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.

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.

Spatiotemporal variation of endogenous cell-generated stresses within 3D multicellular spheroids.

Multicellular spheroids serve as an excellent platform to study tissue behavior and tumor growth in a controlled, three-dimensional (3D) environment. While molecular and cellular studies have long used this platform to study cell behavior in 3D, only recently have studies using multicellular spheroids shown an important role for the mechanics of the microenvironment in a wide range of cellular processes, including during tumor progression. Despite the well-established relevance of mechanical cues to cell behavior and the numerous studies on mechanics using 2D cell culture systems, the spatial and temporal variations in endogenous cellular forces within growing multicellular aggregates remain unknown. Using cell-sized oil droplets with controlled physicochemical properties as force transducers in mesenchymal cell aggregates, we show that the magnitude of cell-generated stresses varies only weakly with spatial location within the spherical aggregate, but it increases considerably over time during aggregate compaction and growth. Moreover, our results indicate that the temporal increase in cellular stresses is due to increasing cell pulling forces transmitted via integrin-mediated cell adhesion, consistent with the need for larger intercellular pulling forces to compact cell aggregates.

Generation of biocompatible droplets for in vivo and in vitro measurement of cell-generated mechanical stresses.

Here we describe a detailed protocol to produce biocompatible droplets that permit the measurement of mechanical stresses at cell and tissue scales. The droplets can be used as force transducers in vivo, ex vivo, and in vitro, to measure mechanical stresses in situ, in three dimensions and time. Versatile and modular droplet coatings using biotinylated molecules, such as ligands for specific adhesion receptors, enable the targeting of specific tissues or cells. Droplet sizes can be varied to measure forces at different scales (tissue and cell scales) and the range of measurable mechanical stresses ranges within approximately 0.3-100 kPa. The protocol described in this chapter is divided in three sections. First, we describe the generation and stabilization of biocompatible droplets. Next, we explain the steps necessary to functionalize the droplet surface. Finally, we describe how to characterize the mechanical properties of the droplets, so that they can be used as calibrated mechanical probes. The procedure to generate, stabilize, and functionalize the droplets is straightforward and can be completed in about 3h with basic laboratory resources. The calibration of the droplet's mechanical properties to perform quantitative stress measurements is also straightforward, but requires the proper equipment to measure interfacial tension (such as a tensiometer). Calibrated droplets can be used to quantify cell-generated mechanical stresses by analyzing the tridimensional shape of the droplet.