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Embryos have a remarkable ability to repair wounds rapidly, with no inflammation or scarring. Embryonic wound healing is driven by the collective movement of the surrounding cells to seal the lesion. During embryonic wound closure, the cells adjacent to the wound polarize the cytoskeletal protein actin and the molecular motor non-muscle myosin II, which accumulate at the wound edge forming a supracellular cable around the wound. Adherens junction proteins including E-cadherin are internalized from the interface with the lesion and localize to former tricellular junctions at the wound margin, in a process necessary for cytoskeletal polarity. Using quantitative live microscopy, we found that the cells adjacent to wounds in theDrosophilaepidermis also polarized Talin, a core component of cell-extracellular matrix (ECM) adhesions that links integrins to the actin cytoskeleton. Integrin knock-down and inhibition of integrin binding delayed wound closure and were associated with a reduction in actin levels around the wound. Additionally, disrupting integrins caused a defect in E-cadherin reinforcement at tricellular junctions along the wound edge, suggesting crosstalk between integrin-based and cadherin-based adhesions. Together, our results show that cell-ECM adhesion contributes to embryonic wound repair and reveal an interplay between cell-cell and cell-ECM adhesion in the collective cell movements that drive rapid wound healing.

α-Catenin couples the cadherin–catenin complex to the actin cytoskeleton. The mechanosensitive α-Catenin M region undergoes conformational changes upon application of force to recruit interaction partners. Here, we took advantage of the tension landscape in the Drosophila embryo to define three different states of α-Catenin mechanosensing in support of cell adhesion. Low-, medium-, and high-tension contacts showed a corresponding recruitment of Vinculin and Ajuba, which was dependent on the α-Catenin M region. In contrast, the Afadin homolog Canoe acts in parallel to α-Catenin at bicellular low- and medium-tension junctions but requires an interaction with α-Catenin for its tension-sensitive enrichment at high-tension tricellular junctions. Individual M region domains make complex contributions to cell adhesion through their impact on interaction partner recruitment, and redundancies with the function of Canoe. Our data argue that α-Catenin and its interaction partners are part of a cooperative and partially redundant mechanoresponsive network that supports AJs remodeling during morphogenesis.

Collective cell movements contribute to tissue development and repair, and spread metastatic disease. In epithelia, cohesive cell movements require reorganization of adherens junctions and the actomyosin cytoskeleton. However, the mechanisms that coordinate cell-cell adhesion and cytoskeletal remodelling during collective cell migration in vivo are unclear. We investigated the mechanisms of collective cell migration during wound healing in the Drosophila embryonic epidermis. Upon wounding, the cells adjacent to the wound internalize cell-cell adhesion molecules and polarize actin and the motor protein myosin II to form a supracellular cable around the wound that coordinates cell movements. The cable anchors at former tricellular junctions (TCJs) along the wound edge, and TCJs are reinforced during wound closure. We found that the small GTPase Rap1 was both necessary and sufficient for rapid wound repair. Rap1 promoted actomyosin polarization to the wound edge and E-cadherin accumulation at TCJs. Using embryos expressing a mutant form of the Rap1 effector Canoe/Afadin that cannot bind Rap1, we found that Rap1 signals through Canoe for adherens junction remodelling, but not for actomyosin cable assembly. Rap1 was necessary and sufficient for RhoA/Rho1 activation at the wound edge. Consistent with this, the RhoGEF Ephexin localized to the wound edge in a Rap1-dependent manner, and Ephexin was necessary for myosin polarization and rapid wound repair, but not for E-cadherin redistribution. Together, our data show that Rap1 coordinates the molecular rearrangements that drive embryonic wound healing and independently drives actomyosin cable assembly through Ephexin-Rho1, and E-cadherin redistribution through Canoe, thus enabling rapid collective cell migration in vivo.

Among the defining features of the animal kingdom is the ability of cells to change shape and move. This underlies embryonic and postembryonic development, tissue homeostasis, regeneration, and wound healing. Cell shape change and motility require linkage of the cell’s force-generating machinery to the plasma membrane at cell–cell and cell–extracellular matrix junctions. Connections of the actomyosin cytoskeleton to cell–cell adherens junctions need to be both resilient and dynamic, preventing tissue disruption during the dramatic events of embryonic morphogenesis. In the past decade, new insights radically altered the earlier simple paradigm that suggested simple linear linkage via the cadherin–catenin complex as the molecular mechanism of junction–cytoskeleton interaction. In this Perspective we provide a brief overview of our current state of knowledge and then focus on selected examples highlighting what we view as the major unanswered questions in our field and the approaches that offer exciting new insights at multiple scales from atomic structure to tissue mechanics.

