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Collective migration of vascular endothelial cells is central for embryonic development, angiogenesis, and wound closure. Although physical confinement of cell assemblies has been shown to elicit specific patterns of collective movement in various cell types, endothelial migration in vivo often occurs without confinement. Here we show that unconfined endothelial cell monolayers on microgrooved substrates that mimic the anisotropic organization of the extracellular matrix exhibit a new type of collective movement that takes the form of a periodic pattern of anti-parallel cell streams. We further establish that the development of these streams requires intact cell-cell junctions and that stream sizes are particularly sensitive to groove depth. Finally, we show that modeling the endothelial cell sheet as an active fluid with the microgrooves acting as constraints on cell orientation predicts the occurrence of the periodic anti-parallel cell streams as well as their lengths and widths. We posit that in unconfined cell assemblies, physical factors that constrain or bias cellular orientation such as anisotropic extracellular matrix cues or directed flow-derived shear forces dictate the pattern of collective cell movement.

International audience; One contribution of 14 to a theme issue 'Multi-scale analysis and modelling of collective migration in biological systems'. Collective migration has become a paradigm for emergent behaviour in systems of moving and interacting individual units resulting in coherent motion. In biology, these units are cells or organisms. Collective cell migration is important in embryonic development, where it underlies tissue and organ formation, as well as pathological processes, such as cancer invasion and metastasis. In animal groups, collective movements may enhance individuals' decisions and facilitate navigation through complex environments and access to food resources. Mathematical models can extract unifying principles behind the diverse manifestations of collective migration. In biology, with a few exceptions, collective migration typically occurs at a 'mesoscopic scale' where the number of units ranges from only a few dozen to a few thousands, in contrast to the large systems treated by statistical mechanics. Recent developments in multi-scale analysis have allowed linkage of mesoscopic to micro-and macroscopic scales, and for different biological systems. The articles in this theme issue on 'Multi-scale analysis and modelling of collective migration' compile a range of mathematical modelling ideas and multi-scale methods for the analysis of collective migration. These approaches (i) uncover new unifying organization principles of collective behaviour, (ii) shed light on the transition from single to collective migration, and (iii) allow us to define similarities and differences of collective behaviour in groups of cells and organisms. As a common theme, self-organized collective migration is the result of ecological and evolutionary constraints both at the cell and organismic levels. Thereby, the rules governing physiological collective behaviours also underlie pathological processes, albeit with different upstream inputs and consequences for the group. This article is part of the theme issue 'Multi-scale analysis and modelling of collective migration in biological systems'.

