Forces requises pour les changements de forme au cours de la morphogenèse des tissus sont générés par d’actine et de myosine. Durant ma thèse, je étudié le rôle de la réglementation MyoII par la voie Rho1-Rok durant l’élongation de l’ectoderme ventro-latéral par intercalation cellulaire. Les pulsations de MyoII médio-apicale se déplacent de manière anisotrope vers les jonctions parallèles avec l’axe dorso-ventral (ou jonctions verticales). Ceci provoque le rétrécissement graduel des jonctions qui sont stabilisées par une population de MyoII polarisée dans le plan du tissu et enrichie au niveau de ces jonctions. Les mécanismes cellulaires qui régulent la pulsatilité, la stabilité et la polarité de la myosine II restent à élucider. J’ai identifié deux propriétés cruciales de la dynamique de la myosine II régie par phospho- à savoir la cinétique d’échange gouvernée par les cycles de phosphorylation-déphosphorylation des chaines légères régulatrices de la MyoII (RLC) et l’advection due à la contraction des moteurs sur le réseau de F-actine. Contrôle spatial sur le chiffre d'affaires MyoII établit 2 régimes stables des taux élevés et faibles dissociation résultant dans MyoII polarité. Pulsatilité est un comportement auto-organisé qui émerge à taux de dissociation intermédiaires permettant d'advection MyoII et les régulateurs en amont. Dans la deuxième partie de ma thèse, je l'ai montré que la protéine GPCR- GRsmog et la brume, et la voie G-protéines en aval permettent l'activation progressive des MyoII, établissant pulsatilité et de la stabilité pour produire des déformations de forme polarisées cours de la morphogenèse. / Forces required to power shape changes during tissue morphogenesis are generated by non-muscle MyosinII (MyoII) pulling filamentous actin. During my PhD, I investigated the role of MyoII regulation through the conserved Rho1-Rok pathway during Drosophila germband extension. The morphogenetic process is powered by cell intercalation involving shrinkage of junctions in the dorsal-ventral axis (‘vertical junctions’) followed by junction extension in the anterior-posterior axis. Advances in light microscopy revealed that the actomyosin networks exhibit pulsed contractions to power junction shrinkage, and alternate with steps of stabilization by MyoII enriched on vertical junctions (planar-polarity) to result in irreversible shape changes. Although described in many different contexts, the underlying mechanisms of this ratchet-like behavior remained unclear. Using genetic and biophysical tools, quantitative imaging and subtle perturbations, I identified 2 critical properties underlying MyoII dynamics- turnover governed by phospho-cycling of the MyoII Regulatory Light Chain, and advection due to contraction of the motors on actin networks. Spatial control over MyoII turnover establishes 2 stable regimes of high and low dissociation rates resulting in MyoII planar polarity. Pulsatility is a self-organized behavior that emerges at intermediate dissociation rates enabling advection of MyoII and upstream regulators. In the second part of my thesis, I showed that G protein coupled receptors- GRsmog and Mist, and the downstream G-protein pathway allow step-wise activation of MyoII, establishing pulsatility and stability, to drive polarized shape deformations during morphogenesis.

