FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity.     

Collective Epithelial and Mesenchymal Cell Migration During Gastrulation

Gastrulation, the process that puts the three major germlayers, the ectoderm, mesoderm and endoderm in their correct topological position in the developing embryo, is characterised by extensive highly organised collective cell migration of epithelial and mesenchymal cells. We discuss current knowledge and insights in the mechanisms controlling these cell behaviours during gastrulation in the chick embryo. We discuss several ideas that have been proposed to explain the observed large scale vortex movements of epithelial cells in the epiblast during formation of the primitive streak. We review current insights in the control and execution of the epithelial to mesenchymal transition (EMT) underlying the formation of the hypoblast and the ingression of the mesendoderm cells through the streak. We discuss the mechanisms by which the mesendoderm cells move, the nature and dynamics of the signals that guide these movements, as well as the interplay between signalling and movement that result in tissue patterning and morphogenesis. We argue that instructive cell-cell signaling and directed chemotactic movement responses to these signals are instrumental in the execution of all phases of gastrulation.     

Control of directed cell migration in vivo by membrane-to-cortex attachment

Cell shape and motility are primarily controlled by cellular mechanics. The attachment of the plasma membrane to the underlying actomyosin cortex has been proposed to be important for cellular processes involving membrane deformation. However, little is known about the actual function of membrane-to-cortex attachment (MCA) in cell protrusion formation and migration, in particular in the context of the developing embryo. Here, we use a multidisciplinary approach to study MCA in zebrafish mesoderm and endoderm (mesendoderm) germ layer progenitor cells, which migrate using a combination of different protrusion types, namely, lamellipodia, filopodia, and blebs, during zebrafish gastrulation. By interfering with the activity of molecules linking the cortex to the membrane and measuring resulting changes in MCA by atomic force microscopy, we show that reducing MCA in mesendoderm progenitors increases the proportion of cellular blebs and reduces the directionality of cell migration. We propose that MCA is a key parameter controlling the relative proportions of different cell protrusion types in mesendoderm progenitors, and thus is key in controlling directed migration during gastrulation.     

A mechanoresponsive cadherin-keratin complex directs polarized protrusive behavior and collective cell migration

Collective cell migration requires maintenance of adhesive contacts between adjacent cells, coordination of polarized cell protrusions, and generation of propulsive traction forces. We demonstrate that mechanical force applied locally to C-cadherins on single Xenopus mesendoderm cells is sufficient to induce polarized cell protrusion and persistent migration typical of individual cells within a collectively migrating tissue. Local tension on cadherin adhesions induces reorganization of the keratin intermediate filament network toward these stressed sites. Plakoglobin, a member of the catenin family, is localized to cadherin adhesions under tension and is required for both mechanoresponsive cell behavior and assembly of the keratin cytoskeleton at the rear of these cells. Local tugging forces on cadherins occur in vivo through interactions with neighboring cells, and these forces result in coordinate changes in cell protrusive behavior. Thus, cadherin-dependent force-inducible regulation of cell polarity in single mesendoderm cells represents an emergent property of the intact tissue.     

The cytoplasmic tyrosine kinase Arg regulates gastrulation via control of actin organization

Coordinated cell movements are crucial for vertebrate gastrulation and are controlled by multiple signals. Although many factors are shown to mediate non-canonical Wnt pathways to regulate cell polarity and intercalation during gastrulation, signaling molecules acting in other pathways are less investigated and the connections between various signals and cytoskeleton are not well understood. In this study, we show that the cytoplasmic tyrosine kinase Arg modulates gastrulation movements through control of actin remodeling. Arg is expressed in the dorsal mesoderm at the onset of gastrulation, and both gain- and loss-of-function of Arg disrupted axial development in Xenopus embryos. Arg controlled migration of anterior mesendoderm, influenced cell decision on individual versus collective migration, and modulated spreading and protrusive activities of anterior mesendodermal cells. Arg also regulated convergent extension of the trunk mesoderm by influencing cell intercalation behaviors. Arg modulated actin organization to control dynamic F-actin distribution at the cell-cell contact or in membrane protrusions. The functions of Arg required an intact tyrosine kinase domain but not the actin-binding motifs in its carboxyl terminus. Arg acted downstream of receptor tyrosine kinases to regulate phosphorylation of endogenous CrkII and paxillin, adaptor proteins involved in activation of Rho family GTPases and actin reorganization. Our data demonstrate that Arg is a crucial cytoplasmic signaling molecule that controls dynamic actin remodeling and mesodermal cell behaviors during Xenopus gastrulation.     

