Modélisation et simulation 3D de la morphogenèse

The embryo of the Drosophila Melanogaster undergoes a series of cell movements during its early development. Gastrulation is the process describing the segregation of the future internal tissues into the interior of the developing embryo. Gastrulation starts with the formation of the ventral furrow, a process commonly known as the ventral furrow invagination. During this process, the most ventrally located blastoderm cells flatten and progressively constrict their apical sides until they are wedge shaped. As a result of these cell-shape changes, the blastoderm epithelium first forms an indentation, the ventral furrow, which is then completely internalized. We focus on the study of the mechanisms that drive the invagination. The main questions that gave birth to this thesis are: “What is the role of the apical constriction of the ventral cells in the invagination?” and “Once the ventral cells are internalized, what is the mechanism that drives the ventral closure?” We attempt to answer to these two questions from a biomechanical point of view. For this purpose, a 3D mesh of the embryo of the Drosophila Melanogaster has been created. Based on this mesh, two “a minima” biomechanical models of the Drosophila embryo have been created, a physically based discrete model and a model based on the Finite Element Method. The results of the simulations in both models show that the geometry of the embryo plays a crucial role in the internalization of the ventral cells. The two models efficiently simulate the internalization of the ventral cells but are incapable of reproducing the ventral closure. We hypothesize that the ventral closure can be explained by the interplay of forces developed in the embryo once the internalized ventral cells undergo cell division. We propose an approach to divide elements in a Finite Element Mesh and we integrate it to the Finite Element Model of the Drosophila Melanogaster.L'embryon de la Drosophila Melanogaster subit une série des mouvements cellulaires pendant son développement. La gastrulation est le processus qui décrit la différentiation des futurs tissus à l'intérieur de l'embryon. La gastrulation commence par la formation du sillon ventral, un processus connu sous le nom de “Ventral Furrow Invagination”. Pendant ce processus, les cellules de la blastoderme positionnées dans la région ventrale de l'embryon, aplatissent et contractent leur surface apicale jusqu'à ce qu'elles deviennent prismatiques. Ce changement de forme cellulaire aboutit à un enfoncement au niveau de la région ventrale, le sillon ventral, qui est ensuite totalement intériorisé. Nous focalisons notre étude sur les mécanismes qui conduisent à l'invagination. Les questions principales auxquelles ce travail de thèse essaie de répondre sont: “Quel est le rôle de la contraction apicale des cellules ventrales dans l'invagination?” et “Quel est le mécanisme qui conduit à la clôture ventrale, une fois les cellules ventrales intériorisées?”. Nous essayons de répondre à ces questions d'un point de vue biomécanique. Dans ce but, un maillage 3D de l'embryon de la Drosophila Melanogaster a été créé. Basés sur ce maillage, deux modèles biomécaniques “a minima” de l'embryon de la Drosophila ont été créés: un modèle physique discret et un modèle basé sur la Méthode des Eléments Finis. Les résultats des simulations des deux modèles montrent que la géométrie joue un rôle décisif dans l'intériorisation des cellules ventrales. Les deux modèles ont permis de simuler l'intériorisation des cellules ventrales mais se trouvent incapables de simuler la clôture ventrale. Notre hypothèse est que la clôture ventrale peut s'expliquer par l'intéraction des forces développées à l'intérieur de l'embryon, une fois que les cellules ventrales commencent à proliférer. Nous proposons une méthode pour diviser des éléments dans un maillage d'éléments finis et ensuite nous expliquons l'intégration de cette méthode dans le modèle des Eléments Finis pour l'embryon de la Drosophila Melanogaster

Amélioration de l'image et la segmentation (applications en imagerie médicale)

