Integrin-based adhesions promote cell-cell junction and cytoskeletal remodelling to drive embryonic wound healing.

Embryos repair wounds rapidly, with no inflammation or scarring. Embryonic wound healing is driven by the collective movement of the cells around the lesion. 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 wound edge and localize to former tricellular junctions at the wound margin, in a process necessary for cytoskeletal polarity. We found that the cells adjacent to wounds in the Drosophila embryonic epidermis polarized Talin, a core component of cell-extracellular matrix (ECM) adhesions, which preferentially accumulated at the wound edge. Integrin knockdown and inhibition of integrin binding delayed wound closure and reduced actin polarization and dynamics 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. 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.

Should I shrink or should I grow: cell size changes in tissue morphogenesis.

Cells change shape, move, divide, and die to sculpt tissues. Common to all these cell behaviours are cell size changes, which have recently emerged as key contributors to tissue morphogenesis. Cells can change their mass-the number of macromolecules they contain-or their volume-the space they encompass. Changes in cell mass and volume occur through different molecular mechanisms and at different timescales, slow for changes in mass and rapid for changes in volume. Therefore, changes in cell mass and cell volume, which are often linked, contribute to the development and shaping of tissues in different ways. Here, we review the molecular mechanisms by which cells can control and alter their size, and we discuss how changes in cell mass and volume contribute to tissue morphogenesis. The role that cell size control plays in developing embryos is only starting to be elucidated. Research on the signals that control cell size will illuminate our understanding of the cellular and molecular mechanisms that drive tissue morphogenesis.

PyJAMAS: open-source, multimodal segmentation and analysis of microscopy images.

SUMMARY: Our increasing ability to resolve fine details using light microscopy is matched by an increasing need to quantify images in order to detect and measure phenotypes. Despite their central role in cell biology, many image analysis tools require a financial investment, are released as proprietary software, or are implemented in languages not friendly for beginners, and thus are used as black boxes. To overcome these limitations, we have developed PyJAMAS, an open-source tool for image processing and analysis written in Python. PyJAMAS provides a variety of segmentation tools, including watershed and machine learning-based methods; takes advantage of Jupyter notebooks for the display and reproducibility of data analyses; and can be used through a cross-platform graphical user interface or as part of Python scripts via a comprehensive application programming interface. AVAILABILITY AND IMPLEMENTATION: PyJAMAS is open-source and available at https://bitbucket.org/rfg_lab/pyjamas. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Role of α-Catenin and its mechanosensing properties in regulating Hippo/YAP-dependent tissue growth.

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.

DDR1 (Discoidin Domain Receptor-1)-RhoA (Ras Homolog Family Member A) Axis Senses Matrix Stiffness to Promote Vascular Calcification.

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.

A stepwise model of reaction-diffusion and positional information governs self-organized human peri-gastrulation-like patterning.

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.

Automated cell tracking identifies mechanically oriented cell divisions during Drosophila axis elongation.

Embryos extend their anterior-posterior (AP) axis in a conserved process known as axis elongation. Drosophila axis elongation occurs in an epithelial monolayer, the germband, and is driven by cell intercalation, cell shape changes, and oriented cell divisions at the posterior germband. Anterior germband cells also divide during axis elongation. We developed image analysis and pattern-recognition methods to track dividing cells from confocal microscopy movies in a generally applicable approach. Mesectoderm cells, forming the ventral midline, divided parallel to the AP axis, while lateral cells displayed a uniform distribution of division orientations. Mesectoderm cells did not intercalate and sustained increased AP strain before cell division. After division, mesectoderm cell density increased along the AP axis, thus relieving strain. We used laser ablation to isolate mesectoderm cells from the influence of other tissues. Uncoupling the mesectoderm from intercalating cells did not affect cell division orientation. Conversely, separating the mesectoderm from the anterior and posterior poles of the embryo resulted in uniformly oriented divisions. Our data suggest that mesectoderm cells align their division angle to reduce strain caused by mechanical forces along the AP axis of the embryo.