Cell migration is essential in various biological processes such as embryogenesis,wound healing, metastatic invasions or immune response. The objective of this thesiswas to identify and extract parameters useful for the establishment of physical modelsand the biological understanding of collective migration. To do this, we study cellmigration around an obstacle. Indeed, the presence of an obstacle in a flow induces heterogeneities which are discriminating for the establishment of models and we wantedto add to the information of velocities and deformations those of the forces exerted bythe cells on the substrate. Initally, the protein pattern geometry on which our cells are deposited is that of a band with an obstacle. Cells are confined by a block which, once removed, allows the tissue to invade free space and flow around the obstacle. The first part of this thesis studies this configuration, from a kinematic and proteinic levels point of view. In particular,we obtain master curves in velocity and deformation, a correlation between velocity and density, and an enrichment of the vimentin at the edge of the confined pattern andat the migration front. A set of relationships between actin, vimentin levels, density,deformation, and velocity is also found. Finally, it is shown that the direction of polarizationdefined by the core to center of mass vector of the cell is on average orthogonalto the direction of velocity. The strip geometry has the advantage of forcing the flow and being very controlled but has the disadvantage of requiring the installation of a block. It is not possible to lay the block on a substrate of low rigidity for force measurements, and to get around this problem we closed the boundary conditions and worked on a racetrack geometry that has two obstacles in the middle of each arm. We wagered on spontaneous generationof large-scale flow, which proved to be correct. The third part of the results studies theracetracks on a hard substrate (as opposed to a soft substrate for forces). The possibilityof generating a long-range flow and the presence of vimentin at the edge of the pattern are retained. We also note that the velocity- density relationship is no longer valid in this geometry, which attests that when it exists, it is in fact a consequence of the spreading dynamics of the monolayer and not of the migration dynamics as such. There is also a relationship between actin and vimentin which illustrates three possible cell states. The first corresponds to very deformed and vimentin-rich cells, the second to very dense and actin-rich cells, and the third to intermediate states of deformation and density where the cells move and where actin and vimentin vary together. The last part of this thesis consists in studying the forces (soft substrate) by traction force microscopy. These experiments confirm that a migrating monolayer is contractile and we show that it is equivalent to measure the displacement of beads in the substrate or the displacement of cells at small scale on phase images. In other words, the instantaneous velocity (the one used by the theoreticians but not experimentally measurable) is equal to the bead position derivative.We conclude that there is continuity of motion between the substrate and the cell. Keeping in mind our first goal to study the migration around an obstacle, the development of a large-scale system oracetracksallows us to better understand the underlying ingredients of migration while developingthe experimental framework that allows quantitative comparisons between systems.; La migration cellulaire est essentielle dans divers processus biologiques tels que l’embryogenèse, la cicatrisation ou les invasions métastatiques. L’objectif de cette thèse était d’identifier et d’extraire les paramètres utiles à l’établissement de modèles physiques ainsi qu’à la compréhension biologique de la migration collective. Pour cela,nous étudions la migration cellulaire autour d’un obstacle. Cela induit des hétérogénéités qui sont discriminantes pour l’établissement de modèles. Nous voulions mesurer dans cette géométrie les champs de vitesses, de déformations et de forces exercées par les cellules sur le substrat.La géométrie initiale du motif protéique sur lequel nos cellules sont déposées est celle d’une bande avec un obstacle sur laquelle des cellules sont confinées par un bloc qui,une fois retiré, permet au tissu d’envahir l’espace libre. La première partie de cette thèse étudie cette configuration, d’un point de vue cinématique et protéique. On retient en particulier l’obtention de courbes maîtresses en vitesse et déformation, la corrélation entre vitesse et densité, et l’enrichissement de la vimentine en bord de motif confiné et en front de migration. Un ensemble de relations entre niveaux d’actine, de vimentine, densité, déformation, et vitesse est également trouvé. Enfin, on montre que la direction de la polarisation définie par le vecteur centre de masse du noyau à centre de masse de la cellule est en moyenne orthogonale à la direction de la vitesse.La géométrie en bande à l’avantage de forcer l’écoulement et d’être très contrôlée mais a le désavantage de nécessiter la pose d’un bloc qui n’est pas possible sur un substrat de faible rigidité pour l’étude des forces. Pour contourner ce problème nous avons fermé les conditions aux limites et travaillé sur une géométrie en hippodrome qui possède deux obstacles aux milieux de chacun des bras. Nous avons parié sur la génération spontanée d’un écoulement à grande échelle, ce qui s’est révélé exact. La troisième partie des résultats étudie les hippodromes sur un substrat dur (en opposition au substrat mou pour les forces). On retient la possibilité de générer un flot de longue portée et la présence de vimentine en bord de motif. On retient également que la relation vitesse densité n’est plus valable dans cette géométrie, ce qui atteste que lors qu’elle existe, elle est en fait une conséquence de la dynamique d’étalement du tissu et non de la dynamique de migration en tant que tel. On trouve également une relation entre actine et vimentine qui met en évidence trois états cellulaires possibles. Le premier correspond aux cellules très déformées et riches en vimentine, le second aux cellules très denses et riches en actine, le troisième aux états intermédiaires de déformation et densité où les cellules bougent et où actine et vimentine varient ensemble.La dernière partie de cette thèse consiste à étudier les forces (substrat mou) par microscopie de force de traction. Ces expériences confirment qu’une monocouche en migration est contractile et nous montrons qu’il est équivalent de mesurer le déplacement des billes dans le substrat ou le déplacement des cellules à petite échelle sur des images de phase. En d’autre terme, la vitesse instantanée (celle utilisée par les théoriciens mais non mesurable expérimentalement) est égale à la dérivée des positions des billes. Nous concluons qu’il y a continuité du déplacement entre le substrat et la cellule.À partir d’un objectif initial qui était d’étudier la migration autour d’un obstacle, le développement d’un système à grande échelle sur des hippodromes permet de mieux comprendre les ingrédients sous-jacents de la migration, tout en développant le cadre expérimental qui permet de faire des comparaisons quantitatives entre systèmes.

