Nonmuscle myosin II generates forces that transmit tension and drive contraction in multiple tissues during dorsal closure

BACKGROUND: The morphogenic movements that characterize embryonic development require the precise temporal and spatial control of cell-shape changes. Drosophila dorsal closure is a well-established model for epithelial sheet morphogenesis, and mutations in more than 60 genes cause defects in closure. Closure requires that four forces, derived from distinct tissues, be precisely balanced. The proteins responsible for generating each of the forces have not been determined. RESULTS: We document dorsal closure in living embryos to show that mutations in nonmuscle myosin II (encoded by zipper; zip/MyoII) disrupt the integrity of multiple tissues during closure. We demonstrate that MyoII localization is distinct from, but overlaps, F-actin in the supracellular purse string, whereas in the amnioserosa and lateral epidermis each has similar, cortical distributions. In zip/MyoII mutant embryos, we restore MyoII function either ubiquitously or specifically in the leading edge, amnioserosa, or lateral epidermis and find that zip/MyoII function in any one tissue can rescue closure. Using a novel, transgenic mosaic approach, we establish that contractility of the supracellular purse string in leading-edge cells requires zip/MyoII-generated forces; that zip/MyoII function is responsible for the apical contraction of amnioserosa cells; that zip/MyoII is important for zipping; and that defects in zip/MyoII contractility cause the misalignment of the lateral-epidermal sheets during seam formation. CONCLUSIONS: We establish that zip/MyoII is responsible for generating the forces that drive cell-shape changes in each of the force-generating tissues that contribute to closure. This highly conserved contractile protein likely drives cell-sheet movements throughout phylogeny.     

DRhoGEF2 regulates cellular tension and cell pulsations in the Amnioserosa during Drosophila dorsal closure

Coordination of apical constriction in epithelial sheets is a fundamental process during embryogenesis. Here, we show that DRhoGEF2 is a key regulator of apical pulsation and constriction of amnioserosal cells during Drosophila dorsal closure. Amnioserosal cells mutant for DRhoGEF2 exhibit a consistent decrease in amnioserosa pulsations whereas overexpression of DRhoGEF2 in this tissue leads to an increase in the contraction time of pulsations. We probed the physical properties of the amnioserosa to show that the average tension in DRhoGEF2 mutant cells is lower than wild-type and that overexpression of DRhoGEF2 results in a tissue that is more solid-like than wild-type. We also observe that in the DRhoGEF2 overexpressing cells there is a dramatic increase of apical actomyosin coalescence that can contribute to the generation of more contractile forces, leading to amnioserosal cells with smaller apical surface than wild-type. Conversely, in DRhoGEF2 mutants, the apical actomyosin coalescence is impaired. These results identify DRhoGEF2 as an upstream regulator of the actomyosin contractile machinery that drives amnioserosa cells pulsations and apical constriction.     

Contractile and Mechanical Properties of Epithelia with Perturbed Actomyosin Dynamics

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.     

Innexin 3, a New Gene Required for Dorsal Closure in Drosophila Embryo

Background

Dorsal closure is a morphogenetic event that occurs during mid-embryogenesis in many insects including Drosophila, during which the ectoderm migrates on the extraembryonic amnioserosa to seal the embryo dorsally. The contribution of the ectoderm in this event has been known for a long time. However, amnioserosa tension and contractibility have recently been shown also to be instrumental to the closure. A critical pre-requisite for dorsal closure is integrity of these tissues that in part is mediated by cell-cell junctions and cell adhesion. In this regard, mutations impairing junction formation and/or adhesion lead to dorsal closure. However, no role for the gap junction proteins Innexins has so far been described.

Results and Discussion

Here, we show that Innexin 1, 2 and 3, are present in the ectoderm but also in the amnioserosa in plaques consistent with gap junctions. However, only the loss of Inx3 leads to dorsal closure defects that are completely rescued by overexpression of inx3::GFP in the whole embryo. Loss of Inx3 leads to the destabilisation of Inx1, Inx2 and DE-cadherin at the plasma membrane, suggesting that these four proteins form a complex. Accordingly, in addition to the known interaction of Inx2 with DE-cadherin, we show that Inx3 can bind to DE-cadherin. Furthermore, Inx3-GFP overexpression recruits DE-cadherin from its wildtype plasma membrane domain to typical Innexin plaques, strengthening the notion that they form a complex. Finally, we show that Inx3 stability is directly dependent on tissue tension. Taken together, we propose that Inx3 is a critical factor for dorsal closure and that it mediates the stability of Inx1, 2 and DE-cadherin by forming a complex.     

