RESUMEN

Apical constriction powers amnioserosa contraction during Drosophila dorsal closure. The nucleation, movement and dispersal of apicomedial actomyosin complexes generates pulsed apical constrictions during early closure. Persistent apicomedial and circumapical actomyosin complexes drive unpulsed constrictions that follow. Here, we show that the microtubule end-binding proteins EB1 and Patronin pattern constriction dynamics and contraction kinetics by coordinating the balance of actomyosin forces in the apical plane. We find that microtubule growth from moving Patronin platforms governs the spatiotemporal dynamics of apicomedial myosin through the regulation of RhoGTPase signaling by transient EB1-RhoGEF2 interactions. We uncover the dynamic reorganization of a subset of short non-centrosomally nucleated apical microtubules that surround the coalescing apicomedial myosin complex, trail behind it as it moves and disperse as the complex dissolves. We demonstrate that apical microtubule reorganization is sensitive to Patronin levels. Microtubule depolymerization compromised apical myosin enrichment and altered constriction dynamics. Together, our findings uncover the importance of reorganization of an intact apical microtubule meshwork, by moving Patronin platforms and growing microtubule ends, in enabling the spatiotemporal modulation of actomyosin contractility and, through it, apical constriction.

Asunto(s)

Actomiosina , Proteínas de Drosophila , Animales , Actomiosina/metabolismo , Constricción , Proteínas Portadoras/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Drosophila/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Drosophila/metabolismo

RESUMEN

Epithelial fusion establishes continuity between the separated flanks of epithelial sheets. Despite its importance in creating resilient barriers, the mechanisms that ensure stable continuity and preserve morphological and molecular symmetry upon fusion remain unclear. Using the segmented embryonic epidermis whose flanks fuse during Drosophila dorsal closure, we demonstrate that epidermal flanks modulate cell numbers and geometry of their fusing fronts to achieve fusion fidelity. While fusing flanks become more matched for both parameters before fusion, differences persisting at fusion are corrected by modulating fusing front width within each segment to ensure alignment of segment boundaries. We show that fusing cell interfaces are remodelled from en-face contacts at fusion to an interlocking arrangement after fusion, and demonstrate that changes in interface length and geometry are dependent on the spatiotemporal regulation of cytoskeletal tension and Bazooka/Par3. Our work uncovers genetically constrained and mechanically triggered adaptive mechanisms contributing to fusion fidelity and epithelial continuity.

Asunto(s)

Proteínas de Drosophila/metabolismo , Drosophila/embriología , Embrión no Mamífero , Desarrollo Embrionario , Epidermis/embriología , Células Epiteliales/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fenómenos Mecánicos , Animales

RESUMEN

How cues that trigger the wound response result in tissue healing is a question of immense biological and medical importance. Here we uncover roles for mitochondrial reactive oxygen species (mtROS) during Drosophila dorsal closure, a model for wound healing. By using real-time visualization of ROS activity and single-cell perturbation strategies, we demonstrate that stochasticities in ROS generation in the amnioserosa are necessary and sufficient to trigger cell delamination. We identify dose-dependent effects of mtROS on actomyosin and mitochondrial architecture, dynamics, and activity that mediate both stochasticities in cell behavior and the phases of tissue dynamics accompanying dorsal closure. Our results establish that ROS levels tune cell behavior and tissue dynamics qualitatively and quantitatively. They identify a pathway triggered by ROS and mediated by the Rho effector ROCK and its substrates that influences tissue patterning and homeostasis through the coordinate regulation of both mitochondrial morphology and tissue tension.

