RESUMO
Epithelial cells contain polarity complexes on the lateral membrane and are organized in a hexagon-dominated polygonal array. The mechanisms regulating the organization of polygonal architecture in metazoan embryogenesis are not completely understood. Drosophila embryogenesis enables mechanistic analysis of epithelial polarity formation and its impact on polygonal organization. The plasma membrane (PM) of syncytial Drosophila blastoderm embryos is organized as a polygonal array with pseudocleavage furrow formation during the almost synchronous cortical division cycles. We find that polygonal (PM) organization arises in the metaphase (MP) of division cycle 11, and hexagon dominance occurs with an increase in furrow length in the metaphase of cycle 12. There is a decrease in cell shape index in metaphase from cycles 11 to 13. This coincides with Drosophila E-cad (DE-cadherin) and Bazooka enrichment at the edges and the septin, Peanut at the vertices of the furrow. We further assess the role of polarity and adhesion proteins in pseudocleavage furrow formation and its organization as a polygonal array. We find that DE-cadherin depletion leads to decreased furrow length, loss of hexagon dominance, and increased cell shape index. Bazooka and Peanut depletion lead to decreased furrow length, delay in onset of hexagon dominance from cycle 12 to 13, and increased cell shape index. Hexagon dominance occurs with an increase in furrow length in cycle 13 and increased DE-cadherin, possibly due to the inhibition of endocytosis. We conclude that polarity protein recruitment and regulation of endocytic pathways enable pseudocleavage furrow stability and the formation of a hexagon-dominated polygon array.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Drosophila/metabolismo , Blastoderma/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Membrana Celular/metabolismo , Caderinas/genética , Caderinas/metabolismo , Drosophila melanogaster/metabolismoRESUMO
In the germarium of the Drosophila ovary, developing germline cysts are surrounded by a population of somatic escort cells that are known to function as the niche cells for germline differentiation;1 however, the underlying molecular mechanisms of this niche function remain poorly understood. Through single-cell gene expression profiling combined with genetic analyses, we here demonstrate that the escort cells can be spatially and functionally divided into two successive domains. The anterior escort cells (aECs) specifically produce ecdysone, which acts on the cystoblast to promote synchronous cell division, whereas the posterior escort cells (pECs) respond to ecdysone signaling and regulate soma-germline cell adhesion to promote the transition from 16-cell cyst-to-egg chamber formation. The patterning of the aEC and pEC domains is independent of the germline but is dependent on JAK/STAT signaling activity, which emanates from the posterior. Thus, a heterogeneous population of escort cells constitutes a stepwise niche environment to orchestrate cystoblast division and differentiation toward egg chamber formation.
Assuntos
Cistos , Proteínas de Drosophila , Ecdisona , Animais , Diferenciação Celular , Drosophila , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ecdisona/metabolismo , Feminino , Células Germinativas , Ovário , Células-TroncoRESUMO
Cell shape morphogenesis, from spherical to polygonal, occurs in epithelial cell formation in metazoan embryogenesis. In syncytial Drosophila embryos, the plasma membrane incompletely surrounds each nucleus and is organized as a polygonal epithelial-like array. Each cortical syncytial division cycle shows a circular to polygonal plasma membrane transition along with furrow extension between adjacent nuclei from interphase to metaphase. In this study, we assess the relative contribution of DE-cadherin (also known as Shotgun) and Myosin II (comprising Zipper and Spaghetti squash in flies) at the furrow to polygonal shape transition. We show that polygonality initiates during each cortical syncytial division cycle when the furrow extends from 4.75 to 5.75â µm. Polygon plasma membrane organization correlates with increased junctional tension, increased DE-cadherin and decreased Myosin II mobility. DE-cadherin regulates furrow length and polygonality. Decreased Myosin II activity allows for polygonality to occur at a lower length than controls. Increased Myosin II activity leads to loss of lateral furrow formation and complete disruption of the polygonal shape transition. Our studies show that DE-cadherin-Myosin II balance regulates an optimal lateral membrane length during each syncytial cycle for polygonal shape transition.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Caderinas/genética , Membrana Celular , Proteínas de Drosophila/genética , Embrião não Mamífero , Miosina Tipo II/genéticaRESUMO
Planar cell polarity and anisotropic cell behavior play critical roles in large-scale epithelial morphogenesis, homeostasis, wound repair, and regeneration. Cell-Cell communication and mechano-transduction in the second to minute scale mediated by E-cadherin complexes play a central role in the coordination and self-organization of cellular activities, such as junction dynamics, cell shape changes, and cell rearrangement. Here we review the current understanding in the interplay of cell polarity and cell dynamics during body axis elongation and dorsal closure in Drosophila embryos with a focus on E-cadherin dynamics in linking cell and tissue polarization and tissue-scale shape changes.
