RESUMEN
Migratory cells - either individually or in cohesive groups - are critical for spatiotemporally regulated processes such as embryonic development and wound healing. Their dysregulation is the underlying cause of formidable health problems such as congenital abnormalities and metastatic cancers. Border cell behavior during Drosophila oogenesis provides an effective model to study temporally regulated, collective cell migration in vivo. Developmental timing in flies is primarily controlled by the steroid hormone ecdysone, which acts through a well-conserved, nuclear hormone receptor complex. Ecdysone signaling determines the timing of border cell migration, but the molecular mechanisms governing this remain obscure. We found that border cell clusters expressing a dominant-negative form of ecdysone receptor extended ineffective protrusions. Additionally, these clusters had aberrant spatial distributions of E-cadherin (E-cad), apical domain markers and activated myosin that did not overlap. Remediating their expression or activity individually in clusters mutant for ecdysone signaling did not restore proper migration. We propose that ecdysone signaling synchronizes the functional distribution of E-cadherin, atypical protein kinase C (aPKC), Discs large (Dlg1) and activated myosin post-transcriptionally to coordinate adhesion, polarity and contractility and temporally control collective cell migration.
Asunto(s)
Proteínas de Drosophila , Animales , Proteínas de Drosophila/metabolismo , Ecdisona/metabolismo , Drosophila/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Movimiento Celular/fisiología , Miosinas/metabolismo , Drosophila melanogaster/metabolismo , Polaridad Celular/fisiología , Adhesión CelularRESUMEN
Cell migration is essential in animal development and co-opted during metastasis and inflammatory diseases. Some cells migrate collectively, which requires them to balance epithelial characteristics such as stable cell-cell adhesions with features of motility like rapid turnover of adhesions and dynamic cytoskeletal structures. How this is regulated is not entirely clear but important to understand. While investigating Drosophila oogenesis, we found that the putative E3 ubiquitin ligase, Mind bomb 2 (Mib2), is required to promote epithelial stability and the collective cell migration of border cells. Through biochemical analysis, we identified components of Mib2 complexes, which include E-cadherin and α- and ß-catenins, as well as actin regulators. We also found that three Mib2 interacting proteins, RhoGAP19D, Supervillin, and Myosin heavy chain-like, affect border cell migration. mib2 mutant main body follicle cells have drastically reduced E-cadherin-based adhesion complexes and diminished actin filaments. We conclude that Mib2 acts to stabilize E-cadherin-based adhesion complexes and promote a robust actin cytoskeletal network, which is important for maintenance of epithelial integrity. The interaction with cadherin adhesion complexes and other cytoskeletal regulators contribute to its role in collective cell migration. Since Mib2 is well conserved, it may have similar functional significance in other organisms.
Asunto(s)
Actinas , Cadenas Pesadas de Miosina , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular , Movimiento Celular/fisiología , Drosophila/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Cell migration is a key component in development, homeostasis, immune function, and pathology. It is important to understand the molecular activity that allows some cells to migrate. Drosophila melanogaster is a useful model system because its genes are largely conserved with humans and it is straightforward to study biologically. The well-conserved transcriptional regulator Signal Transducer and Activator of Transcription (STAT) promotes cell migration, but its signaling is modulated by downstream targets Apontic (APT) and Slow Border Cells (SLBO). Inhibition of STAT activity by APT and cross-repression of APT and SLBO determines whether an epithelial cell in the Drosophila egg chamber becomes motile or remains stationary. Through mathematical modeling and analysis, we examine how the interaction of STAT, APT, and SLBO creates bistability in the Janus Kinase (JAK)/STAT signaling pathway. In this paper, we update and analyze earlier models to represent mechanistically the processes of the JAK/STAT pathway. We utilize parameter, bifurcation, and phase portrait analyses, and make reductions to the system to produce a minimal three-variable quantitative model. We analyze the manifold between migratory and stationary steady states in this minimal model and show that when the initial conditions of our model are near this manifold, cell migration can be delayed.
RESUMEN
Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.