The planar polarity pathway promotes coordinated cell migration during Drosophila oogenesis

Cell migration is fundamental in both animal morphogenesis and disease. The migration of individual cells is relatively well-studied; however, in vivo, cells often remain joined by cell-cell junctions and migrate in cohesive groups. How such groups of cells coordinate their migration is poorly understood. The planar polarity pathway coordinates the polarity of non-migrating cells in epithelial sheets and is required for cell rearrangements during vertebrate morphogenesis. It is therefore a good candidate to play a role in the collective migration of groups of cells. Drosophila border cell migration is a well-characterised and genetically tractable model of collective cell migration, during which a group of about six to ten epithelial cells detaches from the anterior end of the developing egg chamber and migrates invasively towards the oocyte. We find that the planar polarity pathway promotes this invasive migration, acting both in the migrating cells themselves and in the non-migratory polar follicle cells that they carry along. Disruption of planar polarity signalling causes abnormalities in actin-rich processes on the cell surface and leads to less-efficient migration. This is apparently due, in part, to a loss of regulation of Rho GTPase activity by the planar polarity receptor Frizzled, which itself becomes localised to the migratory edge of the border cells. We conclude that, during collective cell migration, the planar polarity pathway can mediate communication between motile and non-motile cells, which enhances the efficiency of migration via the modulation of actin dynamics.     

Mesendoderm extension and mantle closure in Xenopus laevis gastrulation: combined roles for integrin alpha(5)beta(1), fibronectin, and tissue geometry

We describe mesendoderm morphogenesis during gastrulation in the frog Xenopus laevis and investigate the mechanics of these movements with tissue explants. When a dorsal marginal zone explant is plated onto fibronectin, the mesendoderm moves away from the dorsal axial tissues as an intact sheet. Mesendodermal cells within these explants display monopolar protrusive activity and radially intercalate during explant extension. Live time-lapse confocal sequences of actin dynamics at the margin of these extending explants prompt us to propose that integrin-mediated traction drives these movements. We demonstrate that integrin alpha(5)beta(1) recognition of the synergy site located within the type III(9) repeat of fibronectin is required for mesendoderm extension. Normal mesendoderm morphogenesis occurs with a unique "cup-shaped" geometry of the extending mesendodermal mantle and coincides with a higher rate of tissue extension than that seen in the simpler dorsal marginal zone explant. These higher rates can be reconstituted with "in-the-round" configurations of several explants. We propose several mechanically based hypotheses to explain both the initial fibronectin-dependent extension of the mesendoderm and additional requirement of tissue geometry during the high-velocity closure of the mesendodermal mantle.     

Roles of leader and follower cells in collective cell migration

Collective cell migration is a widely observed phenomenon during animal development, tissue repair, and cancer metastasis. Considering its broad involvement in biological processes, it is essential to understand the basics behind the collective movement. Based on the topology of migrating populations, tissue-scale kinetics, called the “leader–follower” model, has been proposed for persistent directional collective movement. Extensive in vivo and in vitro studies reveal the characteristics of leader cells, as well as the special mechanisms leader cells employ for maintaining their positions in collective migration. However, follower cells have attracted increasing attention recently due to their important contributions to collective movement. In this Perspective, the current understanding of the molecular mechanisms behind the “leader–follower” model is reviewed with a special focus on the force transmission and diverse roles of leaders and followers during collective cell movement.     