Supplemental Digital Content is available in the text., Objective: Vascular calcification is a pathology characterized by arterial mineralization, which is a common late-term complication of atherosclerosis that independently increases the risk of adverse cardiovascular events by fourfold. A major source of calcifying cells is transdifferentiating vascular smooth muscle cells (VSMCs). Previous studies showed that deletion of the collagen-binding receptor, DDR1 (discoidin domain receptor-1), attenuated VSMC calcification. Increased matrix stiffness drives osteogenesis, and DDR1 has been implicated in stiffness sensing in other cell types; however, the role of DDR1 as a mechanosensor in VSMCs has not been investigated. Here, we test the hypothesis that DDR1 senses increased matrix stiffness and promotes VSMC transdifferentiation and calcification. Approach and Results: Primary VSMCs isolated from Ddr1+/+ (wild-type) and Ddr1−/− (knockout) mice were studied on collagen-I–coated silicon substrates of varying stiffness, culturing in normal or calcifying medium. DDR1 expression and phosphorylation increased with increasing stiffness, as did in vitro calcification, nuclear localization of Runx2 (Runt-related transcription factor 2), and expression of other osteochondrocytic markers. By contrast, DDR1 deficient VSMCs were not responsive to stiffness and did not undergo transdifferentiation. DDR1 regulated stress fiber formation and RhoA (ras homolog family member A) activation through the RhoGEF (rho guanine nucleotide exchange factor), Vav2. Inhibition of actomyosin contractility reduced Runx2 activation and attenuated in vitro calcification in wild-type VSMCs. Finally, a novel positive feedforward loop was uncovered between DDR1 and actomyosin contractility, important in regulating DDR1 expression, clustering, and activation. Conclusions: This study provides mechanistic insights into DDR1 mechanosignaling and shows that DDR1 activity and actomyosin contractility are interdependent in mediating stiffness-dependent increases in VSMC calcification.

The sarco-endoplasmic reticulum (SR/ER) is the largest membrane-bound organelle in eukaryotic cells and plays important roles in essential cellular processes, and in development and progression of many cardiac diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. In a recently published study led by Lee et al. (Nat Commun 11(1):965), we reported a cardiac enriched SR/ER membrane protein REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes resulted in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants showed sensitized cardiac dysfunction to heart failure induction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrated cardiac dysfunction with dilated cardiac chambers, increased cardiac fibrosis, and reduced ejection fraction. These results demonstrate the critical role of REEP5 in SR/ER organization and function.

Heart development begins with the formation of a tube, as cardiac progenitors migrate from opposite sides of the embryo and meet medially. Defective cardiac progenitor movements cause congenital heart defects. However, the mechanisms of cell migration during early heart development remain poorly understood. We investigated the mechanisms of movement of the Drosophila cardiac progenitors, the cardioblasts. Using quantitative time-lapse microscopy, we found that cardioblasts did not advance monotonically. Instead, cardioblasts took periodic forward and backward steps as they migrated. The forward steps were greater in both amplitude and duration, resulting in net forward movement of the cells. The molecular motor non-muscle myosin II displayed an alternating pattern of localization between the leading and trailing ends of migrating cardioblasts, forming oscillatory waves that traversed the cells. The alternating pattern of myosin polarity was associated with the alternative contraction and relaxation of the leading and trailing edges of the cell. Mathematical modelling predicted that forward migration requires the presence of a stiff boundary at the trailing edge of the cardioblasts. Consistent with this, we found a supracellular actin cable at the trailing edge of the cardioblasts. When we reduced the tension sustained by the actin cable, the amplitude of the backward steps of cardioblasts increased, thus reducing the net forward speed of migration. Together, our results indicate that periodic cell shape changes coupled with a polarized actin cable produce asymmetrical forces that guide cardioblast migration.