Asunto(s)

Citoesqueleto/metabolismo , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Quinasas Asociadas a rho/metabolismo , Actomiosina/metabolismo , Animales , Células Cultivadas , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero/citología , Técnicas para Inmunoenzimas , Microscopía Confocal , Cicatrización de Heridas/fisiología

RESUMEN

Epithelial-to-mesenchymal transition (EMT) is typically accompanied by downregulation of epithelial (E-) cadherin, and is often additionally accompanied by upregulation of a mesenchymal or neuronal (N-) cadherin. Snail represses transcription of the E-cadherin gene both during normal development and during tumour spreading. The formation of the mesodermal germ layer in Drosophila, considered a paradigm of a developmental EMT, is associated with Snail-mediated repression of E-cadherin and the upregulation of N-cadherin. By using genetic manipulation to remove or overexpress the cadherins, we show here that the complementarity of cadherin expression is not necessary for the segregation or the dispersal of the mesodermal germ layer in Drosophila. However, we discover different effects of E- and N-cadherin on the differentiation of subsets of mesodermal derivatives, which depend on Wingless signalling from the ectoderm, indicating differing abilities of E- and N-cadherin to bind to and sequester the common junctional and signalling effector ß-catenin. These results suggest that the downregulation of E-cadherin in the mesoderm might be required to facilitate optimal levels of Wingless signalling.

Asunto(s)

Cadherinas/biosíntesis , Proteínas de Drosophila/biosíntesis , Drosophila/metabolismo , Animales , Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Drosophila/genética , Transición Epitelial-Mesenquimal/fisiología , Mesodermo/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética

RESUMEN

How robust patterns of tissue dynamics emerge from heterogeneities, stochasticities, and asynchronies in cell behavior is an outstanding question in morphogenesis. A clear understanding of this requires examining the influence of the behavior of single cells on tissue patterning. Here we develop single-cell manipulation strategies to uncover the origin of patterned cell behavior in the amnioserosa during Drosophila dorsal closure. We show that the formation and dissolution of contractile, medial actomyosin networks previously shown to underlie pulsed apical constrictions in the amnioserosa are apparently asynchronous in adjacent cells. We demonstrate for the first time that mechanical stresses and Rho1 GTPase control myosin dynamics qualitatively and quantitatively, in amplitude and direction, both cell autonomously and nonautonomously. We then demonstrate that interfering with myosin-dependent contractility in single cells also influences pulsed constrictions cell nonautonomously. Our results suggest that signals and stresses can feedback regulate the amplitude and spatial propagation of pulsed constrictions through their influence on tension and geometry. We establish the relevance of these findings to native closure by showing that cell delamination represents a locally patterned and collective transition from pulsed to unpulsed constriction that also relies on the nonautonomous feedback control of myosin dynamics.

Asunto(s)

Forma de la Célula/fisiología , Miosinas/metabolismo , Animales , Fenómenos Biomecánicos , Membrana Celular/metabolismo , Polaridad Celular , Forma de la Célula/efectos de la radiación , Citoesqueleto/metabolismo , Drosophila/citología , Drosophila/embriología , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/citología , Retroalimentación Fisiológica , Rayos Láser , Morfogénesis , Análisis de la Célula Individual , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

Dead cells in most epithelia are eliminated by cell extrusion. Here, we explore whether cell delamination in the amnioserosa, a seemingly stochastic event that results in the extrusion of a small fraction of cells and known to provide a force for dorsal closure, is contingent upon the receipt of an apoptotic signal. Through the analysis of mutant combinations and the profiling of apoptotic signals in situ, we establish spatial, temporal and molecular hierarchies in the link between death and delamination. We show that although an apoptotic signal is necessary and sufficient to provide cell-autonomous instructions for delamination, its induction during natural delamination occurs downstream of mitochondrial fragmentation. We further show that apoptotic regulators can influence both delamination and dorsal closure cell non-autonomously, presumably by influencing tissue mechanics. The spatial heterogeneities in delamination frequency and mitochondrial morphology suggest that mechanical stresses may underlie the activation of the apoptotic cascade through their influence on mitochondrial dynamics. Our results document for the first time the temporal propagation of an apoptotic signal in the context of cell behaviours that accomplish morphogenesis during development. They highlight the importance of mitochondrial dynamics and tissue mechanics in its regulation. Together, they provide novel insights into how apoptotic signals can be deployed to pattern tissues.

