Regulation of intercellular viscosity by E-cadherin-dependent phosphorylation of EGFR in collective cell migration.

Collective cell migration is crucial in various physiological processes, including wound healing, morphogenesis, and cancer metastasis. Adherens Junctions (AJs) play a pivotal role in regulating cell cohesion and migration dynamics during tissue remodeling. While the role and origin of the junctional mechanical tension at AJs have been extensively studied, the influence of the actin cortex structure and dynamics on junction plasticity remains incompletely understood. Moreover, the mechanisms underlying stress dissipation at junctions are not well elucidated. Here, we found that the ligand-independent phosphorylation of epithelial growth factor receptor (EGFR) downstream of de novo E-cadherin adhesion orchestrates a feedback loop, governing intercellular viscosity via the Rac pathway regulating actin dynamics. Our findings highlight how the E-cadherin-dependent EGFR activity controls the migration mode of collective cell movements independently of intercellular tension. This modulation of effective viscosity coordinates cellular movements within the expanding monolayer, inducing a transition from swirling to laminar flow patterns while maintaining a constant migration front speed. Additionally, we propose a vertex model with adjustable junctional viscosity, capable of replicating all observed cellular flow phenotypes experimentally.

A microfluidic platform to investigate the role of mechanical constraints on tissue reorganization.

Mechanical constraints have a high impact on development processes, and there is a need for new tools to investigate the role of mechanosensitive pathways in tissue reorganization during development. We present here experiments in which embryonic cell aggregates are aspired through constrictions in microfluidic channels, generating highly heterogeneous flows and large cell deformations that can be imaged using two-photon microscopy. This approach provides a way to measure in situ local viscoelastic properties of 3D tissues and connect them to intracellular and intercellular events, such as cell shape changes and cell rearrangements. These methods could be applied to organoids to investigate and quantify rheological properties of tissues, and to understand how constraints affect development.

Aster repulsion drives short-ranged ordering in the Drosophila syncytial blastoderm.

Biological systems are highly complex, yet notably ordered structures can emerge. During syncytial stage development of the Drosophila melanogaster embryo, nuclei synchronously divide for nine cycles within a single cell, after which most of the nuclei reach the cell cortex. The arrival of nuclei at the cortex occurs with remarkable positional order, which is important for subsequent cellularisation and morphological transformations. Yet, the mechanical principles underlying this lattice-like positional order of nuclei remain untested. Here, using quantification of nuclei position and division orientation together with embryo explants, we show that short-ranged repulsive interactions between microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. Such ordered nuclear positioning still occurs with the loss of actin caps and even the loss of the nuclei themselves; the asters can self-organise with similar distribution to nuclei in the wild-type embryo. The explant assay enabled us to deduce the nature of the mechanical interaction between pairs of nuclei. We used this to predict how the nuclear division axis orientation changes upon nucleus removal from the embryo cortex, which we confirmed in vivo with laser ablation. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters are important for ordering in the early Drosophila embryo and minimising positional irregularity.

Activation of butterfly eyespots by Distal-less is consistent with a reaction-diffusion process.

Eyespots on the wings of nymphalid butterflies represent colorful examples of pattern formation, yet the developmental origins and mechanisms underlying eyespot center differentiation are still poorly understood. Using CRISPR-Cas9 we re-examine the function of Distal-less (Dll) as an activator or repressor of eyespots, a topic that remains controversial. We show that the phenotypic outcome of CRISPR mutations depends upon which specific exon is targeted. In Bicyclus anynana, exon 2 mutations are associated with both missing and ectopic eyespots, and also exon skipping. Exon 3 mutations, which do not lead to exon skipping, produce only null phenotypes, including missing eyespots, lighter wing coloration and loss of scales. Reaction-diffusion modeling of Dll function, using Wnt and Dpp as candidate morphogens, accurately replicates these complex crispant phenotypes. These results provide new insight into the function of Dll as a potential activator of eyespot development, scale growth and melanization, and suggest that the tuning of Dll expression levels can generate a diversity of eyespot phenotypes, including their appearance on the wing.This article has an associated 'The people behind the papers' interview.

Roadmap for the multiscale coupling of biochemical and mechanical signals during development.

The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.

Amoeboid Swimming Is Propelled by Molecular Paddling in Lymphocytes.

Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like crawling of fibroblasts, relies on maturation of adhesion complexes and actin fiber traction, whereas the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on two-dimensional and in three-dimensional solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming. We show here experimental and computational evidence that leukocytes do swim, and that efficient propulsion is not fueled by waves of cell deformation but by a rearward and inhomogeneous treadmilling of the cell external membrane. Our model consists of a molecular paddling by transmembrane proteins linked to and advected by the actin cortex, whereas freely diffusing transmembrane proteins hinder swimming. Furthermore, continuous paddling is enabled by a combination of external treadmilling and selective recycling by internal vesicular transport of cortex-bound transmembrane proteins. This mechanism explains observations that swimming is five times slower than the retrograde flow of cortex and also that lymphocytes are motile in nonadherent confined environments. Resultantly, the ubiquitous ability of mammalian amoeboid cells to migrate in two dimensions or three dimensions and with or without adhesion can be explained for lymphocytes by a single machinery of heterogeneous membrane treadmilling.

Pcdh18a regulates endocytosis of E-cadherin during axial mesoderm development in zebrafish.

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not fully understood how these processes act together in a coordinated way during notochord formation. The prechordal plate is an actively migrating cell population in the central mesoderm anterior to the trailing notochordal plate cells. We show that prechordal plate cells express Protocadherin 18a (Pcdh18a), a member of the cadherin superfamily. We find that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms prechordal plate cells into a cohesive and fast migrating cell group. In turn, the prechordal plate cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation and extension of the posterior, axial cells. Indeed, a grafting experiment validated the prediction and local Pcdh18a expression induced an ectopic prechordal plate-like cell group migrating towards the animal pole. Our findings indicate that the Pcdh18a is important for prechordal plate formation, which influences the trailing mesodermal cell sheet by orchestrating the morphogenesis of the notochord.

Influence of proliferation on the motions of epithelial monolayers invading adherent strips.

Biological systems integrate dynamics at many scales, from molecules, protein complexes and genes, to cells, tissues and organisms. At every step of the way, mechanics, biochemistry and genetics offer complementary approaches to understand these dynamics. At the tissue scale, in vitro monolayers of epithelial cells provide a model to capture the influence of various factors on the motions of the tissue, in order to understand in vivo processes from morphogenesis, cancer progression and tissue remodelling. Ongoing efforts include research aimed at deciphering the roles of the cytoskeleton, of cell-substrate and cell-cell adhesions, and of cell proliferation-the point we investigate here. We show that confined to adherent strips, and on the time scale of a day or two, monolayers move with a characteristic front speed independent of proliferation, but that the motion is accompanied by persistent velocity waves, only in the absence of cell divisions. Here we show that the long-range transmission of physical signals is strongly coupled to cell density and proliferation. We interpret our results from a kinematic and mechanical perspective. Our study provides a framework to understand density-driven mechanisms of collective cell migration.

Colloquium: Mechanical formalisms for tissue dynamics.

The understanding of morphogenesis in living organisms has been renewed by tremendous progress in experimental techniques that provide access to cell scale, quantitative information both on the shapes of cells within tissues and on the genes being expressed. This information suggests that our understanding of the respective contributions of gene expression and mechanics, and of their crucial entanglement, will soon leap forward. Biomechanics increasingly benefits from models, which assist the design and interpretation of experiments, point out the main ingredients and assumptions, and ultimately lead to predictions. The newly accessible local information thus calls for a reflection on how to select suitable classes of mechanical models. We review both mechanical ingredients suggested by the current knowledge of tissue behaviour, and modelling methods that can help generate a rheological diagram or a constitutive equation. We distinguish cell scale ("intra-cell") and tissue scale ("inter-cell") contributions. We recall the mathematical framework developed for continuum materials and explain how to transform a constitutive equation into a set of partial differential equations amenable to numerical resolution. We show that when plastic behaviour is relevant, the dissipation function formalism appears appropriate to generate constitutive equations; its variational nature facilitates numerical implementation, and we discuss adaptations needed in the case of large deformations. The present article gathers theoretical methods that can readily enhance the significance of the data to be extracted from recent or future high throughput biomechanical experiments.

The positioning mechanics of microtubule asters in Drosophila embryo explants.

Microtubule asters are essential in localizing the action of microtubules in processes including mitosis and organelle positioning. In large cells, such as the one-cell sea urchin embryo, aster dynamics are dominated by hydrodynamic pulling forces. However, in systems with more densely positioned nuclei such as the early Drosophila embryo, which packs around 6000 nuclei within the syncytium in a crystalline-like order, it is unclear what processes dominate aster dynamics. Here, we take advantage of a cell cycle regulation Drosophila mutant to generate embryos with multiple asters, independent from nuclei. We use an ex vivo assay to further simplify this biological system to explore the forces generated by and between asters. Through live imaging, drug and optical perturbations, and theoretical modeling, we demonstrate that these asters likely generate an effective pushing force over short distances.

Multicellular aggregates: a model system for tissue rheology.