Summary: Embryos repair wounds rapidly, with no inflammation or scarring, in a process that involves polarization of the actomyosin cytoskeleton. Actomyosin polarization results in the assembly of a contractile cable around the wound that drives wound closure. Here, we demonstrate that a contractile actomyosin cable is not sufficient for rapid wound repair in Drosophila embryos. We show that wounding causes activation of the serine/threonine kinase p38 mitogen-activated protein kinase (MAPK) in the cells adjacent to the wound. p38 activation reduces the levels of wound-induced reactive oxygen species in the cells around the wound, limiting wound size. In addition, p38 promotes an increase in volume in the cells around the wound, thus facilitating the collective cell movements that drive rapid wound healing. Our data indicate that p38 regulates cell volumes through the sodium-potassium-chloride cotransporter NKCC1. Our work reveals cell growth and cell survival as cell behaviors critical for embryonic wound repair.

Collective cell movements play a central role in embryonic development, tissue repair, and metastatic disease. Cell movements are often coordinated by supracellular networks formed by the cytoskeletal protein actin and the molecular motor nonmuscle myosin II. During wound closure in the embryonic epidermis, the cells around the wound migrate collectively into the damaged region. In Drosophila embryos, mechanical tension stabilizes myosin at the wound edge, facilitating the assembly of a supracellular myosin cable around the wound that coordinates cell migration. Here, we show that actin is also stabilized at the wound edge. However, loss of tension or myosin activity does not affect the dynamics of actin at the wound margin. Conversely, pharmacological stabilization of actin does not affect myosin levels or dynamics around the wound. Together, our data suggest that actin and myosin are independently regulated during embryonic wound closure, thus conferring robustness to the embryonic wound healing response.

Small Src homology domain 2 (SH2) and 3 (SH3) adapter proteins regulate cell fate and behavior by mediating interactions between cell surface receptors and downstream signaling effectors in many signal transduction pathways. The CT10 regulator of kinase (Crk) family has tissue-specific roles in phagocytosis, cell migration, and neuronal development and mediates oncogenic signaling in pathways like that of Abelson kinase. However, redundancy among the two mammalian family members and the position of the Drosophila gene on the fourth chromosome precluded assessment of Crk’s full role in embryogenesis. We circumvented these limitations with short hairpin RNA and CRISPR technology to assess Crk’s function in Drosophila morphogenesis. We found that Crk is essential beginning in the first few hours of development, where it ensures accurate mitosis by regulating orchestrated dynamics of the actin cytoskeleton to keep mitotic spindles in syncytial embryos from colliding. In this role, it positively regulates cortical localization of the actin-related protein 2/3 complex (Arp2/3), its regulator suppressor of cAMP receptor (SCAR), and filamentous actin to actin caps and pseudocleavage furrows. Crk loss leads to the loss of nuclei and formation of multinucleate cells. We also found roles for Crk in embryonic wound healing and in axon patterning in the nervous system, where it localizes to the axons and midline glia. Thus, Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics.

Embryonic tissues heal wounds rapidly and without scarring, in a process conserved across species and driven by collective cell movements. The mechanisms of coordinated cell movement during embryonic wound closure also drive tissue development and cancer metastasis; therefore, embryonic wound repair has received considerable attention as a model of collective cell migration. During wound closure, a supracellular actomyosin cable at the wound edge coordinates cells, while actin-based protrusions contribute to cell crawling and seamless wound healing. Other cytoskeletal networks are reorganized during wound repair: microtubules extend into protrusions and along cell–cell boundaries as cells stretch into damaged regions, septins accumulate at the wound margin, and intermediate filaments become polarized in the cells adjacent to the wound. Thus, diverse cytoskeletal networks work in concert to maintain tissue structure, while also driving and organizing cell movements to promote rapid repair. Understanding the signals that coordinate the dynamics of different cytoskeletal networks, and how adhesions between cells or with the extracellular matrix integrate forces across cells, will be important to elucidate the mechanisms of efficient embryonic wound healing and may have far-reaching implications for developmental and cancer cell biology.

The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. Here, we show that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1– PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1 and PP2A contributes to the functional plasticity of Crumbs. Overall, our data increase our understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.

