Neuronal migration is an essential process in the development of the nervous system. Neurons are born in one location and migrate sizable distances to their final location. In many other developmental processes, cells migrate as collectives, where the migration of one cell influences the migration of another cell; this process has yet to be shown in the developing central nervous system. Using the conserved tangential migration of facial branchiomotor neurons (FBMNs), I aim to determine the nature of the collective migration in the developing nervous system. Here, two models of FBMN collective migration are tested: the “Pioneer” model, where following FBMNs migrate intimately on the axon of the first neuron to migrate and the “Contact inhibition of locomotion (CIL)” model, where transient cell-cell contacts are the driving influence of the proper caudal migration of FBMNs. Using fixed tissue imaging, it was found that early born FBMNs do not contact the axon. In contrast, they are more likely to make soma-soma contact and display morphology typical of CIL. FBMNs that do contact the axon do not display an elongated morphology that is predicted of a cell using the leader axon as a substrate for migration. Further, wild-type FBMNs are able to rescue PCP-deficient FBMNs. Therefore, blastula-stage transplantation of PCP-deficient neurons into wild-type hosts allows us to live image the method of collective migration. CIL events were observed between PCP-deficient neurons and wild-type neurons, indicating that PCP is not required for CIL. In addition, PCP-deficient neurons making sustained contact with wildtype axons were not rescued, arguing against the Pioneer model. Taken together, these observations are more consistent with the “CIL” model of FBMN collective migration in which transient soma-soma interactions are required for the coordinated movement of neurons as they migrate in the developing nervous system.

As a physical system, a cell interacts with its environment through physical and chemical processes. The cell can change these interactions through modification of its environment or its own composition. This dissertation presents the overarching hypothesis that both biochemical regulation of intercellular adhesion and physical interaction between cells are required to account for the emergence of cluster migration and collective dynamics observed in epithelial cells. Collective migration is defined as the displacement of a group of cells with transient or permanent cell-cell contacts. One mode, cluster migration, plays an important role during embryonic development and in cancer metastasis. Despite its importance, collective migration is a slow process and hard to visualize, and therefore it has not been thoroughly studied in three dimensions (3D). Based on known information about cluster migration from 2D studies of epithelial sheets and 3D single cell migration, this dissertation presents theoretical and experimental techniques to assess the independent contribution of physical and biochemical factors to 3D cluster migration. It first develops two computational models that explore the interaction between cells and the ECM and epithelial discohesion. These discrete mechanistic models reveal the need to account for intracellular regulation of adherens junctions in space and time within a cluster. Consequently, a differential algebraic model is developed that accounts for cross-reactivity of three pathways in a regulatory biochemical network: Wnt/β-catenin signaling, protein N-glycosylation, and E-cadherin adhesion. The model is tested by matching predictions to Wnt/β-catenin inhibition in MDCK cells. The model is then incorporated into a self-propelled particle (SPP) model, creating the first SPP model for study of adhesive mammalian cellular systems. MDCK cell clusters with fluorescent nuclei are grown, seeded, and tracked in 3D collagen gels using confocal microscopy. They provide data on individual cell dynamics within clusters. Borrowed from the field of complex systems, normalized velocity is used to quantify the order of both in vitro and simulated clusters. An analysis of sensitivity of cluster dynamics on factors describing physical and biochemical processes provides new quantitative insights into mechanisms underlying collective cell migration and explains temporal and spatial heterogeneity of cluster behavior.

A cell's ability to sense and respond to mechanical signals highlights the significance of physical forces in biology; however, to date most biomedical research has focused on genetics and biochemical signaling. We sought to further understand the physical mechanisms that guide the cellular migrations that occur in a number of biological processes, such as tissue development and regeneration, bacterial infections and cancer metastasis. We investigated the migration of single cells and determined whether the biomechanics of these cells could be used to elucidate multi-cellular mechanisms. We first studied Borrelia burgdorferi (Bb), the bacterium that causes Lyme disease. We created a mathematical model based on the mechanical interactions between the flagella and cell body that explained the rotation and undulation of the cell body that occurs as the bacterium swims. This model further predicts how the swimming dynamics could be affected by alterations in flagellar or cell wall stiffnesses. Fitting the model to experimental data allowed us to calculate the flagellar torque and drag for Bb, and showed that Treponema pallidum (Tp), the syphilis pathogen, is biomechanically similar to Bb. Next, we used experimentally-determined parameters of Bb's motility to develop a population-level model that accounts for the morphology and spreading of the "bulls-eye" rash that is typically the first indicator of Lyme disease. This work supported clinical findings on the efficacy of antibiotic treatment regimes. Finally, we investigated the dynamics of epithelial monolayers. We found that intracellular contractile stress is the primary driving force behind collective dynamics in epithelial layers, a result previously predicted from a biophysical model. Taken together, these findings identify the relevance of physics in cellular migration and a role of mechanical signaling in biomedical science.