Morphogenetic processes involve cell flows. The mechanical response of a tissue to active forces is linked to its effective viscosity. In order to decouple this mechanical response from the complex genetic changes occurring in a developing organism, we perform rheometry experiments on multicellular aggregates, which are good models for tissues. We observe a cell softening behavior when submitting to stresses. As our technique is very sensitive, we were able to get access to the measurement of a yield point above which a creep regime is observed obtained for strains above 12%. To explain our rheological curves we propose a model for the cytoskeleton that we represent as a dynamic network of parallel springs, which will break under stress and reattach at null strain. Such a simple model is able to reproduce most of the important behavior of cells under strain. We highlight here the importance of considering cells as complex fluids whose properties will vary with time according to the history of applied stress.

Learning the mechanobiology of development from gastruloids.

Gastruloids acquire their organization and shape through cell biochemical and mechanical activities. Such activities determine the physical forces and changes in material properties that transform simple spherical aggregates into organized tissues. In this Perspective, we discuss why the concepts and approaches of mechanobiology, a discipline that focuses on cell and tissue mechanics and its contribution to the organization and functions of living systems, are essential to the gastruloid field and, in turn, what gastruloids may teach us about mechanobiology.

Condensation of the Drosophila nerve cord is oscillatory and depends on coordinated mechanical interactions.

During development, organs reach precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is the condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. Here, we characterize the condensation of the embryonic ventral nerve cord (VNC) at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process but instead occurs through oscillatory contractions. The VNC mechanical properties spatially and temporally vary, and forces along its longitudinal axis are spatially heterogeneous. We demonstrate that the process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. These outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays and effective frictional interactions. In summary, we have defined the progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size.

Cell-state transitions and collective cell movement generate an endoderm-like region in gastruloids.

Shaping the animal body plan is a complex process that involves the spatial organization and patterning of the different germ layers. Recent advances in live imaging have started to unravel the cellular choreography underlying this process in mammals, however, the sequence of events transforming an unpatterned cell ensemble into structured territories is largely unknown. Here, using gastruloids -3D aggregates of mouse embryonic stem cells- we study the formation of one of the three germ layers, the endoderm. We show that the endoderm is generated from an epiblast-like homogeneous state by a three-step mechanism: (i) a loss of E-cadherin mediated contacts in parts of the aggregate leading to the appearance of islands of E-cadherin expressing cells surrounded by cells devoid of E-cadherin, (ii) a separation of these two populations with islands of E-cadherin expressing cells flowing toward the aggregate tip, and (iii) their differentiation into an endoderm population. During the flow, the islands of E-cadherin expressing cells are surrounded by cells expressing T-Brachyury, reminiscent of the process occurring at the primitive streak. Consistent with recent in vivo observations, the endoderm formation in the gastruloids does not require an epithelial-to-mesenchymal transition, but rather a maintenance of an epithelial state for a subset of cells coupled with fragmentation of E-cadherin contacts in the vicinity, and a sorting process. Our data emphasize the role of signaling and tissue flows in the establishment of the body plan.

Collective cell migration without proliferation: density determines cell velocity and wave velocity.

Collective cell migration contributes to embryogenesis, wound healing and tumour metastasis. Cell monolayer migration experiments help in understanding what determines the movement of cells far from the leading edge. Inhibiting cell proliferation limits cell density increase and prevents jamming; we observe long-duration migration and quantify space-time characteristics of the velocity profile over large length scales and time scales. Velocity waves propagate backwards and their frequency depends only on cell density at the moving front. Both cell average velocity and wave velocity increase linearly with the cell effective radius regardless of the distance to the front. Inhibiting lamellipodia decreases cell velocity while waves either disappear or have a lower frequency. Our model combines conservation laws, monolayer mechanical properties and a phenomenological coupling between strain and polarity: advancing cells pull on their followers, which then become polarized. With reasonable values of parameters, this model agrees with several of our experimental observations. Together, our experiments and model disantangle the respective contributions of active velocity and of proliferation in monolayer migration, explain how cells maintain their polarity far from the moving front, and highlight the importance of strain-polarity coupling and density in long-range information propagation.

Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway.

During animal development, several planar cell polarity (PCP) pathways control tissue shape by coordinating collective cell behavior. Here, we characterize by means of multiscale imaging epithelium morphogenesis in the Drosophila dorsal thorax and show how the Fat/Dachsous/Four-jointed PCP pathway controls morphogenesis. We found that the proto-cadherin Dachsous is polarized within a domain of its tissue-wide expression gradient. Furthermore, Dachsous polarizes the myosin Dachs, which in turn promotes anisotropy of junction tension. By combining physical modeling with quantitative image analyses, we determined that this tension anisotropy defines the pattern of local tissue contraction that contributes to shaping the epithelium mainly via oriented cell rearrangements. Our results establish how tissue planar polarization coordinates the local changes of cell mechanical properties to control tissue morphogenesis.