Sculpting with stiffness: rigidity as a regulator of morphogenesis.

From a physical perspective, morphogenesis of tissues results from interplay between their material properties and the mechanical forces exerted on them. The importance of mechanical forces in influencing cell behaviour is widely recognised, whereas the importance of tissue material properties in vivo, like stiffness, has only begun to receive attention in recent years. In this mini-review, we highlight key themes and concepts that have emerged related to how tissue stiffness, a fundamental material property, guides various morphogenetic processes in living organisms.

Collective durotaxis along a self-generated stiffness gradient in vivo.

Collective cell migration underlies morphogenesis, wound healing and cancer invasion1,2. Most directed migration in vivo has been attributed to chemotaxis, whereby cells follow a chemical gradient3-5. Cells can also follow a stiffness gradient in vitro, a process called durotaxis3,4,6-8, but evidence for durotaxis in vivo is lacking6. Here we show that in Xenopus laevis the neural crest-an embryonic cell population-self-generates a stiffness gradient in the adjacent placodal tissue, and follows this gradient by durotaxis. The gradient moves with the neural crest, which is continually pursuing a retreating region of high substrate stiffness. Mechanistically, the neural crest induces the gradient due to N-cadherin interactions with the placodes and senses the gradient through cell-matrix adhesions, resulting in polarized Rac activity and actomyosin contractility, which coordinates durotaxis. Durotaxis synergizes with chemotaxis, cooperatively polarizing actomyosin machinery of the cell group to prompt efficient directional collective cell migration in vivo. These results show that durotaxis and dynamic stiffness gradients exist in vivo, and gradients of chemical and mechanical signals cooperate to achieve efficient directional cell migration.

Durotaxis: The Hard Path from In Vitro to In Vivo.

Durotaxis, the process by which cells follow gradients of extracellular mechanical stiffness, has been proposed as a mechanism driving directed migration. Despite the lack of evidence for its existence in vivo, durotaxis has become an active field of research, focusing on the mechanism by which cells respond to mechanical stimuli from the environment. In this review, we describe the technical and conceptual advances in the study of durotaxis in vitro, discuss to what extent the evidence suggests durotaxis may occur in vivo, and emphasize the urgent need for in vivo demonstration of durotaxis.

All Roads Lead to Directional Cell Migration.

Directional cell migration normally relies on a variety of external signals, such as chemical, mechanical, or electrical, which instruct cells in which direction to move. Many of the major molecular and physical effects derived from these cues are now understood, leading to questions about whether directional cell migration is alike or distinct under these different signals, and how cells might be directed by multiple simultaneous cues, which would be expected in complex in vivo environments. In this review, we compare how different stimuli are spatially distributed, often as gradients, to direct cell movement and the mechanisms by which they steer cells. A comparison of the downstream effectors of directional cues suggests that different external signals regulate a common set of components: small GTPases and the actin cytoskeleton, which implies that the mechanisms downstream of different signals are likely to be closely related and underlies the idea that cell migration operates by a common set of physical principles, irrespective of the input.

Rules of collective migration: from the wildebeest to the neural crest.

Collective migration, the movement of groups in which individuals affect the behaviour of one another, occurs at practically every scale, from bacteria up to whole species' populations. Universal principles of collective movement can be applied at all levels. In this review, we will describe the rules governing collective motility, with a specific focus on the neural crest, an embryonic stem cell population that undergoes extensive collective migration during development. We will discuss how the underlying principles of individual cell behaviour, and those that emerge from a supracellular scale, can explain collective migration. This article is part of the theme issue 'Multi-scale analysis and modelling of collective migration in biological systems'.

Author Correction: An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells.

The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to control structural and functional integrity of the epidermal layer in developing Xenopus embryos.

Integrating chemical and mechanical signals in neural crest cell migration.

Neural crest cells are a multipotent embryonic stem cell population that migrate large distances to contribute a variety of tissues. The cranial neural crest, which contribute to tissues of the face and skull, undergo collective migration whose movement has been likened to cancer metastasis. Over the last few years, a variety of mechanisms for the guidance of collective cranial neural crest cell migration have been described: mostly chemical, but more recently mechanical. Here we review these different mechanisms and attempt to integrate them to provide a unified model of collective cranial neural crest cell migration.