Grimsley-Myers et al. use mouse genetic and in vitro approaches to determine the role of the VE-cadherin-p120-catenin complex in vascular development. The results indicate that p120 stabilization of VE-cadherin is essential for vascular barrier function, whereas VE-cadherin endocytosis modulates polarized endothelial cell migration and angiogenesis., Tissue morphogenesis requires dynamic intercellular contacts that are subsequently stabilized as tissues mature. The mechanisms governing these competing adhesive properties are not fully understood. Using gain- and loss-of-function approaches, we tested the role of p120-catenin (p120) and VE-cadherin (VE-cad) endocytosis in vascular development using mouse mutants that exhibit increased (VE-cadGGG/GGG) or decreased (VE-cadDEE/DEE) internalization. VE-cadGGG/GGG mutant mice exhibited reduced VE-cad-p120 binding, reduced VE-cad levels, microvascular hemorrhaging, and decreased survival. By contrast, VE-cadDEE/DEE mutants exhibited normal vascular permeability but displayed microvascular patterning defects. Interestingly, VE-cadDEE/DEE mutant mice did not require endothelial p120, demonstrating that p120 is dispensable in the context of a stabilized cadherin. In vitro, VE-cadDEE mutant cells displayed defects in polarization and cell migration that were rescued by uncoupling VE-cadDEE from actin. These results indicate that cadherin endocytosis coordinates cell polarity and migration cues through actin remodeling. Collectively, our results indicate that regulated cadherin endocytosis is essential for both dynamic cell movements and establishment of stable tissue architecture.

In many developmental contexts, epithelial folding is accompanied by neighboring tissue flows towards the invagination site. To assess the idea of a long-range coupling between invagination and tissue flow, we have studied two macroscopic morphogenetic events happening during Drosophila metamorphosis: the invagination of the head-to-thorax boundary and the anterior dorsal thorax flow. Using large-scale laser ablations or locally disrupting MyosinII contractility in the invaginating region, we have found that head-thorax interface invagination is an active process which is providing a pulling force locally driving thorax flow. However, we have discovered that thorax flow is also partly autonomous, and thereby can actively provide a pushing force towards the invagination site. Using forward genetic methods, we have uncovered that thorax flow rely on collective migration on the apical extracellular matrix (ECM), depending on the secretion of the ZP protein Dumpy and on planar polarity cues. We have furthermore shown in vivo that regions of active thorax migration are specifically associated with low apical ECM deformation. Taken together, my PhD work uncovers a new mechanism coordinating tissue folding and tissue flow, and emphasizes the importance of tissue-matrix interaction in epithelial mechanics and morphogenesis., Dans de nombreux contextes développementaux, la formation d’un pli dans un épithélium est accompagnée d’un déplacement des tissus adjacents en direction de la zone d’invagination. Afin de tester l’idée d’un couplage entre un processus d’invagination et un flux tissulaire, nous avons étudié deux mouvements morphogénétiques qui surviennent durant la métamorphose de la drosophile : l’invagination de la frontière séparant la tête du thorax et un déplacement coordonné des cellules du thorax antérieur. En utilisant des techniques d’ablation laser ou de perturbations localisées de la contractilité de la Myosine II dans la région qui se plie, nous avons découvert que l’invagination de l’interface entre la tête et le thorax est un processus actif qui fournit une force de traction qui met localement en mouvement le thorax. Nous avons également découvert que le flux directionnel de thorax est en partie autonome, et qu’il peut être à l’origine d’une force de poussée active vers la zone d’invagination. Nous avons observé que le flux de thorax dépend d’une migration collective des cellules du thorax sur la matrice extracellulaire apicale. Cette migration s’appuie sur la sécrétion de la protéine Dumpy et sur des informations de polarité planaire. Nous avons montré in vivo que les régions thoraciques migrant activement sont spécifiquement associées à de faibles déformations de la matrice extracellulaire apicale. En conclusion, mes travaux de thèse mettent en exergue l’existence d’un nouveau mécanisme de couplage entre invagination et flux de tissu et illustrent l’importance de l’interaction entre le tissu et les matrices extracellulaires dans la mécanique et la morphogenèse épithéliales.