Asunto(s)
Envejecimiento/fisiología , Bioensayo/métodos , Drosophila melanogaster/citología , Hemocitos/citología , Fagocitos/citología , Fagocitosis , Animales , Disección , Femenino , Colorantes Fluorescentes/metabolismo , Hemocitos/metabolismo , Concentración de Iones de Hidrógeno , Procesamiento de Imagen Asistido por Computador , Inyecciones , Fagocitos/metabolismo , Coloración y Etiquetado , Fijación del TejidoRESUMEN
In diverse developmental contexts, certain cells must migrate to fulfill their roles. Many questions remain unanswered about the genetic and physical properties that govern cell migration. While the simplest case of a single cell moving alone has been well-studied, additional complexities arise in considering how cohorts of cells move together. Significant differences exist between models of collectively migrating cells. We explore the experimental model of migratory border cell clusters in Drosophila melanogaster egg chambers, which are amenable to direct observation and precise genetic manipulations. This system involves two special characteristics that are worthy of attention: border cell clusters contain a limited number of both migratory and non-migratory cells that require coordination, and they navigate through a heterogeneous three-dimensional microenvironment. First, we review how clusters of motile border cells are specified and guided in their migration by chemical signals and the physical impact of adjacent tissue interactions. In the second part, we examine questions around the 3D structure of the motile cluster and surrounding microenvironment in understanding the limits to cluster size and speed of movement through the egg chamber. Mathematical models have identified sufficient gene regulatory networks for specification, the key forces that capture emergent behaviors observed in vivo, the minimal regulatory topologies for signaling, and the distribution of key signaling cues that direct cell behaviors. This interdisciplinary approach to studying border cells is likely to reveal governing principles that apply to different types of cell migration events.
Asunto(s)
Movimiento Celular , Drosophila melanogaster/citología , Modelos Biológicos , Ovario/citología , Animales , Femenino , Ovario/metabolismoRESUMEN
Physical resiliency declines with age and comorbid conditions. In humans, angiotensin-converting enzyme (ACE) has been associated with attenuation of the decline in physical performance with age. ACE-inhibitor compounds, commonly prescribed for hypertension, often have beneficial effects on physical performance however the generality of these effects are unclear. Here, we tested the effects of the ACE-inhibitor Lisinopril on life span, and age-specific speed, endurance, and strength using three genotypes of the Drosophila melanogaster Genetic Reference Panel. We show that age-related decline in physical performance and survivorship varies with genetic background. Lisinopril treatment increased mean life span in all Drosophila Genetic Reference Panel lines, but its effects on life span, speed, endurance, and strength depended on genotype. We show that genotypes with increased physical performance on Lisinopril treatment experienced reduced age-related protein aggregation in muscle. Knockdown of skeletal muscle-specific Ance, the Drosophila ortholog of ACE, abolished the effects of Lisinopril on life span, implying a role for skeletal muscle Ance in survivorship. Using transcriptome profiling, we identified genes involved in stress response that showed expression changes associated with genotype and age-dependent responsiveness to Lisinopril. Our results demonstrate that Ance is involved in physical decline and demonstrate genetic variation in phenotypic responses to an ACE inhibitor.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efectos de los fármacos , Lisinopril/farmacología , Longevidad/efectos de los fármacos , Peptidil-Dipeptidasa A/metabolismo , Animales , Drosophila melanogaster/genética , Genotipo , Masculino , Fenotipo , TranscriptomaRESUMEN
Over the past three-decades, Janus kinase (Jak) and signal transducer and activator of transcription (STAT) signaling has emerged as a paradigm to understand the involvement of signal transduction in development and disease pathology. At the molecular level, cytokines and interleukins steer Jak/STAT signaling to transcriptional regulation of target genes, which are involved in cell differentiation, migration, and proliferation. Jak/STAT signaling is involved in various types of blood cell disorders and cancers in humans, and its activation is associated with carcinomas that are more invasive or likely to become metastatic. Despite immense information regarding Jak/STAT regulation, the signaling network has numerous missing links, which is slowing the progress towards developing drug therapies. In mammals, many components act in this cascade, with substantial cross-talk with other signaling pathways. In Drosophila, there are fewer pathway components, which has enabled significant discoveries regarding well-conserved regulatory mechanisms. Work across species illustrates the relevance of these regulators in humans. In this review, we showcase fundamental Jak/STAT regulation mechanisms in blood cells, stem cells, and cell motility. We examine the functional relevance of key conserved regulators from Drosophila to human cancer stem cells and metastasis. Finally, we spotlight less characterized regulators of Drosophila Jak/STAT signaling, which stand as promising candidates to be investigated in cancer biology. These comparisons illustrate the value of using Drosophila as a model for uncovering the roles of Jak/STAT signaling and the molecular means by which the pathway is controlled.