A Highlights from MBoC Selection: Complete canthi removal reveals that forces from the amnioserosa alone are sufficient to drive dorsal closure in Drosophila

Drosophila''s dorsal closure provides an excellent model system with which to analyze biomechanical processes during morphogenesis. During native closure, the amnioserosa, flanked by two lateral epidermal sheets, forms an eye-shaped opening with canthi at each corner. The dynamics of amnioserosa cells and actomyosin purse strings in the leading edges of epidermal cells promote closure, whereas the bulk of the lateral epidermis opposes closure. Canthi maintain purse string curvature (necessary for their dorsalward forces), and zipping at the canthi shortens leading edges, ensuring a continuous epithelium at closure completion. We investigated the requirement for intact canthi during closure with laser dissection approaches. Dissection of one or both canthi resulted in tissue recoil and flattening of each purse string. After recoil and a temporary pause, closure resumed at approximately native rates until slowing near the completion of closure. Thus the amnioserosa alone can drive closure after dissection of one or both canthi, requiring neither substantial purse string curvature nor zipping during the bulk of closure. How the embryo coordinates multiple, large forces (each of which is orders of magnitude greater than the net force) during native closure and is also resilient to multiple perturbations are key extant questions.     

A Cell-Level Biomechanical Model of Drosophila Dorsal Closure

We report a model describing the various stages of dorsal closure of Drosophila. Inspired by experimental observations, we represent the amnioserosa by 81 hexagonal cells that are coupled mechanically through the position of the nodes and the elastic forces on the edges. In addition, each cell has radial spokes representing actin filaments on which myosin motors can attach and exert contractile forces on the nodes, the attachment being controlled by a signaling molecule. Thus, the model couples dissipative cell and tissue motion with kinetic equations describing the myosin and signal dynamics. In the early phase, amnioserosa cells oscillate as a result of coupling among the chemical signaling, myosin attachment/detachment, and mechanical deformation of neighboring cells. In the slow phase, we test two ratcheting mechanisms suggested by experiments: an internal ratchet by the apical and junctional myosin condensates, and an external one by the supracellular actin cables encircling the amnioserosa. Within the range of parameters tested, the model predictions suggest the former as the main contributor to cell and tissue area reduction in this stage. In the fast phase of dorsal closure, cell pulsation is arrested, and the cell and tissue areas contract consistently. This is realized in the model by gradually shrinking the resting length of the spokes. Overall, the model captures the key features of dorsal closure through the three distinct phases, and its predictions are in good agreement with observations.     

Remodeling Tissue Interfaces and the Thermodynamics of Zipping during Dorsal Closure in Drosophila

Dorsal closure during Drosophila embryogenesis is an important model system for investigating the biomechanics of morphogenesis. During closure, two flanks of lateral epidermis (with actomyosin-rich purse strings near each leading edge) close an eye-shaped opening that is filled with amnioserosa. At each canthus (corner of the eye) a zipping process remodels the tissue interfaces between the leading edges of the lateral epidermis and the amnioserosa. We investigated zipping dynamics and found that apposing leading edge cells come together at their apical ends and then square off basally to form a lateral junction. Meanwhile, the purse strings act as contractile elastic rods bent toward the embryo interior near each canthus. We propose that a canthus-localized force contributes to both bending the ends of the purse strings and the formation of lateral junctions. We developed a thermodynamic model for zipping based on three-dimensional remodeling of the tissue interfaces and the reaction dynamics of adhesion molecules in junctions and elsewhere, which we applied to zipping during unperturbed wild-type closure and to laser or genetically perturbed closure. We identified two processes that can contribute to the zipping mechanism, consistent with experiments, distinguished by whether amnioserosa dynamics do or do not augment canthus adhesion dynamics.     

Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila

The molecular and cellular bases of cell shape change and movement during morphogenesis and wound healing are of intense interest and are only beginning to be understood. Here, we investigate the forces responsible for morphogenesis during dorsal closure with three approaches. First, we use real-time and time-lapsed laser confocal microscopy to follow actin dynamics and document cell shape changes and tissue movements in living, unperturbed embryos. We label cells with a ubiquitously expressed transgene that encodes GFP fused to an autonomously folding actin binding fragment from fly moesin. Second, we use a biomechanical approach to examine the distribution of stiffness/tension during dorsal closure by following the response of the various tissues to cutting by an ultraviolet laser. We tested our previous model (Young, P.E., A.M. Richman, A.S. Ketchum, and D.P. Kiehart. 1993. Genes Dev. 7:29-41) that the leading edge of the lateral epidermis is a contractile purse-string that provides force for dorsal closure. We show that this structure is under tension and behaves as a supracellular purse-string, however, we provide evidence that it alone cannot account for the forces responsible for dorsal closure. In addition, we show that there is isotropic stiffness/tension in the amnioserosa and anisotropic stiffness/tension in the lateral epidermis. Tension in the amnioserosa may contribute force for dorsal closure, but tension in the lateral epidermis opposes it. Third, we examine the role of various tissues in dorsal closure by repeated ablation of cells in the amnioserosa and the leading edge of the lateral epidermis. Our data provide strong evidence that both tissues appear to contribute to normal dorsal closure in living embryos, but surprisingly, neither is absolutely required for dorsal closure. Finally, we establish that the Drosophila epidermis rapidly and reproducibly heals from both mechanical and ultraviolet laser wounds, even those delivered repeatedly. During healing, actin is rapidly recruited to the margins of the wound and a newly formed, supracellular purse-string contracts during wound healing. This result establishes the Drosophila embryo as an excellent system for the investigation of wound healing. Moreover, our observations demonstrate that wound healing in this insect epidermal system parallel wound healing in vertebrate tissues in situ and vertebrate cells in culture (for review see Kiehart, D.P. 1999. Curr. Biol. 9:R602-R605).     