Avancement dans l'acquisition d'image et le progrès dans les méthodes de traitement d'image ont apporté les mathématiciens et les informaticiens dans les domaines qui sont d'une importance énorme pour les médecins et les biologistes. Le diagnostic précoce de maladies (comme la cécité, le cancer et les problèmes digestifs) ont été des domaines d'intérêt en médecine. Développement des équipements comme microscope bi-photonique à balayage laser et microscope de fluorescence par réflexion totale interne fournit déjà une bonne idée des caractéristiques très intéressantes sur l'objet observé. Cependant, certaines images ne sont pas appropriés pour extraire suffisamment d'informations sur de cette image. Les méthodes de traitement d'image ont été fournit un bon soutien à extraire des informations utiles sur les objets d'intérêt dans ces images biologiques. Rapide méthodes de calcul permettent l'analyse complète, dans un temps très court, d'une série d'images, offrant une assez bonne idée sur les caractéristiques souhaitées. La thèse porte sur l'application de ces méthodes dans trois séries d'images destinées à trois différents types de diagnostic ou d'inférence. Tout d'abord, Images de RP-muté rétine ont été traités pour la détection des cônes, où il n'y avait pas de bâtonnets présents. Le logiciel a été capable de détecter et de compter le nombre de cônes dans chaque image. Deuxièmement, un processus de gastrulation chez la drosophile a été étudié pour observer toute la mitose et les résultats étaient cohérents avec les recherches récentes. Enfin, une autre série d'images ont été traités où la source était une vidéo à partir d'un microscopie photonique à balayage laser. Dans cette vidéo, des objets d'intérêt sont des cellules biologiques. L'idée était de suivre les cellules si elles subissent une mitose. La position de la cellule, la dispersion spatiale et parfois le contour de la membrane cellulaire sont globalement les facteurs limitant la précision dans cette vidéo. Des méthodes appropriées d'amélioration de l'image et de segmentation ont été choisies pour développer une méthode de calcul pour observer cette mitose. L'intervention humaine peut être requise pour éliminer toute inférence fausse.Advancement in Image Acquisition Equipment and progress in Image Processing Methods have brought the mathematicians and computer scientists into areas which are of huge importance for physicians and biologists. Early diagnosis of diseases like blindness, cancer and digestive problems have been areas of interest in medicine. Development of Laser Photon Microscopy and other advanced equipment already provides a good idea of very interesting characteristics of the object being viewed. Still certain images are not suitable to extract sufficient information out of that image. Image Processing methods have been providing good support to provide useful information about the objects of interest in these biological images. Fast computational methods allow complete analysis, in a very short time, of a series of images, providing a reasonably good idea about the desired characteristics. The thesis covers application of these methods in 3 series of images intended for 3 different types of diagnosis or inference. Firstly, Images of RP-mutated retina were treated for detection of rods, where there were no cones present. The software was able to detect and count the number of cones in each frame. Secondly, a gastrulation process in drosophila was studied to observe any mitosis and results were consistent with recent research. Finally, another series of images were treated where biological cells were observed to undergo mitosis. The source was a video from a photon laser microscope. In this video, objects of interest were biological cells. The idea was to track the cells if they undergo mitosis. Cell position, spacing and sometimes contour of the cell membrane are broadly the factors limiting the accuracy in this video. Appropriate method of image enhancement and segmentation were chosen to develop a computational method to observe this mitosis. Cases where human intervention may be required have been proposed to eliminate any false inference.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Two new distinct mechanisms drive epithelial folding in Drosophila wing imaginal discs