Asunto(s)
Drosophila/metabolismo , Neoplasias/metabolismo , Transducción de Señal , Animales , Células Sanguíneas/metabolismo , Movimiento Celular , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Células Madre/metabolismoRESUMEN
How vesicle trafficking components actively contribute to regulation of paracrine signaling is unclear. We genetically uncovered a requirement for α-soluble NSF attachment protein (α-Snap) in the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway during Drosophila egg development. α-Snap, a well-conserved vesicle trafficking regulator, mediates association of N-ethylmaleimide-sensitive factor (NSF) and SNAREs to promote vesicle fusion. Depletion of α-Snap or the SNARE family member Syntaxin1A in epithelia blocks polar cells maintenance and prevents specification of motile border cells. Blocking apoptosis rescues polar cell maintenance in α-Snap-depleted egg chambers, indicating that the lack of border cells in mutants is due to impaired signaling. Genetic experiments implicate α-Snap and NSF in secretion of a STAT-activating cytokine. Live imaging suggests that changes in intracellular Ca2+ are linked to this event. Our data suggest a cell-type specific requirement for particular vesicle trafficking components in regulated exocytosis during development. Given the central role for STAT signaling in immunity, this work may shed light on regulation of cytokine release in humans.
Asunto(s)
Citocinas/metabolismo , Exocitosis/fisiología , Quinasas Janus/metabolismo , Ovario/metabolismo , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Animales , Drosophila , Femenino , Transducción de SeñalRESUMEN
Cell migration is essential during animal development. In the Drosophila ovary, the steroid hormone ecdysone coordinates nutrient sensing, growth, and the timing of morphogenesis events including border cell migration. To identify downstream effectors of ecdysone signaling, we profiled gene expression in wild-type follicle cells compared to cells expressing a dominant negative Ecdysone receptor or its coactivator Taiman. Of approximately 400 genes that showed differences in expression, we validated 16 candidate genes for expression in border and centripetal cells, and demonstrated that seven responded to ectopic ecdysone activation by changing their transcriptional levels. We found a requirement for seven putative targets in effective cell migration, including two other nuclear hormone receptors, a calcyphosine-encoding gene, and a prolyl hydroxylase. Thus, we identified multiple new genetic regulators modulated at the level of transcription that allow cells to interpret information from the environment and coordinate cell migration in vivo.
Asunto(s)
Movimiento Celular/genética , Ecdisona/genética , Morfogénesis/genética , Transcripción Genética , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Regulación del Desarrollo de la Expresión Génica , Ovario/crecimiento & desarrolloRESUMEN
Drosophila border cells undergo a straightforward and stereotypical collective migration during egg development. However, a complex genetic program underlies this process. A variety of approaches, including biochemical, genetic, and imaging strategies have identified many regulatory components, revealing layers of control. This complexity suggests that the active processes of evaluating the environment, remodeling the cytoskeleton, and coordinating movements among cells, demand rapid systems for modulating cell behaviors. Multiple signaling inputs, nodes of integration, and feedback loops act as molecular rheostats to fine-tune gene expression levels and physical responses. Since key genetic regulators of border cell migration have been shown to be required in other types of cell migration, this model system continues to provide an important avenue for genetic discovery.