Cell traction in collective cell migration and morphogenesis: The chase and run mechanism

Directional collective cell migration plays an important role in development, physiology, and disease. An increasing number of studies revealed key aspects of how cells coordinate their movement through distances surpassing several cell diameters. While physical modeling and measurements of forces during collective cell movements helped to reveal key mechanisms, most of these studies focus on tightly connected epithelial cultures. Less is known about collective migration of mesenchymal cells. A typical example of such behavior is the migration of the neural crest cells, which migrate large distances as a group. A recent study revealed that this persistent migration is aided by the interaction between the neural crest and the neighboring placode cells, whereby neural crest chase the placodes via chemotaxis, but upon contact both populations undergo contact inhibition of locomotion and a rapid reorganization of cellular traction. The resulting asymmetric traction field of the placodes forces them to run away from the chasers. We argue that this chase and run interaction may not be specific only to the neural crest system, but could serve as the underlying mechanism for several morphogenetic processes involving collective cell migration.     

Light-sheet microscopy-based 3D single-cell tracking reveals a correlation between cell cycle and the start of endoderm cell internalization in early zebrafish development

Controlling the initiation of cell migration plays a fundamental role in shaping the tissue during embryonic development. During gastrulation in zebrafish, some mesendoderm cells migrate inward to form the endoderm as the innermost germ layer along the yolk syncytial layer. However, how the initiation of inward migration is regulated is poorly understood. In this study, we performed light-sheet microscopy-based 3D single-cell tracking consisting of (a) whole-embryo time-lapse imaging with light-sheet microscopy and (b) three-dimensional single cell tracking in the zebrafish gastrula in which cells are marked with histone H2A-mCherry (nuclei) and the sox17:EGFP transgene (expressed in endoderm cells). We analyzed the correlation between the timing of cell internalization and cell division. Most cells that differentiated into endoderm cells began to internalize during the first half of the cell cycle, where the length of a cell cycle was defined by the period between two successive cell divisions. By contrast, the timing of other internalized cells was not correlated with a certain phase of the cell cycle. These results suggest the possibility that cell differentiation is associated with the relationship between cell cycle progression and the start of internalization. Moreover, the 3D single-cell tracking approach is useful for further investigating how cell migration is integrated with cell proliferation to shape tissues in zebrafish embryos.     

Mechanisms of mesendoderm internalization in the Xenopus gastrula: lessons from the ventral side.

Two main processes are involved in driving ventral mesendoderm internalization in the Xenopus gastrula. First, vegetal rotation, an active movement of the vegetal cell mass, initiates gastrulation by rolling the peripheral blastocoel floor against the blastocoel roof. In this way, the leading edge of the internalized mesendoderm is established, that remains separated from the blastocoel roof by Brachet's cleft. Second, in a process of active involution, blastopore lip cells translocate on arc-like trails around the tip of Brachet's cleft. Hereby the lower, Xbra-negative part of the lip moves toward the interior, to contribute mainly to endoderm. In contrast, the upper, Xbra-expressing part moves toward the blastocoel roof-apposed surface of the involuted mesoderm, and eventually becomes inserted into this surface. Vegetal rotation and active mesoderm surface insertion persist over much of gastrulation ventrally. Both processes are also active dorsally. In fact, internalization processes generally spread from dorsal to ventral, though at different rates, which suggests that they are independently controlled. Ventrally and laterally, mesoderm occurs not only in the marginal zone, but also in the adjacent blastocoel roof. Such blastocoel roof mesoderm shares properties with the remaining, ectodermal roof, that are related to its function as substratum for mesendoderm migration. It repels involuted mesoderm, thus contributing to separation of cell layers, and it assembles a fibronectin matrix. These properties change as the blastocoel roof mesoderm moves into the blastopore lip during gastrulation.     

Leader cell positioning drives wound-directed collective migration in TGFβ-stimulated epithelial sheets

During wound healing and cancer metastasis, cells are frequently observed to migrate in collective groups. This mode of migration relies on the cooperative guidance of leader and follower cells throughout the collective group. The upstream determinants and molecular mechanisms behind such cellular guidance remain poorly understood. We use live-cell imaging to track the behavior of epithelial sheets of keratinocytes in response to transforming growth factor β (TGFβ), which stimulates collective migration primarily through extracellular regulated kinase 1/2 (Erk1/2) activation. TGFβ-treated sheets display a spatial pattern of Erk1/2 activation in which the highest levels of Erk1/2 activity are concentrated toward the leading edge of a sheet. We show that Erk1/2 activity is modulated by cellular density and that this functional relationship drives the formation of patterns of Erk1/2 activity throughout sheets. In addition, we determine that a spatially constrained pattern of Erk1/2 activity results in collective migration that is primarily wound directed. Conversely, global elevation of Erk1/2 throughout sheets leads to stochastically directed collective migration throughout sheets. Our study highlights how the spatial patterning of leader cells (cells with elevated Erk1/2 activity) can influence the guidance of a collective group of cells during wound healing.     

