RESUMEN
To gain insight into the transcription programs activated during the formation of Drosophila larval structures, we carried out single cell RNA sequencing during two periods of Drosophila embryogenesis: stages 10-12, when most organs are first specified and initiate morphological and physiological specialization; and stages 13-16, when organs achieve their final mature architectures and begin to function. Our data confirm previous findings with regards to functional specialization of some organs - the salivary gland and trachea - and clarify the embryonic functions of another - the plasmatocytes. We also identify two early developmental trajectories in germ cells and uncover a potential role for proteolysis during germline stem cell specialization. We identify the likely cell type of origin for key components of the Drosophila matrisome and several commonly used Drosophila embryonic cell culture lines. Finally, we compare our findings with other recent related studies and with other modalities for identifying tissue-specific gene expression patterns. These data provide a useful community resource for identifying many new players in tissue-specific morphogenesis and functional specialization of developing organs.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Transcriptoma/genética , Organogénesis , Proteínas de Drosophila/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
In mammals, the serine protease plasmin degrades extracellular proteins during blood clot removal, tissue remodeling, and cell migration. The zymogen plasminogen is activated into plasmin by two serine proteases: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), a process regulated by plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor that specifically inhibits tPA and uPA. Plasmodium gametes and sporozoites use tPA and uPA to activate plasminogen and parasite-bound plasmin degrades extracellular matrices, facilitating parasite motility in the mosquito and the mammalian host. Furthermore, inhibition of plasminogen activation by PAI-1 strongly blocks infection in both hosts. To block parasite utilization of plasmin, we engineered Anopheles stephensi transgenic mosquitoes constitutively secreting human PAI-1 (huPAI-1) in the midgut lumen, in the saliva, or both. Mosquitoes expressing huPAI-1 strongly reduced rodent and human Plasmodium parasite transmission to mosquitoes, showing that co-opting plasmin for mosquito infection is a conserved mechanism among Plasmodium species. huPAI-1 expression in saliva induced salivary gland deformation which affects sporozoite invasion and P. berghei transmission to mice, resulting in significant levels of protection from malaria. Targeting the interaction of malaria parasites with the fibrinolytic system using genetically engineered mosquitoes could be developed as an intervention to control malaria transmission.
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Anopheles , Malaria , Plasmodium , Animales , Animales Modificados Genéticamente , Anopheles/parasitología , Fibrinolisina , Humanos , Malaria/parasitología , Mamíferos , Ratones , Mosquitos Vectores/genética , Plasminógeno , Inhibidor 1 de Activador Plasminogénico/genética , Plasmodium/fisiología , EsporozoítosRESUMEN
Cell growth is well defined for late (postembryonic) stages of development, but evidence for early (embryonic) cell growth during postmitotic morphogenesis is limited. Here, we report early cell growth as a key characteristic of tubulogenesis in the Drosophila embryonic salivary gland (SG) and trachea. A BTB/POZ domain nuclear factor, Ribbon (Rib), mediates this early cell growth. Rib binds the transcription start site of nearly every SG-expressed ribosomal protein gene (RPG) and is required for full expression of all RPGs tested. Rib binding to RPG promoters in vitro is weak and not sequence specific, suggesting that specificity is achieved through cofactor interactions. Accordingly, we demonstrate Rib's ability to physically interact with each of the three known regulators of RPG transcription. Surprisingly, Rib-dependent early cell growth in another tubular organ, the embryonic trachea, is not mediated by direct RPG transcription. These findings support a model of early cell growth customized by transcriptional regulatory networks to coordinate organ form and function.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regiones Promotoras Genéticas , Proteínas Ribosómicas/genética , Glándulas Salivales/metabolismo , Animales , Proteínas de Drosophila/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.