Epithelial folding is an important morphogenetic process that is essential in transforming simple sheets of cells into complex three-dimensional tissues and organs during animal development (Davidson, 2012). Epithelial folding has been shown to rely on constriction forces generated by the apical actomyosin network (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). However, the contributions of mechanical forces acting along lateral and basal cell surfaces to epithelial folding remain poorly understood. Here we combine live imaging with force measurements of epithelial mechanics to analyze the formation of two epithelial folds in the Drosophila larval wing imaginal disc. We show that these two neighboring folds form via two distinct mechanisms. These two folds are driven either by decrease of basal tension or increase of lateral tension, none of them depends on apical constriction. In the first fold, a local decrease in extracellular matrix (ECM) density in prefold cells results in a reduction of mechanical tension on the basal cell surface, leading to basal expansion and fold formation. Consistent with that, a local reduction of ECM by overexpression of Matrix metalloproteinase II is sufficient to induce ectopic folding. In the second fold a different mechanism is at place. Here basal tension is not different with neighboring cells, but pulsed dynamic F-actin accumulations along the lateral interface of prefold cells lead to increased lateral tension, which drives cell shortening along the apical-basal axis and fold formation. In this thesis I described two distinct mechanisms driving epithelial folding, both basal decrease and lateral increase in tension can generate similar morphological changes and promote epithelial folding in the Drosophila wing discs.Die Faltung von Epithelien ist ein wichtiger morphogenetischer Prozess, der die Entstehung komplexer, dreidimensionaler Gewebe und Organe aus einfachen Zellschichten ermöglicht (Davidson, 2012). Es ist bekannt, dass Kräfte erzeugt durch das apikale Aktomyosin-Netzwerk wichtig sind für die erfolgreiche Faltung von Epithelien (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). Die Rolle von mechanischen Kräften, die entlang der lateralen und basalen Seite wirken, ist jedoch kaum verstanden. Wir verbinden Lebendmikroskopie mit der Messung von mechanischen Eigenschaften, um die Entstehung von 2 Epithelfalten in den Imaginalscheiben von Drosophila zu verstehen. Wir können dadurch zeigen, dass die beiden Falten durch unterschiedliche Mechanismen entstehen. Sie entstehen entweder durch eine Verringerung der Spannung auf der basalen Seite oder durch eine Erhöhung der Spannung auf der lateralen Seite, aber keine von beiden entsteht durch zusammenziehende Kräfte auf der apikalen Seite. Die erste Falte entsteht durch eine lokale Verringerung der extrazellulären Matrix in den Vorläuferzellen, was zu einer Reduktion der Spannung auf der basalen Seite und zur Ausbildung der Falte führt. Die zweite Falte wird durch einen anderen Mechanismus ausgebildet. Hier ist nicht die Spannung auf der basalen Seite reduziert sondern dynamische Anreicherungen von F-Aktin auf der lateralen Seite resultieren in einer erhöhten lateralen Spannung, die zu einer Verkürzung der Zellen und damit zur Ausbildung einer Falte führt. In meiner Arbeit zeige ich 2 neue Mechanismen zur Entstehung von Epithelfalten auf, durch Absenken der Spannung auf der basalen oder Erhöhen auf der lateralen Seite

Molecular, Cellular and Mechanical basis of Epithelial Morphogenesis during Tribolium Embryogenesis

