Adherens junctions as molecular regulators of emergent tissue mechanics.

Tissue and organ development during embryogenesis relies on the collective and coordinated action of many cells. Recent studies have revealed that tissue material properties, including transitions between fluid and solid tissue states, are controlled in space and time to shape embryonic structures and regulate cell behaviours. Although the collective cellular flows that sculpt tissues are guided by tissue-level physical changes, these ultimately emerge from cellular-level and subcellular-level molecular mechanisms. Adherens junctions are key subcellular structures, built from clusters of classical cadherin receptors. They mediate physical interactions between cells and connect biochemical signalling to the physical characteristics of cell contacts, hence playing a fundamental role in tissue morphogenesis. In this Review, we take advantage of the results of recent, quantitative measurements of tissue mechanics to relate the molecular and cellular characteristics of adherens junctions, including adhesion strength, tension and dynamics, to the emergent physical state of embryonic tissues. We focus on systems in which cell-cell interactions are the primary contributor to morphogenesis, without significant contribution from cell-matrix interactions. We suggest that emergent tissue mechanics is an important direction for future research, bridging cell biology, developmental biology and mechanobiology to provide a holistic understanding of morphogenesis in health and disease.

Conserved physical mechanisms of cell and tissue elongation.

Living organisms have the ability to self-shape into complex structures appropriate for their function. The genetic and molecular mechanisms that enable cells to do this have been extensively studied in several model and non-model organisms. In contrast, the physical mechanisms that shape cells and tissues have only recently started to emerge, in part thanks to new quantitative in vivo measurements of the physical quantities guiding morphogenesis. These data, combined with indirect inferences of physical characteristics, are starting to reveal similarities in the physical mechanisms underlying morphogenesis across different organisms. Here, we review how physics contributes to shape cells and tissues in a simple, yet ubiquitous, morphogenetic transformation: elongation. Drawing from observed similarities across species, we propose the existence of conserved physical mechanisms of morphogenesis.

Mechanics of the cellular microenvironment as probed by cells in vivo during zebrafish presomitic mesoderm differentiation.

Tissue morphogenesis, homoeostasis and repair require cells to constantly monitor their three-dimensional microenvironment and adapt their behaviours in response to local biochemical and mechanical cues. Yet the mechanical parameters of the cellular microenvironment probed by cells in vivo remain unclear. Here, we report the mechanics of the cellular microenvironment that cells probe in vivo and in situ during zebrafish presomitic mesoderm differentiation. By quantifying both endogenous cell-generated strains and tissue mechanics, we show that individual cells probe the stiffness associated with deformations of the supracellular, foam-like tissue architecture. Stress relaxation leads to a perceived microenvironment stiffness that decreases over time, with cells probing the softest regime. We find that most mechanical parameters, including those probed by cells, vary along the anteroposterior axis as mesodermal progenitors differentiate. These findings expand our understanding of in vivo mechanosensation and might aid the design of advanced scaffolds for tissue engineering applications.

A fluid-to-solid jamming transition underlies vertebrate body axis elongation.

Just as in clay moulding or glass blowing, physically sculpting biological structures requires the constituent material to locally flow like a fluid while maintaining overall mechanical integrity like a solid. Disordered soft materials, such as foams, emulsions and colloidal suspensions, switch from fluid-like to solid-like behaviours at a jamming transition1-4. Similarly, cell collectives have been shown to display glassy dynamics in 2D and 3D5,6 and jamming in cultured epithelial monolayers7,8, behaviours recently predicted theoretically9-11 and proposed to influence asthma pathobiology8 and tumour progression12. However, little is known about whether these seemingly universal behaviours occur in vivo13 and, specifically, whether they play any functional part during embryonic morphogenesis. Here, by combining direct in vivo measurements of tissue mechanics with analysis of cellular dynamics, we show that during vertebrate body axis elongation, posterior tissues undergo a jamming transition from a fluid-like behaviour at the extending end, the mesodermal progenitor zone, to a solid-like behaviour in the presomitic mesoderm. We uncover an anteroposterior, N-cadherin-dependent gradient in yield stress that provides increasing mechanical integrity to the presomitic mesoderm, consistent with the tissue transiting from a wetter to a dryer foam-like architecture. Our results show that cell-scale stresses fluctuate rapidly (within about 1 min), enabling cell rearrangements and effectively 'melting' the tissue at the growing end. Persistent (more than 0.5 h) stresses at supracellular scales, rather than cell-scale stresses, guide morphogenetic flows in fluid-like tissue regions. Unidirectional axis extension is sustained by the reported rigidification of the presomitic mesoderm, which mechanically supports posterior, fluid-like tissues during remodelling before their maturation. The spatiotemporal control of fluid-like and solid-like tissue states may represent a generic physical mechanism of embryonic morphogenesis.