In many developmental contexts, epithelial folding is accompanied by neighboring tissue flows towards the invagination site. To assess the idea of a long-range coupling between invagination and tissue flow, we have studied two macroscopic morphogenetic events happening during Drosophila metamorphosis: the invagination of the head-to-thorax boundary and the anterior dorsal thorax flow. Using large-scale laser ablations or locally disrupting MyosinII contractility in the invaginating region, we have found that head-thorax interface invagination is an active process which is providing a pulling force locally driving thorax flow. However, we have discovered that thorax flow is also partly autonomous, and thereby can actively provide a pushing force towards the invagination site. Using forward genetic methods, we have uncovered that thorax flow rely on collective migration on the apical extracellular matrix (ECM), depending on the secretion of the ZP protein Dumpy and on planar polarity cues. We have furthermore shown in vivo that regions of active thorax migration are specifically associated with low apical ECM deformation. Taken together, my PhD work uncovers a new mechanism coordinating tissue folding and tissue flow, and emphasizes the importance of tissue-matrix interaction in epithelial mechanics and morphogenesis.; Dans de nombreux contextes développementaux, la formation d’un pli dans un épithélium est accompagnée d’un déplacement des tissus adjacents en direction de la zone d’invagination. Afin de tester l’idée d’un couplage entre un processus d’invagination et un flux tissulaire, nous avons étudié deux mouvements morphogénétiques qui surviennent durant la métamorphose de la drosophile : l’invagination de la frontière séparant la tête du thorax et un déplacement coordonné des cellules du thorax antérieur. En utilisant des techniques d’ablation laser ou de perturbations localisées de la contractilité de la Myosine II dans la région qui se plie, nous avons découvert que l’invagination de l’interface entre la tête et le thorax est un processus actif qui fournit une force de traction qui met localement en mouvement le thorax. Nous avons également découvert que le flux directionnel de thorax est en partie autonome, et qu’il peut être à l’origine d’une force de poussée active vers la zone d’invagination. Nous avons observé que le flux de thorax dépend d’une migration collective des cellules du thorax sur la matrice extracellulaire apicale. Cette migration s’appuie sur la sécrétion de la protéine Dumpy et sur des informations de polarité planaire. Nous avons montré in vivo que les régions thoraciques migrant activement sont spécifiquement associées à de faibles déformations de la matrice extracellulaire apicale. En conclusion, mes travaux de thèse mettent en exergue l’existence d’un nouveau mécanisme de couplage entre invagination et flux de tissu et illustrent l’importance de l’interaction entre le tissu et les matrices extracellulaires dans la mécanique et la morphogenèse épithéliales.

Collective cell migration is fundamental throughout development, during wound healing and in many diseases. Although much effort has focused on cell–cell junctions, a role for physical confinement in collective cell migration remains unclear. Here, we used adhesive microstripes of varying widths to mimic the spatial confinement experienced by follower cells within epithelial tissues. Our results reveal that the substrate area confinement is sufficient to modulate the three-dimensional cellular morphology without the need for intercellular adhesive cues. Our findings show a direct correlation between the migration velocity of confined cells and their cell–substrate adhesive area. Closer examination revealed that adhesive area confinement reduces lamellipodial protrusive forces, decreases the number of focal complexes at the leading edge and prevents the maturation of focal adhesions at the trailing edge, together leading to less effective forward propelling forces. The release of follower confinement required for the emergence of leader cells is associated with a threefold increase in contractile stress and a tenfold increase in protrusive forces, together providing a sufficient stress to generate highly motile mesenchymal cells. These findings demonstrate that epithelial confinement alone can induce follower-like behaviours and identify substrate adhesive area confinement as a key determinant of cell velocity in collective migration. Cells migrating within a collective naturally have restricted access to their surroundings. Experiments on micropatterned substrates now show that this confinement can regulate epithelial migration—governing cell morphology, forces and velocity.

International audience; The metastatic progression of cancer is a multi-step process initiated by the local invasion of the peritumoral stroma. To identify the mechanisms underlying colorectal carcinoma (CRC) invasion, we collected live human primary cancer specimens at the time of surgery and monitored them ex vivo. This revealed that conventional adenocarcinomas undergo collective invasion while retaining their epithelial glandular architecture with an inward apical pole delineating a luminal cavity. To identify the underlying mechanisms , we used microscopy-based assays on 3D organotypic cultures of Caco-2 cysts as a model system. We performed two siRNA screens targeting Rho-GTPases effectors and guanine nucleotide exchange factors. These screens revealed that ROCK2 inhibition triggers the initial leader/follower polarization of the CRC cell cohorts and induces collective invasion. We further identified FARP2 as the Rac1 GEF necessary for CRC collective invasion. However, FARP2 activation is not sufficient to trigger leader cell formation and the concomitant inhibition of Myosin-II is required to induce invasion downstream of ROCK2 inhibition. Our results contrast with ROCK pro-invasive function in other cancers, stressing that the molecular mechanism of metastatic spread likely depends on tumour types and invasion mode.