RESUMO
Proper stem cell activity in tissues ensures the correct balance between proliferation and differentiation, thus allowing tissue homeostasis and repair. The Drosophila ovary develops well-defined niches that contain on average 2-4 germline stem cells (GSCs), whose maintenance depends on systemic signals and local factors. A known player in the decline of tissue homeostasis is ageing, which correlates with the waning of resident stem cell populations. In Drosophila, ovaries from old females contain fewer GSCs than those from young flies. We isolated niche cells of aged ovaries, performed a transcriptomic analysis and identified mastermind (mam) as a factor for Drosophila ovarian niche functionality during ageing. We show that mam is upregulated in aged niche cells and that we can induce premature GSC loss by overexpressing mam in otherwise young niche cells. High mam levels in niche cells induce reduced Hedgehog amounts, a decrease in cadherin levels and a likely increase in reactive oxygen species, three scenarios known to provoke GSC loss. Mam is a canonical co-activator of the Notch pathway in many Drosophila tissues. However, we present evidence to support a Notch-independent role for mam in the ovarian germline niche.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Envelhecimento , Animais , Senescência Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Proteínas Nucleares/genética , Ovário/citologia , Ovário/fisiologia , Nicho de Células-Tronco , TranscriptomaRESUMO
The actomyosin network is involved in crucial cellular processes including morphogenesis, cell adhesion, apoptosis, proliferation, differentiation, and collective cell migration in Drosophila, Caenorhabditiselegans, and mammals. Here, we demonstrate that Drosophila larval blood stem-like progenitors require actomyosin activity for their maintenance. Genetic loss of the actomyosin network from progenitors caused a decline in their number. Likewise, the progenitor population increased upon sustained actomyosin activation via phosphorylation by Rho-associated kinase. We show that actomyosin positively regulates larval blood progenitors by controlling the maintenance factor Cubitus interruptus (Ci). Overexpression of the maintenance signal via a constitutively activated construct (ci.HA) failed to sustain Ci-155 in the absence of actomyosin components like Zipper (zip) and Squash (sqh), thus favoring protein kinase A (PKA)-independent regulation of Ci activity. Furthermore, we demonstrate that a change in cortical actomyosin assembly mediated by DE-cadherin modulates Ci activity, thereby determining progenitor status. Thus, loss of cell adhesion and downstream actomyosin activity results in desensitization of the progenitors to Hh signaling, leading to their differentiation. Our data reveal how cell adhesion and the actomyosin network cooperate to influence patterning, morphogenesis, and maintenance of the hematopoietic stem-like progenitor pool in the developing Drosophila hematopoietic organ.