Macromolecular Crowding as an Intracellular Stimulus for Responsive Nanomaterials.

Stimuli-responsive materials are exploited in biological, materials, and sensing applications. We introduce a new endogenous stimulus, biomacromolecule crowding, which we achieve by leveraging changes in thermoresponsive properties of polymers upon high concentrations of crowding agents. We prepare poly(2-oxazoline) amphiphiles that exhibit lower critical solution temperatures (LCST) in serum above physiological temperature. These amphiphiles stabilize oil-in-water nanoemulsions at temperatures below the LCST but are ineffective surfactants above the LCST, resulting in emulsion fusion. We find that the transformations observed upon heating nanoemulsions above their surfactant's LCST can instead be induced at physiological temperatures through the addition of polymers and protein, rendering thermoresponsive materials "crowding responsive." We demonstrate that the cytosol is a stimulus for nanoemulsions, with droplet fusion occurring upon injection into cells of living zebrafish embryos. This report sets the stage for classes of thermoresponsive materials to respond to macromolecule concentration rather than temperature changes.

Physical constraints on early blastomere packings.

At very early embryonic stages, when embryos are composed of just a few cells, establishing the correct packing arrangements (contacts) between cells is essential for the proper development of the organism. As early as the 4-cell stage, the observed cellular packings in different species are distinct and, in many cases, differ from the equilibrium packings expected for simple adherent and deformable particles. It is unclear what are the specific roles that different physical parameters, such as the forces between blastomeres, their division times, orientation of cell division and embryonic confinement, play in the control of these packing configurations. Here we simulate the non-equilibrium dynamics of cells in early embryos and systematically study how these different parameters affect embryonic packings at the 4-cell stage. In the absence of embryo confinement, we find that cellular packings are not robust, with multiple packing configurations simultaneously possible and very sensitive to parameter changes. Our results indicate that the geometry of the embryo confinement determines the packing configurations at the 4-cell stage, removing degeneracy in the possible packing configurations and overriding division rules in most cases. Overall, these results indicate that physical confinement of the embryo is essential to robustly specify proper cellular arrangements at very early developmental stages.

Coordinating cell polarization and morphogenesis through mechanical feedback.

Many cellular processes require cell polarization to be maintained as the cell changes shape, grows or moves. Without feedback mechanisms relaying information about cell shape to the polarity molecular machinery, the coordination between cell polarization and morphogenesis, movement or growth would not be possible. Here we theoretically and computationally study the role of a genetically-encoded mechanical feedback (in the Cell Wall Integrity pathway) as a potential coordination mechanism between cell morphogenesis and polarity during budding yeast mating projection growth. We developed a coarse-grained continuum description of the coupled dynamics of cell polarization and morphogenesis as well as 3D stochastic simulations of the molecular polarization machinery in the evolving cell shape. Both theoretical approaches show that in the absence of mechanical feedback (or in the presence of weak feedback), cell polarity cannot be maintained at the projection tip during growth, with the polarization cap wandering off the projection tip, arresting morphogenesis. In contrast, for mechanical feedback strengths above a threshold, cells can robustly maintain cell polarization at the tip and simultaneously sustain mating projection growth. These results indicate that the mechanical feedback encoded in the Cell Wall Integrity pathway can provide important positional information to the molecular machinery in the cell, thereby enabling the coordination of cell polarization and morphogenesis.

Fluorous Soluble Cyanine Dyes for Visualizing Perfluorocarbons in Living Systems.

The bioorthogonal nature of perfluorocarbons provides a unique platform for introducing dynamic nano- and microdroplets into cells and organisms. To monitor the localization and deformation of the droplets, fluorous soluble fluorophores that are compatible with standard fluorescent protein markers and applicable to cells, tissues, and small organisms are necessary. Here, we introduce fluorous cyanine dyes that represent the most red-shifted fluorous soluble fluorophores to date. We study the effect of covalently appended fluorous tags on the cyanine scaffold and evaluate the changes in photophysical properties imparted by the fluorous phase. Ultimately, we showcase the utility of the fluorous soluble pentamethine cyanine dye for tracking the localization of perfluorocarbon nanoemulsions in macrophage cells and for measurements of mechanical forces in multicellular spheroids and zebrafish embryonic tissues. These studies demonstrate that the red-shifted cyanine dyes offer spectral flexibility in multiplexed imaging experiments and enhanced precision in force measurements.