Zebrafish Dkk1 functions in forebrain specification and axial mesendoderm formation

We identified a zebrafish homologue of Dickkopf-1 (Dkk1), which was previously identified in Xenopus as a Wnt inhibitor with potent head-inducing activity. Zebrafish dkk1 is expressed in the dorsal marginal blastoderm and also in the dorsal yolk syncytial layer after mid-blastula transition. At later blastula stages, the expression expands to the entire blastoderm margin. During gastrulation, dkk1-expressing cells are confined to the embryonic shield and later to the anterior axial mesendoderm, prospective prechordal plate. Embryos, in which dkk1 was ectopically expressed, exhibited enlarged forebrain, eyes, and axial mesendoderm such as prechordal plate and notochord. dkk1 expression in the dorso-anterior mesendoderm during gastrulation was prominently reduced in zebrafish mutants bozozok (boz), squint (sqt), and one-eyed pinhead (oep), which all display abnormalities in the formation and function of the Spemann organizer and axial mesendoderm. dkk1 expression was normal in these embryos during the blastula period, indicating that zygotic functions of these genes are required for maintenance but not establishment of dkk1 expression. Overexpression of dkk1 suppressed defects in the development of forebrain, eyes, and notochord in boz mutants. Overexpression of dkk1 promoted anterior neuroectoderm development in the embryos injected with antivin RNA, which lack most of the mesoderm and endoderm, suggesting that Dkk1 can affect regionalization of neuroectoderm independently of dorso-anterior mesendoderm. These data indicate that Dkk1, expressed in dorsal mesendoderm, functions in the formation of both the anterior nervous system and the axial mesendoderm in zebrafish.     

Propagating Waves of Directionality and Coordination Orchestrate Collective Cell Migration

The ability of cells to coordinately migrate in groups is crucial to enable them to travel long distances during embryonic development, wound healing and tumorigenesis, but the fundamental mechanisms underlying intercellular coordination during collective cell migration remain elusive despite considerable research efforts. A novel analytical framework is introduced here to explicitly detect and quantify cell clusters that move coordinately in a monolayer. The analysis combines and associates vast amount of spatiotemporal data across multiple experiments into transparent quantitative measures to report the emergence of new modes of organized behavior during collective migration of tumor and epithelial cells in wound healing assays. First, we discovered the emergence of a wave of coordinated migration propagating backward from the wound front, which reflects formation of clusters of coordinately migrating cells that are generated further away from the wound edge and disintegrate close to the advancing front. This wave emerges in both normal and tumor cells, and is amplified by Met activation with hepatocyte growth factor/scatter factor. Second, Met activation was found to induce coinciding waves of cellular acceleration and stretching, which in turn trigger the emergence of a backward propagating wave of directional migration with about an hour phase lag. Assessments of the relations between the waves revealed that amplified coordinated migration is associated with the emergence of directional migration. Taken together, our data and simplified modeling-based assessments suggest that increased velocity leads to enhanced coordination: higher motility arises due to acceleration and stretching that seems to increase directionality by temporarily diminishing the velocity components orthogonal to the direction defined by the monolayer geometry. Spatial and temporal accumulation of directionality thus defines coordination. The findings offer new insight and suggest a basic cellular mechanism for long-term cell guidance and intercellular communication during collective cell migration.     