Asunto(s)

Apoptosis/fisiología , Adhesión Celular/fisiología , Drosophila/embriología , Embrión no Mamífero/embriología , Epitelio/embriología , Transducción de Señal/fisiología , Animales , Caspasas/metabolismo , Drosophila/genética , Técnica del Anticuerpo Fluorescente , Microscopía Confocal

RESUMEN

Tissue patterning relies on cellular reorganization through the interplay between signaling pathways and mechanical stresses. Their integration and spatiotemporal coordination remain poorly understood. Here we investigate the mechanisms driving the dynamics of cell delamination, diversely deployed to extrude dead cells or specify distinct cell fates. We show that a local mechanical stimulus (subcellular laser perturbation) releases cellular prestress and triggers cell delamination in the amnioserosa during Drosophila dorsal closure, which, like spontaneous delamination, results in the rearrangement of nearest neighbors around the delaminating cell into a rosette. We demonstrate that a sequence of "emergent cytoskeletal polarities" in the nearest neighbors (directed myosin flows, lamellipodial growth, polarized actomyosin collars, microtubule asters), triggered by the mechanical stimulus and dependent on integrin adhesion, generate active stresses that drive delamination. We interpret these patterns in the language of active gels as asters formed by active force dipoles involving surface and body stresses generated by each cell and liken delamination to mechanical yielding that ensues when these stresses exceed a threshold. We suggest that differential contributions of adhesion, cytoskeletal, and external stresses must underlie differences in spatial pattern.

Asunto(s)

Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Integrinas/metabolismo , Actomiosina/química , Animales , Adhesión Celular , Linaje de la Célula , Citoplasma/metabolismo , Drosophila , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal/métodos , Modelos Biológicos , Factores de Tiempo , Cicatrización de Heridas

RESUMEN

Transcription factors of the Grainy head (Grh) family are required in epithelia to generate the impermeable apical layer that protects against the external environment. This function is conserved in vertebrates and invertebrates, despite the differing molecular composition of the protective barrier. Epithelial cells also have junctions that create a paracellular diffusion barrier (tight or septate junctions). To examine whether Grh has a role in regulating such characteristics, we used an epidermal layer in the Drosophila embryo that has no endogenous Grh and lacks septate junctions, the amnioserosa. Expression of Grh in the amnioserosa caused severe defects in dorsal closure, a process similar to wound closure, and induced robust expression of the septate junction proteins Coracle, Fasciclin 3 and Sinuous. Grh-binding sites are present within the genes encoding these proteins, consistent with them being direct targets. Removal of Grh from imaginal disc cells caused a reduction in Fasciclin 3 and Coracle levels, suggesting that Grh normally fine tunes their epithelial expression and hence contributes to barrier properties. The fact that ectopic Grh arrests dorsal closure also suggests that this dynamic process relies on epithelia having distinct adhesive properties conferred by differential deployment of Grh.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/fisiología , Epitelio/embriología , Proteínas de la Membrana/genética , Factores de Transcripción/fisiología , Animales , Sitios de Unión , Adhesión Celular , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Células Epidérmicas , Epidermis/embriología , Células Epiteliales/citología , Epitelio/metabolismo , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/metabolismo , Morfogénesis , Mutación , Factores de Transcripción/genética