Compartment boundaries prevent cell mixing during animal development. In the early Drosophila embryo, the mesectoderm is a group of glial precursors that separate ectoderm and mesoderm, forming the ventral midline. Mesectoderm cells undergo one round of oriented divisions during axis elongation and are eventually internalized 6 h later. Using spinning disk confocal microscopy and image analysis, we found that after dividing, mesectoderm cells reversed their planar polarity. The polarity factor Bazooka was redistributed to mesectoderm-mesectoderm cell interfaces, and the molecular motor non-muscle Myosin II and its upstream activator Rho-kinase (Rok) accumulated at mesectoderm-ectoderm (ME) interfaces, forming supracellular cables flanking the mesectoderm on either side of the tissue. Laser ablation revealed the presence of increased tension at ME cables, where Myosin was stabilized, as shown by fluorescence recovery after photobleaching. We used laser nanosurgery to reduce tension at the ME boundary, and we found that Myosin fluorescence decreased rapidly, suggesting a role for tension in ME boundary maintenance. Mathematical modelling predicted that increased tension at the ME boundary was necessary to prevent the premature establishment of contacts between the two ectodermal sheets on opposite sides of the mesectoderm, thus controlling the timing of mesectoderm internalization. We validated the model in vivo: Myosin inhibition disrupted the linearity of the ME boundary and resulted in early internalization of the mesectoderm. Our results suggest that the redistribution of Rok polarizes Myosin and Bazooka within the mesectoderm to establish tissue boundaries, and that ME boundaries control the timely internalization of the mesectoderm as embryos develop.

Coordinated cell movements drive embryonic development and tissue repair, and can also spread disease. Time-lapse microscopy is an integral part in the study of the cell biology of collective cell movements. Advances in imaging techniques enable monitoring dynamic cellular and molecular events in real time within living animals. Here, we demonstrate the use of spinning disk confocal microscopy to investigate coordinated cell movements and epithelial-to-mesenchymal-like transitions during embryonic wound closure in Drosophila. We describe image-based metrics to quantify the efficiency of collective cell migration. Finally, we show the application of super-resolution radial fluctuation microscopy to obtain multidimensional, super-resolution images of protrusive activity in collectively moving cells in vivo. Together, the methods presented here constitute a toolkit for the modern analysis of collective cell migration in living animals.

Coordinated cell movements drive embryonic development and tissue repair, and can also spread disease. Time-lapse microscopy is an integral part in the study of the cell biology of collective cell movements. Advances in imaging techniques enable monitoring dynamic cellular and molecular events in real time within living animals. Here, we demonstrate the use of spinning disk confocal microscopy to investigate coordinated cell movements and epithelial-to-mesenchymal-like transitions during embryonic wound closure in Drosophila. We describe image-based metrics to quantify the efficiency of collective cell migration. Finally, we show the application of super-resolution radial fluctuation microscopy to obtain multidimensional, super-resolution images of protrusive activity in collectively moving cells in vivo. Together, the methods presented here constitute a toolkit for the modern analysis of collective cell migration in living animals.

Embryonic wounds heal rapidly, in a process driven by coordinated cell movements. Polarization of actin and the molecular motor non-muscle myosin II in the cells adjacent to the wound results in the formation of a supracellular cable around the wound that drives repair. In Drosophila embryos, the distribution of actin around wounds is heterogeneous, with regions of high and low actin density, and actin heterogeneity is necessary for rapid repair. Here, we demonstrate that actin and myosin display stochastic patterns around embryonic wounds, and that contractile forces around wounds are heterogeneous. Mathematical modelling suggests that actomyosin heterogeneity favours wound closure if myosin is regulated by tension and strain, a hypothesis that we validate experimentally. We show that inhibition of stretch-activated ion channels disrupts myosin dynamics and tissue repair. Together, our results indicate that staggered contractile events, mechanical signals and force-regulated myosin dynamics coordinate cell behaviours to drive efficient wound closure. The motor proteins and contractile forces involved in wound closure are both shown to be heterogeneously distributed around a wound. Theory suggests that this heterogeneity speeds up wound closure, as long as the proteins are mechanically regulated.