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Anopheles , Animales , Disección , Larva , Proteómica , Glándulas SalivalesRESUMEN
Filamins are highly conserved actin-crosslinking proteins that regulate organization of the actin cytoskeleton. As key components of versatile signaling scaffolds, filamins are implicated in developmental anomalies and cancer. Multiple isoforms of filamins exist, raising the possibility of distinct functions for each isoform during development and in disease. Here, we provide an initial characterization of jitterbug (jbug), which encodes one of the two filamin-type proteins in Drosophila. We generate Jbug antiserum that recognizes all of the spliced forms and reveals differential expression of different Jbug isoforms during development, and a significant maternal contribution of Jbug protein. To reveal the function of Jbug isoforms, we create new genetic tools, including a null allele that deletes all isoforms, hypomorphic alleles that affect only a subset, and UAS lines for Gal4-driven expression of the major isoforms. Using these tools, we demonstrate that Jbug is required for viability and that specific isoforms are required in the formation of actin-rich protrusions including thoracic bristles in adults and ventral denticles in the embryo. We also show that specific isoforms of Jbug show differential localization within epithelia and that maternal and zygotic loss of jbug disrupts Crumbs (Crb) localization in several epithelial cell types.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Morfogénesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMEN
Secretory organs are critical for organismal survival. Yet, the transcriptional regulatory mechanisms governing their development and maintenance remain unclear for most model secretory organs. The Drosophila embryonic salivary gland (SG) remedies this deficiency as one of the few organs wherein direct connections from the expression of the early patterning genes to cell specification to organ architecture and functional specialization can be made. Few other models of secretion can be accorded this distinction. Studies from the past three decades have made enormous strides in parsing out the roles of distinct transcription factors (TFs) that direct major steps in furnishing this secretory organ. In the first step of specifying the salivary gland, the activity of the Hox factors Sex combs reduced, Extradenticle, and Homothorax activate expression of fork head (fkh), sage, and CrebA, which code for the major suite of TFs that carry forward the task of organ building and maintenance. Then, in the second key step of building the SG, the program for cell fate maintenance and morphogenesis is deployed. Fkh maintains the secretory cell fate by regulating its own expression and that of sage and CrebA. Fkh and Sage maintain secretory cell viability by actively blocking apoptotic cell death. Fkh, along with two other TFs, Hkb and Rib, also coordinates organ morphogenesis, transforming two plates of precursor cells on the embryo surface into elongated internalized epithelial tubes. Acquisition of functional specialization, the third key step, is mediated by CrebA and Fkh working in concert with Sage and yet another TF, Sens. CrebA directly upregulates expression of all of the components of the secretory machinery as well as other genes (e.g., Xbp1) necessary for managing the physiological stress that inexorably accompanies high secretory load. Secretory cargo specificity is controlled by Sage and Sens in collaboration with Fkh. Investigations have also uncovered roles for various signaling pathways, e.g., Dpp signaling, EGF signaling, GPCR signaling, and cytoskeletal signaling, and their interactions within the gene regulatory networks that specify, build, and specialize the SG. Collectively, studies of the SG have expanded our knowledge of secretory dynamics, cell polarity, and cytoskeletal mechanics in the context of organ development and function. Notably, the embryonic SG has made the singular contribution as a model system that revealed the core function of CrebA in scaling up secretory capacity, thus, serving as the pioneer system in which the conserved roles of the mammalian Creb3/3L-family orthologues were first discovered.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Transcripción Forkhead/metabolismo , Mamíferos/metabolismo , Proteínas Nucleares/genética , Glándulas Salivales/metabolismoRESUMEN
The Tre1 G-protein coupled receptor (GPCR) was discovered to be required for Drosophila germ cell (GC) coalescence almost two decades ago, yet the molecular events both upstream and downstream of Tre1 activation remain poorly understood. To gain insight into these events, we describe a bona fide null allele and both untagged and tagged versions of Tre1. We find that the primary defect with complete Tre1 loss is the failure of GCs to properly navigate, with GC mis-migration occurring from early stages. We find that Tre1 localizes with F-actin at the migration front, along with PI(4,5)P2; dPIP5K, an enzyme that generates PI(4,5)P2; and dWIP, a protein that binds activated Wiskott-Aldrich syndrome protein (WASP), which stimulates F-actin polymerization. We show that Tre1 is required for polarized accumulation of F-actin, PI(4,5)P2, and dPIP5K. Smoothened also localizes with F-actin at the migration front, and Hh, through Smo, increases levels of Tre1 at the plasma membrane and Tre1's association with dPIP5K.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinales Embrionarias/metabolismo , Proteínas Hedgehog/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Citoesqueleto de Actina/genética , Animales , Animales Modificados Genéticamente , Movimiento Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Proteínas Hedgehog/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Factores de Tiempo , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Specialization of many cells, including the acinar cells of the salivary glands and pancreas, milk-producing cells of mammary glands, mucus-secreting goblet cells, antibody-producing plasma cells, and cells that generate the dense extracellular matrices of bone and cartilage, requires scaling up both secretory machinery and cell-type specific secretory cargo. Using tissue-specific genome-scale analyses, we determine how increases in secretory capacity are coordinated with increases in secretory load in the Drosophila salivary gland (SG), an ideal model for gaining mechanistic insight into the functional specialization of secretory organs. Our findings show that CrebA, a bZIP transcription factor, directly binds genes encoding the core secretory machinery, including protein components of the signal recognition particle and receptor, ER cargo translocators, Cop I and Cop II vesicles, as well as the structural proteins and enzymes of these organelles. CrebA directly binds a subset of SG cargo genes and CrebA binds and boosts expression of Sage, a SG-specific transcription factor essential for cargo expression. To further enhance secretory output, CrebA binds and activates Xbp1 and Tudor-SN. Thus, CrebA directly upregulates the machinery of secretion and additional factors to increase overall secretory capacity in professional secretory cells; concomitant increases in cargo are achieved both directly and indirectly.