Asunto(s)
Movimiento Celular/genética , Drosophila melanogaster/genética , Óvulo/crecimiento & desarrollo , Animales , Linaje de la Célula/genética , Polaridad Celular/genética , Citoesqueleto/genética , Drosophila melanogaster/crecimiento & desarrollo , Regulación de la Expresión Génica/genética , Transducción de SeñalRESUMEN
The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway is an essential regulator of cell migration both in mammals and fruit flies. Cell migration is required for normal embryonic development and immune response but can also lead to detrimental outcomes, such as tumor metastasis. A cluster of cells termed "border cells" in the Drosophila ovary provides an excellent example of a collective cell migration, in which two different cell types coordinate their movements. Border cells arise within the follicular epithelium and are required to invade the neighboring cells and migrate to the oocyte to contribute to a fertilizable egg. Multiple components of the STAT signaling pathway are required during border cell specification and migration; however, the functions and identities of other potential regulators of the pathway during these processes are not yet known. To find new components of the pathway that govern cell invasiveness, we knocked down 48 predicted STAT modulators using RNAi expression in follicle cells, and assayed defective cell movement. We have shown that seven of these regulators are involved in either border cell specification or migration. Examination of the epistatic relationship between candidate genes and Stat92E reveals that the products of two genes, Protein tyrosine phosphatase 61F (Ptp61F) and brahma (brm), interact with Stat92E during both border cell specification and migration.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Folículo Ovárico/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/genética , Factores de Transcripción STAT/genética , Transactivadores/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Epistasis Genética , Femenino , Técnicas de Silenciamiento del Gen , Oogénesis/genética , Folículo Ovárico/citología , Folículo Ovárico/crecimiento & desarrollo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Interferencia de ARN , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Transactivadores/metabolismoRESUMEN
BACKGROUND: Microenvironments called niches maintain resident stem cell populations by balancing self-renewal with differentiation, but the genetic regulation of this process is unclear. The niche of the Drosophila testis is well-characterized and genetically tractable, making it ideal for investigating the molecular regulation of stem cell biology. The JAK/STAT pathway, activated by signals from a niche component called the hub, maintains both germline and somatic stem cells. RESULTS: This study investigated the molecular regulation of the JAK/STAT pathway in the stem cells of the Drosophila testis. We determined that the transcriptional regulator Apontic (Apt) acts in the somatic (cyst) stem cells (CySCs) to balance differentiation and maintenance. We found Apt functions as a negative feedback inhibitor of STAT activity, which enables cyst cell maturation. Simultaneous loss of the STAT regulators apt and Socs36E, or the Stat92E-targeting microRNA miR-279, expanded the somatic stem cell-like population. CONCLUSIONS: Genetic analysis revealed that a conserved genetic regulatory network limits JAK/STAT activity in the somatic stem cells of Drosophila testis. In these cells, we determined JAK/STAT signaling promotes apt expression. Then, Apt functions through Socs36E and miR-279 to attenuate pathway activation, which is required for timely CySC differentiation. We propose that Apt acts as a core component of a STAT-regulatory circuit to prevent stem cell overpopulation and allow stem cell maturation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Células Madre/citología , Testículo/citología , Factores de Transcripción/metabolismo , Animales , Recuento de Células , Diferenciación Celular , Retroalimentación Fisiológica , Redes Reguladoras de Genes , Masculino , Modelos Biológicos , Factores de Transcripción STAT/metabolismoRESUMEN
Drosophila oogenesis provides many examples of essential processes in development. A myriad of genetic tools combined with recent advances in culturing egg chambers ex vivo has revealed several surprising mechanisms that govern how this tissue develops, and which could not have been determined in fixed tissues. Here we describe a straightforward protocol for dissecting ovaries, culturing egg chambers, and observing egg development in real time by fluorescent microscopy. This technique is suitable for observation of early- or late-stage egg development, and can be adapted to study a variety of cellular, molecular, or developmental processes. Ongoing analysis of oogenesis in living egg chambers has tremendous potential for discovery of new developmental mechanisms.