In vivo quantification of spatially varying mechanical properties in developing tissues.

The mechanical properties of the cellular microenvironment and their spatiotemporal variations are thought to play a central role in sculpting embryonic tissues, maintaining organ architecture and controlling cell behavior, including cell differentiation. However, no direct in vivo and in situ measurement of mechanical properties within developing 3D tissues and organs has yet been performed. Here we introduce a technique that employs biocompatible, magnetically responsive ferrofluid microdroplets as local mechanical actuators and allows quantitative spatiotemporal measurements of mechanical properties in vivo. Using this technique, we show that vertebrate body elongation entails spatially varying tissue mechanics along the anteroposterior axis. Specifically, we find that the zebrafish tailbud is viscoelastic (elastic below a few seconds and fluid after just 1 min) and displays decreasing stiffness and increasing fluidity toward its posterior elongating region. This method opens new avenues to study mechanobiology in vivo, both in embryogenesis and in disease processes, including cancer.

Mechanical feedback coordinates cell wall expansion and assembly in yeast mating morphogenesis.

The shaping of individual cells requires a tight coordination of cell mechanics and growth. However, it is unclear how information about the mechanical state of the wall is relayed to the molecular processes building it, thereby enabling the coordination of cell wall expansion and assembly during morphogenesis. Combining theoretical and experimental approaches, we show that a mechanical feedback coordinating cell wall assembly and expansion is essential to sustain mating projection growth in budding yeast (Saccharomyces cerevisiae). Our theoretical results indicate that the mechanical feedback provided by the Cell Wall Integrity pathway, with cell wall stress sensors Wsc1 and Mid2 increasingly activating membrane-localized cell wall synthases Fks1/2 upon faster cell wall expansion, stabilizes mating projection growth without affecting cell shape. Experimental perturbation of the osmotic pressure and cell wall mechanics, as well as compromising the mechanical feedback through genetic deletion of the stress sensors, leads to cellular phenotypes that support the theoretical predictions. Our results indicate that while the existence of mechanical feedback is essential to stabilize mating projection growth, the shape and size of the cell are insensitive to the feedback.

The effect of cell geometry on polarization in budding yeast.

The localization (or polarization) of proteins on the membrane during the mating of budding yeast (Saccharomyces cerevisiae) is an important model system for understanding simple pattern formation within cells. While there are many existing mathematical models of polarization, for both budding and mating, there are still many aspects of this process that are not well understood. In this paper we set out to elucidate the effect that the geometry of the cell can have on the dynamics of certain models of polarization. Specifically, we look at several spatial stochastic models of Cdc42 polarization that have been adapted from published models, on a variety of tip-shaped geometries, to replicate the shape change that occurs during the growth of the mating projection. We show here that there is a complex interplay between the dynamics of polarization and the shape of the cell. Our results show that while models of polarization can generate a stable polarization cap, its localization at the tip of mating projections is unstable, with the polarization cap drifting away from the tip of the projection in a geometry dependent manner. We also compare predictions from our computational results to experiments that observe cells with projections of varying lengths, and track the stability of the polarization cap. Lastly, we examine one model of actin polarization and show that it is unlikely, at least for the models studied here, that actin dynamics and vesicle traffic are able to overcome this effect of geometry.

A toolbox to explore the mechanics of living embryonic tissues.

The sculpting of embryonic tissues and organs into their functional morphologies involves the spatial and temporal regulation of mechanics at cell and tissue scales. Decades of in vitro work, complemented by some in vivo studies, have shown the relevance of mechanical cues in the control of cell behaviors that are central to developmental processes, but the lack of methodologies enabling precise, quantitative measurements of mechanical cues in vivo have hindered our understanding of the role of mechanics in embryonic development. Several methodologies are starting to enable quantitative studies of mechanics in vivo and in situ, opening new avenues to explore how mechanics contributes to shaping embryonic tissues and how it affects cell behavior within developing embryos. Here we review the present methodologies to study the role of mechanics in living embryonic tissues, considering their strengths and drawbacks as well as the conditions in which they are most suitable.

