The zonula adherens matura redefines the apical junction of intestinal epithelia.

Cell-cell apical junctions of epithelia consist of multiprotein complexes that organize as belts regulating cell-cell adhesion, permeability, and mechanical tension: the tight junction (zonula occludens), the zonula adherens (ZA), and the macula adherens. The prevailing dogma is that at the ZA, E-cadherin and catenins are lined with F-actin bundles that support and transmit mechanical tension between cells. Using super-resolution microscopy on human intestinal biopsies and Caco-2 cells, we show that two distinct multiprotein belts are basal of the tight junctions as the intestinal epithelia mature. The most apical is populated with nectins/afadin and lined with F-actin; the second is populated with E-cad/catenins. We name this dual-belt architecture the zonula adherens matura. We find that the apical contraction apparatus and the dual-belt organization rely on afadin expression. Our study provides a revised description of epithelial cell-cell junctions and identifies a module regulating the mechanics of epithelia.

Two-point optical manipulation reveals mechanosensitive remodeling of cell-cell contacts in vivo.

Biological tissues acquire reproducible shapes during development through dynamic cell behaviors. Most of these behaviors involve the remodeling of cell-cell contacts. During epithelial morphogenesis, contractile actomyosin networks remodel cell-cell contacts by shrinking and extending junctions between lateral cell surfaces. However, actomyosin networks not only generate mechanical stresses but also respond to them, confounding our understanding of how mechanical stresses remodel cell-cell contacts. Here, we develop a two-point optical manipulation method to impose different stress patterns on cell-cell contacts in the early epithelium of the Drosophila embryo. The technique allows us to produce junction extension and shrinkage through different push and pull manipulations at the edges of junctions. We use these observations to expand classical vertex-based models of tissue mechanics, incorporating negative and positive mechanosensitive feedback depending on the type of remodeling. In particular, we show that Myosin-II activity responds to junction strain rate and facilitates full junction shrinkage. Altogether our work provides insight into how stress produces efficient deformation of cell-cell contacts in vivo and identifies unanticipated mechanosensitive features of their remodeling.

Genetic induction and mechanochemical propagation of a morphogenetic wave.

Tissue morphogenesis arises from coordinated changes in cell shape driven by actomyosin contractions. Patterns of gene expression regionalize cell behaviours by controlling actomyosin contractility. Here we report two modes of control over Rho1 and myosin II (MyoII) activation in the Drosophila endoderm. First, Rho1-MyoII are induced in a spatially restricted primordium via localized transcription of the G-protein-coupled receptor ligand Fog. Second, a tissue-scale wave of Rho1-MyoII activation and cell invagination progresses anteriorly away from the primordium. The wave does not require sustained gene transcription, and is not governed by regulated Fog delivery. Instead, MyoII inhibition blocks Rho1 activation and propagation, revealing a mechanical feedback driven by MyoII. We find that MyoII activation and invagination in each row of cells drives adhesion to the vitelline membrane mediated by integrins, apical spreading, MyoII activation and invagination in the next row. Endoderm morphogenesis thus emerges from local transcriptional initiation and a mechanically driven cycle of cell deformation.

Force generation, transmission, and integration during cell and tissue morphogenesis.

Cell shape changes underlie a large set of biological processes ranging from cell division to cell motility. Stereotyped patterns of cell shape changes also determine tissue remodeling events such as extension or invagination. In vitro and cell culture systems have been essential to understanding the fundamental physical principles of subcellular mechanics. These are now complemented by studies in developing organisms that emphasize how cell and tissue morphogenesis emerge from the interplay between force-generating machines, such as actomyosin networks, and adhesive clusters that transmit tensile forces at the cell cortex and stabilize cell-cell and cell-substrate interfaces. Both force production and transmission are self-organizing phenomena whose adaptive features are essential during tissue morphogenesis. A new era is opening that emphasizes the similarities of and allows comparisons between distant dynamic biological phenomena because they rely on core machineries that control universal features of cytomechanics.

Sculpting with stem cells: how models of embryo development take shape.

During embryogenesis, organisms acquire their shape given boundary conditions that impose geometrical, mechanical and biochemical constraints. A detailed integrative understanding how these morphogenetic information modules pattern and shape the mammalian embryo is still lacking, mostly owing to the inaccessibility of the embryo in vivo for direct observation and manipulation. These impediments are circumvented by the developmental engineering of embryo-like structures (stembryos) from pluripotent stem cells that are easy to access, track, manipulate and scale. Here, we explain how unlocking distinct levels of embryo-like architecture through controlled modulations of the cellular environment enables the identification of minimal sets of mechanical and biochemical inputs necessary to pattern and shape the mammalian embryo. We detail how this can be complemented with precise measurements and manipulations of tissue biochemistry, mechanics and geometry across spatial and temporal scales to provide insights into the mechanochemical feedback loops governing embryo morphogenesis. Finally, we discuss how, even in the absence of active manipulations, stembryos display intrinsic phenotypic variability that can be leveraged to define the constraints that ensure reproducible morphogenesis in vivo.

