RESUMO
Rho signaling is a conserved mechanism for generating forces through activation of contractile actomyosin. How this pathway can produce different cell morphologies is poorly understood. In the Drosophila embryonic epithelium, we investigate how Rho signaling controls force asymmetry to drive morphogenesis. We study a distinct morphogenetic process termed 'alignment'. This process results in striking columns of rectilinear cells connected by aligned cell-cell contacts. We found that this is driven by contractile actomyosin cables that elevate tension along aligning interfaces. Our data show that polarization of Rho effectors, Rok and Dia, directs formation of these cables. Constitutive activation of these effectors causes aligning cells to instead invaginate. This suggests that moderating Rho signaling is essential to producing the aligned geometry. Therefore, we tested for feedback that could fine-tune Rho signaling. We discovered that F-actin exerts negative feedback on multiple nodes in the pathway. Further, we present evidence that suggests that Rok in part mediates feedback from F-actin to Rho in a manner independent of Myo-II. Collectively, our work suggests that multiple feedback mechanisms regulate Rho signaling, which may account for diverse morphological outcomes.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Epitélio/fisiologia , Morfogênese , Transdução de Sinais , Proteínas rho de Ligação ao GTP/fisiologia , Citoesqueleto de Actina/fisiologia , Actinas/fisiologia , Actomiosina/fisiologia , Animais , Polaridade Celular , Desenvolvimento Embrionário , Miosina Tipo II/fisiologiaRESUMO
Adult stem cells are often found in specialized niches, where the constituent cells direct self-renewal of their stem cell pool. The niche is therefore crucial for both normal homeostasis and tissue regeneration. In many mammalian tissues, niche cells have classically been difficult to identify, which has hampered any understanding of how tissues first construct niches during development. Fortunately, the Drosophila germline stem cell (GSC) niche is well defined, allowing for unambiguous identification of both niche cells and resident stem cells. The testis niche first forms in the early embryo, during a late stage of gonadogenesis. Here, using live-imaging both in vivo and ex vivo, we follow pro-niche cells as they assemble and assume their final form. We show that after ex vivo culture the niche appears fully functional, as judged by enrichment of adhesion proteins, the ability to activate STAT in adjacent GSCs, and to direct GSCs to divide orthogonally to the niche, just as they would in situ. Collectively, our imaging has generated several novel insights on niche morphogenesis that could not be inferred from fixed images alone. We identify dynamic processes that constitute an assembly phase and a compaction phase during morphogenesis. The compaction phase correlates with cell neighbor exchange among the assembled pro-niche cells, as well as a burst of divisions among newly recruited stem cells. Before compaction, an assembly phase involves the movement of pro-niche cells along the outer periphery of the gonad, using the extracellular matrix (ECM) to assemble at the anterior of the gonad. Finally, live-imaging in integrin mutants allows us to define the role of pro-niche cell-ECM interaction with regard to the new assembly and compaction dynamics revealed here.
Assuntos
Células Germinativas/metabolismo , Nicho de Células-Tronco , Células-Tronco/metabolismo , Testículo/metabolismo , Imagem com Lapso de Tempo/métodos , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnicas de Cultura Embrionária/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Microscopia Confocal , Morfogênese , Testículo/citologia , Testículo/embriologiaRESUMO
The formation of complex tissues from simple epithelial sheets requires the regional subdivision of the developing tissue. This is initially accomplished by a sequence of gene regulatory hierarchies that set up distinct fates within adjacent territories, and rely on cross-regulatory interactions to do so. However, once adjacent territories are established, cells that confront one another across territorial boundaries must actively participate in maintaining separation from each other. Classically, it was assumed that adhesive differences would be a primary means of sorting cells to their respective territories. Yet it is becoming clear that no single, simple mechanism is at play. In the few instances studied, an emergent theme along developmental boundaries is the generation of asymmetry in cell mechanical properties. The repertoire of ways in which cells might establish and then put mechanical asymmetry to work is not fully appreciated since only a few boundaries have been molecularly studied. Here, we characterize once such boundary in the develop leg epithelium of Drosophila. The region of the pretarsus / tarsus is a known gene expression boundary that also exhibits a lineage restriction (Sakurai et al., 2007). We now show that the interface comprising this boundary is strikingly aligned compared to other cell interfaces across the disk. The boundary also exhibits an asymmetry for both Myosin II accumulation as well as one of its activators, Rho Kinase. Furthermore, the enrichment correlates with increased mechanical tension across that interface, and that tension is Rho Kinase-dependent. Lastly, interfering with actomyosin contractility, either by depletion of myosin heavy chain or expression of a phosphomimetic variant of regulatory light chain causes defects in alignment of the interfaces. These data suggest strongly that mechanical asymmetries are key in establishing and maintaining this developmental boundary.