L'induction expérimentale de jumeaux (oursin) ou de siamois (poisson zébré) sollicite la plasticité et la robustesse de l'embryon en développement. Nous avons montré que les individus jumeaux et les paires de siamois suivent des voies de développement différentes de celles du type normal. Nous émettons l’hypothèse que ces différents chemins révèlent la plasticité développementale de l’organisme. Chez les deux espèces, la formation de jumeaux ou siamois est interprétée comme reposant sur i) la labilité des gradients de morphogènes déterminant les axes embryonnaires, ii) le répertoire des comportements cellulaires au cours de la blastulation et de la gastrulation en réponse aux gradients de morphogènes et aux signaux biomécaniques et/ou iii) les processus de régulation de la taille des champs morphogénétiques. Nous avons montré que le développement des jumeaux d'oursins explore un domaine de possibilités différent de celui des embryons typiques. Les jumeaux se développent d'abord sous forme d'une couche cellulaire unique et plate avant de se compacter en véritable blastula. Les jumeaux en développement appartiennent à 3 catégories phénotypiques en fonction du nombre de précurseurs de feuillets embryonnaires (nombre correct ou non) et de leurs contacts (contacts corrects ou non). Cependant, les 3 classes conduisent à un pourcentage similaire de larves de pluteus normales. Nous démontrons qu’une réorganisation cellulaire qui rétablirait des contacts normaux entre les différents types cellulaires n'est pas nécessaire, ce qui implique que le développement passe par des changements de destinées cellulaires. En outre, les larves de jumeaux ont un nombre normal de cellules mésenchymateuses primaires, sans division cellulaire supplémentaire. Cette situation est réminiscente de l'ablation des micromères dont la perte est compensée par un changement de destinée cellulaires des cellules mésenchymateuses secondaires. Les poissons zébrés siamois peuvent être obtenus par la surexpression de la voie nodale, la transplantation de l'organisateur de Spemann au début de la gastrulation, ou chez le mutant homozygote maternel Janus. Notre travail s'est concentré sur le premier cas. L’interaction des voies de signalisation dans la détermination des axes embryonnaires et dans l’instauration d’un centre d’organisation de type Spemann a fait l’objet de nombreuses études chez le poisson zébré. Cependant, l’état de l’art n’explique pas le lien entre les signaux biochimiques et les comportements cellulaires individuels, ni l’émergence de champs morphogénétiques et la régulation de leur taille. Lorsque organisateur endogène et organisateur induit ne sont pas en positions parfaitement opposées, l’étendue de la régulation de la taille des champs morphogénétique dépend de la position de l'organisateur induit. Le lignage cellulaire de poissons zébrés siamois reconstruit à partir de d’imagerie 3D + temps in toto révèle les comportements cellulaires sous-jacents à la fusion de champs morphogénétiques identiques. / The experimental induction of twins (sea urchin) or Siamese (zebrafish) challenges the plasticity and robustness of the developing embryo. We showed that individuals in twin and Siamese pairs follow different developmental paths that are in fact quite different than wild-type. We hypothesize that these different paths reveal the organism’s developmental plasticity. In both species, twinning is interpreted to rely on i) the lability of morphogen gradients setting the embryonic axes, ii) the repertoire of cell behaviors during blastulation and gastrulation in response to morphogen gradients and biomechanical cues and/or iii) the regulation of morphogenetic field size. We showed that the development of sea urchin twins explores a realm of possibilities different from that of typical embryos. The twins develop first as a single and flat cell layer before compacting into a bona fide blastula. Developing twins belong to 3 phenotypic categories depending on the number of germ layer precursors (correct number or not) and their contacts (correct contacts or not). However, the 3 classes lead to similar percentage of normal pluteus larvae. We demonstrate that cellular reorganization is not required to restore the normal contacts between the different cell types implying that development proceeds through fate changes. In addition, larvae from twins have a normal number of primary mesenchymal cells and this does not involve further cell division. This situation is reminiscent of the ablation of micromeres with transfating compensating for the loss, leading to a proper number of PMCs and is thus likely to involve the transfating of SMCs.Siamese zebrafish are observed through Nodal pathway overexpression, transplantation of the Spemann organizer at the onset of gastrulation, or in the maternal homozygous mutant Janus. Our work focused on the first case. The interplay of signaling pathways in setting embryonic axes and inducing a Spemann-type organizing center has been extensively studied in zebrafish. However, the current state of art does not explain the link between biochemical cues and individual cell behaviors, and from them the emergence of morphogenetic fields and the regulation of their size. If not in opposite positions, the extent of field size regulation is dependent on the position of the induced or transplanted organizer. The cell lineage of siamese zebrafish reconstructed from 3D+time live imaging reveals cell behaviors underlying the fusion of identical fields.