Embryonic development entails a series of morphogenetic events which require a precise coordination of molecular mechanisms coupled with cellular dynamics. Phyla such as arthropods show morphological and gene expression similarities during middle embryogenesis (at the phylotypic germband stage), yet early embryogenesis adopts diverse developmental strategies. In an effort towards understanding patterns of conservation and divergence during development, investigations are required beyond the traditional model systems. Therefore, in the past three decades, several insect species representing various insect orders have been established as experimental model systems for comparative developmental studies. Among these, the red flour beetle Tribolium castaneum has emerged as the best studied holometabolous insect model after the fruit fly Drosophila melanogaster. Unlike Drosophila, Tribolium is a short-germ insect that retains many ancestral characters common to most insects. The early embryogenesis of Tribolium shows dynamic epithelial rearrangements with an epibolic expansion of the extraembryonic tissue serosa over the embryo, the folding of the embryo in between the serosa and the second extra embryonic tissue amnion and the folding of the amnion underneath the embryo. These extensive tissues are evolutionarily conserved epithelia that undergo different tissue movements and are present in varying proportions in different insects, providing exceptional material to compare and contrast morphogenesis during early embryogenesis. However, most of the previous work on insects including Tribolium have largely focused on the conservation and divergence of gene expression patterns and on gene regulatory interactions. Consequently, very little studies on dynamic cell behaviour have been done and we lack detailed information about the cellular and tissue dynamics during these early morphogenetic events. During my PhD, I first established a live imaging and data analysis pipeline for studying Tribolium embryogenesis in 4-D. I combined live confocal and lightsheet imaging of transgenic or transiently labelled embryos with mechanical or genetic perturbations using laser ablations and gene knockdowns. Using this pipeline quantifications of cell dynamics and tissue behaviours can be done to compare different regions of the embryo as the development proceeds. In the second and third part of my thesis, I describe the actomyosin dynamics and associated cell behaviours during the stages of serosa epibolic expansion, amniotic fold formation and serosa window closure. I cloned and characterised the cellular dynamics of the Tribolium spaghetti squash gene (Tc-squash) - the non-muscle Myosin II regulatory light chain, which is the main molecular force generator in epithelial cells. Interestingly, the analysis of Tc-squash dynamics indicates a conserved role of Myosin II in controlling similar cell behaviours across short germ and long germ embryos. In the last part of the thesis, I report the dynamics of an actomyosin cable that emerges at the interface of the serosa and amnion. This cable increases in tension during development, concomitant with serosa tissue expansion and increased tensions in the serosa. It behaves as a modified purse string as it’s circumference shrinks due to a decrease in the number of cable forming cells over time. This shrinkage is an individual contractile property of the cells forming the cable. This indicates that a supracellular and contractile actomyosin cable might be functional during serosa window closure in insects with distinct serosa and amnion tissues. Further, the tension in the cable might depend on the relative proportion of the serosa, amnion and embryonic regions. Using these integrated approaches, I have correlated global cellular dynamics during early embryogenesis with actomyosin behaviours, and then performed a high-resolution analysis and perturbations of selected events. The established imaging, image processing and perturbation tools can serve as an important basis for future investigations into the tissue mechanics underlying Tribolium embryogenesis and can also be adapted for comparisons of morphogenesis in other insect embryos. More broadly, correlating the existing genetic, mechanical and biochemical understanding of developmental processes from Drosophila with species such as Tribolium, could help identify deeply conserved design principles that lead to different morphologies through differences in underlying regulation.:Page List of Tables v List of Figures vii 1 Introduction 1 1.1 Evo-Devo of insects 3 1.2 Tribolium castaneum 5 1.3 Fluorescence live imaging and lightsheet microscopy 10 1.4 Morphogenesis 15 1.5 Thesis objective 29 2 4D lightsheet imaging and analysis pipeline of Tribolium embryos 33 2.1 Standardisation of an injection protocol for sample mounting and imaging with the Zeiss LZ1 SPIM 35 2.2 Double labelling of Tribolium embryos 37 2.3 Image processing with Fiji 37 2.4 Long term timelapse imaging of Tribolium embryogenesis with SPIM 44 2.5 2D cartographic projections of 3D data as a method to visualise and analyse SPIM data 47 2.6 Summary 59 3 Cellular dynamics of the non muscle Myosin II regulatory light chain - Tc-Squash 61 3.1 Tc-Squash dynamics during Tribolium embryogenesis 64 3.2 Myosin drives basal cell closure during blastoderm cellularisation 66 3.3 Myosin shows planar polarity in the embryonic tissue 69 3.4 Myosin accumulation and apical constriction of putative germ cells at the posterior pole 71 3.5 Myosin pulses during apical constriction of mesoderm cells 74 3.6 Myosin accumulates at the extraembryonic-embryonic boundary to form a contractile supracellular cable 77 3.7 Summary 77 4 A supracellular actomyosin cable operates during serosa epiboly 79 4.1 Actin and Myosin accumulate at the extraembryonic-embryonic boundary 81 4.2 The actomyosin assembly migrates ventrally till it forms the rim of the serosa window 82 4.3 The actomyosin cable shows dynamic shape changes during serosa window closure 87 4.4 Serosa cells increase in area till circular serosa window stage 89 4.5 Tension in the serosa tissue increases during epibolic expansion 89 4.6 Serosa cells decrease their apical areas after laser ablation 92 4.7 Tension in the actomyosin cable increases during serosa epiboly 93 4.8 Myosin dynamics at the cable changes between early and serosa window stage 96 4.9 Individual cell membrane shrinkage and cell rearrangements decrease the cable circumference 98 4.10 Myosin dynamics at the cable during serosa window closure 101 4.11 Tension in the cable is not relieved after multiple laser cuts 103 4.12 Analysis of the actomyosin cable in Tc-zen 1 knockdown 105 4.13 Summary 109 5 Discussion 111 5.1 Reconstruction of insect embryogenesis using lightsheet microscopy and tissue cartography 111 5.2 Conserved Myosin II behaviours and its implications on morphogenesis across insects 114 5.3 A contractile supracellular actomyosin cable functions serosa window closure in Tribolium 119 6 Materials and Methods 123 6.1 Tribolium stock maintenance 123 6.2 RNA extraction and cDNA synthesis 124 6.3 Cloning of templates for mRNA synthesis and transgenesis 124 6.4 dsRNA synthesis for RNAi experiments 126 6.5 Capped, single stranded RNA synthesis 126 6.6 Fluorescence image acquisition 27 A Appendix 131 Bibliography 14