Assuntos
Actomiosina/metabolismo , Drosophila melanogaster/metabolismo , Extremidades/embriologia , Animais , Padronização Corporal , Proteínas de Drosophila/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
The niche directs key behaviors of its resident stem cells, and is thus crucial for tissue maintenance, repair and longevity. However, little is known about the genetic pathways that guide niche specification and development. The male germline stem cell niche in Drosophila houses two stem cell populations and is specified within the embryonic gonad, thus making it an excellent model for studying niche development. The hub cells that form the niche are specified early by Notch activation. Over the next few hours, these individual cells then cluster together and take up a defined position before expressing markers of hub cell differentiation. This timing suggests that there are other factors for niche development yet to be defined. Here, we have identified a role for the large Maf transcription factor Traffic jam (Tj) in hub cell specification downstream of Notch. Tj downregulation is the first detectable effect of Notch activation in hub cells. Furthermore, Tj depletion is sufficient to generate ectopic hub cells that can recruit stem cells. Surprisingly, ectopic niche cells in tj mutants remain dispersed in the absence of Notch activation. This led us to uncover a branched pathway downstream of Notch in which Bowl functions to direct hub cell assembly in parallel to Tj downregulation.
Assuntos
Linhagem da Célula , Proteínas de Drosophila/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch/metabolismo , Nicho de Células-Tronco , Células-Tronco/citologia , Animais , Diferenciação Celular , Regulação para Baixo , Drosophila melanogaster/citologia , Células Germinativas/citologia , Gônadas/citologia , Masculino , Modelos Biológicos , Mutação/genética , Transdução de Sinais , Células-Tronco/metabolismoRESUMO
Atypical cadherins Dachsous (Ds) and Fat coordinate the establishment of planar polarity, essential for the patterning of complex tissues and organs. The precise mechanisms by which this system acts, particularly in cases where Ds and Fat act independently of the 'core' frizzled system, are still the subject of investigation. Examining the deployment of the Ds-Fat system in different tissues of the model organism Drosophila, has provided insights into the general mechanisms by which polarity is established and propagated to coordinate outcomes across a field of cells. The Drosophila embryonic epidermis provides a simple model epithelia where the establishment of polarity can be observed from start to finish, and in the absence of proliferation, over a fixed number of cells. Using the asymmetric placement of f-actin during denticle assembly as a read-out of polarity, we examine the requirement for Ds and Fat in establishing polarity across the denticle field. Comparing detailed phenotypic analysis with steady state protein enrichment revealed a spatially restricted requirement for the Ds-Fat system within the posterior denticle field. Ectopic Ds signaling provides evidence for a model whereby Ds acts to asymmetrically enrich Fat in a neighboring cell, in turn polarizing the cell to specify the position of the actin-based protrusions at the cell cortex.
Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Polaridade Celular , Extensões da Superfície Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Epitélio/embriologia , Actinas/metabolismo , Animais , Padronização Corporal , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/citologia , Epitélio/metabolismo , Mutação/genética , Fenótipo , Transdução de SinaisRESUMO
Cells that comprise tissues often need to coordinate cytoskeletal events to execute morphogenesis properly. For epithelial tissues, some of that coordination is accomplished by polarization of the cells within the plane of the epithelium. Two groups of genes--the Dachsous (Ds) and Frizzled (Fz) systems--play key roles in the establishment and maintenance of such polarity. There has been great progress in uncovering the how these genes work together to produce planar polarity, yet fundamental questions remain unanswered. Here, we study the Drosophila larval ventral epidermis to begin to address several of these questions. We show that ds and fz contribute independently to polarity and that they do so over spatially distinct domains. Furthermore, we find that the requirement for the Ds system changes as field size increases. Lastly, we find that Ds and its putative receptor Fat (Ft) are enriched in distinct patterns in the epithelium during embryonic development.