During gastrulation, a set of highly coordinated morphogenetic movements creates the shape and internal organization of the embryo. In teleostean fishes, these morphogenetic movements involve not only the embryonic progenitor cells (deep cells) but also two extra-embryonic tissues: an outer sheet of epithelial cells (EVL) and a yolk syncytial layer (YSL). Epiboly is characterized by the spreading of the blastoderm (deep cells and EVL) to cover the large yolk cell, whereas convergence and extension leads, respectively, to mediolateral narrowing and anteroposterior elongation of the embryo. Recent studies have shown that the nuclei of the YSL undergo epiboly and convergence and extension movements similarly to the overlying deep cells, suggesting that these tissues interact during gastrulation. However, it is so far not clear whether and how the movements of YSL nuclei and deep cells influence each other. In the first part of this thesis, the convergence and extension movement of YSL nuclei was quantitatively compared to the movement of the overlying mesendodermal progenitor (or “hypoblast)” cells. This revealed that, besides the similarity in the overall direction of movement, YSL nuclei and hypoblast cell movements display differences in speed and directionality. Next, the interaction between YSL and hypoblast was addressed. The movement of the blastoderm was analyzed when YSL nuclei movement was impaired by interfering with the YSL microtubule cytoskeleton. We found that YSL and blastoderm epiboly were strongly reduced, while convergence and extension were only mildly affected, suggesting that YSL microtubules and YSL nuclei movement are required for epiboly, but not essential for convergence and extension of the blastoderm. We also addressed whether blastodermal cells can influence YSL nuclei movement. In maternal-zygotic one-eyed pinhead (MZoep) mutant embryos, which lack hypoblast cells, YSL nuclei do not undergo proper convergence movement. Moreover, transplantation of wild type hypoblast cells into these mutants locally rescued the YSL nuclei convergence phenotype, indicating that hypoblast cells can control the movement of YSL nuclei. Finally, we propose that the hypoblast influences YSL nuclei movement as a result of shape changes caused by the collective movement of cells, and that this process requires the adhesion molecule E-cadherin.

During zebrafish gastrulation, highly coordinated cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. Recent studies have identified silberblick (slb/wnt11) as a key molecule that regulates gastrulation movement through a conserved pathway, which shares significant similarity with a signalling pathway that establishes epithelial planar cell polarity (PCP) in Drosophila (Heisenberg et al., 2000; Veeman et al., 2003), suggesting a role for cell polarity in regulating gastrulation movements. However, the cellular and molecular mechanisms by which slb/wnt11 functions during zebrafish gastrulation are still not fully understood. In the first part of the thesis, the three-dimensional movement and morphology of individual cells in living embryos during the course of gastrulation were recorded and analysed using high resolution confocal microscopy. It was shown that in slb/wnt11 mutant embryos, hypoblast cells within the forming germ ring display slower, less directed migratory movements at the onset of gastrulation, which are accompanied by defects in the orientation of cellular processes along the individual movement directions of these cells. The net movement direction of the cells is not changed, suggesting that slb/wnt11-mediated orientation of cellular processes serves to facilitate and stabilize cell movements during gastrulation. By using an in vitro reaggregation assay on mesendodermal cells, combined with an analysis of the endogenous expression levels and distribution of E-cadherin in zebrafish embryos at the onset of gastrulation, E-cadherin mediated adhesion was found to be a downstream mechanism regulating slb/wnt11 function during gastrulation. Interestingly, the effects of slb/wnt11 on cell adhesion appear to be dependent on Rab5-mediated endocytosis, suggesting endocytic turnover of cell-cell contacts as one possible mechanism through which slb/wnt11 mediates its effects on gastrulation movements. - Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: QuickTimeMovies (ca. 23 MB)- Übersicht über Inhalte siehe Dissertation S. 92 - 93"

Gastrulation leads to the formation of the embryonic germ layers, ectoderm, mesoderm and endoderm, and is the first key morphogenetic process that occurs in development. Gastrulation provides a unique developmental assay system in which to study cellular movements and rearrangements in vivo. The different cell movements occurring during gastrulation take place in a highly coordinated spatial and temporal manner, indicating that they must be controlled by a complex interplay of morphogenetic and inductive events. Generally, cell movement constitutes a highly integrated program of different cellular behaviors including sensing, polarization, cytoskeletal reorganization, and changes in adhesion and cell shape. During migration, these different behaviors require a continuous regulation and feedback control to direct and coordinate them. In this work, we analyze the cellular and molecular mechanisms underlying the different types of cell behaviors during gastrulation in zebrafish. Specifically, we focus on the role of the adhesive and mechanical properties of germ layer progenitors in the regulation of gastrulation movements. In the first part of the project, we investigated the role of the adhesive and mechanical properties of the different germ layer progenitor cell types for germ layer separation and stratification. In the second part of this study, we applied the same methodology to determine the function of germ layer progenitor cell adhesion in collective cell migration. Tissue organization is thought to depend on the adhesive and mechanical properties of the constituent cells. However, it has been difficult to determine the precise contribution of these different properties due to the lack of tools to measure them. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of the different germ layer progenitor cell types. Applying this methodology, we demonstrate that mesoderm and endoderm progenitors are more adhesive than ectoderm cells and that E-cadherin is the main adhesion molecule regulating this differential adhesion. In contrast, ectoderm progenitors exhibit a higher actomyosin-dependent cell cortex tension than mesoderm and endoderm progenitors. Combining these data with tissue self-assembly in vitro and in vivo, we provide evidence that the combinatorial activities of cell adhesion and cell cortex tension direct germ layer separation and stratification. It has been hypothesized that the directionality of cell movement during collective migration results from a collective property. Using a single cell transplantation assay, we show that individual progenitor cells are capable of normal directed migration when moving as single cells, but require cell-cell adhesion to participate in coordinated and directed migration when moving collectively. These findings contribute to the understanding of the gastrulation process. Cell-cell adhesion is required for collective germ layer progenitor cell migration, and cell cortex tension is critical for germ layer separation and stratification. However, many questions still have to be solved. Future studies will have to explore the interaction between the adhesive and mechanical progenitor cell properties, as well as the role of these properties for cell protrusion formation, cell polarization, interaction with extracellular matrix, and their regulation by different signaling pathways.