Geometrical characterization of fluorescently labelled surfaces from noisy 3D microscopy data.

Modern fluorescence microscopy enables fast 3D imaging of biological and inert systems alike. In many studies, it is important to detect the surface of objects and quantitatively characterize its local geometry, including its mean curvature. We present a fully automated algorithm to determine the location and curvatures of an object from 3D fluorescence images, such as those obtained using confocal or light-sheet microscopy. The algorithm aims at reconstructing surface labelled objects with spherical topology and mild deformations from the spherical geometry with high accuracy, rather than reconstructing arbitrarily deformed objects with lower fidelity. Using both synthetic data with known geometrical characteristics and experimental data of spherical objects, we characterize the algorithm's accuracy over the range of conditions and parameters typically encountered in 3D fluorescence imaging. We show that the algorithm can detect the location of the surface and obtain a map of local mean curvatures with relative errors typically below 2% and 20%, respectively, even in the presence of substantial levels of noise. Finally, we apply this algorithm to analyse the shape and curvature map of fluorescently labelled oil droplets embedded within multicellular aggregates and deformed by cellular forces.

Quantifying cell-generated mechanical forces within living embryonic tissues.

Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. Little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. We present a method to quantify cell-generated mechanical stresses exerted locally within living embryonic tissues, using fluorescent, cell-sized oil microdroplets with defined mechanical properties and coated with adhesion receptor ligands. After a droplet is introduced between cells in a tissue, local stresses are determined from droplet shape deformations, measured using fluorescence microscopy and computerized image analysis. Using this method, we quantified the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates, and we confirmed that these stresses (3.4 nN µm(-2)) are dependent on myosin II activity and are more than twofold larger than stresses generated by cells of embryonic tooth mesenchyme, either within cultured aggregates or in developing whole mouse mandibles.

Improved calibration of the nonlinear regime of a single-beam gradient optical trap.

We report an improved method for calibrating the nonlinear region of a single-beam gradient optical trap. Through analysis of the position fluctuations of a trapped object that is displaced from the trap center by controlled flow we measure the local trap stiffness in both the linear and nonlinear regimes without knowledge of the magnitude of the applied external forces. This approach requires only knowledge of the system temperature, and is especially useful for measurements involving trapped objects of unknown size, or objects in a fluid of unknown viscosity.

Closely related bird species demonstrate flexibility between beak morphology and underlying developmental programs.

The astonishing variation in the shape and size of bird beaks reflects a wide range of dietary specializations that played an important role in avian diversification. Among Darwin's finches, ground finches (Geospiza spp.) have beaks that represent scaling variations of the same shape, which are generated by alterations in the signaling pathways that regulate growth of the two skeletal components of the beak: the prenasal cartilage (pnc) and the premaxillary bone (pmx). Whether this developmental mechanism is responsible for variation within groups of other closely related bird species, however, has remained unknown. Here, we report that the Caribbean bullfinches (Loxigilla spp.), which are closely related to Darwin's finches, have independently evolved beaks of a novel shape, different from Geospiza, but also varying from each other only in scaling. However, despite sharing the same beak shape, the signaling pathways and tissues patterning Loxigilla beaks differ among the three species. In Loxigilla noctis, as in Geospiza, the pnc develops first, shaped by Bmp4 and CaM signaling, followed by the development of the pmx, regulated by TGFßIIr, ß-catenin, and Dkk3 signaling. In contrast, beak morphogenesis in Loxigilla violacea and Loxigilla portoricensis is generated almost exclusively by the pmx through a mechanism in which Ihh and Bmp4 synergize to promote expansion of bone tissue. Together, our results demonstrate high flexibility in the relationship between morphology and underlying developmental causes, where different developmental programs can generate identical shapes, and similar developmental programs can pattern different shapes.

Proliferation-driven mechanical compression induces signalling centre formation during mammalian organ development.