Assuntos
Actomiosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Caderinas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Drosophila melanogaster , Hematopoese , Proteínas de Membrana/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/metabolismo , Multimerização Proteica , Quinases Associadas a rho/metabolismoRESUMO
Phosphorylation of a highly conserved serine cluster in the intracellular domain of E-Cadherin is essential for binding to ß-Catenin in vitro In cultured cells, phosphorylation of specific serine residues within the cluster is also required for regulation of adherens junction (AJ) stability and dynamics. However, much less is known about how such phosphorylation of E-Cadherin regulates AJ formation and dynamics in vivo In this report, we generated an extensive array of Drosophila E-Cadherin (DE-Cad) endogenous knock-in alleles that carry mutations targeting this highly conserved serine cluster. Analyses of these mutations suggest that the overall phosphorylation potential, rather than the potential site-specific phosphorylation, of the serine cluster enhances the recruitment of ß-Catenin by DE-Cad in vivo Moreover, phosphorylation potential of the serine cluster only moderately increases the level of ß-Catenin in AJs and is in fact dispensable for AJ formation in vivo Nonetheless, phosphorylation-dependent recruitment of ß-Catenin is essential for development, probably by enhancing the interactions between DE-Cad and α-Catenin. In addition, several phospho-mutations dramatically reduced the biosynthetic turnover rate of DE-Cad during apical-basal polarization, and such biosynthetically stable DE-Cad mutants specifically rescued the polarity defects in embryonic epithelia lacking the polarity proteins Stardust and Crumbs.
Assuntos
Junções Aderentes/metabolismo , Vias Biossintéticas , Caderinas/química , Caderinas/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Polaridade Celular , Sequência Conservada , Embrião não Mamífero/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas Mutantes/metabolismo , Fosforilação , Domínios Proteicos , Estabilidade Proteica , Serina/metabolismo , Relação Estrutura-Atividade , alfa Catenina/metabolismo , beta Catenina/metabolismoRESUMO
Correct positioning of stem cells within their niche is essential for tissue morphogenesis and homeostasis. How stem cells acquire and maintain niche position remains largely unknown. Here, we show that a subset of brain neuroblasts (NBs) in Drosophila utilize Phosphoinositide 3-kinase (PI3-kinase) and DE-cadherin to build adhesive contact for NB niche positioning. NBs remain within their native microenvironment when levels of PI3-kinase activity and DE-cadherin are elevated in NBs. This occurs through PI3-kinase-dependent regulation of DE-Cadherin-mediated cell adhesion between NBs and neighboring cortex glia, and between NBs and their ganglion mother cell daughters. When levels of PI3-kinase activity and/or DE-Cadherin are reduced in NBs, NBs lose niche position and relocate to a non-native brain region that is rich in neurosecretory neurons, including those that secrete some of the Drosophila insulin-like peptides. Linking levels of PI3-kinase activity to the strength of adhesive attachment could provide cancer stem cells and hematopoietic stem cells with a means to cycle from trophic-poor to trophic-rich microenvironments.
Assuntos
Encéfalo/embriologia , Caderinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Adesão Celular , Proliferação de Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Mitose , Morfogênese , Neuroglia/metabolismo , Neurônios/citologiaRESUMO
We have investigated how cell contractility and adhesion are functionally integrated during epithelial morphogenesis. To this end, we have analysed the role of α-Catenin, a key molecule linking E-Cadherin-based adhesion and the actomyosin cytoskeleton, during Drosophila embryonic dorsal closure, by studying a newly developed allelic series. We find that α-Catenin regulates pulsatile apical contraction in the amnioserosa, the main force-generating tissue driving closure of the embryonic epidermis. α-Catenin controls actomyosin dynamics by stabilising and promoting the formation of actomyosin foci, and also stabilises DE-Cadherin (Drosophila E-Cadherin, also known as Shotgun) at the cell membrane, suggesting that medioapical actomyosin contractility regulates junction stability. Furthermore, we uncover a genetic interaction between α-Catenin and Vinculin, and a tension-dependent recruitment of Vinculin to amniosersoa apical cell membranes, suggesting the existence of a mechano-sensitive module operating in this tissue.