Assuntos
Caderinas/metabolismo , Polaridade Celular/fisiologia , Proteínas de Drosophila/metabolismo , Células Epidérmicas , Epiderme/metabolismo , Receptores Frizzled/metabolismo , Animais , Caderinas/genética , Polaridade Celular/genética , Drosophila , Proteínas de Drosophila/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Imunofluorescência , Receptores Frizzled/genética , Regulação da Expressão Gênica no Desenvolvimento , Larva/citologia , Larva/metabolismoRESUMO
Interactions between niche cells and stem cells are vital for proper control over stem cell self-renewal and differentiation. However, there are few tissues where the initial establishment of a niche has been studied. The Drosophila testis houses two stem cell populations, which each lie adjacent to somatic niche cells. Although these niche cells sustain spermatogenesis throughout life, it is not understood how their fate is established. Here, we show that Notch signaling is necessary to specify niche cell fate in the developing gonad. Surprisingly, our results indicate that adjacent endoderm is the source of the Notch-activating ligand Delta. We also find that niche cell specification occurs earlier than anticipated, well before the expression of extant markers for niche cell fate. This work further suggests that endoderm plays a dual role in germline development. The endoderm assists both in delivering germ cells to the somatic gonadal mesoderm, and in specifying the niche where these cells will subsequently develop as stem cells. Because in mammals primordial germ cells also track through endoderm on their way to the genital ridge, our work raises the possibility that conserved mechanisms are employed to regulate germline niche formation.
Assuntos
Proteínas de Drosophila/metabolismo , Endoderma/metabolismo , Células Germinativas/metabolismo , Proteínas de Membrana/metabolismo , Mesoderma/metabolismo , Receptores Notch/metabolismo , Nicho de Células-Tronco/metabolismo , Testículo/embriologia , Animais , Animais Geneticamente Modificados , Drosophila/citologia , Drosophila/embriologia , Drosophila/metabolismo , Endoderma/citologia , Células Germinativas/citologia , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Mesoderma/citologia , Transdução de Sinais/fisiologia , Espermatogênese/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Testículo/citologia , Testículo/metabolismoRESUMO
To function properly, tissue-specific stem cells must reside in a niche. The Drosophila testis niche is one of few niches studied in vivo. Here, a single niche, comprising ten hub cells, maintains both germline stem cells (GSC) and somatic stem cells (CySC). Here, we show that lines is an essential CySC factor. Surprisingly, lines-depleted CySCs adopted several characteristics of hub cells, including the recruitment of new CySCs. This led us to examine the developmental relationship between CySCs and hub cells. In contrast to a previous report, we did not observe significant conversion of steady-state CySC progeny to hub fate. However, we found that these two cell types derive from a common precursor pool during gonadogenesis. Furthermore, lines mutant embryos exhibited gonads containing excess hub cells, indicating that lines represses hub cell fate during gonadogenesis. In many tissues, lines acts antagonistically to bowl, and we found that this is true for hub specification, establishing bowl as a positively acting factor in the development of the testis niche.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Nicho de Células-Tronco , Testículo/citologia , Testículo/embriologia , Fatores de Transcrição/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Proteínas de Transporte/genética , Desdiferenciação Celular/genética , Desdiferenciação Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Proteínas de Ligação a DNA/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Embrião não Mamífero , Células Germinativas/citologia , Células Germinativas/metabolismo , Masculino , Testículo/metabolismo , Fatores de Transcrição/genéticaRESUMO
Stem cells often rely on signals from a niche, which in many tissues adopts a precise morphology. What remains elusive is how niches are formed and how morphology impacts function. To address this, we leverage the Drosophila gonadal niche, which affords genetic tractability and live-imaging. We have previously shown mechanisms dictating niche cell migration to their appropriate position within the gonad and the resultant consequences on niche function. Here, we show that once positioned, niche cells robustly polarize filamentous actin (F-actin) and non-muscle myosin II (MyoII) toward neighboring germ cells. Actomyosin tension along the niche periphery generates a highly reproducible smoothened contour. Without contractility, niches are misshapen and exhibit defects in their ability to regulate germline stem cell behavior. We additionally show that germ cells aid in polarizing MyoII within niche cells and that extrinsic input is required for niche morphogenesis and function. Our work reveals a feedback mechanism where stem cells shape the niche that guides their behavior.