The development of new microscopy techniques for super-resolved, long-term monitoring of cellular and subcellular dynamics in living organisms is revealing new fundamental aspects of tissue development and repair. However, new microscopy approaches present several challenges. In addition to unprecedented requirements for data storage, the analysis of high resolution, time-lapse images is too complex to be done manually. Machine learning techniques are ideally suited for the (semi-)automated analysis of multidimensional image data. In particular, support vector machines (SVMs), have emerged as an efficient method to analyze microscopy images obtained from animals. Here, we discuss the use of SVMs to analyze in vivo microscopy data. We introduce the mathematical framework behind SVMs, and we describe the metrics used by SVMs and other machine learning approaches to classify image data. We discuss the influence of different SVM parameters in the context of an algorithm for cell segmentation and tracking. Finally, we describe how the application of SVMs has been critical to study protein localization in yeast screens, for lineage tracing in C. elegans, or to determine the developmental stage of Drosophila embryos to investigate gene expression dynamics. We propose that SVMs will become central tools in the analysis of the complex image data that novel microscopy modalities have made possible. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

Epithelial tissues form and repair in complex processes influenced by molecular and physical factors. Recent years have witnessed the development of new microscopy modalities that push the limits of spatial resolution, and enable long-term monitoring of developing animals. Increasingly, methods from the physical sciences are used to investigate the role of mechanical forces in living organisms. The application of these new technologies to developmental biology has led to ever-expanding volumes of data that must be interpreted and integrated. For these reasons, computer models are being applied to investigate tissue morphogenesis. Here, we discuss the use of vertex models to study the morphogenesis of epithelial tissues. We motivate the use of computational models and consider their advantages and limitations. We provide an introduction to the theoretical foundation of vertex models and describe how they can integrate mechanical and biochemical dynamics. Finally, we review recent advances in the application of vertex models to investigate dorsal closure, a morphogenetic process in the Drosophila embryo with parallels to both embryonic development and wound repair in vertebrate organisms.

Cell–extracellular matrix (ECM) and cell–cell adhesion are interdependent during dorsal closure in the fly. Cell–ECM adhesion is required for normal myosin dynamics and organization of both cell–cell adhesions and actin networks during dorsal closure. Loss of cell–cell adhesion affects cell–ECM adhesion and tissue biomechanics., Tissue morphogenesis relies on the coordinated action of actin networks, cell–cell adhesions, and cell–extracellular matrix (ECM) adhesions. Such coordination can be achieved through cross-talk between cell–cell and cell–ECM adhesions. Drosophila dorsal closure (DC), a morphogenetic process in which an extraembryonic tissue called the amnioserosa contracts and ingresses to close a discontinuity in the dorsal epidermis of the embryo, requires both cell–cell and cell–ECM adhesions. However, whether the functions of these two types of adhesions are coordinated during DC is not known. Here we analyzed possible interdependence between cell–cell and cell–ECM adhesions during DC and its effect on the actomyosin network. We find that loss of cell–ECM adhesion results in aberrant distributions of cadherin-mediated adhesions and actin networks in the amnioserosa and subsequent disruption of myosin recruitment and dynamics. Moreover, loss of cell–cell adhesion caused up-regulation of cell–ECM adhesion, leading to reduced cell deformation and force transmission across amnioserosa cells. Our results show how interdependence between cell–cell and cell–ECM adhesions is important in regulating cell behaviors, force generation, and force transmission critical for tissue morphogenesis.

Drosophila neural stem cells, or neuroblasts, ingress from the neuroepithelium in an EMT-like process, during which the apical cell domain is lost. Apical constriction of neuroblasts and the serial loss of cell–cell contacts require periodic pulses of actomyosin that cause progressively stronger ratcheted contractions of the neuroblast apical cortex., Epithelial–mesenchymal transitions play key roles in development and cancer and entail the loss of epithelial polarity and cell adhesion. In this study, we use quantitative live imaging of ingressing neuroblasts (NBs) in Drosophila melanogaster embryos to assess apical domain loss and junctional disassembly. Ingression is independent of the Snail family of transcriptional repressors and down-regulation of Drosophila E-cadherin (DEcad) transcription. Instead, the posttranscriptionally regulated decrease in DEcad coincides with the reduction of cell contact length and depends on tension anisotropy between NBs and their neighbors. A major driver of apical constriction and junctional disassembly are periodic pulses of junctional and medial myosin II that result in progressively stronger cortical contractions during ingression. Effective contractions require the molecular coupling between myosin and junctions and apical relaxation of neighboring cells. Moreover, planar polarization of myosin leads to the loss of anterior–posterior junctions before the loss of dorsal–ventral junctions. We conclude that planar-polarized dynamic actomyosin networks drive apical constriction and the anisotropic loss of cell contacts during NB ingression.

