females. In this study, we investigated the role of eye span in a more primitive species, Sphyracephala detrahens, in tribe Sphyracephalini with less pronounced sexual dimorphism. Male-male, female-female, and male-female pairs showed similar contests influenced by eye span, which was correlated with nutrition and reproductive ability in both sexes. During mating, males did not distinguish between sexes and chose individuals with larger eye spans, whereas females did not choose males. However, males with larger eye spans copulated repeatedly. These results indicate that, in this species, eye span with a small sexual difference does not function in sex recognition but affects contest and reproductive outcomes, suggesting the primitive state of sexual dimorphism.
Asunto(s)
Dípteros/fisiología , Modelos Biológicos , Conducta Sexual Animal , Animales , Evolución Biológica , Dípteros/anatomía & histología , Ojo/anatomía & histología , Femenino , Masculino , Caracteres SexualesRESUMEN
Multicellular organisms repair injured epithelium by evolutionarily conserved biological processes including activation of c-Jun N-terminal kinase (JNK) signaling. Here, we show in Drosophila imaginal epithelium that physical injury leads to the emergence of dying cells, which are extruded from the wounded tissue by JNK-induced Slit-Roundabout2 (Robo2) repulsive signaling. Reducing Slit-Robo2 signaling in the wounded tissue suppresses extrusion of dying cells and generates aberrant cells with highly upregulated growth factors Wingless (Wg) and Decapentaplegic (Dpp). The inappropriately elevated Wg and Dpp impairs wound repair, as halving one of these growth factor genes cancelled wound healing defects caused by Slit-Robo2 downregulation. Our data suggest that JNK-mediated Slit-Robo2 signaling contributes to epithelial wound repair by promoting extrusion of dying cells from the wounded tissue, which facilitates transient and appropriate induction of growth factors for proper wound healing.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Transducción de Señal/fisiología , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiologíaRESUMEN
The adult male accessory gland in insects is an internal reproductive organ analogous to the mammalian prostate, and secretes various components in the seminal fluid. Products of the accessory gland in the fruit fly Drosophila melanogaster are known to control reproductive behaviors in mated females, such as food uptake, oviposition rate, and rejection of re-mating with other males, all of which increase male reproductive capacity. Production of larger amounts of accessory gland products is thus thought to result in higher male reproductive success. The epithelium of the Drosophila accessory gland lobe is composed of a unique population of binucleate cells. We previously predicted, based on measurements of cell size in mono/binucleate mosaic accessory glands, that binucleation results in a higher plasticity in cell shape, enabling more effective ejection of seminal fluid. However, the actual effect of binucleation on ejection of seminal fluid or reproductive capacity remained unclear, as we were unable to generate an organ with uniformly mononucleate cells. In the present study, we generated organs in which most of the epithelial cells are mononucleate by manipulating aurora B or fizzy-related to block binucleation. Mononucleation resulted in a less elastic accessory gland lobe, which decreased ejection volume and the oviposition of mated females; these effects were particularly pronounced over the long term. These results suggest that binucleation in accessory gland epithelial cells contributes to higher plasticity in the volume of this organ, and enhances male reproductive success through enabling ejection of larger amounts of seminal fluid.
Asunto(s)
Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/fisiología , Genitales Masculinos/anatomía & histología , Genitales Masculinos/fisiología , Animales , Animales Modificados Genéticamente , Mapeo Cromosómico , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Masculino , Conducta Sexual AnimalRESUMEN
The Drosophila adult has an intestine composed of a series of differentiated cells and tissue stem cells, all of which are similar to the mammalian intestinal cells. The aged adult intestine shows apparent characteristics such as multilayering of absorptive cells, misexpression of cell type-specific genes, and hyperproliferation of stem cells. Recent studies have revealed various gene networks responsible for progression of these aged phenotypes. The molecular mechanism for senescence of the Drosophila adult midgut and its relation with the corresponding mechanism in mammals are overviewed. In addition, a basic method for observing aged phenotypes of the midgut is described.