Pendant la morphogenèse, la réparation des tissus et le cancer, les cellules peuvent migrer en manière mésenchymateuse et collective. Le cytosquelette est essentiel pour la migration, mais alors que l'actine et les microtubules ont été largement étudiés, le rôle des filaments intermédiaires (FIs) est encore largement inconnu. La déplétion des FI diminue souvant la vitesse de migration et les FI sont fréquemment surexprimé dans les tumeurs invasives. Pour ces propriétés, nous supposons que les FIs peuvent jouer un rôle clé dans la mécanique cellulaire pendant la migration.Pour étudier le rôle des FI dans la migration collective, nous avons utilisé des astrocytes, les principales cellules gliales du système nerveux central. Les astrocytes migrent collectivement pendant le développement et l'astrogliose en réponse à des signaux pathologiques ou traumatiques. Les astrocytes expriment trois principales FI cytoplasmiques: nestine, GFAP (protéine acide fibrillaire fibreuse) et vimentine, qui forment un réseau dense. Les FI sont surexprimé pendant l'astrogliose et dans les glioblastomes, des tumeurs cérébrales hautement invasives et létales. On ignore si la surexpression des FI est responsable de l'invasion du glioblastome.Au cours de la migration collective dans un test de blessure, les FI contrôlent le positionnement du noyau, la polarité et la migration. On montre que les FI régulent la migration collective dirigée de manière dépendante de la rigidité. Ils agissent avec la protéine connecteur cytoplasmique plectine pour contrôler les point focaux et les jonctions adhérentes. Les FI contrôlent la dynamique et l'organisation de l'actine et régulent la distribution des tractions cellulaires et des contraintes dans la monocouche migrante. Ces résultats démontrent le rôle crucial des FI dans les propriétés mécaniques des cellules migrantes. / During morphogenesis, tissue repair and cancer, cells can migrate in a mesenchymal and collective manner. The cytoskeleton is essential for migration, but whereas actin and microtubules have been extensively studied, the role of intermediate filaments (IFs) is still largely unknown. IF depletion generally decreases migration speed and IF proteins are frequently found upregulated in invasive tumours. Because of IF properties, we hypothesise that they may be key players in cell mechanics during migration. To study the role of IFs in collective migration we used astrocytes, the main glial cells of the central nervous system. Astrocytes migrate collectively during development and astrogliosis in response to pathological or traumatic signals. Astrocytes express three main cytoplasmic IFs: nestin, GFAP (Glial Fibrillary Acidic Protein) and vimentin, which form a dense network. IF proteins are upregulated during astrogliosis and glioblastomas, highly invasive and lethal brain tumours. Whether upregulation of IFs is responsible for glioblastoma invasion is still unknown. During wound-induced collective migration, IFs control nuclear positioning, polarisation and migration. We found that IFs regulate collective directed migration in a stiffness-dependent manner. They act in concert with the cytolinker protein plectin to control focal adhesions and adherens junctions. IFs control actin dynamics and organisation and regulate the distribution of cell tractions and stresses across the migrating cell monolayer. These results demonstrate the crucial role of IFs in the mechanical properties of migrating cells.

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of many studies, the effects that environmental changes have on this form of movement are poorly understood. In certain pathologies where the cornea exists in a chronic hypoxic state, wound healing is delayed. The goal of this work is to examine the changes in corneal structure in hypoxic corneas that may affect migration and to determine the effects that changes in basement membrane stiffness and focal adhesion turnover have on epithelial cell migration. We analyzed migration after injury in organ cultures and determined that hypoxic corneas exhibited alterations in leading edge morphology. Under hypoxia, fibronectin localization to the apical epithelium and anterior stroma was reduced. Investigators have suggested that alterations in basal lamina composition may increase the stiffness of the membrane. To examine the effect that increased stiffness has on collective migration we performed traction force microscopy. Using multi-layered corneal epithelial sheets, we developed a novel method to analyze the generation of cellular traction forces and the directionality of sheet movement on polyacrylamide gels. We determined that the leading edges of corneal epithelial sheets undergo contraction prior to migration. Alterations in stiffness affected the amount of force exerted by cells at the leading edge. On stiffer surfaces, individual cells within sheets exhibited greater movement compared to cells on softer substrates. To further assess sheet dynamics, we examined the activation of focal adhesion proteins in hypoxic corneas and in human corneal limbal epithelial (HCLE) cells seeded onto soft and rigid substrates. Wounded hypoxic corneas displayed alterations in the localization of the focal adhesion proteins paxillin and vinculin. In HCLE cells cultured on stiff substrates, there was an elevation in vinculin pY1065 phosphorylation after injury, a reduction in vinculin-positive focal adhesions, and decreased actin bundle thickness. Our results demonstrate that changes in membrane stiffness may affect cellular tractions and vinculin dynamics, possibly contributing to the delayed healing response associated with certain corneal pathologies.