α-catenin is a key protein of adherens junctions (AJs) with mechanosensory properties. It also acts as a tumor suppressor that limits tissue growth. Here we analyzed the function of Drosophila α-Catenin (α-Cat) in growth regulation of the wing epithelium. We found that different α-Cat levels led to a differential activation of Hippo/Yorkie or JNK signaling causing tissue overgrowth or degeneration, respectively. α-Cat can modulate Yorkie-dependent tissue growth through recruitment of Ajuba, a negative regulator of Hippo signaling to AJs but also through a mechanism independent of Ajuba recruitment to AJs. Both mechanosensory regions of α-Cat, the M region and the actin-binding domain (ABD), contribute to growth regulation. Whereas M is dispensable for α-Cat function in the wing, individual M domains (M1, M2, M3) have opposing effects on growth regulation. In particular, M1 limits Ajuba recruitment. Loss of M1 causes Ajuba hyper-recruitment to AJs, promoting tissue-tension independent overgrowth. Although M1 binds Vinculin, Vinculin is not responsible for this effect. Moreover, disruption of mechanosensing of the α-Cat ABD affects tissue growth, with enhanced actin interactions stabilizing junctions and leading to tissue overgrowth. Together, our findings indicate that α-Cat acts through multiple mechanisms to control tissue growth, including regulation of AJ stability, mechanosensitive Ajuba recruitment, and dynamic direct F-actin interactions., Author summary We explore the regulation of tissue and organ size which is an important consideration in normal development and health. During development, tissues reach specific sizes in proportion to the rest of the body. Uncontrolled growth can lead to malformations or promote tumor growth. Recent findings have emphasized an important role for mechanical cues in the regulation of tissue growth. Mechanical signals can, for example, arise from cytoskeletal contraction that increases tension, or from compression due to proliferation and a resulting increase in cell density that would lower tension. Mechanosensory molecules that are sensitive to changes in tissue tension can convert mechanical cues into biochemical signals that enhance or slow proliferation or cell death to adjust overall tissue size. One such mechanosensory molecule is α-Catenin which is a key component of cell adhesion structures that physically link cells together and couples these structures to the cytoskeleton within cells. We clarify several molecular parameters of how α-Catenin regulates signalling pathways that control cell proliferation and cell death.

How position-dependent cell fate acquisition occurs during embryogenesis is a central question in developmental biology. To study this process, we developed a defined, high-throughput assay to induce peri-gastrulation-associated patterning in geometrically confined human pluripotent stem cell (hPSC) colonies. We observed that, upon BMP4 treatment, phosphorylated SMAD1 (pSMAD1) activity in the colonies organized into a radial gradient. We developed a reaction-diffusion (RD)-based computational model and observed that the self-organization of pSMAD1 signaling was consistent with the RD principle. Consequent fate acquisition occurred as a function of both pSMAD1 signaling strength and duration of induction, consistent with the positional-information (PI) paradigm. We propose that the self-organized peri-gastrulation-like fate patterning in BMP4-treated geometrically confined hPSC colonies arises via a stepwise model of RD followed by PI. This two-step model predicted experimental responses to perturbations of key parameters such as colony size and BMP4 dose. Furthermore, it also predicted experimental conditions that resulted in RD-like periodic patterning in large hPSC colonies, and rescued peri-gastrulation-like patterning in colony sizes previously thought to be reticent to this behavior., Summary: A high-throughput in vitro system allowing the induction of peri-gastrulation-like fates in geometrically confined hPSC colonies reveals that a two-step process underlies the observed self-organization and subsequent fate acquisition.