Assuntos
Actomiosina/metabolismo , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , alfa Catenina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Adesão Celular , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Junções Intercelulares/metabolismo , Mutação/genética , Vinculina/metabolismoRESUMO
The homeostatic turnover of adult organs and their regenerative capacity following injury depend on a careful balance between stem cell self-renewal (to maintain or enlarge the stem cell pool) and differentiation (to replace lost tissue). We have recently characterized the role of the Drosophila Snail family transcription factor escargot (esg) in testis cyst stem cells (CySCs) (1,2) and intestinal stem cells (ISCs). (3,4) CySCs mutant for esg are not maintained as stem cells, but they remain capable of differentiating normally along the cyst cell lineage. In contrast, esg mutant CySCs that give rise to a closely related lineage, the apical hub cells, cannot maintain hub cell identity. Similarly, Esg maintains stemness of ISCs while regulating the terminal differentiation of progenitor cells into absorptive enterocytes or secretory enteroendocrine cells. Therefore, our findings suggest that Esg may play a conserved and pivotal regulatory role in adult stem cells, controlling both their maintenance and terminal differentiation. Here we propose that this dual regulatory role is due to simultaneous control by Esg of overlapping genetic programs and discuss the exciting challenges and opportunities that lie ahead to explore the underlying mechanisms experimentally.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/citologia , Células-Tronco/citologia , Animais , Caderinas/metabolismo , Diferenciação Celular , Drosophila/metabolismo , Trato Gastrointestinal/citologia , Trato Gastrointestinal/metabolismo , Masculino , Células-Tronco/metabolismo , Testículo/citologia , Testículo/metabolismoRESUMO
Notch signaling pathway represents a principal cellular communication system that plays a pivotal role during development of metazoans. Drosophila misshapen (msn) encodes a protein kinase, which is related to the budding yeast Ste20p (sterile 20 protein) kinase. In a genetic screen, using candidate gene approach to identify novel kinases involved in Notch signaling, we identified msn as a novel regulator of Notch signaling. Data presented here suggest that overexpression of kinase active form of Msn exhibits phenotypes similar to Notch loss-of-function condition and msn genetically interacts with components of Notch signaling pathway. Kinase active form of Msn associates with Notch receptor and regulate its signaling activity. We further show that kinase active Misshapen leads to accumulation of membrane-tethered form of Notch. Moreover, activated Msn also depletes Armadillo and DE-Cadherin from adherens junctions. Thus, this study provides a yet unknown mode of regulation of Notch signaling by Misshapen.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Notch/metabolismo , Junções Aderentes , Animais , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Caderinas/genética , Caderinas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Imunofluorescência , Técnicas Imunoenzimáticas , Longevidade , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Notch/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Hox genes are highly conserved selector genes controlling tissue identity and organogenesis. Recent work indicates that Hox genes also controls cell segregation and segmental boundary in various species, however the underlying cellular mechanisms involved in this function are poorly understood. In Drosophila melanogaster, the Hox gene Deformed (Dfd) is required for specification and organogenesis of the adult Maxillary (Mx) palp. Here, we demonstrate that differential Dfd expression control Mx morphogenesis through the formation of a physical boundary separating the Mx field and the Peripodial Epithelium (PE). We show that this boundary relies on DE-cadherin (DE-cad) basal accumulation in Mx cells controlled by differential Dfd expression. Indeed, Dfd controls boundary formation through cell autonomous basal redistribution of DE-cad which leads to subsequent fold at the Dfd expression border. Finally, the loss of Mx DE-cad basal accumulation and hence of Mx-PE folding is sufficient to prevent Mx organogenesis thus revealing the crucial role of boundaries in organ differentiation. Altogether, these results reveal that Hox coordination of tissue morphogenesis relies on boundary fold formation through the modulation of DE-cad positioning.