Asunto(s)
Proteínas de Drosophila , Animales , Proteína de Unión al Elemento de Respuesta al AMP Cíclico , Drosophila , Proteínas de Drosophila/genética , Glándulas Salivales , Factores de TranscripciónRESUMEN
Plasmodium sporozoites (SPZs) must traverse the mosquito salivary glands (SGs) to reach a new vertebrate host and continue the malaria disease cycle. Although SGs can harbor thousands of sporozoites, only 10 to 100 are deposited into a host during probing. To determine how the SGs might function as a bottleneck in SPZ transmission, we have characterized Anopheles stephensi SGs infected with the rodent malaria parasite Plasmodium berghei using immunofluorescence confocal microscopy. Our analyses corroborate findings from previous electron microscopy studies and provide new insights into the invasion process. We identified sites of SPZ accumulation within SGs across a range of infection intensities. Although SPZs were most often seen in the distal lateral SG lobes, they were also observed in the medial and proximal lateral lobes. Most parasites were associated with either the basement membrane or secretory cavities. SPZs accumulated at physical barriers, including fused salivary ducts and extensions of the chitinous salivary duct wall into the distal lumen. SPZs were observed only rarely within salivary ducts. SPZs appeared to contact each other in many different quantities, not just in the previously described large bundles. Within parasite bundles, all of the SPZs were oriented in the same direction. We found that moderate levels of infection did not necessarily correlate with major SG disruptions or abundant SG cell death. Altogether, our findings suggest that SG architecture largely acts as a barrier to SPZ transmission.IMPORTANCE Malaria continues to have a devastating impact on human health. With growing resistance to insecticides and antimalarial drugs, as well as climate change predictions indicating expansion of vector territories, the impact of malaria is likely to increase. Additional insights regarding pathogen migration through vector mosquitoes are needed to develop novel methods to prevent transmission to new hosts. Pathogens, including the microbes that cause malaria, must invade the salivary glands (SGs) for transmission. Since SG traversal is required for parasite transmission, SGs are ideal targets for transmission-blocking strategies. The work presented here highlights the role that mosquito SG architecture plays in limiting parasite traversal, revealing how the SG transmission bottleneck is imposed. Further, our data provide unprecedented detail about SG-sporozoite interactions and gland-to-gland variation not provided in previous studies.
Asunto(s)
Anopheles/parasitología , Malaria/transmisión , Mosquitos Vectores/parasitología , Plasmodium berghei/crecimiento & desarrollo , Animales , Anopheles/fisiología , Femenino , Humanos , Malaria/parasitología , Masculino , Ratones , Mosquitos Vectores/fisiología , Plasmodium berghei/fisiología , Glándulas Salivales/parasitología , Esporozoítos/crecimiento & desarrollo , Esporozoítos/fisiologíaRESUMEN
BACKGROUND: CG4552/tbc1 was identified as a downstream target of Fork head (Fkh), the single Drosophila member of the FoxA family of transcription factors and a major player in salivary gland formation and homeostasis. Tbc1 and its orthologues have been implicated in phagocytosis, the innate immune response, border cell migration, cancer and an autosomal recessive form of non-degenerative Pontocerebellar hypoplasia. Recently, the mammalian Tbc1 orthologue, Tbc1d23, has been shown to bind both the conserved N-terminal domains of two Golgins (Golgin-97 and Golgin-245) and the WASH complex on endosome vesicles. Through this activity, Tbc1d23 has been proposed to link endosomally-derived vesicles to their appropriate target membrane in the trans Golgi (TGN). RESULTS: In this paper, we provide an initial characterization of Drosophila orthologue, we call tbc1. We show that, like its mammalian orthologue, Tbc1 localizes to the trans Golgi. We show that it also colocalizes with a subset of Rabs associated with both early and recycling endosomes. Animals completely missing tbc1 survive, but females have fertility defects. Consistent with the human disease, loss of tbc1 reduces optic lobe size and increases response time to mechanical perturbation. Loss and overexpression of tbc1 in the embryonic salivary glands leads to secretion defects and apical membrane irregularities. CONCLUSIONS: These findings support a role for tbc1 in endocytic/membrane trafficking, consistent with its activities in other systems.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Glándulas Salivales/embriología , Alelos , Animales , Drosophila melanogaster/metabolismo , Endosomas/metabolismo , Factores de Transcripción Forkhead/metabolismo , GTP Fosfohidrolasas/metabolismo , Expresión Génica , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Sistemas de Lectura Abierta/genética , Lóbulo Óptico de Animales no Mamíferos/metabolismo , Glándulas Salivales/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Red trans-Golgi/metabolismoRESUMEN