Asunto(s)
Envejecimiento/patología , Drosophila , Intestinos/patología , Modelos Animales , Animales , HumanosRESUMEN
Nutrient conditions affect the reproductive potential and lifespan of many organisms through the insulin signaling pathway. Although this is well characterized in female oogenesis, nutrient-dependent regulation of fertility/fecundity in males is not known. Seminal fluid components synthesized in the accessory gland are required for high fecundity in Drosophila males. The accessory gland is composed of two types of binucleated cells: a main cell and a secondary cell (SC). The transcription factors Defective proventriculus (Dve) and Abdominal-B (Abd-B) are strongly expressed in adult SCs, whose functions are essential for male fecundity. We found that gene expression of both Dve and Abd-B was down-regulated under nutrient-poor conditions. In addition, nutrient conditions during the pupal stage affected the size and number of SCs. These morphological changes clearly correlated with fecundity, suggesting that SCs act as nutrient sensors. Here, we provide evidence that Dve associates nutrient conditions with optimal reproductive potential in a target of rapamycin signaling-dependent manner.
Asunto(s)
Proteínas de Drosophila/fisiología , Fertilidad/efectos de los fármacos , Genitales/metabolismo , Proteínas de Homeodominio/fisiología , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Genitales/fisiología , Proteínas de Homeodominio/genética , Insulina/metabolismo , Masculino , Semen/metabolismo , Semen/fisiología , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Enteroendocrine cells (EEs) are evolutionarily conserved gastrointestinal secretory cells that show scattered distribution in the intestinal epithelium. These cells classified into several subtypes based on the hormones they produce in both mammals and insects. In the fruit fly Drosophila, it has been suggested that nearly equal numbers of two subtypes of EEs (Allatostatin A: AstA and Diuretic hormone 31 : Dh31) are alternately produced from the intestinal stem cells in the posterior midgut. However, we found that these two subtypes are not always present in this manner, but are rather distributed in a complementary frequency gradient along the posterior midgut. We show that midgut-preferential RNA knockdown of the peptide hormones AstA or Dh31 respectively results in decreased or increased adult lifespan. This effect on longevity is apparently correlated with the midgut senescence phenotypes as a result of direct hormone action through both hormone receptors expressed in the enteroblasts or other midgut cell types. However, gut senescence does not appear to be the direct cause for longevity regulation, as knockdown of both hormone receptors did not affect adult lifespan. Furthermore, these senescence phenotypes appear to be independent of insulin signaling and manifest in an organ-specific manner. These results indicate that the two intestinal secretory peptides antagonistically regulate adult lifespan and intestinal senescence through multiple pathways, irrespective of insulin, which implicates a complementary gradient distribution of each of the hormone-producing EEs, consistent with local requirements for cell activity along the posterior midgut.
Asunto(s)
Envejecimiento , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Hormonas de Insectos/genética , Neuropéptidos/genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Tracto Gastrointestinal/metabolismo , Hormonas de Insectos/metabolismo , Longevidad , Neuropéptidos/metabolismoRESUMEN
Adult intestinal tissues, exposed to the external environment, play important roles including barrier and nutrient-absorption functions. These functions are ensured by adequately controlled rapid-cell metabolism. GATA transcription factors play essential roles in the development and maintenance of adult intestinal tissues both in vertebrates and invertebrates. We investigated the roles of GATAe, the Drosophila intestinal GATA factor, in adult midgut homeostasis with its first-generated knock-out mutant as well as cell type-specific RNAi and overexpression experiments. Our results indicate that GATAe is essential for proliferation and maintenance of intestinal stem cells (ISCs). Also, GATAe is involved in the differentiation of enterocyte (EC) and enteroendocrine (ee) cells in both Notch (N)-dependent and -independent manner. The results also indicate that GATAe has pivotal roles in maintaining normal epithelial homeostasis of the Drosophila adult midgut through interaction of N signaling. Since recent reports showed that mammalian GATA-6 regulates normal and cancer stem cells in the adult intestinal tract, our data also provide information on the evolutionally conserved roles of GATA factors in stem-cell regulation.