RESUMEN

To understand how transcription factors direct developmental events, it is necessary to know their target or 'effector' genes whose products mediate the downstream cell biological events. Whereas loss of a single target may partially or fully recapitulate the phenotype of loss of the transcription factor, this does not mean that this target is the only direct mediator. For a complete understanding of the pathway it is necessary to identify the full set of targets that together are sufficient to carry out the programme initiated by the transcription factor, which has not yet been attempted for any pathway. In the case of the transcriptional activator Twist, which acts at the top of the mesodermal developmental cascade in Drosophila, two targets, Snail and Fog, are known to be necessary for the first morphogenetic event, the orderly invagination of the mesoderm. We use a system of reconstituting loss of Twist function by transgenes expressing Snail and Fog independently of Twist to analyse the sufficiency of these factors-a loss of function assay for additional gene functions to assess what further functions might be needed downstream of Twist. Confirming and extending previous studies, we show that Snail plays an essential role, allowing basic cell shape changes to take place. Fog and at least two other genes are needed to accelerate and coordinate shape changes. Furthermore, this study represents the first step in the systematic reconstruction of the morphogenetic programme downstream of Twist.

Asunto(s)

Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Mesodermo/fisiología , Morfogénesis/fisiología , Animales , Gástrula/fisiología

RESUMEN

Dorsal closure during Drosophila embryogenesis provides a valuable model for epithelial morphogenesis and wound healing. Previous studies have focused on two cell populations, the dorsal epidermis and the extraembryonic amnioserosa. Here, we demonstrate that there is an additional player, the large yolk cell. We find that integrins are expressed in the amnioserosa and yolk cell membrane and that they are required for three processes: (1) assembly of an intervening extracellular matrix, (2) attachment between these two cell layers, and (3) contraction of the amnioserosa cells. We also provide evidence for integrin-extracellular matrix interactions occurring between the lateral surfaces of the amnioserosa cell and the leading edge epidermis that effectively mediate cell-cell adhesion. Thus, dorsal closure shares mechanistic similarities with vertebrate epithelial morphogenetic events, including epiboly, that also employ an underlying substrate.

Asunto(s)

Drosophila/embriología , Integrinas/metabolismo , Saco Vitelino/metabolismo , Animales , Adhesión Celular/fisiología , Epidermis/metabolismo , Epitelio/embriología , Epitelio/metabolismo , Matriz Extracelular/metabolismo , Inmunohistoquímica , Membranas/metabolismo , Microscopía Confocal , Morfogénesis

RESUMEN

We report the functional characterization of the Drosophila ortholog of tensin, a protein implicated in linking integrins to the cytoskeleton and signaling pathways. A tensin null was generated and is viable with wing blisters, a phenotype characteristic of loss of integrin adhesion. In tensin mutants, mechanical abrasion is required during wing expansion to cause wing blisters, suggesting that tensin strengthens integrin adhesion. The localization of tensin requires integrins, talin, and integrin-linked kinase. The N-terminal domain and C-terminal PTB domain of tensin provide essential recruitment signals. The intervening SH2 domain is not localized on its own. We suggest a model where tensin is recruited to sites of integrin adhesion via its PTB and N-terminal domains, localizing the SH2 domain so that it can interact with phosphotyrosine-containing proteins, which stabilize the integrin link to the cytoskeleton.

Asunto(s)

Adhesión Celular/fisiología , Integrinas/fisiología , Proteínas de Microfilamentos/fisiología , Animales , Animales Modificados Genéticamente , Adhesión Celular/genética , Citoesqueleto/metabolismo , Drosophila/genética , Embrión no Mamífero , Proteína-Tirosina Quinasas de Adhesión Focal , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto/genética , Proteínas Fluorescentes Verdes , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Proteínas de Insectos/metabolismo , Integrinas/genética , Larva , Proteínas Luminiscentes/metabolismo , Proteínas de Microfilamentos/genética , Modelos Biológicos , Mutación , Fosfotirosina/metabolismo , Proteína de Unión al Tracto de Polipirimidina , Unión Proteica , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Estrés Fisiológico/genética , Estrés Fisiológico/metabolismo , Talina/fisiología , Tensinas , Tirosina/metabolismo , Alas de Animales/embriología , Alas de Animales/metabolismo , Dominios Homologos src/fisiología