Bruce Baker, a preeminent Drosophila geneticist who made fundamental contributions to our understanding of the molecular genetic basis of sex differences, passed away July 1, 2018 at the age of 72. Members of Bruce's laboratory remember him as an intensely dedicated, rigorous, creative, deep-thinking, and fearless scientist. His trainees also remember his strong commitment to teaching students at every level. Bruce's career studying sex differences had three major epochs, where the laboratory was focused on: (1) sex determination and dosage compensation, (2) the development of sex-specific structures, and (3) the molecular genetic basis for sex differences in behavior. Several members of the Baker laboratory have come together to honor Bruce by highlighting some of the laboratory's major scientific contributions in these areas.
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Drosophila/genética , Genética/historia , Procesos de Determinación del Sexo/genética , Animales , Compensación de Dosificación (Genética) , Evolución Molecular , Historia del Siglo XX , Historia del Siglo XXI , Mentores , Conducta SexualRESUMEN
Mosquito-borne diseases cause one million deaths and hundreds of millions of human infections yearly. With all such diseases, the pathogen must traverse the mosquito salivary gland (SG) for transmission to a new host, making the SGs ideal targets for genetic strategies to block transmission. Prior studies have elucidated details of SG structure by light and electron microscopy and have deeply explored the salivary transcriptome and proteome. Very little is known, however, about how the unique functional architecture of mosquito SGs is achieved. Using immunohistochemistry and confocal microscopy, we address two questions regarding SGs of the malaria vector Anopheles gambiae. How does the distinct cup-shaped morphology of SG secretory cells arise? And, how does the salivary duct, the structure through which saliva and parasites exit the glands, form? We demonstrate that SG cells begin as cuboidal-shaped cells surrounding a matrix-filled lumen that mature into cup-shaped cells through the formation and fusion of a large pre-apical compartment (PAC) to the apical surface. The secretory duct begins as buds of chitin at the apical surface of individual secretory cells. Further chitin deposition connects these chitin buds to form a contiguous duct that largely separates from the apical surface during PAC fusion.
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Anopheles/embriología , Anopheles/crecimiento & desarrollo , Organogénesis , Glándulas Salivales/embriología , Glándulas Salivales/crecimiento & desarrollo , Animales , Anopheles/parasitología , Biomarcadores , Humanos , Inmunohistoquímica , Malaria/parasitología , Malaria/transmisión , Masculino , Modelos Biológicos , Fenotipo , Glándulas Salivales/citología , Glándulas Salivales/parasitología , Factores de TiempoRESUMEN
Apical constriction is a widely utilized cell shape change linked to folding, bending and invagination of polarized epithelia. It remains unclear how apical constriction is regulated spatiotemporally during tissue invagination and how this cellular process contributes to tube formation in different developmental contexts. Using Drosophila salivary gland (SG) invagination as a model, we show that regulation of folded gastrulation expression by the Fork head transcription factor is required for apicomedial accumulation of Rho kinase and non-muscle myosin II, which coordinate apical constriction. We demonstrate that neither loss of spatially coordinated apical constriction nor its complete blockage prevent internalization and tube formation, although such manipulations affect the geometry of invagination. When apical constriction is disrupted, compressing force generated by a tissue-level myosin cable contributes to SG invagination. We demonstrate that fully elongated polarized SGs can form outside the embryo, suggesting that tube formation and elongation are intrinsic properties of the SG.