RESUMO
Niches are often found in specific positions in tissues relative to the stem cells they support. Consistency of niche position suggests that placement is important for niche function. However, the complexity of most niches has precluded a thorough understanding of how their proper placement is established. To address this, we investigated the formation of a genetically tractable niche, the Drosophila Posterior Signaling Center (PSC), the assembly of which had not been previously explored. This niche controls hematopoietic progenitors of the lymph gland (LG). PSC cells were previously shown to be specified laterally in the embryo, but ultimately reside dorsally, at the LG posterior. Here, using live-imaging, we show that PSC cells migrate as a tight collective and associate with multiple tissues during their trajectory to the LG posterior. We find that Slit emanating from two extrinsic sources, visceral mesoderm and cardioblasts, is required for the PSC to remain a collective, and for its attachment to cardioblasts during migration. Without proper Slit-Robo signaling, PSC cells disperse, form aberrant contacts, and ultimately fail to reach their stereotypical position near progenitors. Our work characterizes a novel example of niche formation and identifies an extrinsic signaling relay that controls precise niche positioning.
RESUMO
Stem cells often rely on signals from a niche, which in many tissues adopts a precise morphology. What remains elusive is how niches are formed, and how morphology impacts function. To address this, we leverage the Drosophila gonadal niche, which affords genetic tractability and live-imaging. We have previously shown mechanisms dictating niche cell migration to their appropriate position within the gonad, and the resultant consequences on niche function. Here, we show that once positioned, niche cells robustly polarize filamentous actin (F-actin) and Non-muscle Myosin II (MyoII) towards neighboring germ cells. Actomyosin tension along the niche periphery generates a highly reproducible smoothened contour. Without contractility, niches are misshapen and exhibit defects in their ability to regulate germline stem cell behavior. We additionally show that germ cells aid in polarizing MyoII within niche cells, and that extrinsic input is required for niche morphogenesis and function. Our work reveals a feedback mechanism where stem cells shape the niche that guides their behavior.
RESUMO
Proper control of epithelial morphogenesis is vital to development and is often disrupted in disease. After germ band extension, the cells of the Drosophila ventral embryonic epidermis are packed in a two-dimensional polygonal array. Although epithelial cell rearrangements are being studied productively in several tissues, the ventral epidermis is of particular interest as the final cell arrangement is, uniquely, far from equilibrium. We show that over the course of several hours, a subset of cells within each parasegment adopts a rectilinear configuration and aligns into parallel columns. Live imaging shows that this is accomplished by the shrinkage of select cell interfaces, as three-cell junctions are converted to four-cell junctions. Additionally, we show that non-muscle Myosin II and the polarity proteins Discs large (Dlg) and Bazooka are enriched along cell interfaces in a complex but reproducible pattern that suggests their involvement in junctional conversion and cell alignment. Indeed, depletion of Myosin II or dlg disrupts these processes. These results show that tight spatial regulation of actomyosin contractility is required to produce this high-energy arrangement of cells.
Assuntos
Actomiosina/fisiologia , Drosophila/embriologia , Células Epiteliais/fisiologia , Animais , Anticorpos , Caderinas/análise , Forma Celular , Drosophila/fisiologia , Embrião não Mamífero/citologia , Embrião não Mamífero/imunologia , Embrião não Mamífero/fisiologia , Células Epidérmicas , Epiderme/fisiologia , Células Epiteliais/citologia , Junções Intercelulares/fisiologia , Junções Intercelulares/ultraestrutura , Morfogênese , Miosina Tipo II/fisiologiaRESUMO
Certain Drosophila embryonic epidermal cells construct actin-based protrusions, called denticles, which exhibit stereotyped, column-specific differences in size, density and hook orientation. This precise denticle pattern is conserved throughout all drosophilids yet studied, and screening for mutations that affect this pattern has been used to identify genes involved in development and signaling. However, how column-specific differences are specified and the mechanism(s) involved have remained elusive. Here, we show that the transcription factor Stripe is required for multiple aspects of this column-specific denticle pattern, including denticle hook orientation. The induction of stripe expression in certain denticle field cells appears to be the primary mechanism by which developmental pathways assign denticle hook orientation. Furthermore, we show that the cytoskeletal linker protein Short stop (Shot) functions both cell-autonomously and non-autonomously to specify denticle hook orientation via interaction with the microtubule cytoskeleton. We propose that stripe mediates its effect on hook orientation, in part, via upregulation of shot.
Assuntos
Drosophila/embriologia , Embrião não Mamífero/fisiologia , Animais , Tamanho Celular , Drosophila/anatomia & histologia , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Embrião não Mamífero/citologia , Células Epidérmicas , Fator de Crescimento Epidérmico/genética , Epiderme/fisiologia , Variação Genética , Proteínas Hedgehog/genética , Imuno-Histoquímica , Proteínas de Membrana/genética , Mutação , Fenótipo , RNA de Cadeia Dupla/genéticaRESUMO
Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here, we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study. With over 44,000 nuclei and 6000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.