Localized sources of morphogens, called signalling centres, play a fundamental role in coordinating tissue growth and cell fate specification during organogenesis. However, how these signalling centres are established in tissues during embryonic development is still unclear. Here we show that the main signalling centre orchestrating development of rodent incisors, the enamel knot (EK), is specified by a cell proliferation-driven buildup in compressive stresses (mechanical pressure) in the tissue. Direct mechanical measurements indicate that the stresses generated by cell proliferation are resisted by the surrounding tissue, creating a circular pattern of mechanical anisotropy with a region of high compressive stress at its centre that becomes the EK. Pharmacological inhibition of proliferation reduces stresses and suppresses EK formation, and application of external pressure in proliferation-inhibited conditions rescues the formation of the EK. Mechanical information is relayed intracellularly through YAP protein localization, which is cytoplasmic in the region of compressive stress that establishes the EK and nuclear in the stretched anisotropic cells that resist the pressure buildup around the EK. Together, our data identify a new role for proliferation-driven mechanical compression in the specification of a model signalling centre during mammalian organ development.

Synthesis of Fluorous Ferrofluids and Effects of the Nanoparticle Coatings on Field- and Temperature-Dependent Magnetizations.

Ferrofluids have been extensively employed in industrial, environmental, and biomedical areas. Among them, fluorous ferrofluids are of particular interest because of the biorthogonal nature of perfluorocarbons (PFCs). However, the noninteracting nature of PFCs as well as challenges in functionalization of nanoparticle surfaces with fluorous ligands has limited their applications, especially in biomedicine. In particular, commercially available fluorous ferrofluids are stabilized using ionic surfactants with charged groups that physically interact with a wide range of charged biological molecules. In this paper, we developed a unique two-phase ligand attachment strategy to render stable fluorous ferrofluids using nonionic surfactants. The superparamagnetic Fe3O4 or MnFe2O4 core of the magnetic nanoparticles, the magnetic component of the ferrofluid, was coated with a silica shell containing abundant surface hydroxyl groups, thereby enabling the installation of fluorous ligands through stable covalent, neutral, siloxane bonds. We explored chemistry-material relationships between different ligands and PFC solvents and found that low-molecular-weight ligands can assist with the installation of high-molecular-weight ligands (4000-8000 g/mol), allowing us to systematically control the size and thickness of ligand functionalization on the nanoparticle surface. By zero-field-cooled magnetization measurements, we studied how the ligands affect magnetic dipole orientation forces and observed a curve flattening that is only associated with the ferrofluids. This work provided insight into ferrofluids' dependence on interparticle interactions and contributed a methodology to synthesize fluorous ferrofluids with nonionic surfactants that exhibit both magnetic and chemical stability. We believe that the doped MnFe2O4 fluorous ferrofluid has the highest combination of stability and magnetization reported to date.

In situ quantification of osmotic pressure within living embryonic tissues.

Mechanics is known to play a fundamental role in many cellular and developmental processes. Beyond active forces and material properties, osmotic pressure is believed to control essential cell and tissue characteristics. However, it remains very challenging to perform in situ and in vivo measurements of osmotic pressure. Here we introduce double emulsion droplet sensors that enable local measurements of osmotic pressure intra- and extra-cellularly within 3D multicellular systems, including living tissues. After generating and calibrating the sensors, we measure the osmotic pressure in blastomeres of early zebrafish embryos as well as in the interstitial fluid between the cells of the blastula by monitoring the size of droplets previously inserted in the embryo. Our results show a balance between intracellular and interstitial osmotic pressures, with values of approximately 0.7 MPa, but a large pressure imbalance between the inside and outside of the embryo. The ability to measure osmotic pressure in 3D multicellular systems, including developing embryos and organoids, will help improve our understanding of its role in fundamental biological processes.

Actin network growth under load.

Many processes in eukaryotic cells, including the crawling motion of the whole cell, rely on the growth of branched actin networks from surfaces. In addition to their well-known role in generating propulsive forces, actin networks can also sustain substantial pulling loads thanks to their persistent attachment to the surface from which they grow. The simultaneous network elongation and surface attachment inevitably generate a force that opposes network growth. Here, we study the local dynamics of a growing actin network, accounting for simultaneous network elongation and surface attachment, and show that there exist several dynamical regimes that depend on both network elasticity and the kinetic parameters of actin polymerization. We characterize this in terms of a phase diagram and provide a connection between mesoscopic theories and the microscopic dynamics of an actin network at a surface. Our framework predicts the onset of instabilities that lead to the local detachment of the network and translate to oscillatory behavior and waves, as observed in many cellular phenomena and in vitro systems involving actin network growth, such as the saltatory dynamics of actin-propelled oil drops.