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Drosophila/embriología , Morfogénesis , Glándulas Salivales/embriología , Animales , Miosina Tipo II/metabolismoRESUMEN
Determining how organs attain precise positioning within an organism is a crucial facet of developmental biology. The Fox family winged-helix transcription factors are known to play key roles in development of multiple organs. Drosophila FoxL1 (aka Fd64A) is dynamically expressed in embryos but its function is completely uncharacterized. FoxL1 is expressed in a single group of body wall - muscles in the 2nd and 3rd thoracic segments, in homologous abdominal muscles at earlier stages, and in the hindgut mesoderm from early through late embryogenesis. We show that FoxL1 expression in T2 and T3 is in VIS5, which is not a single muscle spanning the entire thorax, as previously published, but is, instead, three individual muscles, each spanning a single thoracic segment. We generate mutations in foxL1 and show that, surprisingly, none of the tissues that express FoxL1 are affected by its loss. Instead, loss of foxL1 results in defects in salivary gland positioning and morphology, as well as defects in the migration of hemocytes, germ cells and Malpighian tubules. We also show that FoxL1-dependent expression of secreted Sema2a in T3 VIS5 is required for normal salivary gland positioning. Altogether, these findings suggest that Drosophila FoxL1 functions like its mammalian counterpart in non-autonomously orchestrating the behaviors of surrounding tissues.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/embriología , Factores de Transcripción Forkhead/fisiología , Organogénesis/fisiología , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrión no Mamífero/ultraestructura , Células Germinales Embrionarias/citología , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Hemocitos/citología , Túbulos de Malpighi/embriología , Músculos/embriología , Músculos/ultraestructura , Especificidad de Órganos , Organogénesis/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Glándulas Salivales/embriología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tórax/embriología , Tórax/ultraestructuraRESUMEN
Transcription factors affect spatiotemporal patterns of gene expression often regulating multiple aspects of tissue morphogenesis, including cell-type specification, cell proliferation, cell death, cell polarity, cell shape, cell arrangement and cell migration. In this work, we describe a distinct role for Ribbon (Rib) in controlling cell shape/volume increases during elongation of the Drosophila salivary gland (SG). Notably, the morphogenetic changes in rib mutants occurred without effects on general SG cell attributes such as specification, proliferation and apoptosis. Moreover, the changes in cell shape/volume in rib mutants occurred without compromising epithelial-specific morphological attributes such as apicobasal polarity and junctional integrity. To identify the genes regulated by Rib, we performed ChIP-seq analysis in embryos driving expression of GFP-tagged Rib specifically in the SGs. To learn if the Rib binding sites identified in the ChIP-seq analysis were linked to changes in gene expression, we performed microarray analysis comparing RNA samples from age-matched wild-type and rib null embryos. From the superposed ChIP-seq and microarray gene expression data, we identified 60 genomic sites bound by Rib likely to regulate SG-specific gene expression. We confirmed several of the identified Rib targets by qRT-pCR and/or in situ hybridization. Our results indicate that Rib regulates cell growth and tissue shape in the Drosophila salivary gland via a diverse array of targets through both transcriptional activation and repression. Furthermore, our results suggest that autoregulation of rib expression may be a key component of the SG morphogenetic gene network.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Morfogénesis/genética , Proteínas Represoras/metabolismo , Glándulas Salivales/embriología , Activación Transcripcional/genética , Animales , Apoptosis/genética , Secuencia de Bases , Sitios de Unión , División Celular/genética , Polaridad Celular/genética , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Secuencia de Consenso , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Motivos de Nucleótidos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de los Órganos , Unión Proteica , Reproducibilidad de los Resultados , Glándulas Salivales/citología , Glándulas Salivales/metabolismo , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: Anopheles mosquitoes are vectors for malaria, a disease with continued grave outcomes for human health. Transmission of malaria from mosquitoes to humans occurs by parasite passage through the salivary glands (SGs). Previous studies of mosquito SG architecture have been limited in scope and detail. METHODS: We developed a simple, optimized protocol for fluorescence staining using dyes and/or antibodies to interrogate cellular architecture in Anopheles stephensi adult SGs. We used common biological dyes, antibodies to well-conserved structural and organellar markers, and antibodies against Anopheles salivary proteins to visualize many individual SGs at high resolution by confocal microscopy. RESULTS: These analyses confirmed morphological features previously described using electron microscopy and uncovered a high degree of individual variation in SG structure. Our studies provide evidence for two alternative models for the origin of the salivary duct, the structure facilitating parasite transport out of SGs. We compare SG cellular architecture in An. stephensi and Drosophila melanogaster, a fellow Dipteran whose adult SGs are nearly completely unstudied, and find many conserved features despite divergence in overall form and function. Anopheles salivary proteins previously observed at the basement membrane were localized either in SG cells, secretory cavities, or the SG lumen. Our studies also revealed a population of cells with characteristics consistent with regenerative cells, similar to muscle satellite cells or midgut regenerative cells. CONCLUSIONS: This work serves as a foundation for linking Anopheles stephensi SG cellular architecture to function and as a basis for generating and evaluating tools aimed at preventing malaria transmission at the level of mosquito SGs.
