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.     

Cell adhesion molecule Echinoid associates with unconventional myosin VI/Jaguar motor to regulate cell morphology during dorsal closure in Drosophila

Echinoid (Ed) is a homophilic immunoglobulin domain-containing cell adhesion molecule (CAM) that localizes to adherens junctions (AJs) and cooperates with Drosophila melanogaster epithelial (DE)-cadherin to mediate cell adhesion. Here we show that Ed takes part in many processes of dorsal closure, a morphogenetic movement driven by coordinated cell shape changes and migration of epidermal cells to cover the underlying amnioserosa. Ed is differentially expressed, appearing in epidermis but not in amnioserosa cells. Ed functions independently from the JNK signaling pathway and is required to regulate cell morphology, and for assembly of actomyosin cable, filopodial protrusion and coordinated cell migration in dorsal-most epidermal cells. The effect of Ed on cell morphology requires the presence of the intracellular domain (Ed^intra). Interestingly, Ed forms homodimers in vivo and Ed^intra monomer directly associates with unconventional myosin VI/Jaguar (Jar) motor protein. We further show that ed genetically interacts with jar to control cell morphology. It has previously been shown that myosin VI is monomeric in vitro and that its dimeric form can associate with and travel processively along actin filaments. Thus, we propose that Ed mediates the dimerization of myosin VI/Jar in vivo which in turn regulates the reorganization and/or contraction of actin filaments to control changes in cell shape. Consistent with this, we found that ectopic ed expression in the amnioserosa induces myosin VI/Jar-dependent apical constriction of this tissue.     

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).     

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.     

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.     

Cell ingression and apical shape oscillations during dorsal closure in Drosophila

Programmed patterns of gene expression, cell-cell signaling, and cellular forces cause morphogenic movements during dorsal closure. We investigated the apical cell-shape changes that characterize amnioserosa cells during dorsal closure in Drosophila embryos with in vivo imaging of green-fluorescent-protein-labeled DE-cadherin. Time-lapsed, confocal images were assessed with a novel segmentation algorithm, Fourier analysis, and kinematic and dynamical modeling. We found two generic processes, reversible oscillations in apical cross-sectional area and cell ingression characterized by persistent loss of apical area. We quantified a time-dependent, spatially-averaged sum of intracellular and intercellular forces acting on each cell's apical belt of DE-cadherin. We observed that a substantial fraction of amnioserosa cells ingress near the leading edges of lateral epidermis, consistent with the view that ingression can be regulated by leading-edge cells. This is in addition to previously observed ingression processes associated with zipping and apoptosis. Although there is cell-to-cell variability in the maximum rate for decreasing apical area (0.3-9.5 μm(2)/min), the rate for completing ingression is remarkably constant (0.83 cells/min, r(2) > 0.99). We propose that this constant ingression rate contributes to the spatiotemporal regularity of mechanical stress exerted by the amnioserosa on each leading edge during closure.     

Dynamic analysis of actin cable function during Drosophila dorsal closure

Throughout development, a series of epithelial movements and fusions occur that collectively shape the embryo. They are dependent on coordinated reorganizations and contractions of the actin cytoskeleton within defined populations of epithelial cells. One paradigm morphogenetic movement, dorsal closure in the Drosophila embryo, involves closure of a dorsal epithelial hole by sweeping of epithelium from the two sides of the embryo over the exposed extraembryonic amnioserosa to form a seam where the two epithelial edges fuse together. The front row cells exhibit a thick actin cable at their leading edge. Here, we test the function of this cable by live analysis of GFP-actin-expressing embryos in which the cable is disrupted by modulating Rho1 signaling or by loss of non-muscle myosin (Zipper) function. We show that the cable serves a dual role during dorsal closure. It is contractile and thus can operate as a "purse string," but it also restricts forward movement of the leading edge and excess activity of filopodia/lamellipodia. Stripes of epithelium in which cable assembly is disrupted gain a migrational advantage over their wild-type neighbors, suggesting that the cable acts to restrain front row cells, thus maintaining a taut, free edge for efficient zippering together of the epithelial sheets.     

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.     

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.     

A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers’ attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.     

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.     

The Sac1 lipid phosphatase regulates cell shape change and the JNK cascade during dorsal closure in Drosophila

The Sac1 lipid phosphatase dephosphorylates several phosphatidylinositol (PtdIns) phosphates and, in yeast, regulates a diverse range of cellular processes including organization of the actin cytoskeleton and secretion. We have identified mutations in the gene encoding Drosophila Sac1. sac1 mutants die as embryos with defects in dorsal closure (DC). DC involves the migration of the epidermis to close a hole in the dorsal surface of the embryo occupied by the amnioserosa. It requires cell shape change in both the epidermis and amnioserosa and activation of a Jun N-terminal kinase (JNK) MAPK cascade in the leading edge cells of the epidermis [2]. Loss of Sac1 leads to the improper activation of two key events in DC: cell shape change in the amnioserosa and JNK signaling. sac1 interacts genetically with other participants in these two events, and our data suggest that loss of Sac1 leads to upregulation of one or more signals controlling DC. This study is the first report of a role for Sac1 in the development of a multicellular organism.     

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.     

Signaling pathways directing the movement and fusion of epithelial sheets: lessons from dorsal closure in Drosophila

Wound healing in embryos and various developmental events in metazoans require the spreading and fusion of epithelial sheets. The complex signaling pathways regulating these processes are being pieced together through genetic, cell biological, and biochemical approaches. At present, dorsal closure of the Drosophila embryo is the best-characterized example of epithelial sheet movement. Dorsal closure involves migration of the lateral epidermal flanks to close a hole in the dorsal epidermis occupied by an epithelium called the amnioserosa. Detailed genetic studies have revealed a network of interacting signaling molecules regulating this process. At the center of this network is a Jun N-terminal kinase cascade acting at the leading edge of the migrating epidermis that triggers signaling by the TGF-beta superfamily member Decapentaplegic and which interacts with the Wingless pathway. These signaling modules regulate the cytoskeletal reorganization and cell shape change necessary to drive dorsal closure. Activation of this network requires signals from the amnioserosa and input from a variety of proteins at cell-cell junctions. The Rho family of small GTPases is also instrumental, both in activation of signaling and regulation of the cytoskeleton. Many of the proteins regulating dorsal closure have been implicated in epithelial movement in other organisms, and dorsal closure has emerged as an ideal model system for the study of the migration and fusion of epithelial sheets.     

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.     

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.     

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.     

Drosophila Yurt is a new protein-4.1-like protein required for epithelial morphogenesis

Proteins of the 4.1 family play a key role in the integrity of the cytoskeleton and in epithelial tissue movement, as shown by the disruption of the actin cytoskeleton in human erythrocytes caused by genetic loss of protein 4.1, and the failure of epithelial tissue migration during Drosophila embryogenesis caused by genetic loss of the 4.1 homolog Coracle. Here we report the genetic characterization of Yurt, a novel protein 4.1 family member in Drosophila that is associated with the plasma membrane of epithelial cells. Homozygous loss-of-function mutations in the yurt gene cause failure of germ-band retraction, dorsal closure, and head involution, associated with degeneration of the amnioserosa and followed by embryonic lethality. A mammalian homolog of Yurt is up-regulated in metastatic melanoma cells. These novel cytoskeletal proteins appear to play important roles in epithelial cell movements and in the morphogenetic tissue changes that depend on them.