No description available.

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.

Kollektive Migration und Schwarmverhalten sind Beispiele für Selbstorganisation und können in verschiedenen biologischen Systemen beobachtet werden, beispielsweise in Vogel-und Fischschwärmen oder Bakterienpopulationen. Im Zentrum dieser Arbeit steht ein räumlich diskretes und zeitlich stetiges Model, welches das kollektive Migrieren von Individuen mittels eines stochastischen Vielteilchensystems (VTS) beschreibt und analysierbar macht. Das konstruierte Modell ist in keiner Klasse gut untersuchter Vielteilchensysteme enthalten, sodass der größte Teil der Arbeit der Entwicklung von Methoden zur Untersuchung des Langzeitverhaltens bestimmter VTS gewidmet ist. Eine entscheidende Rolle spielen hier Gibbs-Maße, die zu zeitlich invarianten Maßen in Beziehung gesetzt werden. Durch eine Simulationsstudie und die Analyse des Einflusses der Parameter Migrationsgeschwindigkeit, Sensitivität der Individuen und (räumliche) Dichte der Anfangsverteilung können Eigenschaften kollektiver Migration erklärt und Hypothesen für weitere Analysen aufgestellt werden. / Collective migration and swarming behavior are examples of self-organization and can be observed in various biological systems, such as in flocks of birds, schools of fish or populations of bacteria. In the center of this thesis lies a stochastic interacting particle system (IPS), which is a spatially discrete model with a continuous time scale that describes collective migration and which can be treated using analytical methods. The constructed model is not contained in any class of well-understood IPS’s. The largest part of this work is used to develop methods that can be used to study the long-term behavior of certain IPS’s. Thereby Gibbs-Measures play an important role and are related to temporally invariant measures. One can explain the properties of collective migration and propose a hypothesis for further analyses by a simulation study and by analysing the parameters migration velocity, sensitivity of individuals and (spatial) density of the initial distribution.

Kollektive Migration und Schwarmverhalten sind Beispiele für Selbstorganisation und können in verschiedenen biologischen Systemen beobachtet werden, beispielsweise in Vogel-und Fischschwärmen oder Bakterienpopulationen. Im Zentrum dieser Arbeit steht ein räumlich diskretes und zeitlich stetiges Model, welches das kollektive Migrieren von Individuen mittels eines stochastischen Vielteilchensystems (VTS) beschreibt und analysierbar macht. Das konstruierte Modell ist in keiner Klasse gut untersuchter Vielteilchensysteme enthalten, sodass der größte Teil der Arbeit der Entwicklung von Methoden zur Untersuchung des Langzeitverhaltens bestimmter VTS gewidmet ist. Eine entscheidende Rolle spielen hier Gibbs-Maße, die zu zeitlich invarianten Maßen in Beziehung gesetzt werden. Durch eine Simulationsstudie und die Analyse des Einflusses der Parameter Migrationsgeschwindigkeit, Sensitivität der Individuen und (räumliche) Dichte der Anfangsverteilung können Eigenschaften kollektiver Migration erklärt und Hypothesen für weitere Analysen aufgestellt werden. / Collective migration and swarming behavior are examples of self-organization and can be observed in various biological systems, such as in flocks of birds, schools of fish or populations of bacteria. In the center of this thesis lies a stochastic interacting particle system (IPS), which is a spatially discrete model with a continuous time scale that describes collective migration and which can be treated using analytical methods. The constructed model is not contained in any class of well-understood IPS’s. The largest part of this work is used to develop methods that can be used to study the long-term behavior of certain IPS’s. Thereby Gibbs-Measures play an important role and are related to temporally invariant measures. One can explain the properties of collective migration and propose a hypothesis for further analyses by a simulation study and by analysing the parameters migration velocity, sensitivity of individuals and (spatial) density of the initial distribution.

Editorial on the Research Topic. Editorial: Editor’s Pick 2021: Highlights in Cell Adhesion and Migration.