Wnt ligands regulate the asymmetric divisions of neuronal progenitors in C. elegans embryos.

Wnt/ß-catenin signalling has been implicated in the terminal asymmetric divisions of neuronal progenitors in vertebrates and invertebrates. However, the role of Wnt ligands in this process remains poorly characterized. Here, we used the terminal divisions of the embryonic neuronal progenitors in C. elegans to characterize the role of Wnt ligands during this process, focusing on a lineage that produces the cholinergic interneuron AIY. We observed that, during interphase, the neuronal progenitor is elongated along the anteroposterior axis, then divides along its major axis, generating an anterior and a posterior daughter with different fates. Using time-controlled perturbations, we show that three Wnt ligands, which are transcribed at higher levels at the posterior of the embryo, regulate the orientation of the neuronal progenitor and its asymmetric division. We also identify a role for a Wnt receptor (MOM-5) and a cortical transducer APC (APR-1), which are, respectively, enriched at the posterior and anterior poles of the neuronal progenitor. Our study establishes a role for Wnt ligands in the regulation of the shape and terminal asymmetric divisions of neuronal progenitors, and identifies downstream components.

The elmo-mbc complex and rhogap19d couple Rho family GTPases during mesenchymal-to-epithelial-like transitions.

Many metazoan developmental processes require cells to transition between migratory mesenchymal- and adherent epithelial-like states. These transitions require Rho GTPase-mediated actin rearrangements downstream of integrin and cadherin pathways. A regulatory toolbox of GEF and GAP proteins precisely coordinates Rho protein activities, yet defining the involvement of specific regulators within a cellular context remains a challenge due to overlapping and coupled activities. Here we demonstrate that Drosophila dorsal closure is a powerful model for Rho GTPase regulation during transitions from leading edges to cadherin contacts. During these transitions a Rac GEF elmo-mbc complex regulates both lamellipodia and Rho1-dependent, actomyosin-mediated tension at initial cadherin contacts. Moreover, the Rho GAP Rhogap19d controls Rac and Rho GTPases during the same processes and genetically regulates the elmo-mbc complex. This study presents a fresh framework to understand the inter-relationship between GEF and GAP proteins that tether Rac and Rho cycles during developmental processes.

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.

Polarization-resolved microscopy reveals a muscle myosin motor-independent mechanism of molecular actin ordering during sarcomere maturation.

Sarcomeres are stereotyped force-producing mini-machines of striated muscles. Each sarcomere contains a pseudocrystalline order of bipolar actin and myosin filaments, which are linked by titin filaments. During muscle development, these three filament types need to assemble into long periodic chains of sarcomeres called myofibrils. Initially, myofibrils contain immature sarcomeres, which gradually mature into their pseudocrystalline order. Despite the general importance, our understanding of myofibril assembly and sarcomere maturation in vivo is limited, in large part because determining the molecular order of protein components during muscle development remains challenging. Here, we applied polarization-resolved microscopy to determine the molecular order of actin during myofibrillogenesis in vivo. This method revealed that, concomitantly with mechanical tension buildup in the myotube, molecular actin order increases, preceding the formation of immature sarcomeres. Mechanistically, both muscle and nonmuscle myosin contribute to this actin order gain during early stages of myofibril assembly. Actin order continues to increase while myofibrils and sarcomeres mature. Muscle myosin motor activity is required for the regular and coordinated assembly of long myofibrils but not for the high actin order buildup during sarcomere maturation. This suggests that, in muscle, other actin-binding proteins are sufficient to locally bundle or cross-link actin into highly regular arrays.

Measuring forces and stresses in situ in living tissues.

Development, homeostasis and regeneration of tissues result from a complex combination of genetics and mechanics, and progresses in the former have been quicker than in the latter. Measurements of in situ forces and stresses appear to be increasingly important to delineate the role of mechanics in development. We review here several emerging techniques: contact manipulation, manipulation using light, visual sensors, and non-mechanical observation techniques. We compare their fields of applications, their advantages and limitations, and their validations. These techniques complement measurements of deformations and of mechanical properties. We argue that such approaches could have a significant impact on our understanding of the development of living tissues in the near future.

Direct laser manipulation reveals the mechanics of cell contacts in vivo.