Scutoids unveil the three-dimensional packing in curved epithelia

As animals develop, the initial simple planar epithelia of the early embryos must acquire complex three-dimensional architectures to form the final functional tissues of the organism. Epithelial bending is, therefore, a general principle of all developing systems. Scholarly publications depict epithelial cells as prisms where their basal and apical faces resemble polygons with the same number of sides. The accepted view is that, when a tissue bend, the cells of the epithelia modify their shape from columnar to what has been traditionally called “bottle shape”. However, the morphology and packing of curved epithelia remain largely unknown. Here, through mathematical and computational modelling, we show that cells in bent epithelia necessarily undergo intercalations along the apico-basal axis. This event forces cells to exchange their neighbours between their basal and apical surfaces. Therefore, the traditional view of epithelial cells as simple prisms is incompatible with this phenomenon. Consequently, epithelial cells are compelled to adopt a novel geometrical shape that we have named “scutoid”. The in-depth analysis of diverse epithelial tissues and organs confirm the generation of apico-basal transitions among cell during morphogenesis. Using biophysics arguments, we determine that scutoids support the energetic minimization on the tissue and conclude that the transitions along the apico-basal axis stabilize the threedimensional packing of the tissue. Altogether, we argue that scutoids are nature’s solution to bend efficiently epithelia, and the missing piece for developing a unifying and realistic model of epithelial architecture

Investigation of the role of Rho GTPase signalling in cell shape changes during Drosophila morphogenesis

Morphogenesis, the generation of the shape of an organism, requires several cellular processes including cell migration, cell division and cell shape changes. These processes are mainly mediated by the cell cytoskeleton, which is regulated in part by Rho family of small GTPases. One activator of Rho that is known to be important in morphogenetic cell shape change is RhoGEF2. RhoGEF2 is itself activated by Folded gastrulation and Concertina during gastrulation. Genetic interactions between folded gastrulation or concertina and RhoGEF2 were apparent in developmental processes other than gastrulation, showing conservation of a signalling pathway that activates cell shape change. Protein Kinase N and Serum Response Factor, both known targets of Rho signalling, interact, presumably indirectly, with RhoGEF2. Alleles of ten putative novel Rho signalling pathway components also interact with RhoGEF2, indicating the existence of other proteins involved in regulation of cell shape change in morphogenesis. Signalling through Rho results in many diverse outcomes. One major question in the field relates to the mechanism used by this single protein to select a particular outcome. The hypothesis tested here is that the individual guanine nucleotide exchange factor that activates Rho participates in the selection. If this is the case, activation of the exchange factor would be expected always to result in the same outcome. From a series of experiments it is shown that RhoGEF2 promotes shape changes and epithelial folding in all tissues studied, but has no observed effect on any other Rho-mediated processes studied. The cellular and molecular function of RhoGEF2 was analysed during gastrulation. Time-lapse monitoring of the dynamic process of gastrulation in wild type embryos revealed features that have not been observed in fixed tissues. RhoGEF2 appears to be important in the accumulation of myosin, presumably for apical cellular constriction. RhoGEF2 possibly receives several signals during gastrulation, one of which is likely to be from the FGF receptor Heartless. If this is the case, it explains many unanswered questions regarding the regulation of cell shape change in Drosophila gastrulation