Asunto(s)
Diferenciación Celular , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Factores de Transcripción GATA/fisiología , Intestinos/citología , Células Madre/citología , Envejecimiento , Animales , Drosophila melanogaster/citología , Factor de Transcripción GATA4/fisiología , Factor de Transcripción GATA6/fisiologíaRESUMEN
The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left-right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Miosina Tipo I/genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Genitales Masculinos/embriología , Genitales Masculinos/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/embriología , Masculino , Mutación , Miosina Tipo I/metabolismo , Especificidad de ÓrganosRESUMEN
BACKGROUND: In standard cell division, the cells undergo karyokinesis and then cytokinesis. Some cells, however, such as cardiomyocytes and hepatocytes, can produce binucleate cells by going through mitosis without cytokinesis. This cytokinesis skipping is thought to be due to the inhibition of cytokinesis machinery such as the central spindle or the contractile ring, but the mechanisms regulating it are unclear. We investigated them by characterizing the binucleation event during development of the Drosophila male accessory gland, in which all cells are binucleate. RESULTS: The accessory gland cells arrested the cell cycle at 50 hours after puparium formation (APF) and in the middle of the pupal stage stopped proliferating for 5 hours. They then restarted the cell cycle and at 55 hours APF entered the M-phase synchronously. At this stage, accessory gland cells binucleated by mitosis without cytokinesis. Binucleating cells displayed the standard karyokinesis progression but also showed unusual features such as a non-round shape, spindle orientation along the apico-basal axis, and poor assembly of the central spindle. Mud, a Drosophila homolog of NuMA, regulated the processes responsible for these three features, the classical isoform Mud(PBD) and the two newly characterized isoforms Mud(L) and Mud(S) regulated them differently: Mud(L) repressed cell rounding, Mud(PBD) and Mud(S) oriented the spindle along the apico-basal axis, and Mud(S) and Mud(L) repressed central spindle assembly. Importantly, overexpression of Mud(S) induced binucleation even in standard proliferating cells such as those in imaginal discs. CONCLUSIONS: We characterized the binucleation in the Drosophila male accessory gland and examined mechanisms that regulated unusual morphologies of binucleating cells. We demonstrated that Mud, a microtubule binding protein regulating spindle orientation, was involved in this binucleation. We suggest that atypical functions exerted by three structurally different isoforms of Mud regulate cell rounding, spindle orientation and central spindle assembly in binucleation. We also propose that Mud(S) is a key regulator triggering cytokinesis skipping in binucleation processes.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Secuencia de Aminoácidos , Animales , Núcleo Celular/fisiología , Polaridad Celular , Forma de la Célula , Citocinesis , Drosophila melanogaster/citología , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Genitales Masculinos/citología , Masculino , Metafase , Datos de Secuencia Molecular , Isoformas de Proteínas/fisiología , Huso Acromático/metabolismoRESUMEN
Many animals show left-right (LR) asymmetric morphology. The mechanisms of LR asymmetric development are evolutionarily divergent, and they remain elusive in invertebrates. Various organs in Drosophila melanogaster show stereotypic LR asymmetry, including the embryonic gut. The Drosophila embryonic hindgut twists 90° left-handedly, thereby generating directional LR asymmetry. We recently revealed that the hindgut epithelial cell is chiral in shape and other properties; this is termed planar cell chirality (PCC). We previously showed by computer modeling that PCC is sufficient to induce the hindgut rotation. In addition, both the PCC and the direction of hindgut twisting are reversed in Myosin31DF (Myo31DF) mutants. Myo31DF encodes Drosophila MyosinID, an actin-based motor protein, whose molecular functions in LR asymmetric development are largely unknown. Here, to understand how PCC directs the asymmetric cell-shape, we analyzed PCC in genetic mosaics composed of cells homozygous for mutant Myo31DF, some of which also overexpressed wild-type Myo31DF. Wild-type cell-shape chirality only formed in the Myo31DF-overexpressing cells, suggesting that cell-shape chirality was established in each cell and reflects intrinsic PCC. A computer model recapitulating the development of this genetic mosaic suggested that mechanical interactions between cells are required for the cell-shape behavior seen in vivo. Our mosaic analysis also suggested that during hindgut rotation in vivo, wild-type Myo31DF suppresses the elongation of cell boundaries, supporting the idea that cell-shape chirality is an intrinsic property determined in each cell. However, the amount and distribution of F-actin and Myosin II, which are known to help generate the contraction force on cell boundaries, did not show differences between Myo31DF mutant cells and wild-type cells, suggesting that the static amount and distribution of these proteins are not involved in the suppression of cell-boundary elongation. Taken together, our results suggest that cell-shape chirality is intrinsically formed in each cell, and that mechanical force from intercellular interactions contributes to its formation and/or maintenance.