Cell-generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell-cell and cell-ECM contacts to apply local forces on adhesive structures. In epithelia, force imbalance at cell contacts induces cell shape changes, such as apical constriction or polarized junction remodeling, driving tissue morphogenesis. The dynamics of these processes are well-characterized; however, the mechanical basis of cell shape changes is largely unknown because of a lack of mechanical measurements in vivo. We have developed an approach combining optical tweezers with light-sheet microscopy to probe the mechanical properties of epithelial cell junctions in the early Drosophila embryo. We show that optical trapping can efficiently deform cell-cell interfaces and measure tension at cell junctions, which is on the order of 100 pN. We show that tension at cell junctions equilibrates over a few seconds, a short timescale compared with the contractile events that drive morphogenetic movements. We also show that tension increases along cell interfaces during early tissue morphogenesis and becomes anisotropic as cells intercalate during germ-band extension. By performing pull-and-release experiments, we identify time-dependent properties of junctional mechanics consistent with a simple viscoelastic model. Integrating this constitutive law into a tissue-scale model, we predict quantitatively how local deformations propagate throughout the tissue.

Boron Difluoride Curcuminoid Fluorophores with Enhanced Two-Photon Excited Fluorescence Emission and Versatile Living-Cell Imaging Properties.

The synthesis of boron difluoride complexes of a series of curcuminoid derivatives containing various donor end groups is described. Time-dependent (TD)-DFT calculations confirm the charge-transfer character of the second lowest-energy transition band and ascribe the lowest energy band to a "cyanine-like" transition. Photophysical studies reveal that tuning the donor strength of the end groups allows covering a broad spectral range, from the visible to the NIR region, of the UV-visible absorption and fluorescence spectra. Two-photon-excited fluorescence and Z-scan techniques prove that an increase in the donor strength or in the rigidity of the backbone results in a considerable increase in the two-photon cross section, reaching 5000 GM, with predominant two-photon absorption from the S0-S2 charge-transfer transition. Direct comparisons with the hemicurcuminoid derivatives show that the two-photon active band for the curcuminoid derivatives has the same intramolecular charge-transfer character and therefore arises from a dipolar structure. Overall, this structure-relationship study allows the optimization of the two-photon brightness (i.e., 400-900 GM) with one dye that emits in the NIR region of the spectrum. In addition, these dyes demonstrate high intracellular uptake efficiency in Cos7 cells with emission in the visible region, which is further improved by using porous silica nanoparticles as dye vehicles for the imaging of two mammalian carcinoma cells type based on NIR fluorescence emission.

Borondifluoride complexes of hemicurcuminoids as bio-inspired push-pull dyes for bioimaging.

Hemicurcuminoids are based on half of the π-conjugated backbone of curcuminoids. The synthesis of a series of such systems and their borondifluoride complexes is described. The electrochemical and photophysical properties of difluorodioxaborine species were investigated as a function of the nature of electron donor and acceptor groups appended at either terminal positions of the molecular backbone. The emissive character of these dipolar dyes was attributed to an intraligand charge transfer process, leading to fluorescence emission that is strongly dependent on solvent polarity. Quasi-quantitative quenching of fluorescence in high polarity solvents was attributed to photoinduced electron transfer. These dyes were shown to behave as versatile fluorophores. Indeed, they display efficient two-photon excited fluorescence emission leading to high two-photon brightness values. Furthermore, they form nanoparticles in water whose fluorescence emission quantum yield is less than that of the dye in solution, owing to aggregation-induced fluorescence quenching. When cos7 living cells were exposed to these weakly-emitting nanoparticles, one- and two-photon excited fluorescence spectra showed a strong emission within the cytoplasm that originated from the individual molecules. Dye uptake thus involved a disaggregation mechanism at the cell membrane which restored fluorescence emission. This off-on fluorescence switching allows a selective optical monitoring of those molecules that do enter the cell, which offers improved sensitivity and selectivity of detection for bioimaging purposes.

Planar polarized actomyosin contractile flows control epithelial junction remodelling.