Asunto(s)
Biología Molecular/métodos , Oogénesis/genética , Técnicas de Cultivo de Órganos/métodos , Óvulo/crecimiento & desarrollo , Animales , Drosophila melanogaster/genética , Femenino , Microscopía FluorescenteRESUMEN
The Suppressor of Cytokine Signaling (SOCS) proteins are critical, highly conserved feedback inhibitors of signal transduction cascades. The family of SOCS proteins is divided into two groups: ancestral and vertebrate-specific SOCS proteins. Vertebrate-specific SOCS proteins have been heavily studied as a result of their strong mutant phenotypes. However, the ancestral clade remains less studied, a potential result of genetic redundancies in mammals. Use of the genetically tractable organism Drosophila melanogaster enables in vivo assessment of signaling components and mechanisms with less concern about the functional redundancy observed in mammals. In this study, we investigated how the SOCS family member Suppressor of Cytokine Signaling at 36E (Socs36E) attenuates Janus Kinase/Signal Transducer and Activator of Transcription (Jak/STAT) activation during specification of motile border cells in Drosophila oogenesis. We found that Socs36E genetically interacts with the Cullin2 (Cul2) scaffolding protein. Like Socs36E, Cul2 is required to limit the number of motile cells in egg chambers. We demonstrated that loss of Cul2 in the follicle cells significantly increased nuclear STAT protein levels, which resulted in additional cells acquiring invasive properties. Further, reduction of Cul2 suppressed border cell migration defects that occur in a Stat92E-sensitized genetic background. Our data incorporated Cul2 into a previously described Jak/STAT-directed genetic regulatory network that is required to generate a discrete boundary between cell fates. We also found that Socs36E is able to attenuate STAT activity in the egg chamber when it does not have a functional SOCS box. Collectively, this work contributes mechanistic insight to a Jak/STAT regulatory genetic circuit, and suggests that Socs36E regulates Jak/STAT signaling via a Cul2-dependent mechanism, as well as by a Cullin-independent manner, in vivo.
Asunto(s)
Proteínas Cullin/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Factores de Transcripción STAT/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Animales Modificados Genéticamente , Movimiento Celular/genética , Movimiento Celular/fisiología , Proteínas Cullin/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Genes de Insecto , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Quinasas Janus/genética , Quinasas Janus/metabolismo , Mutación , Oogénesis/genética , Oogénesis/fisiología , Ovario/citología , Factores de Transcripción STAT/genética , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
Extracellular signalling molecules control many biological processes, but the influence of tissue architecture on the local concentrations of these factors is unclear. Here we examine this issue in the Drosophila egg chamber, where two anterior cells secrete Unpaired (Upd) to activate Signal transducer and activator of transcription (STAT) signalling in the epithelium. High STAT signalling promotes cell motility. Genetic analysis shows that all cells near the Upd source can respond. However, using upright imaging, we show surprising asymmetries in STAT activation patterns, suggesting that some cells experience different Upd levels than predicted by their location. We develop a three-dimensional mathematical model to characterize the spatio-temporal distribution of the activator. Simulations show that irregular tissue domains can produce asymmetric distributions of Upd, consistent with results in vivo. Mutant analysis substantiates this idea. We conclude that cellular landscape can heavily influence the effect of diffusible activators and should be more widely considered.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Factores de Transcripción STAT/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Drosophila/embriología , Proteínas de Drosophila/genética , Femenino , Masculino , Modelos Biológicos , Óvulo/crecimiento & desarrollo , Receptores Notch/metabolismoRESUMEN
Cell migration is essential in animal development, homeostasis, and disease progression, but many questions remain unanswered about how this process is controlled. While many kinds of individual cell movements have been characterized, less effort has been directed towards understanding how clusters of cells migrate collectively through heterogeneous, cellular environments. To explore this, we have focused on the migration of the border cells during Drosophila egg development. In this case, a cluster of different cell types coalesce and traverse as a group between large cells, called nurse cells, in the center of the egg chamber. We have developed a new model for this collective cell migration based on the forces of adhesion, repulsion, migration and stochastic fluctuation to generate the movement of discrete cells. We implement the model using Identical Math Cells, or IMCs. IMCs can each represent one biological cell of the system, or can be aggregated using increased adhesion forces to model the dynamics of larger biological cells. The domain of interest is filled with IMCs, each assigned specific biophysical properties to mimic a diversity of cell types. Using this system, we have successfully simulated the migration of the border cell cluster through an environment filled with larger cells, which represent nurse cells. Interestingly, our simulations suggest that the forces utilized in this model are sufficient to produce behaviors of the cluster that are observed in vivo, such as rotation. Our framework was developed to capture a heterogeneous cell population, and our implementation strategy allows for diverse, but precise, initial position specification over a three- dimensional domain. Therefore, we believe that this model will be useful for not only examining aspects of Drosophila oogenesis, but also for modeling other two or three-dimensional systems that have multiple cell types and where investigating the forces between cells is of interest.