International audience; Despite extensive knowledge on the mechanisms that drive singlecell migration, those governing the migration of cell clusters, as occurring during embryonic development and cancer metastasis,remain poorly understood. Here, we investigate the collective migration of cell on adhesive gels with variable rigidity, using 3D cellular aggregates as a model system. After initial adhesion to the substrate, aggregates spread by expanding outward a cell monolayer, whose dynamics is optimal in a narrowrange of rigidities. Fast expansion gives rise to the accumulation of mechanical tension that leads to the rupture of cell–cell contacts and the nucleation of holes within the monolayer, which becomes unstable and undergoes dewetting like a liquid film. This leads to a symmetry breaking and causes the entire aggregate to move as a single entity. Varying the substrate rigidity modulates the extent of dewetting and induces different modes of aggregate motion: “giant keratocytes,” where the lamellipodium is a cell monolayer that expands at the front and retracts at the back; “penguins,” characterized by bipedal locomotion; and “running spheroids,” for nonspreading aggregates. We characterize these diversemodes of collectivemigration by quantifying the flows and forces that drive them, andwe unveil the fundamental physical principles that govern these behaviors, which underscore the biological predisposition of living material to migrate, independent of length scale.

During the establishment of left-right asymmetry in the zebrafish brain, a small group of cells, the parapineal, collectively migrates from the dorsal midline of the epithalamus to the left in most wild-type embryos. Parapineal migration requires Fibroblastic Growth Factor 8 (Fgf8), a secreted signal expressed bilaterally in epithalamic tissues surrounding the parapineal. The left bias in the orientation of parapineal migration depends on the activity of Cyclops, a secreted factor of the Nodal/TGFß family that is transiently expressed in the left epithalamus prior to parapineal migration. Therefore, the parapineal provides a powerful new model to understand FGF dependent collective cell migration and to study how other signaling pathways modulate this process. Live imaging of an FGF reporter transgene revealed that the FGF pathway is activated in only few parapineal cells that are usually located at the leading edge of migration. Global expression of a constitutively activated Fgf receptor (CA-FGFR) delays migration in wild-type, while it partially restores both parapineal migration and focal activation of the FGF reporter transgene in fgf8-/- mutant embryos. Importantly, focal activation of FGF signaling in few parapineal cells is sufficient to restore collective migration in fgf8-/- mutants. Finally, Nodal asymmetry contributes to restrict and left-bias the activation of the FGF pathway (Manuscript n°1). Following this work, my thesis project aimed at understanding how the activation of the FGF pathway is restricted to few cells, despite all parapineal cells apparently being competent to activate the pathway. We showed that Notch signaling is able to restrict FGF activity. Loss or gain of function of the Notch pathway respectively triggers an increase or decrease in FGF activity, which correlate with PP migration defects. Moreover, decreasing or increasing FGF activity levels respectively rescues or aggravates parapineal migration defects in Notch loss-of-function context. Our data indicate that Notch signaling restricts the activation of the FGF pathway within parapineal cells to promote their collective migration (Manuscript n°2). We also found that Notch pathway is required for the specification of a correct number of parapineal cells, independently of FGF pathway. In parallel, we analysed the function of MMP2 (Matrix Metalloprotease 2), a protein mosaïcally expressed in the parapineal and a candidate to modulate FGF signaling. However, we found no significant defects in the specification or migration of parapineal cells in mmp2-/- mutant embryos (Manuscript n°3). My PhD work reveals a role for Notch signaling in restricting the activation of FGF signaling within few parapineal cells, a process that is biased by Nodal pathway to the left and required for the migration of the entire parapineal. These data provide insights into the interaction of FGF, Notch and Nodal/TGFb signaling pathways that may be applicable to other models of collective cell migration, such as cancer cells migration for instance.; Lors du développement de l'asymétrie gauche droite dans le cerveau du poisson zèbre, un petit groupe de cellules, le parapinéale, migre collectivement depuis la ligne médiane vers la partie gauche de l'épithalamus. Cette migration est défectueuse dans des mutants pour le gène fgf8, indiquant que le facteur Fgf8 (Fibroblast Growth Factor 8), sécrété de part et d'autre de la ligne médiane, est requis pour la migration. Cependant, l'orientation gauche de la migration dépend de l'activation, plus précocement dans l'épithalamus gauche, de la voie de signalisation Nodal/TGFb (Transforming Growth Factor). Par conséquent, la parapinéale est un modèle de choix pour comprendre comment les cellules migrent collectivement en réponse aux Fgf et pour étudier comment d'autres voies de signalisation modulent ce processus. L'imagerie en temps réel d'un transgène rapporteur de la signalisation FGF a révélé que la voie FGF est activée préférentiellement dans quelques cellules de tête, c'est à dire localisées au front de migration. L'expression globale d'un récepteur aux Fgf activé de façon constitutive (CA-FgfR1) interfère avec la migration de la parapinéale en contexte sauvage mais est capable de restaurer à la fois la migration de la parapinéale et l'activation focale de la voie FGF au front de migration dans les mutants fgf8-/-. De plus, l'activation focale de la voie FGF dans seulement quelques cellules de parapinéale est suffisante pour restaurer la migration de tout le collectif dans les mutants fgf8-/-. Finalement, nos données montrent que la signalisation Nodal contribue à restreindre et à biaiser l'activation de la voie FGF afin d'orienter la migration de la parapinéale vers le côté gauche (Manuscript n°1). Par la suite, mes travaux de thèse ont visé à comprendre comment l'activation de la voie FGF est restreinte à quelques cellules, bien que toutes les cellules de parapinéale semblent compétentes pour activer la voie. Nos résultats montrent que la signalisation Notch est capable de restreindre l'activation de la voie FGF. La perte ou le gain de fonction de la voie Notch entrainent respectivement une augmentation ou une diminution de l'activité FGF, associés à des défauts de migration de la parapinéale dans les deux contextes. De plus, la diminution ou l'augmentation artificielle du niveau d'activation de la voie FGF peut respectivement restaurer la migration de la parapinéale ou aggraver les défauts de migration en absence d'activité Notch. Nos données indiquent que la signalisation Notch restreint l'activation de la voie FGF au sein des cellules de parapinéale pour permettre la migration du collectif (Manuscript n°2). La voie Notch est également requise pour la spécification d'un nombre correct de cellules de parapinéale, indépendamment de la voie FGF. En parallèle, nous avons analysé la fonction de MMP2 (Matrix Metalloprotease 2), une protéine exprimée mosaïquement dans la parapinéale et candidate pour moduler la signalisation FGF. Cependant, nous n'avons observé aucun défaut de spécification ou de migration de la parapinéale dans les embryons mutants pour le gène mmp2 -/- (Manuscript n°3). Mon travail de thèse révèle un rôle de la voie Notch pour restreindre l'activation de la signalisation FGF dans quelques cellules de parapinéale, un processus qui est biaisé par la voie Nodal afin d'orienter la migration du collectif vers la gauche. Ces données pourraient permettre de mieux comprendre les interactions entre les voies de signalisation FGF, Notch et Nodal dans d'autres modèles de migration cellulaire collective comme, par exemple, la migration des cellules cancéreuses.