Force generation by Myosin-II motors on actin filaments drives cell and tissue morphogenesis. In epithelia, contractile forces are resisted at apical junctions by adhesive forces dependent on E-cadherin, which also transmits tension. During Drosophila embryonic germband extension, tissue elongation is driven by cell intercalation, which requires an irreversible and planar polarized remodelling of epithelial cell junctions. We investigate how cell deformations emerge from the interplay between force generation and cortical force transmission during this remodelling in Drosophila melanogaster. The shrinkage of dorsal-ventral-oriented ('vertical') junctions during this process is known to require planar polarized junctional contractility by Myosin II (refs 4, 5, 7, 12). Here we show that this shrinkage is not produced by junctional Myosin II itself, but by the polarized flow of medial actomyosin pulses towards 'vertical' junctions. This anisotropic flow is oriented by the planar polarized distribution of E-cadherin complexes, in that medial Myosin II flows towards 'vertical' junctions, which have relatively less E-cadherin than transverse junctions. Our evidence suggests that the medial flow pattern reflects equilibrium properties of force transmission and coupling to E-cadherin by α-Catenin. Thus, epithelial morphogenesis is not properly reflected by Myosin II steady state distribution but by polarized contractile actomyosin flows that emerge from interactions between E-cadherin and actomyosin networks.

Calcium signaling in developing embryos: focus on the regulation of cell shape changes and collective movements.

During morphogenesis tissues significantly remodel by coordinated cell migrations and cell rearrangements. Central to this problem are cell shape changes that are driven by distinct cytoskeletal reorganization responsible for force generation. Calcium is a versatile and universal messenger that is implicated in the regulation of embryonic development. Although calcium transients accrue clearly and more intensely in tissues undergoing rearrangement/migration, it is far from clear what the role of these calcium signals is. Here we summarize the evidence implicating calcium participation in tissue movements, cell shape changes and the reorganization of contractile cytoskeletal elements in developing embryos. We also discuss a novel hypothesis that short-lived calcium spikes are required in cells and tissues undergoing migration and rearrangements as a fine tuning response mechanism to prevent local, abnormally high fluctuations in cytoskeletal activities.

A two-tiered mechanism for stabilization and immobilization of E-cadherin.

Epithelial tissues maintain a robust architecture which is important for their barrier function, but they are also remodelled through the reorganization of cell-cell contacts. Tissue stability requires intercellular adhesion mediated by E-cadherin, in particular its trans-association in homophilic complexes supported by actin filaments through beta- and alpha-catenin. How alpha-catenin dynamic interactions between E-cadherin/beta-catenin and cortical actin control both stability and remodelling of adhesion is unclear. Here we focus on Drosophila homophilic E-cadherin complexes rather than total E-cadherin, including diffusing 'free' E-cadherin, because these complexes are a better proxy for adhesion. We find that E-cadherin complexes partition in very stable microdomains (that is, bona fide adhesive foci which are more stable than remodelling contacts). Furthermore, we find that stability and mobility of these microdomains depend on two actin populations: small, stable actin patches concentrate at homophilic E-cadherin clusters, whereas a rapidly turning over, contractile network constrains their lateral movement by a tethering mechanism. alpha-Catenin controls epithelial architecture mainly through regulation of the mobility of homophilic clusters and it is largely dispensable for their stability. Uncoupling stability and mobility of E-cadherin complexes suggests that stable epithelia may remodel through the regulated mobility of very stable adhesive foci.

Transfer of polarity information via diffusion of Wnt ligands in C. elegans embryos.

Different signaling mechanisms concur to ensure robust tissue patterning and cell fate instruction during animal development. Most of these mechanisms rely on signaling proteins that are produced, transported, and detected. The spatiotemporal dynamics of signaling molecules are largely unknown, yet they determine signal activity's spatial range and time frame. Here, we use the Caenorhabditis elegans embryo to study how Wnt ligands, an evolutionarily conserved family of signaling proteins, dynamically organize to establish cell polarity in a developing tissue. We identify how Wnt ligands, produced in the posterior half of the embryos, spread extracellularly to transmit information to distant target cells in the anterior half. With quantitative live imaging and fluorescence correlation spectroscopy, we show that Wnt ligands diffuse through the embryo over a timescale shorter than the cell cycle, in the intercellular space, and outside the tissue below the eggshell. We extracted diffusion coefficients of Wnt ligands and their receptor Frizzled and characterized their co-localization. Integrating our different measurements and observations in a simple computational framework, we show how fast diffusion in the embryo can polarize individual cells through a time integration of the arrival of the ligands at the target cells. The polarity established at the tissue level by a posterior Wnt source can be transferred to the cellular level. Our results support a diffusion-based long-range Wnt signaling, which is consistent with the dynamics of developing processes.

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.

Two-Point Optical Manipulation of Cell Junctions in the Early Epithelium of the Drosophila Embryo.

Laser manipulation is widely used to study mechanics from the molecular to the tissue scale. We implemented optical tweezers to directly manipulate single cell-cell junctions in a developing tissue. We further extended the approach to two-point laser manipulation to enable extensive remodeling of cell-cell junctions. Here, we describe two-point laser manipulation and its implementation to probe the mechanics of cell junctions in the Drosophila embryo.