No description available.

Gastrulation leads to the formation of the embryonic germ layers, ectoderm, mesoderm and endoderm, and is the first key morphogenetic process that occurs in development. Gastrulation provides a unique developmental assay system in which to study cellular movements and rearrangements in vivo. The different cell movements occurring during gastrulation take place in a highly coordinated spatial and temporal manner, indicating that they must be controlled by a complex interplay of morphogenetic and inductive events. Generally, cell movement constitutes a highly integrated program of different cellular behaviors including sensing, polarization, cytoskeletal reorganization, and changes in adhesion and cell shape. During migration, these different behaviors require a continuous regulation and feedback control to direct and coordinate them. In this work, we analyze the cellular and molecular mechanisms underlying the different types of cell behaviors during gastrulation in zebrafish. Specifically, we focus on the role of the adhesive and mechanical properties of germ layer progenitors in the regulation of gastrulation movements. In the first part of the project, we investigated the role of the adhesive and mechanical properties of the different germ layer progenitor cell types for germ layer separation and stratification. In the second part of this study, we applied the same methodology to determine the function of germ layer progenitor cell adhesion in collective cell migration. Tissue organization is thought to depend on the adhesive and mechanical properties of the constituent cells. However, it has been difficult to determine the precise contribution of these different properties due to the lack of tools to measure them. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of the different germ layer progenitor cell types. Applying this methodology, we demonstrate that mesoderm and endoderm progenitors are more adhesive than ectoderm cells and that E-cadherin is the main adhesion molecule regulating this differential adhesion. In contrast, ectoderm progenitors exhibit a higher actomyosin-dependent cell cortex tension than mesoderm and endoderm progenitors. Combining these data with tissue self-assembly in vitro and in vivo, we provide evidence that the combinatorial activities of cell adhesion and cell cortex tension direct germ layer separation and stratification. It has been hypothesized that the directionality of cell movement during collective migration results from a collective property. Using a single cell transplantation assay, we show that individual progenitor cells are capable of normal directed migration when moving as single cells, but require cell-cell adhesion to participate in coordinated and directed migration when moving collectively. These findings contribute to the understanding of the gastrulation process. Cell-cell adhesion is required for collective germ layer progenitor cell migration, and cell cortex tension is critical for germ layer separation and stratification. However, many questions still have to be solved. Future studies will have to explore the interaction between the adhesive and mechanical progenitor cell properties, as well as the role of these properties for cell protrusion formation, cell polarization, interaction with extracellular matrix, and their regulation by different signaling pathways.