CNOT3 interacts with the Aurora B and MAPK/ERK kinases to promote survival of differentiating mesendodermal progenitor cells

Mesendoderm cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between CNOT3 and the cell cycle kinase Aurora B that requires sequences in the NOT box domain of CNOT3 and regulates MAPK/ERK signaling during mesendoderm differentiation. Aurora B phosphorylates CNOT3 at two sites located close to a nuclear localization signal and promotes localization of CNOT3 to the nuclei of mouse embryonic stem cells (ESCs) and metastatic lung cancer cells. ESCs that have both sites mutated give rise to embryoid bodies that are largely devoid of mesoderm and endoderm and are composed mainly of cells with ectodermal characteristics. The mutant ESCs are also compromised in their ability to differentiate into mesendoderm in response to FGF2, BMP4, and Wnt3 due to reduced survival and proliferation of differentiating mesendoderm cells. We also show that the double mutation alters the balance of interaction of CNOT3 with Aurora B and with ERK and reduces phosphorylation of ERK in response to FGF2. Our results identify a potential adaptor function for CNOT3 that regulates the Ras/MEK/ERK pathway during embryogenesis.     

Mechanobiology of leader–follower dynamics in epithelial cell migration

Collective cell migration is fundamental to biological form and function. It is also relevant to the formation and repair of organs and to various pathological situations, including metastatic propagation of cancer. Technological, experimental, and computational advancements have allowed the researchers to explore various aspects of collective migration, spanning from biochemical signalling to inter-cellular force transduction. Here, we summarize our current understanding of the mechanobiology of collective cell migration, limiting to epithelial tissues. On the basis of recent studies, we describe how cells sense and respond to guidance signals to orchestrate various modes of migration and identify the determining factors dictating leader–follower interactions. We highlight how the inherent mechanics of dense epithelial monolayers at multicellular length scale might instruct individual cells to behave collectively. On the basis of these findings, we propose that mechanical resilience, obtained by a certain extent of cell jamming, allows the epithelium to perform efficient collective migration during wound healing.     

Three-dimensional collective cell motions in an acinus-like lumen

Collective cell migration is an essential process in embryo development, wound healing, inflammatory response, and cancer invasion. Although cell motions in two-dimensional (2D) monolayers have been studied previously, three-dimensional (3D) collective cell migration, which constantly occurs during embryogenesis such as the establishment of ducts and acini in vivo, remains elusive. In this paper, we develop a cell-based model incorporating cell mechanics and cell motility to address coherent cell motions in a spherical acinus-like lumen with different cell populations. It is found that the interplays between cell persistence, random fluctuation, and geometrical confinement may engender rich and novel migratory modes. In a 3D spherical lumen, two cells may undergo stripe-like or cross-circular coherent rotations, whereas multiple cells can form dynamic twisting or circulating bands, leaving sparse cells at the center or even a hollow cavity in the cell aggregate. The cell density is found to profoundly influence the collective cell migration modes. Our model can reproduce the fundamental features observed in experiments and highlight the role of mechanics in steering 3D collective cell dynamics during mammary acinar morphogenesis.     

On the Mechanical Interplay Between Intra- and Inter-Synchronization During Collective Cell Migration: A Numerical Investigation

Collective cell migration is a fundamental process that takes place during several biological phenomena such as embryogenesis, immunity response, and tumorogenesis, but the mechanisms that regulate it are still unclear. Similarly to collective animal behavior, cells receive feedbacks in space and time, which control the direction of the migration and the synergy between the cells of the population, respectively. While in single cell migration intra-synchronization (i.e. the synchronization between the protrusion-contraction movement of the cell and the adhesion forces exerted by the cell to move forward) is a sufficient condition for an efficient migration, in collective cell migration the cells must communicate and coordinate their movement between each other in order to be as efficient as possible (i.e. inter-synchronization). Here, we propose a 2D mechanical model of a cell population, which is described as a continuum with embedded discrete cells with or without motility phenotype. The decomposition of the deformation gradient is employed to reproduce the cyclic active strains of each single cell (i.e. protrusion and contraction). We explore different modes of collective migration to investigate the mechanical interplay between intra- and inter-synchronization. The main objective of the paper is to evaluate the efficiency of the cell population in terms of covered distance and how the stress distribution inside the cohort and the single cells may in turn provide insights regarding such efficiency.