Asunto(s)
Anopheles/citología , Insectos Vectores , Animales , Asia , Drosophila melanogaster/citología , Microscopía Confocal , Microscopía Fluorescente , Glándulas Salivales/química , Glándulas Salivales/citología , Proteínas y Péptidos Salivales/análisisRESUMEN
Multiple inositol polyphosphate phosphatase (Mipp), a highly conserved but poorly understood histidine phosphatase, dephosphorylates higher-order IPs (IP4-IP6) to IP3. To gain insight into the biological roles of these enzymes, we have characterized Drosophila mipp1. mipp1 is dynamically expressed in the embryonic trachea, specifically in the leading cells of migrating branches at late stages, where Mipp1 localizes to the plasma membrane and filopodia. FGF signaling activates mipp1 expression in these cells, where extensive filopodia form to drive migration and elongation by cell intercalation. We show that Mipp1 facilitates formation and/or stabilization of filopodia in leading cells through its extracellular activity. mipp1 loss decreases filopodia number, whereas mipp1 overexpression increases filopodia number in a phosphatase-activity-dependent manner. Importantly, expression of Mipp1 gives cells a migratory advantage for the lead position in elongating tracheal branches. Altogether, these findings suggest that extracellular pools of inositol polyphosphates affect cell behavior during development.
Asunto(s)
Movimiento Celular , Drosophila/embriología , Drosophila/metabolismo , Células Epiteliales/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Tráquea/embriología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Western Blotting , Movimiento Celular/fisiología , Embrión no Mamífero , Espacio Extracelular/metabolismo , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Filogenia , Conformación Proteica , ARN Interferente Pequeño , TransfecciónRESUMEN
G-protein-coupled receptors (GPCRs) are the largest family of receptors in many organisms, including worms, mice and humans. GPCRs are seven-transmembrane pass proteins that are activated by binding a stimulus (or ligand) in the extracellular space and then transduce that information to the inside of the cell through conformational changes. The conformational changes activate heterotrimeric G-proteins, which execute the downstream signaling pathways through the recruitment and activation of cellular enzymes. The highly specific ligand-GPCR interaction prompts an efficient cellular response, which is vital for the health of the cell and organism. In this Commentary, we review general features of GPCR signaling and then focus on the Drosophila GPCRs, which are not as well-characterized as their worm and mammalian counterparts. We discuss findings that the Drosophila odorant and gustatory receptors are not bona fide GPCRs as is the case for their mammalian counterparts. We also present here a phylogenetic analysis of the bona fide Drosophila GPCRs that suggest potential roles for several family members. Finally, we discuss recently discovered roles of GPCRs in Drosophila embryogenesis, a field we expect will uncover many previously unappreciated functions for GPCRs.
Asunto(s)
Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Animales , Arrestinas/genética , Drosophila , Humanos , Ratones , Modelos Biológicos , Receptores Acoplados a Proteínas G/genética , Relación Estructura-ActividadRESUMEN
Cells of specialized secretory organs expand their secretory pathways to accommodate the increased protein load necessary for their function. The endoplasmic reticulum (ER), the Golgi apparatus and the secretory vesicles, expand not only the membrane components but also the protein machinery required for increased protein production and transport. Increased protein load causes an ER stress response akin to the Unfolded Protein Response (UPR). Recent work has implicated several bZip transcription factors in the regulation of protein components of the early secretory pathway necessary to alleviate this stress. Here, we highlight eight bZip transcription factors in regulating secretory pathway component genes. These include components of the three canonical branches of the UPR-ATF4, XBP1, and ATF6, as well as the five members of the Creb3 family of transcription factors. We review findings from both invertebrate and vertebrate model systems suggesting that all of these proteins increase secretory capacity in response to increased protein load. Finally, we propose that the Creb3 family of factors may have a dual role in secretory cell differentiation by also regulating the pathways necessary for cell cycle exit during terminal differentiation.