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.     

Differential expression of the adhesion molecule Echinoid drives epithelial morphogenesis in Drosophila

Epithelial morphogenesis requires cell movements and cell shape changes coordinated by modulation of the actin cytoskeleton. We identify a role for Echinoid (Ed), an immunoglobulin domain-containing cell-adhesion molecule, in the generation of a contractile actomyosin cable required for epithelial morphogenesis in both the Drosophila ovarian follicular epithelium and embryo. Analysis of ed mutant follicle cell clones indicates that the juxtaposition of wild-type and ed mutant cells is sufficient to trigger actomyosin cable formation. Moreover, in wild-type ovaries and embryos, specific epithelial domains lack detectable Ed, thus creating endogenous interfaces between cells with and without Ed; these interfaces display the same contractile characteristics as the ectopic Ed expression borders generated by ed mutant clones. In the ovary, such an interface lies between the two cell types of the dorsal appendage primordia. In the embryo, Ed is absent from the amnioserosa during dorsal closure, generating an Ed expression border with the lateral epidermis that coincides with the actomyosin cable present at this interface. In both cases, ed mutant epithelia exhibit loss of this contractile structure and subsequent defects in morphogenesis. We propose that local modulation of the cytoskeleton at Ed expression borders may represent a general mechanism for promoting epithelial morphogenesis.     

SCAR/WAVE complex recruitment to a supracellular actomyosin cable by myosin activators and a junctional Arf-GEF during Drosophila dorsal closure

Expansive Arp2/3 actin networks and contractile actomyosin networks can be spatially and temporally segregated within the cell, but the networks also interact closely at various sites, including adherens junctions. However, molecular mechanisms coordinating these interactions remain unclear. We found that the SCAR/WAVE complex, an Arp2/3 activator, is enriched at adherens junctions of the leading edge actomyosin cable during Drosophila dorsal closure. Myosin activators were both necessary and sufficient for SCAR/WAVE accumulation at leading edge junctions. The same myosin activators were previously shown to recruit the cytohesin Arf-GEF Steppke to these sites, and mammalian studies have linked Arf small G protein signaling to SCAR/WAVE activation. During dorsal closure, we find that Steppke is required for SCAR/WAVE enrichment at the actomyosin-linked junctions. Arp2/3 also localizes to adherens junctions of the leading edge cable. We propose that junctional actomyosin activity acts through Steppke to recruit SCAR/WAVE and Arp2/3 for regulation of the leading edge supracellular actomyosin cable during dorsal closure.     

Armadillo/beta-catenin-dependent Wnt signalling is required for the polarisation of epidermal cells during dorsal closure in Drosophila

At the end of germband retraction, the dorsal epidermis of the Drosophila embryo exhibits a discontinuity that is covered by the amnioserosa. The process of dorsal closure (DC) involves a coordinated set of cell-shape changes within the epidermis and the amnioserosa that result in epidermal continuity. Polarisation of the dorsal-most epidermal (DME) cells in the plane of the epithelium is an important aspect of DC. The DME cells of embryos mutant for wingless or dishevelled exhibit polarisation defects and fail to close properly. We have investigated the role of the Wingless signalling pathway in the polarisation of the DME cells and DC. We find that the beta-catenin-dependent Wingless signalling pathway is required for polarisation of the DME cells. We further show that although the DME cells are polarised in the plane of the epithelium and present polarised localisation of proteins associated with the process of planar cell polarity (PCP) in the wing, e.g. Flamingo, PCP Wingless signalling is not involved in DC.     

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.     

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.     

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).     

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.     

The small GTPase Rac plays multiple roles in epithelial sheet fusion--dynamic studies of Drosophila dorsal closure

The coordinated migration and fusion of epithelial sheets is a crucial morphogenetic tool used on numerous occasions during the normal development of an embryo and re-activated as part of the wound healing response. Drosophila dorsal closure, whereby a hole in the embryonic epithelium is zipped closed late in embryogenesis, serves as an excellent, genetically tractable model for epithelial migration. Using live confocal imaging, we have dissected multiple roles for the small GTPase Rac in this process. We show that constitutive activation of Rac1 leads to excessive assembly of lamellipodia and precocious halting of epithelial sweeping, possibly through premature activation of contact-inhibition machinery. Conversely, blocking Rac activity, either by loss-of-function mutations or expression of dominant negative Rac1, disables the assembly of both actin cable and protrusions by epithelial cells. Movies of mutant embryos show that continued contraction of the amnioserosa is sufficient to draw the epithelial edges towards one another, allowing the zipper machinery to bypass non-functioning regions of leading edge. In addition to illustrating the key role of Rac in organization of leading edge actin, loss-of-function mutants also provide substantive proof that Rac acts upstream in the Jun N-terminal kinase (JNK) cascade to direct epithelial cell shape changes during dorsal closure.     

Differential adhesion and actomyosin cable collaborate to drive Echinoid-mediated cell sorting

Cell sorting involves the segregation of two cell populations into `immiscible' adjacent tissues with smooth borders. Echinoid (Ed), a nectin ortholog, is an adherens junction protein in Drosophila, and cells mutant for ed sort out from the surrounding wild-type cells. However, it remains unknown which factors trigger cell sorting. Here, we dissect the sequence of this process and find that cell sorting occurs when differential expression of Ed triggers the assembly of actomyosin cable. Conversely, Ed-mediated cell sorting can be rescued by recruitment of Ed, via homophilic or heterophilic interactions, to the wild-type cell side of the clonal interface, even when differential Ed expression persists. We found, unexpectedly, that when actomyosin cable was largely absent, differential adhesion was sufficient to cause limited cell segregation but with a jagged tissue border (imperfect sorting). We propose that Ed-mediated cell sorting is driven both by differential Ed adhesion that induces cell segregation with a jagged border and by actomyosin cable assembly at the interface that smoothens this border.     

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.     

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.     

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.     

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.     

Coordinated waves of actomyosin flow and apical cell constriction immediately after wounding

Epithelial wound healing relies on tissue movements and cell shape changes. Our work shows that, immediately after wounding, there was a dramatic cytoskeleton remodeling consisting of a pulse of actomyosin filaments that assembled in cells around the wound edge and flowed from cell to cell toward the margin of the wound. We show that this actomyosin flow was regulated by Diaphanous and ROCK and that it elicited a wave of apical cell constriction that culminated in the formation of the leading edge actomyosin cable, a structure that is essential for wound closure. Calcium signaling played an important role in this process, as its intracellular concentration increased dramatically immediately after wounding, and down-regulation of transient receptor potential channel M, a stress-activated calcium channel, also impaired the actomyosin flow. Lowering the activity of Gelsolin, a known calcium-activated actin filament–severing protein, also impaired the wound response, indicating that cleaving the existing actin filament network is an important part of the cytoskeleton remodeling process.     

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 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.