Optogenetic modulation of Delta reveals the role of Notch signalling dynamics during tissue differentiation

Spatio-temporal regulation of signalling pathways plays an important role in generating diverse responses during the development of multicellular organisms. While increasing number studies are uncovering the importance of signalling dynamics in controlling tissue patterning and morphogenesis, the precise role of signal dynamics in transferring information in vivo is incompletely understood owing to the lack of methods to manipulate protein activity at the relevant spatio-temporal scales. In this PhD thesis, I employ genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto-Delta), at its endogenous locus. Light mediated activation of opto-Delta disrupts Notch signalling during different developmental stages. Using clonal analysis, I show that optogenetic activation blocks Notch activation through cis-inhibition in signal-receiving cells. To investigate how a Notch input is dynamically translated into a differentiation output, I focused on mesectoderm specification during early Drosophila embryogenesis. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals different modes of regulation at the tissue and cellular level. While at the tissue-level the duration of Notch signalling determines the probability with which a cellular response will occur, in individual cells Notch activation needs to reach a minimum threshold to generate a response. Taken together these results provide novel insights into the dynamic input-output regulation of Notch signalling, supporting a model in which the Notch receptor is an integrator of (noisy) analog signals that generates a digital switch-like behaviour at the level of target gene expression during tissue differentiation. In order to further test this model, I attempted to develop an optogenetic system to activate Notch in vivo (opto-Notch). Despite showing light-responsive changes in localization, a certain level of Notch is activated even prior to photo-activation, thus necessitating further optimization. Finally, I describe efforts for further characterization of opto-Delta as a tool to spatially perturb signalling, to study Notch signalling during neuroblast delamination, and for adaptation to mammalian cell-culture systems

Regulation of Actomyosin Contraction during Tissue Morphogenesis: Genes and Mechanics

Contraction of cortical actomyosin networks drives cell shape changes and is of fundamental importance during morphogenesis of multicellular organisms. Over the course of the last decade, an increasing number of studies has demonstrated the key role played by localized myosin activation at the apical surface of epithelial cells during a wide range of tissue morphogenetic processes. However, recent in vitro studies and computational models suggest that the architecture of the underlying actin network is also important. Furthermore, whether apical myosin-derived forces alone are sufficient to drive cell shape changes and morphogenesis remains highly debated, as the role of the basolateral surface must also be taken into account. In this thesis, by focusing on the dynamic remodeling of a basally localized actomyosin network required for early Drosophila embryonic development, I provide strong evidence in support of the role of actin network organization in controlling contractility. Using a combination of genetic, biochemical, and optogenetic approaches, I identified a mechanism based on actin crosslinkers, which regulate the spatial organization of actin networks and thereby time actomyosin contraction during cellularization, the transformation of the syncytial embryo in 6000 mononucleated epithelial cells. I further demonstrate that following cellularization, myosin-II activity at the basal surface of ventral cells must be downregulated in order to allow efficient apical constriction, cell wedging and ventral furrow invagination. Collectively the results presented in this thesis provide novel insights into the underlying spatiotemporal organization of actomyosin networks and molecular principles regulating actomyosin contraction during tissue morphogenesis