Assuntos
Células-Tronco Adultas , Testículo , Animais , Masculino , Testículo/metabolismo , Drosophila , RNA-Seq , SêmenRESUMO
Understanding how stem cells are maintained in their microenvironment (the niche) is vital for their application in regenerative medicine. Studies of Drosophila male germline stem cells (GSCs) have served as a paradigm in niche-stem cell biology. It is known that the BMP and JAK-STAT pathways are necessary for the maintenance of GSCs in the testis (Kawase et al., 2004; Kiger et al., 2001; Schulz et al., 2004; Shivdasani and Ingham, 2003; Tulina and Matunis, 2001). However, our recent work strongly suggests that BMP signaling is the primary pathway leading to GSC self-renewal (Leatherman and DiNardo, 2010). Here we show that magu controls GSC maintenance by modulating the BMP pathway. We found that magu was specifically expressed from hub cells, and accumulated at the testis tip. Testes from magu mutants exhibited a reduced number of GSCs, yet maintained a normal population of somatic stem cells and hub cells. Additionally, BMP pathway activity was reduced, whereas JAK-STAT activation was retained in mutant testes. Finally, GSC loss caused by the magu mutation could be suppressed by overactivating the BMP pathway in the germline.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila/metabolismo , Proteínas da Matriz Extracelular/fisiologia , Transdução de Sinais , Nicho de Células-Tronco/citologia , Testículo/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Morfogenéticas Ósseas/genética , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Masculino , Mutação , Nicho de Células-Tronco/metabolismo , Testículo/citologiaRESUMO
Tissue homeostasis often requires a properly placed niche to support stem cells. Morphogenetic processes that position a niche are just being described. For the Drosophila testis, we recently showed that pro-niche cells, specified at disparate positions during early gonadogenesis, must assemble into one collective at the anterior of the gonad. We now find that Slit and FGF signals emanating from adjacent visceral mesoderm regulate assembly. In response to signaling, niche cells express islet, which we find is also required for niche assembly. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. The function of such niches is severely disrupted, with niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. Our work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Masculino , Mesoderma/metabolismo , Nicho de Células-Tronco , Testículo/metabolismoRESUMO
For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Transcriptoma , Animais , Núcleo Celular/metabolismo , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes de Insetos , Masculino , RNA-Seq , Caracteres Sexuais , Análise de Célula Única , Fatores de Transcrição/genéticaRESUMO
The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the cells in their natural gonadal environment.
Assuntos
Dissecação , Drosophila melanogaster/embriologia , Embrião não Mamífero/diagnóstico por imagem , Gônadas/diagnóstico por imagem , Gônadas/embriologia , Imageamento Tridimensional , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Gônadas/citologia , MasculinoRESUMO
Activation of the Wnt signaling cascade provides key signals during development and in disease. Here we provide evidence, by designing a Wnt receptor with ligand-independent signaling activity, that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. We have uncovered a branch of the Wnt pathway in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3beta/Zw3, the kinase normally required for controlling beta-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. We propose that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de LDL/metabolismo , Transdução de Sinais/genética , Transativadores/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Proteínas do Domínio Armadillo , Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Feminino , Receptores Frizzled , Regulação da Expressão Gênica no Desenvolvimento/genética , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Proteínas Relacionadas a Receptor de LDL , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Receptores de LDL/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Fatores de Transcrição , Proteína Wnt1RESUMO
Tissue renewal becomes compromised with age. Although defects in niche and stem cell behavior have been implicated in promoting age-related decline, the causes of early-onset aging defects are unknown. We have identified an early consequence of aging in germline stem cells (GSCs) in the Drosophila testis. Aging disrupts the unique program of GSC cytokinesis, with GSCs failing to abscise from their daughter cells. Abscission failure significantly disrupts both self-renewal and the generation of differentiating germ cells. Extensive live imaging and genetic analyses show that abscission failure is due to inappropriate retention of F-actin at the intercellular bridges between GSC-daughter cells. Furthermore, F-actin is regulated by the Jak/STAT pathway-increasing or decreasing pathway activity can rescue or exacerbate the age-induced abscission defect, respectively. Even subtle decreases to STAT activity are sufficient to precociously age young GSCs and induce abscission failure. Thus, this work has identified the earliest age-related defect in GSCs and has revealed a unique role for an established niche signaling pathway in controlling stem cell cytokinesis and in regulating stem cell behavior with age.