Quantitative modelling of epithelial morphogenesis: integrating cell mechanics and molecular dynamics.

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.

Tension regulates myosin dynamics during Drosophila embryonic wound repair.

Embryos repair epithelial wounds rapidly in a process driven by collective cell movements. Upon wounding, actin and the molecular motor non-muscle myosin II are redistributed in the cells adjacent to the wound, forming a supracellular purse string around the lesion. Purse string contraction coordinates cell movements and drives rapid wound closure. By using fluorescence recovery after photobleaching in Drosophila embryos, we found that myosin turns over as the purse string contracts. Myosin turnover at the purse string was slower than in other actomyosin networks that had a lower level of contractility. Mathematical modelling suggested that myosin assembly and disassembly rates were both reduced by tension at the wound edge. We used laser ablation to show that tension at the purse string increased as wound closure progressed, and that the increase in tension was associated with reduced myosin turnover. Reducing purse string tension by laser-mediated severing resulted in increased turnover and loss of myosin. Finally, myosin motor activity was necessary for its stabilization around the wound and for rapid wound closure. Our results indicate that mechanical forces regulate myosin dynamics during embryonic wound repair.

Modeling cell intercalation during Drosophila germband extension.

Germband extension during Drosophila development is primarily driven by cell intercalation, which involves three key components: planar cell polarity, anisotropic myosin contractile forces on cellular junctions, and cellular deformation and movement. Prior experimental work probed each of these factors in depth, but the connection between them remains unclear. This paper presents an integrated chemomechanical model that combines the three factors into a coherent mathematical framework for studying cell intercalation in the germband tissue. The model produces the planar cell polarization of key proteins, including Rho-kinase, Bazooka and myosin, the development of anisotropic contractile forces, and subsequent cell deformation and rearrangement. Cell intercalation occurs through T1 transitions among four neighboring cells and rosettes involving six cells. Such six-cell rosettes entail stronger myosin-based contractile forces, and on average produce a moderately larger amount of germband extension than the T1 transitions. The resolution of T1 and rosettes is driven by contractile forces on junctions anterior and posterior to the assembly as well as the pulling force of the medial myosin in the anterior and posterior cells. The global stretching due to posterior midgut invagination also plays a minor role. These model predictions are in reasonable agreement with experimental observations.

Shape of my heart: Cell-cell adhesion and cytoskeletal dynamics during Drosophila cardiac morphogenesis.

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.

Automated multidimensional image analysis reveals a role for Abl in embryonic wound repair.

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 ß-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.

(Machine-)Learning to analyze in vivo microscopy: Support vector machines.

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.

A biomechanical model for cell polarization and intercalation during Drosophila germband extension.

Germband extension during Drosophila development features the merging of cells along the dorsal-ventral (DV) axis and their separation along the anterior-posterior (AP) axis. This intercalation process involves planar cell polarity, anisotropic contractile forces along cell edges, and concerted cell deformation and movement. Although prior experiments have probed each of these factors separately, the connection among them remains unclear. This paper presents a chemo-mechanical model that integrates the three factors into a coherent framework. The model predicts the polarization of Rho-kinase, myosin and Bazooka downstream of an anisotropic Shroom distribution. In particular, myosin accumulates on cell edges along the DV axis, causing them to contract into a vertex. Subsequently, medial myosin in the cells anterior and posterior to the vertex helps to elongate it into a new edge parallel to the body axis. Thus, the tissue extends along the AP axis and narrows in the transverse direction through neighbor exchange. Model predictions of the polarity of the proteins and cell and tissue deformation are in good agreement with experimental observations.

Cortical tension promotes Kibra degradation via Par-1.