During the establishment of left-right asymmetry in the zebrafish brain, a small group of cells, the parapineal, collectively migrates from the dorsal midline of the epithalamus to the left in most wild-type embryos. Parapineal migration requires Fibroblastic Growth Factor 8 (Fgf8), a secreted signal expressed bilaterally in epithalamic tissues surrounding the parapineal. The left bias in the orientation of parapineal migration depends on the activity of Cyclops, a secreted factor of the Nodal/TGFß family that is transiently expressed in the left epithalamus prior to parapineal migration. Therefore, the parapineal provides a powerful new model to understand FGF dependent collective cell migration and to study how other signaling pathways modulate this process. Live imaging of an FGF reporter transgene revealed that the FGF pathway is activated in only few parapineal cells that are usually located at the leading edge of migration. Global expression of a constitutively activated Fgf receptor (CA-FGFR) delays migration in wild-type, while it partially restores both parapineal migration and focal activation of the FGF reporter transgene in fgf8-/- mutant embryos. Importantly, focal activation of FGF signaling in few parapineal cells is sufficient to restore collective migration in fgf8-/- mutants. Finally, Nodal asymmetry contributes to restrict and left-bias the activation of the FGF pathway (Manuscript n°1). Following this work, my thesis project aimed at understanding how the activation of the FGF pathway is restricted to few cells, despite all parapineal cells apparently being competent to activate the pathway. We showed that Notch signaling is able to restrict FGF activity. Loss or gain of function of the Notch pathway respectively triggers an increase or decrease in FGF activity, which correlate with PP migration defects. Moreover, decreasing or increasing FGF activity levels respectively rescues or aggravates parapineal migration defects in Notch loss-of-function context. Our data indicate that Notch signaling restricts the activation of the FGF pathway within parapineal cells to promote their collective migration (Manuscript n°2). We also found that Notch pathway is required for the specification of a correct number of parapineal cells, independently of FGF pathway. In parallel, we analysed the function of MMP2 (Matrix Metalloprotease 2), a protein mosaïcally expressed in the parapineal and a candidate to modulate FGF signaling. However, we found no significant defects in the specification or migration of parapineal cells in mmp2-/- mutant embryos (Manuscript n°3). My PhD work reveals a role for Notch signaling in restricting the activation of FGF signaling within few parapineal cells, a process that is biased by Nodal pathway to the left and required for the migration of the entire parapineal. These data provide insights into the interaction of FGF, Notch and Nodal/TGFb signaling pathways that may be applicable to other models of collective cell migration, such as cancer cells migration for instance.; Lors du développement de l'asymétrie gauche droite dans le cerveau du poisson zèbre, un petit groupe de cellules, le parapinéale, migre collectivement depuis la ligne médiane vers la partie gauche de l'épithalamus. Cette migration est défectueuse dans des mutants pour le gène fgf8, indiquant que le facteur Fgf8 (Fibroblast Growth Factor 8), sécrété de part et d'autre de la ligne médiane, est requis pour la migration. Cependant, l'orientation gauche de la migration dépend de l'activation, plus précocement dans l'épithalamus gauche, de la voie de signalisation Nodal/TGFb (Transforming Growth Factor). Par conséquent, la parapinéale est un modèle de choix pour comprendre comment les cellules migrent collectivement en réponse aux Fgf et pour étudier comment d'autres voies de signalisation modulent ce processus. L'imagerie en temps réel d'un transgène rapporteur de la signalisation FGF a révélé que la voie FGF est activée préférentiellement dans quelques cellules de tête, c'est à dire localisées au front de migration. L'expression globale d'un récepteur aux Fgf activé de façon constitutive (CA-FgfR1) interfère avec la migration de la parapinéale en contexte sauvage mais est capable de restaurer à la fois la migration de la parapinéale et l'activation focale de la voie FGF au front de migration dans les mutants fgf8-/-. De plus, l'activation focale de la voie FGF dans seulement quelques cellules de parapinéale est suffisante pour restaurer la migration de tout le collectif dans les mutants fgf8-/-. Finalement, nos données montrent que la signalisation Nodal contribue à restreindre et à biaiser l'activation de la voie FGF afin d'orienter la migration de la parapinéale vers le côté gauche (Manuscript n°1). Par la suite, mes travaux de thèse ont visé à comprendre comment l'activation de la voie FGF est restreinte à quelques cellules, bien que toutes les cellules de parapinéale semblent compétentes pour activer la voie. Nos résultats montrent que la signalisation Notch est capable de restreindre l'activation de la voie FGF. La perte ou le gain de fonction de la voie Notch entrainent respectivement une augmentation ou une diminution de l'activité FGF, associés à des défauts de migration de la parapinéale dans les deux contextes. De plus, la diminution ou l'augmentation artificielle du niveau d'activation de la voie FGF peut respectivement restaurer la migration de la parapinéale ou aggraver les défauts de migration en absence d'activité Notch. Nos données indiquent que la signalisation Notch restreint l'activation de la voie FGF au sein des cellules de parapinéale pour permettre la migration du collectif (Manuscript n°2). La voie Notch est également requise pour la spécification d'un nombre correct de cellules de parapinéale, indépendamment de la voie FGF. En parallèle, nous avons analysé la fonction de MMP2 (Matrix Metalloprotease 2), une protéine exprimée mosaïquement dans la parapinéale et candidate pour moduler la signalisation FGF. Cependant, nous n'avons observé aucun défaut de spécification ou de migration de la parapinéale dans les embryons mutants pour le gène mmp2 -/- (Manuscript n°3). Mon travail de thèse révèle un rôle de la voie Notch pour restreindre l'activation de la signalisation FGF dans quelques cellules de parapinéale, un processus qui est biaisé par la voie Nodal afin d'orienter la migration du collectif vers la gauche. Ces données pourraient permettre de mieux comprendre les interactions entre les voies de signalisation FGF, Notch et Nodal dans d'autres modèles de migration cellulaire collective comme, par exemple, la migration des cellules cancéreuses.