Tissue morphogenesis requires force-generating mechanisms to organize cells into complex structures. Although many such mechanisms have been characterized, we know little about how forces are integrated across developing tissues. We provide evidence that integrin-mediated cell-extracellular matrix (ECM) adhesion modulates the transmission of apically generated tension during dorsal closure (DC) in Drosophila. Integrin-containing adhesive structures resembling focal adhesions were identified on the basal surface of the amnioserosa (AS), an extraembryonic epithelium essential for DC. Genetic modulation of integrin-mediated adhesion results in defective DC. Quantitative image analysis and laser ablation experiments reveal that basal cell-ECM adhesions provide resistance to apical cell displacements and force transmission between neighboring cells in the AS. Finally, we provide evidence for integrin-dependent force transmission to the AS substrate. Overall, we find that integrins regulate force transmission within and between cells, thereby playing an essential role in transmitting tension in developing tissues.

α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis., Cell-cell adhesion mediated by catenin-cadherin complexes plays a critical role in translating the mechanical forces into physiological responses. Here the authors define a mechanism of force-dependent cadherin-actin linkage dynamically regulated through the actin-binding domain of α-catenin.

Summary Embryos have a striking ability to heal wounds rapidly and without scarring. Embryonic wound repair is a conserved process, driven by polarization of cell-cell junctions and the actomyosin cytoskeleton in the cells around the wound. However, the upstream signals that trigger cell polarization around wounds are unknown. We used quantitative in vivo microscopy in Drosophila and zebrafish embryos to identify reactive oxygen species (ROS) as a critical signal that orchestrates cell polarity around wounds. ROS promote trafficking of adherens junctions and accumulation of actin and myosin at the wound edge and are necessary for wound closure. We show that, in Drosophila, ROS drive wound healing in part through an ortholog of Src kinase, Src42A, which we identify as a redox sensor that promotes polarization of junctions and the cytoskeleton around wounds. We propose that ROS are a reparative signal that drives rapid embryonic wound healing in vertebrate and invertebrate species.

Clathrin, dynamin, and ARF6 accumulate around wounds in Drosophila embryos in a calcium- and actomyosin-dependent manner and drive polarized E-cadherin endocytosis, which is necessary for actomyosin remodeling during wound repair., Embryonic epithelia have a remarkable ability to rapidly repair wounds. A supracellular actomyosin cable around the wound coordinates cellular movements and promotes wound closure. Actomyosin cable formation is accompanied by junctional rearrangements at the wound margin. We used in vivo time-lapse quantitative microscopy to show that clathrin, dynamin, and the ADP-ribosylation factor 6, three components of the endocytic machinery, accumulate around wounds in Drosophila melanogaster embryos in a process that requires calcium signaling and actomyosin contractility. Blocking endocytosis with pharmacological or genetic approaches disrupted wound repair. The defect in wound closure was accompanied by impaired removal of E-cadherin from the wound edge and defective actomyosin cable assembly. E-cadherin overexpression also resulted in reduced actin accumulation around wounds and slower wound closure. Reducing E-cadherin levels in embryos in which endocytosis was blocked rescued actin localization to the wound margin. Our results demonstrate a central role for endocytosis in wound healing and indicate that polarized E-cadherin endocytosis is necessary for actomyosin remodeling during embryonic wound repair.

During development and in adult tissues separation of phenotypically distinct cell populations is necessary to ensure proper organization and function of tissues and organs. Various phenomena, such as differential adhesion, differential mechanical tension and cell-cell repulsion, are proposed to cause boundary formation. Moreover, emerging evidence suggests that interplay between multiple such phenomena can underlie boundary formation. Boundary-forming mechanisms are commonly studied in vivo in complex embryo models or in vitro using simple model systems not reflective of in vivo boundary complexity. To better elucidate the interplay between multiple boundary formation mechanism, there is therefore a need for more relevant in vitro model systems that allow quantitative and concomitant studies of the multiple changes in cell/tissue behaviour that lead to boundary establishment. Here, we develop such a model using patterned co-cultures of two cell populations. Using a set of quantitative tools, we demonstrate that our approach allows us to study the mechanisms underlying boundary formation. We demonstrate that in our specific system differential mechanical tension and modulation of migratory behavior of cells accompany boundary formation. The design of our in vitro model system will allow researchers to obtain quantitative, integrative mechanistic data facilitating a faster and more thorough understanding of the fundamental principles underlying boundary formation.