The Hippo pathway is an evolutionarily conserved regulator of tissue growth. Multiple Hippo signaling components are regulated via proteolytic degradation. However, how these degradation mechanisms are themselves modulated remains unexplored. Kibra is a key upstream pathway activator that promotes its own ubiquitin-mediated degradation upon assembling a Hippo signaling complex. Here, we demonstrate that Hippo complex-dependent Kibra degradation is modulated by cortical tension. Using classical genetic, osmotic, and pharmacological manipulations of myosin activity and cortical tension, we show that increasing cortical tension leads to Kibra degradation, whereas decreasing cortical tension increases Kibra abundance. Our study also implicates Par-1 in regulating Kib abundance downstream of cortical tension. We demonstrate that Par-1 promotes ubiquitin-mediated Kib degradation in a Hippo complex-dependent manner and is required for tension-induced Kib degradation. Collectively, our results reveal a previously unknown molecular mechanism by which cortical tension affects Hippo signaling and provide novel insights into the role of mechanical forces in growth control.

Contractile and expansive actin networks in Drosophila: Developmental cell biology controlled by network polarization and higher-order interactions.

Actin networks are central to shaping and moving cells during animal development. Various spatial cues activate conserved signal transduction pathways to polarize actin network assembly at sub-cellular locations and to elicit specific physical changes. Actomyosin networks contract and Arp2/3 networks expand, and to affect whole cells and tissues they do so within higher-order systems. At the scale of tissues, actomyosin networks of epithelial cells can be coupled via adherens junctions to form supracellular networks. Arp2/3 networks typically integrate with distinct actin assemblies, forming expansive composites which act in conjunction with contractile actomyosin networks for whole-cell effects. This review explores these concepts using examples from Drosophila development. First, we discuss the polarized assembly of supracellular actomyosin cables which constrict and reshape epithelial tissues during embryonic wound healing, germ band extension, and mesoderm invagination, but which also form physical borders between tissue compartments at parasegment boundaries and during dorsal closure. Second, we review how locally induced Arp2/3 networks act in opposition to actomyosin structures during myoblast cell-cell fusion and cortical compartmentalization of the syncytial embryo, and how Arp2/3 and actomyosin networks also cooperate for the single cell migration of hemocytes and the collective migration of border cells. Overall, these examples show how the polarized deployment and higher-order interactions of actin networks organize developmental cell biology.

Rap1 coordinates cell-cell adhesion and cytoskeletal reorganization to drive collective cell migration in vivo.

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 remodeling during collective cell migration in vivo are unclear. We investigated the mechanisms of collective cell migration during epidermal wound healing in Drosophila embryos. Upon wounding, the cells adjacent to the wound internalize cell-cell adhesion molecules and polarize actin and the motor protein non-muscle 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 necessary and sufficient for rapid wound repair. Rap1 promoted myosin 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 remodeling, but not for actomyosin cable assembly. Instead, Rap1 was necessary and sufficient for RhoA/Rho1 activation at the wound edge. 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, promoting actomyosin cable assembly through Ephexin-Rho1, and E-cadherin redistribution through Canoe, thus enabling rapid collective cell migration in vivo.

Myosin waves and a mechanical asymmetry guide the oscillatory migration of Drosophila cardiac progenitors.

Heart development begins with the formation of a tube as cardiac progenitors migrate from opposite sides of the embryo. Abnormal cardiac progenitor movements cause congenital heart defects. However, the mechanisms of cell migration during early heart development remain poorly understood. Using quantitative microscopy, we found that in Drosophila embryos, cardiac progenitors (cardioblasts) migrated through a sequence of forward and backward steps. Cardioblast steps were associated with oscillatory non-muscle myosin II waves that induced periodic shape changes and were necessary for timely heart tube formation. Mathematical modeling predicted that forward cardioblast migration required a stiff boundary at the trailing edge. Consistent with this, we found a supracellular actin cable at the trailing edge of the cardioblasts that limited the amplitude of the backward steps, thus biasing the direction of cell movement. Our results indicate that periodic shape changes coupled with a polarized actin cable produce asymmetrical forces that promote cardioblast migration.