International audience; Collective cell migration has a key role during morphogenesis and during wound healing and tissue renewal in the adult, and it is involved in cancer spreading. In addition to displaying a coordinated migratory behaviour, collectively migrating cells move more efficiently than if they migrated separately, which indicates that a cellular interplay occurs during collective cell migration. In recent years, evidence has accumulated confirming the importance of such intercellular communication and exploring the molecular mechanisms involved. These mechanisms are based both on direct physical interactions, which coordinate the cellular responses, and on the collective cell behaviour that generates an optimal environment for efficient directed migration. The recent studies have described how leader cells at the front of cell groups drive migration and have highlighted the importance of follower cells and cell-cell communication, both between followers and between follower and leader cells, to improve the efficiency of collective movement.

The importance of collective cellular migration during embryogenesis and tissue repair asks for a sound understanding of underlying principles and mechanisms. Here, we address recent in vitro experiments on cell monolayers which show that the advancement of the leading edge relies on cell proliferation and protrusive activity at the tissue margin. Within a simple viscoelastic mechanical model amenable to detailed analysis, we identify a key parameter responsible for tissue expansion, and we determine the dependence of the monolayer velocity as a function of measurable rheological parameters. Our results allow us to discuss the effects of pharmacological perturbations on the observed tissue dynamics., 12 pages, 5 figures

Comment in Retrograde flow of cadherins in collective cell migration. Hirata E, Park D, Sahai E. Nat Cell Biol. 2014 Jul;16(7):621-3. doi: 10.1038/ncb2995. PMID: 24981633; International audience; Collective cell migration is essential for both physiological and pathological processes. Adherens junctions (AJs) maintain the integrity of the migrating cell group and promote cell coordination while allowing cellular rearrangements. Here, we show that AJs undergo a continuous treadmilling along the lateral sides of adjacent leading cells. The treadmilling is driven by an actin-dependent rearward movement of AJs and is supported by the polarized recycling of N-cadherin. N-cadherin is mainly internalized at the cell rear and then recycled to the leading edge where it accumulates before being incorporated into forming AJs at the front of lateral cell-cell contacts. The polarized dynamics of AJs is controlled by a front-to-rear gradient of p120-catenin phosphorylation, which regulates polarized trafficking of N-cadherin. Perturbation of the GSK3-dependent phosphorylation of p120-catenin impacts on the stability of AJs, and the polarity and speed of leading cells during collective migration

International audience; 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.

International audience; Collective migration has become a paradigm for emergent behaviour in systems of moving and interacting individual units resulting in coherent motion. In biology, these units are cells or organisms. Collective cell migration is important in embryonic development, where it underlies tissue and organ formation, as well as pathological processes, such as cancer invasion and metastasis. In animal groups, collective movements may enhance individuals' decisions, facilitate navigation through complex environments and access to food resources. Mathematical models can extract unifying principles behind the diverse manifestations of collective migration. In biology, with a few exceptions, collective migration typically occurs at a "mesoscopic scale" where the number of units ranges from only a few dozen to a few thousands, in contrast to the large systems treated by statistical mechanics. Recent developments in multi-scale analysis have allowed to link mesoscopic to micro-and macroscopic scales, and for different biological systems. The articles in this theme issue on "Multi-scale analysis and modelling of collective migration", compile a range of mathematical modelling ideas and multiscale methods for the analysis of collective migration. These approaches (i) uncover new unifying organisation principles of collective behaviour, (ii) shed light on the transition from single to collective migration, and (iii) allow to define similarities and differences of collective behaviour in groups of cells and organisms. As common theme, self-organised collective migration is the result of ecological, and evolutionary constraints both at the cell and organismic levels. Thereby, the rules governing physiological collective behaviours also underlie pathological processes, albeit with different upstream inputs and consequences for the group.

Chemotactic bacteria are known to collectively migrate towards sources of attractants. In confined convectionless geometries, concentration “waves” of swimming Escherichia coli can form and propagate through a self-organized process involving hundreds of thousands of these microorganisms. These waves are observed in particular in microcapillaries or microchannels; they result from the interaction between individual chemotactic bacteria and the macroscopic chemical gradients dynamically generated by the migrating population. By studying individual trajectories within the propagating wave, we show that, not only the mean run length is longer in the direction of propagation, but also that the directional persistence is larger compared to the opposite direction. This modulation of the reorientations significantly improves the efficiency of the collective migration. Moreover, these two quantities are spatially modulated along the concentration profile. We recover quantitatively these microscopic and macroscopic observations with a dedicated kinetic model.

International audience; The authors review how cancer cells may cooperate in metastasis by means of microenvironmental changes. The main mechanisms underlying this cooperation are clustered migration of cancer cells, extracellular matrix degradation, paracrine loops of released signaling factors and/or induction of adhesion molecules on stromal cells. Another critical factor could be temporal cooperation: successive waves of cancer cells may induce progressive conditioning of the microenvironment. The "class action" of cancer cells against the microenvironment involves successive steps of the metastatic process: invasion of the primary tumor microenvironment, collective migration through the extracellular matrix, blood vessel disruption, vascular or lymphatic tumor emboli, establishment of a premetastatic niche by secreted factors and endothelial precursor recruitment, induction of cell adhesion molecule expression in endothelial cells, extravasation, micrometastasis dormancy and establishment of a new growth in distant sites. As a result, after completion of the metastatic process, the series of microenvironmental changes from the primary tumor to the metastatic site may promote colonization of metastases by nonmetastatic cancer cells of the primary tumor.