Assuntos
Caderinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Epitélio/embriologia , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Maxila/embriologia , Microscopia Confocal , Mitose , Organogênese , Dobramento de Proteína , Interferência de RNARESUMO
Adherens junctions are known for their role in mediating cell-cell adhesion. DE-cadherin and Echinoid are the principle adhesion molecules of adherens junctions in Drosophila epithelia. Here, using live imaging to trace the movement of endocytosed Echinoid vesicles in the epithelial cells of Drosophila embryos, we demonstrate that Echinoid vesicles co-localize and move with Rab5-or Rab11-positive endosomes. Surprisingly, these Echinoid-containing endosomes undergo directional cell-to-cell movement, through adherens junctions. Consistent with this, cell-to-cell movement of Echinoid vesicles requires the presence of DE-cadherin at adherens junctions. Live imaging further revealed that Echinoid vesicles move along adherens junction-associated microtubules into adjacent cells, a process requiring a kinesin motor. Importantly, DE-cadherin- and EGFR-containing vesicles also exhibit intercellular movement. Together, our results unveil a transport function of adherens junctions. Furthermore, this adherens junctions-based intercellular transport provides a platform for the exchange of junctional proteins and signaling receptors between neighboring cells.
Assuntos
Junções Aderentes/fisiologia , Drosophila/metabolismo , Animais , Transporte Biológico , Caderinas/metabolismo , Drosophila/embriologia , Endossomos/metabolismo , Receptores ErbB/metabolismo , Proteínas de Fluorescência Verde/metabolismoRESUMO
Border cell cluster (BCC) migration in the Drosophila ovary is an excellent system to study the gene regulatory network that enables collective cell migration. Here, we identify the large Maf transcription factor Traffic jam (Tj) as an important regulator of BCC migration. Tj has a multifaceted impact on the known core cascade that enables BCC motility, consisting of the Jak/Stat signaling pathway, the C/EBP factor Slow border cells (Slbo), and the downstream effector DE-cadherin (DEcad). The initiation of BCC migration coincides with a Slbo-dependent decrease in Tj expression. This reduction of Tj is required for normal BCC motility, as high Tj expression strongly impedes migration. At high concentration, Tj has a tripartite negative effect on the core pathway: a decrease in Slbo, an increase in the Jak/Stat inhibitor Socs36E, and a Slbo-independent reduction of DEcad. However, maintenance of a low expression level of Tj in the BCC during migration is equally important, as loss of tj function also results in a significant delay in migration concomitant with a reduction of Slbo and consequently of DEcad. Taken together, we conclude that the regulatory feedback loop between Tj and Slbo is necessary for achieving the correct activity levels of migration-regulating factors to ensure proper BCC motility.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Oogênese/fisiologia , Ovário/embriologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Caderinas/genética , Caderinas/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Proteínas de Drosophila/genética , Feminino , Fatores de Transcrição Maf Maior/genética , Oogênese/genética , Ovário/citologia , Ovário/metabolismo , Proteínas Proto-Oncogênicas/genéticaRESUMO
Drosophila Hrp38, a homolog of human hnRNP A1, has been shown to regulate splicing, but its function can be modified by poly(ADP-ribosyl)ation. Notwithstanding such findings, our understanding of the roles of poly(ADP-ribosyl)ated Hrp38 on development is limited. Here, we have demonstrated that Hrp38 is essential for fly eye development based on a rough-eye phenotype with disorganized ommatidia observed in adult escapers of the hrp38 mutant. We also observed that poly(ADP-ribose) glycohydrolase (Parg) loss-of-function, which caused increased Hrp38 poly(ADP-ribosyl)ation, also resulted in the rough-eye phenotype with disrupted ommatidial lattice and reduced number of photoreceptor cells. In addition, ectopic expression of DE-cadherin, which is required for retinal morphogenesis, fully rescued the rough-eye phenotype of the hrp38 mutant. Similarly, Parg mutant eye clones had decreased expression level of DE-cadherin with orientation defects, which is reminiscent of DE-cadherin mutant eye phenotype. Therefore, our results suggest that Hrp38 poly(ADP-ribosyl)ation controls eye pattern formation via regulation of DE-cadherin expression, a finding which has implications for understanding the pathogenic mechanisms of Hrp38-related Fragile X syndrome and PARP1-related retinal degeneration diseases.