The fruit fly Drosophila melanogaster has recently emerged as an excellent system to investigate the genetics of cardiovascular development and disease. Drosophila provides an inexpensive and genetically-tractable in vivo system with a large number of conserved features. In addition, the Drosophila embryo is transparent, and thus amenable to time-lapse fluorescence microscopy, as well as biophysical and pharmacological manipulations. One of the conserved aspects of heart development from Drosophila to humans is the initial assembly of a tube. Here, we review the cellular behaviours and molecular dynamics important for the initial steps of heart morphogenesis in Drosophila, with particular emphasis on the cell-cell adhesion and cytoskeletal networks that cardiac precursors use to move, coordinate their migration, interact with other tissues and eventually sculpt a beating heart.

Collective cell movements drive embryonic development and tissue repair, and can cause disease. However, the mechanisms that coordinate the migration of groups of cells in vivo are unclear. Cells generate, transmit and sense mechanical forces to align their movements. Therefore, the machinery used by cells to generate force (cytoskeleton) and to transmit and sense mechanical signals (cell-cell adhesion) is critical for collective movement. Here, we review the components and organization of the cytoskeletal and cell-cell adhesive machineries, and how they are organized to promote collective cell movements in living animals. We discuss the signals that orchestrate molecular rearrangements necessary for coordinated cell motility, and we provide specific examples of movements both in the plane of the tissue (wound healing) and perpendicular to that plane (apical constriction).

The embryonic epidermis displays a remarkable ability to repair wounds rapidly. Embryonic wound repair is driven by the evolutionary conserved redistribution of cytoskeletal and junctional proteins around the wound. Drosophila has emerged as a model to screen for factors implicated in wound closure. However, genetic screens have been limited by the use of manual analysis methods. We introduce MEDUSA, a novel image-analysis tool for the automated quantification of multicellular and molecular dynamics from time-lapse confocal microscopy data. We validate MEDUSA by quantifying wound closure in Drosophila embryos, and we show that the results of our automated analysis are comparable to analysis by manual delineation and tracking of the wounds, while significantly reducing the processing time. We demonstrate that MEDUSA can also be applied to the investigation of cellular behaviors in three and four dimensions. Using MEDUSA, we find that the conserved nonreceptor tyrosine kinase Abelson (Abl) contributes to rapid embryonic wound closure. We demonstrate that Abl plays a role in the organization of filamentous actin and the redistribution of the junctional protein β-catenin at the wound margin during embryonic wound repair. Finally, we discuss different models for the role of Abl in the regulation of actin architecture and adhesion dynamics at the wound margin.

Cells are thought to divide along their longest axis. Now, two studies in Developmental Cell (Scarpa et al., 2018) and EMBO Journal (Finegan et al., 2018) reveal that in many instances, cell division orientation in vivo is not determined by cell shape, but rather by local anisotropies in cell mechanics.

In early Drosophila embryos, wound closure is driven by the apical constriction of the wounded cells, which assemble actomyosin networks that lose actin and myosin as they contract. In late embryos, wound closure is mediated by the cells around the wound, which assemble an actomyosin purse string that contracts with constant actomyosin levels., Epithelial tissues are protective barriers that display a remarkable ability to repair wounds. Wound repair is often associated with an accumulation of actin and nonmuscle myosin II around the wound, forming a purse string. The role of actomyosin networks in generating mechanical force during wound repair is not well understood. Here we investigate the mechanisms of force generation during wound repair in the epidermis of early and late Drosophila embryos. We find that wound closure is faster in early embryos, where, in addition to a purse string around the wound, actomyosin networks at the medial cortex of the wounded cells contribute to rapid wound repair. Laser ablation demonstrates that both medial and purse-string actomyosin networks generate contractile force. Quantitative analysis of protein localization dynamics during wound closure indicates that the rapid contraction of medial actomyosin structures during wound repair in early embryos involves disassembly of the actomyosin network. By contrast, actomyosin purse strings in late embryos contract more slowly in a mechanism that involves network condensation. We propose that the combined action of two force-generating structures—a medial actomyosin network and an actomyosin purse string—contributes to the increased efficiency of wound repair in the early embryo.

SummaryThe bending and internalization of tissues during embryonic development is a conserved process driven by dramatic cell-shape changes. A recent study details the molecules required for mesoderm internalization in Drosophila and their unique spatial localization pattern.