Novel functions for integrins in epithelial morphogenesis

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.     

Asymmetric distribution of Echinoid defines the epidermal leading edge during Drosophila dorsal closure

During Drosophila melanogaster dorsal closure, lateral sheets of embryonic epidermis assemble an actomyosin cable at their leading edge and migrate dorsally over the amnioserosa, converging at the dorsal midline. We show that disappearance of the homophilic cell adhesion molecule Echinoid (Ed) from the amnioserosa just before dorsal closure eliminates homophilic interactions with the adjacent dorsal-most epidermal (DME) cells, which comprise the leading edge. The resulting planar polarized distribution of Ed in the DME cells is essential for the localized accumulation of actin regulators and for actomyosin cable formation at the leading edge and for the polarized localization of the scaffolding protein Bazooka/PAR-3. DME cells with uniform Ed fail to assemble a cable and protrude dorsally, suggesting that the cable restricts dorsal migration. The planar polarized distribution of Ed in the DME cells thus provides a spatial cue that polarizes the DME cell actin cytoskeleton, defining the epidermal leading edge and establishing its contractile properties.     

Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents anoikis

BACKGROUND: Two extraembryonic tissues form early in Drosophila development. One, the amnioserosa, has been implicated in the morphogenetic processes of germ band retraction and dorsal closure. The developmental role of the other, the yolk sac, is obscure. RESULTS: By using live-imaging techniques, we report intimate interactions between the amnioserosa and the yolk sac during germ band retraction and dorsal closure. These tissue interactions fail in a subset of myospheroid (mys: betaPS integrin) mutant embryos, leading to failure of germ band retraction and dorsal closure. The Drosophila homolog of mammalian basigin (EMMPRIN, CD147)-an integrin-associated transmembrane glycoprotein-is highly enriched in the extraembryonic tissues. Strong dominant genetic interactions between basigin and mys mutations cause severe defects in dorsal closure, consistent with basigin functioning together with betaPS integrin in extraembryonic membrane apposition. During normal development, JNK signaling is upregulated in the amnioserosa, as midgut closure disrupts contact with the yolk sac. Subsequently, the amnioserosal epithelium degenerates in a process that is independent of the reaper, hid, and grim cell death genes. In mys mutants that fail to establish contact between the extraembryonic membranes, the amnioserosa undergoes premature disintegration and death. CONCLUSIONS: Intimate apposition of the amnioserosa and yolk sac prevents anoikis of the amnioserosa. Survival of the amnioserosa is essential for germ band retraction and dorsal closure. We hypothesize that during normal development, loss of integrin-dependent contact between the extraembryonic tissues results in JNK-dependent amnioserosal disintegration and death, thus representing an example of developmentally programmed anoikis.     

Specialized extraembryonic cells connect embryonic and extraembryonic epidermis in response to Dpp during dorsal closure in Drosophila

Dorsal closure in Drosophila embryogenesis involves expansion of the dorsal epidermis, followed by closure of the opposite epidermal edges. This process is driven by contractile force generated by an extraembryonic epithelium covering the yolk syncytium known as the amnioserosa. The secreted signaling molecule Dpp is expressed in the leading edge of the dorsal epidermis and is essential for dorsal closure. We found that the outermost row of amnioserosa cells (termed pAS) maintains a tight basolateral cell-cell adhesion interface with the leading edge of dorsal epidermis throughout the dorsal closure process. pAS was subject to altered cell motility in response to Dpp emanating from the dorsal epidermis, and this response was essential for dorsal closure. alphaPS3 and betaPS integrin subunits accumulated in the interface between pAS and dorsal epidermis, and were both required for dorsal closure. Looking at alphaPS3, type I Dpp receptor, and JNK mutants, we found that pAS cell motility was altered and pAS and dorsal epidermis adhesion failed under the mechanical stress of dorsal closure, suggesting that a Dpp-mediated mechanism connects the squamous pAS to the columnar dorsal epidermis to form a single coherent epithelial layer.     

The dorsal-open group gene raw is required for restricted DJNK signaling during closure.