Asunto(s)
Movimiento Celular/fisiología , Drosophila melanogaster/citología , Modelos Biológicos , Modelos Estadísticos , Oocitos/citología , Oogénesis/fisiología , Animales , Adhesión Celular , Linaje de la Célula/fisiología , Polaridad Celular , Simulación por Computador , Drosophila melanogaster/crecimiento & desarrollo , Oocitos/fisiología , Procesos EstocásticosRESUMEN
Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.
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Drosophila melanogaster/anatomía & histología , Animales , Femenino , Microscopía/métodos , Oogénesis , Folículo Ovárico/citología , Óvulo/citologíaRESUMEN
Most multicellular organisms show a physiological decline in immune function with age. However, little is known about the mechanisms underlying these changes. We examined Drosophila melanogaster, an important model for identifying genes affecting innate immunity and senescence, to explore the role of phagocytosis in age-related immune dysfunction. We characterized the localized response of immune cells at the dorsal vessel to bacterial infection in 1-week- and 5-week-old flies. We developed a quantitative phagocytosis assay for adult Drosophila and utilized this to characterize the effect of age on phagocytosis in transgenic and natural variant lines. We showed that genes necessary for bacterial engulfment in other contexts are also required in adult flies. We found that blood cells from young and old flies initially engulf bacteria equally well, while cells from older flies accumulate phagocytic vesicles and thus are less capable of destroying pathogens. Our results have broad implications for understanding how the breakdown in cellular processes influences immune function with age.
Asunto(s)
Envejecimiento/inmunología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/inmunología , Hemocitos/citología , Fagocitosis/inmunología , Animales , Bioensayo , Recuento de Células , Drosophila melanogaster/genética , Escherichia coli/fisiología , Femenino , Fluorescencia , Genotipo , Corazón/fisiología , Hemocitos/inmunología , Fagocitosis/genéticaRESUMEN
The Janus kinase/Signal transducers and activators of transcription (JAK/STAT) pathway determines cell fates by regulating gene expression. One example is the specification of the motile cells called border cells during Drosophila oogenesis. It has been established that too much or too little STAT activity disrupts follicle cell identity and cell motility, which suggests the signaling must be precisely regulated. Here, we find that Suppressor of cytokine signaling at 36E (Socs36E) is a necessary negative regulator of JAK/STAT signaling during border cell specification. We find when STAT signaling is too low to induce migration in the presumptive border cell population, nearby follicle cells uncharacteristically become invasive to enable efficient migration of the cluster. We generated a genetic null allele that reveals Socs36E is required in the anterior follicle cells to limit invasive behavior to an optimal number of cells. We further show Socs36E genetically interacts with the required STAT feedback inhibitor apontic (apt) and APT's downstream target, mir-279, and provide evidence that suggests APT directly regulates Socs36E transcriptionally. Our work shows Socs36E plays a critical role in a genetic circuit that establishes a boundary between the motile border cell cluster and its non-invasive epithelial neighbors through STAT attenuation.