Asunto(s)
Tipificación del Cuerpo/fisiología , Polaridad Celular/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Miosina Tipo I/fisiología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Polaridad Celular/genética , Forma de la Célula/genética , Forma de la Célula/fisiología , Simulación por Computador , Sistema Digestivo/citología , Sistema Digestivo/embriología , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes de Insecto , Mecanotransducción Celular/genética , Modelos Biológicos , Mosaicismo , Mutación , Miosina Tipo I/genéticaRESUMEN
Proper control of adult stem cells including their proliferation and differentiation is crucial in maintaining homeostasis of well-organized tissues/organs throughout an organism's life. The Drosophila adult midgut has intestinal stem cells (ISCs), which have been exploited as a simple model system to investigate mechanisms controlling adult tissue homeostasis. Here, we found that a viable mutant of ßν integrin (ßint-ν), encoding one of two Drosophila integrin ß subunits, showed a short midgut and abnormal multilayered epithelia accompanied by an increase in ISC proliferation and misdifferentiation defects. The increase in ISC proliferation and misdifferentiation was due to frequent ISC duplication expanding a pool of ISCs, which was caused by depression of the Notch signalling, and up-regulation of unpaired (upd), a gene encoding an extracellular ligand in the JAK/STAT signalling pathway. In addition, we observed that abnormally high accumulation of filamentous actin (F-actin) was caused in the ßint-ν mutant enterocytes. Furthermore, the defects were rescued by suppressing c-Jun N-terminal kinase (JNK) signalling, which was up-regulated in a manner correlated with the defect levels in the above-mentioned ßint-ν mutant phenotype. These symptoms observed in young ßint-ν mutant midgut were very similar to those in the aged midgut in wild type. Our results suggested that ßint-ν has a novel function for the Drosophila adult midgut homeostasis under normal conditions and provided a new insight into possible age-related diseases caused by latent abnormality of an integrin function.
Asunto(s)
Senescencia Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Cadenas beta de Integrinas/metabolismo , Intestinos/citología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Células Madre/citología , Animales , Apoptosis , Proliferación Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Ensayo de Inmunoadsorción Enzimática , Técnicas para Inmunoenzimas , Hibridación in Situ , Cadenas beta de Integrinas/genética , Mucosa Intestinal/metabolismo , Mutación/genética , Fenotipo , Filogenia , Células Madre/metabolismoRESUMEN
Animals often show left-right (LR) asymmetry in their body structures. In some vertebrates, the mechanisms underlying LR symmetry breaking and the subsequent signals responsible for LR asymmetric development are well understood. However, in invertebrates, the molecular bases of these processes are largely unknown. Therefore, we have been studying the genetic pathway of LR asymmetric development in Drosophila. The embryonic gut is the first organ that shows directional LR asymmetry during Drosophila development. We performed a genetic screen to identify mutations affecting LR asymmetric development of the embryonic gut. From this screen, we isolated pebble (pbl), which encodes a homolog of a mammalian RhoGEF, Ect2. The laterality of the hindgut was randomized in embryos homozygous for a null mutant of pbl. Pbl is a multi-functional protein required for cytokinesis and the epithelial-to-mesenchymal transition in Drosophila. Consistent with Pbl's role in cytokinesis, we found reduced numbers of cells in the hindgut epithelium in pbl homozygous embryos. The specific expression of pbl in the hindgut epithelium, but not in other tissues, rescued the LR defects and reduced cell number in embryonic pbl homozygotes. Embryos homozygous for string (stg), a mutant that reduces cell number through a different mechanism, also showed LR defects of the hindgut. However, the reduction in cell number in the pbl mutants was not accompanied by defects in the specification of hindgut epithelial tissues or their integrity. Based on these results, we speculate that the reduction in cell number may be one reason for the LR asymmetry defect of the pbl hindgut, although we cannot exclude contributions from other functions of Pbl, including regulation of the actin cytoskeleton through its RhoGEF activity.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Tracto Gastrointestinal/embriología , Factores de Intercambio de Guanina Nucleótido/genética , Alelos , Animales , Tipificación del Cuerpo/genética , Recuento de Células , Polaridad Celular , Citocinesis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Células Epiteliales/fisiología , Epitelio/embriología , Epitelio/metabolismo , Tracto Gastrointestinal/citología , Eliminación de Gen , Expresión Génica , Factores de Intercambio de Guanina Nucleótido/metabolismo , Homocigoto , Especificidad de Órganos , Mutación Puntual , Sitios de Empalme de ARN , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Mosaic analysis is used to assess gene function and cell autonomy in a subset of cells in an organism, and has been extensively applied in Drosophila studies. However, it is difficult to generate mosaic cells in Drosophila embryonic tissues using existing methods. Therefore, we developed a new method for generating genetic mosaic embryos using a modified Cre/loxP system. In this report, we also characterized the capabilities and limitations of this novel method. RESULTS: We first constructed a novel cassette combining loxP with the Actin 5C enhancer and Gal4 cDNA, and generated a transgenic fly carrying this construct (Aloxg-Gal4). In Aloxg-Gal4, the activation of Gal4 expression is suppressed by the gypsy insulator. Once the gypsy insulator is removed, however, Gal4 is expressed when site-specific recombination between loxP sites is induced by Cre recombinase. This system allowed the mosaic expression of Gal4 in Drosophila embryonic tissues (epidermis, amnioserosa, tracheal system, malpighian tubules, foregut, hindgut, midgut, and neuron), leading to the Gal4-dependent activation of arbitrary genes under the control of the upstream activation sequence (UAS). CONCLUSIONS: This practical method can be used to generate mosaic cells in Drosophila embryonic tissues and can be applied to any gene without specialized equipment.