During dorsal closure in Drosophila melanogaster, cells of the lateral epidermis migrate over the amnioserosa to encase the embryo. At least three classes of dorsal-open group gene products are necessary for this morphogenetic movement. Class I genes code for structural proteins that effect changes in epidermal cell shape and motility. Class II and III genes code for regulatory components of closure: Class II genes encode Drosophila Jun amino (N)-terminal kinase (DJNK) signaling molecules and Class III genes encode Decapentaplegic-mediated signaling molecules. All characterized dorsal-open group gene products function in the epidermis. Here we report a molecular and genetic characterization of raw, a newly defined member of the Class II dorsal-open group genes. We show that the novel protein encoded by raw is required for restriction of DJNK signaling to leading edge epidermal cells as well as for proper development of the amnioserosa. Taken together, our results demonstrate a role for Raw in restriction of epidermal signaling during closure and suggest that this effect may be mediated via the amnioserosa.     

Emergent properties during dorsal closure in Drosophila morphogenesis

Dorsal closure is an essential stage of Drosophila development that is a model system for research in morphogenesis and biological physics. Dorsal closure involves an orchestrated interplay between gene expression and cell activities that produce shape changes, exert forces and mediate tissue dynamics. We investigate the dynamics of dorsal closure based on confocal microscopic measurements of cell shortening in living embryos. During the mid-stages of dorsal closure we find that there are fluctuations in the width of the leading edge cells but the time-averaged analysis of measurements indicate that there is essentially no net shortening of cells in the bulk of the leading edge, that contraction predominantly occurs at the canthi as part of the process for zipping together the two leading edges of epidermis and that the rate constant for zipping correlates with the rate of movement of the leading edges. We characterize emergent properties that regulate dorsal closure, i.e., a velocity governor and the coordination and synchronization of tissue dynamics.     

dPak is required for integrity of the leading edge cytoskeleton during Drosophila dorsal closure but does not signal through the JNK cascade

The Pak kinases are effectors for the small GTPases Rac and Cdc42 and are divided into two subfamilies. Group I Paks possess an autoinhibitory domain that can suppress their kinase activity in trans. In Drosophila, two Group I kinases have been identified, dPak and Pak3. Rac and Cdc42 participate in dorsal closure of the embryo, a process in which a hole in the dorsal epidermis is sealed through migration of the epidermal flanks over a tissue called the amnioserosa. Dorsal closure is driven in part by an actomyosin contractile apparatus at the leading edge of the epidermis, and is regulated by a Jun amino terminal kinase (JNK) cascade. Impairment of dPak function using either loss-of-function mutations or expression of a transgene encoding the autoinhibitory domain of dPak led to disruption of the leading edge cytoskeleton and defects in dorsal closure but did not affect the JNK cascade. Group I Pak kinase activity in the amnioserosa is required for correct morphogenesis of the epidermis, and may be a component of the signaling known to occur between these two tissues. We conclude that dorsal closure requires Group I Pak function in both the amnioserosa and the epidermis.     

Embryonic wound healing by apical contraction and ingression in Xenopus laevis

We have characterized excisional wounds in the animal cap of early embryos of the frog Xenopus laevis and found that these wounds close accompanied by three distinct processes: (1) the assembly of an actin purse-string in the epithelial cells at the wound margin, (2) contraction and ingression of exposed deep cells, and (3) protrusive activity of epithelial cells at the margin. Microsurgical manipulation allowing fine control over the area and depth of the wound combined with videomicroscopy and confocal analysis enabled us to describe the kinematics and challenge the mechanics of the closing wound. Full closure typically occurs only when the deep, mesenchymal cell-layer of the ectoderm is left intact; in contrast, when deep cells are removed along with the superficial, epithelial cell-layer of the ectoderm, wounds do not close. Actin localizes to the superficial epithelial cell-layer at the wound margin immediately after wounding and forms a contiguous "purse-string" in those cells within 15 min. However, manipulation and closure kinematics of shaped wounds and microsurgical cuts made through the purse-string rule out a major force-generating role for the purse-string. Further analysis of the cell behaviors within the wound show that deep, mesenchymal cells contract their apical surfaces and ingress from the exposed surface. High resolution time-lapse sequences of cells at the leading edge of the wound show that these cells undergo protrusive activity only during the final phases of wound closure as the ectoderm reseals. We propose that assembly of the actin purse-string works to organize and maintain the epithelial sheet at the wound margin, that contraction and ingression of deep cells pulls the wound margins together, and that protrusive activity of epithelial cells at the wound margin reseals the ectoderm and re-establishes tissue integrity during wound healing in the Xenopus embryonic ectoderm.     

Local,cell-nonautonomous feedback regulation of myosin dynamics patterns transitions in cell behavior: a role for tension and geometry?

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.