In Vivo and In Vitro Quantitative Analysis of Neural Crest Cell Migration.

The neural crest is an embryonic cell population induced at the border of the neural plate from where it delaminates and migrates long distances across the embryo. Due to its extraordinary migratory capabilities, the neural crest has become a powerful system to study cellular and molecular aspects of collective and single cell migration both in vivo and in vitro. Here we provide detailed protocols used to perform quantitative analysis of molecular and cellular aspects of Xenopus laevis neural crest cell migration, both in vivo and in vitro.

Supracellular migration - beyond collective cell migration.

Collective cell migration is a highly complex process in which groups of cells move together. A fundamental question is how cell ensembles can migrate efficiently. In some cases, the group is no more than a collection of individual cells. In others, the group behaves as a supracellular unit, whereby the cell group could be considered as a giant 'supracell', the concept of which was conceived over a century ago. The development of recent tools has provided considerable evidence that cell collectives are highly cooperative, and their migration can better be understood at the tissue level, rather than at the cell level. In this Review, we will define supracellular migration as a type of collective cell migration that operates at a scale higher than the individual cells. We will discuss key concepts of supracellular migration, review recent evidence of collectives exhibiting supracellular features and argue that many seemingly complex collective movements could be better explained by considering the participating cells as supracellular entities.

Supracellular contraction at the rear of neural crest cell groups drives collective chemotaxis.

Collective cell chemotaxis, the directed migration of cell groups along gradients of soluble chemical cues, underlies various developmental and pathological processes. We use neural crest cells, a migratory embryonic stem cell population whose behavior has been likened to malignant invasion, to study collective chemotaxis in vivo. Studying Xenopus and zebrafish, we have shown that the neural crest exhibits a tensile actomyosin ring at the edge of the migratory cell group that contracts in a supracellular fashion. This contractility is polarized during collective cell chemotaxis: It is inhibited at the front but persists at the rear of the cell cluster. The differential contractility drives directed collective cell migration ex vivo and in vivo through the intercalation of rear cells. Thus, in neural crest cells, collective chemotaxis works by rear-wheel drive.

Chemotaxis during neural crest migration.

Chemotaxis refers to the directional migration of cells towards external, soluble factors along their gradients. It is a process that is used by many different cell types during development for tissue organisation and the formation of embryonic structures, as well as disease like cancer metastasis. The neural crest (NC) is a multipotent, highly migratory cell population that contribute to a range of tissues. It has been hypothesised that NC migration, at least in part, is reliant on chemotactic signals. This review will explore the current evidence for proposed chemoattractants of NC cells, and outline mechanisms for the chemotactic response of the NC to them.

Endorepellin affects angiogenesis by antagonizing diverse vascular endothelial growth factor receptor 2 (VEGFR2)-evoked signaling pathways: transcriptional repression of hypoxia-inducible factor 1α and VEGFA and concurrent inhibition of nuclear factor of activated T cell 1 (NFAT1) activation.

Endorepellin, the angiostatic C-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits angiogenesis by simultaneously binding to the α2ß1 integrin and the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) on endothelial cells. This interaction triggers the down-regulation of both receptors and the concurrent activation of the tyrosine phosphatase SHP-1, which leads to a signaling cascade resulting in angiostasis. Here, we provide evidence that endorepellin is capable of attenuating both the PI3K/PDK1/Akt/mTOR and the PKC/JNK/AP1 pathways. We show that hypoxia-inducible factor 1α (HIF-1α) transcriptional activity induced by VEGFA was inhibited by endorepellin independent of oxygen concentration and that only a combination of both PI3K and calcineurin inhibitors completely blocked the suppressive activity evoked by endorepellin on HIF1A and VEGFA promoter activity. Moreover, endorepellin inhibited the PKC/JNK/AP1 axis induced by the recruitment of phospholipase γ and attenuated the VEGFA-induced activation of NFAT1, a process dependent on calcineurin activity. Finally, endorepellin inhibited VEGFA-evoked nuclear translocation of NFAT1 and promoted NFAT1 stability. Thus, we provide evidence for a novel downstream signaling axis for an angiostatic fragment and for the key components involved in the dual antagonistic activity of endorepellin, highlighting its potential use as a therapeutic agent.