Asunto(s)
Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Integrasas/genética , Mosaicismo , Animales , Animales Modificados Genéticamente , Drosophila/embriología , Expresión Génica , Genes ReporterosRESUMEN
The Drosophila male accessory gland has functions similar to those of the mammalian prostate gland and the seminal vesicle, and secretes accessory gland proteins into the seminal fluid. Each of the two lobes of the accessory gland is composed of two types of binucleate cell: about 1,000 main cells and 40 secondary cells. A well-known accessory gland protein, sex peptide, is secreted from the main cells and induces female postmating response to increase progeny production, whereas little is known about physiological significance of the secondary cells. The homeodomain transcriptional repressor Defective proventriculus (Dve) is strongly expressed in adult secondary cells, and its mutation resulted in loss of secondary cells, mononucleation of main cells, and reduced size of the accessory gland. dve mutant males had low fecundity despite the presence of sex peptide, and failed to induce the female postmating responses of increased egg laying and reduced sexual receptivity. RNAi-mediated dve knockdown males also had low fecundity with normally binucleate main cells. We provide the first evidence that secondary cells are crucial for male fecundity, and also that Dve activity is required for survival of the secondary cells. These findings provide new insights into a mechanism of fertility/fecundity.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Glándulas Exocrinas/metabolismo , Genitales Masculinos/metabolismo , Proteínas de Homeodominio/metabolismo , Análisis de Varianza , Animales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Glándulas Exocrinas/citología , Glándulas Exocrinas/crecimiento & desarrollo , Femenino , Fertilidad/fisiología , Perfilación de la Expresión Génica , Vectores Genéticos/genética , Genitales Masculinos/citología , Genitales Masculinos/crecimiento & desarrollo , Proteínas de Homeodominio/genética , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular , Masculino , Péptidos/metabolismo , Interferencia de ARN , Semen/metabolismo , Semen/fisiología , Conducta Sexual Animal/fisiología , Factores de Transcripción/genéticaRESUMEN
Many animals develop left-right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Therefore, we studied the genetic pathway of LR asymmetric development in Drosophila. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. In this study, we found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 (fz2) and Wnt4, which encode a receptor and ligand of Wnt signaling, respectively, were required for the LR asymmetric development of the AMG. arrow (arr), an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Our genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 were expressed in the visceral muscles of the midgut. Consistent with these results, we observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. Our results also suggest that canonical Wnt/ß-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/ß-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/ß-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates.
Asunto(s)
Sistema Digestivo/embriología , Drosophila melanogaster/embriología , Músculo Liso/embriología , Vía de Señalización Wnt , Animales , Análisis Mutacional de ADN , Sistema Digestivo/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Músculo Liso/citología , Músculo Liso/metabolismo , Mutación , Miocitos del Músculo Liso/metabolismo , Especificidad de Órganos , Organogénesis , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Some organs in animals display left-right (LR) asymmetry. To better understand LR asymmetric morphogenesis in Drosophila, we studied LR directional rotation of the hindgut epithelial tube. Hindgut epithelial cells adopt a LR asymmetric (chiral) cell shape within their plane, and we refer to this cell behavior as planar cell-shape chirality (PCC). Drosophila E-cadherin (DE-Cad) is distributed to cell boundaries with LR asymmetry, which is responsible for the PCC formation. Myosin ID switches the LR polarity found in PCC and in DE-Cad distribution, which coincides with the direction of rotation. An in silico simulation showed that PCC is sufficient to induce the directional rotation of this tissue. Thus, the intrinsic chirality of epithelial cells in vivo is an underlying mechanism for LR asymmetric tissue morphogenesis.