An early event in animal development is the formation of the three primary germ layers that define the body plan. During gastrulation, cells migrate through the primitive streak of the embryo and undergo changes in morphology and gene expression, thus creating the mesodermal and endodermal cell layers. Gastrulation requires expression of Fibroblast Growth Factor (FGF), Wnt, and Platelet-Derived Growth Factor (PDGF). Embryos treated with FGF inhibitors fail to gastrulate, as cell migration is completely halted. During gastrulation, 44 microRNAs are expressed in the primitive streak of G. gallus embryos, and six (microRNAs -let7b, -9, -19b, -107, -130b, and -218) are strongly upregulated when FGF signaling is blocked. The abundance of these six FGF-regulated microRNAs is controlled at various stages of processing: most are regulated transcriptionally, and three of them (let7b, 9, and 130b) are blocked by the presence of Lin28B, an RNA-binding protein upregulated by FGF signaling. These microRNAs target various serine/threonine and tyrosine kinase receptors. We propose a novel pathway by which FGF signaling downregulates several key microRNAs (partially through Lin28B), upregulating gene targets such as PDGFRA, which permits and directs cell migration during gastrulation. These findings add new layers of complexity to the role that FGF signaling plays during embryogenesis. FGF signaling is also required for the formation of the heartfields, and has an overlapping pattern of expression with BMP (Bone Morphogenetic Protein). A microarray experiment using inhibitors of FGF and BMP found that thousands of genes in pre-cardiac mesoderm are affected by FGF signaling, BMP signaling, or a cooperative effect of the two. The promoter regions of similarly regulated genes were queried for over-represented transcription factor binding sites or novel DNA motifs. Cluster analysis of over-represented sites determined candidate transcriptional modules that were tested in primary cardiac myocyte and fibroblast cultures. About 75% of predicted modules in FGF-upregulated genes proved to be functional enhancers or repressors. Functional enhancers among FGF-upregulated genes contained clusters of CdxA and NFY sites, and increased transcription in the presence of a constitutively active FGF receptor.

Mon travail de thèse a été principalement de comprendre comment les voies de signalisations contrôlent la différenciation et la morphogenèse de l'embryon de vertébré. Les communications entre cellules sont à la base du développement des métazoaires et de leurs évolutions et sont souvent impliquées dans les pathologies humaines. J'ai profité de la puissance des approches fonctionnelles chez le xenope pour essayer de comprendre comment les signaux BMP, FGF, Nodal et Notch sont intégrés dans le temps et l'espace afin de coordonnées différentes décisions cellulaires. Premièrement, nous avons montré que la voie Nodal est active avant la transition mid-blastuléene et permet l'induction du mesedoderme à travers l'auto régulation de l'expression de ces ligands Xnr5 et Xnr6 (Skirkanish et al. soumis à Development). Deuxièmement, j'ai montré que différent ligand de la voie Nodal contrôlent séquentiellement l'induction du mesendoderm et les mouvements de gastrulation (Luxardi et al., Development, 2010). Troisièmement, j'ai montré qu'un cinquième ligand de la voie Nodal, Xnr4, contrôle la régionalisation médio latérale de la plaque neurale ouverte et la neurogenèse. Quatrièmement, nous avons montré qu'une famille de microARN, nir449, contrôle la différenciation des cellules multi-ciliées à travers son action sur un ligand de la voie Notch, Delta-1 (Marcet et al. Nature Cell Biology, en révision). Enfin, j'ai découvert une nouvelle fonction des signaux BMP dans le control de la spécification des épithéliums muco cilié. / My PhD work generally addressed how signaling pathways control differentiation and morphogenesis in the vertebrate embryo. intercellular communication is the basis of metazoan development and evolution and is often involved in human pathologies. I take advantage of the power of functional approaches in the Xenopus embryo, to try and understand how BMP, FGF, Nodal and Notch signals are intragrated in time ans space to coordinate vatious cellular decisions. First, we showed that Nodal signaling is activated before the mid blastula transition and allow mesendoderm induction through the auro regulation of the expression of its ligands Xnr5 and Xnr6 (Skirkanish et al., submitted to development). Second, I have demonstrated that in a gastrulation movements (Luxardi et al., Development, 2010). Third, I have demonstrated that a fifth Nodal ligand, Xnr4, control medio-lateral patterning of the open neural plate and neurogenesis. Froth, we showed that a microRNA family, mir449, controls differenciation of multiciliated cells through the regulation of the Notch ligand Delta-1 (Marcet et al. Nature Cell Biology, in revision). Last, I have discovered a novel function of the BMP pathway in the control of cell type specification within the epidermal mucocialiary epithelium

Coordinated inductive and morphogenetic processes of gastrulation establish the zebrafish body plan. Gastrulation includes massive cell rearrangements to generate the three germ layers and shape the embryonic body. Three modes of cell migration must occur during vertebrate gastrulation and include: epiboly, internalization of the presumptive mesendoderm and convergent extension (C&E). C&E movements narrow the germ layers mediolaterally (convergence) and elongate them anteroposteriorly (extension) to define the embryonic axis. The molecular mechanisms regulating coordinated cell migrations remain poorly understand and studying these has become of great interest to researchers. Understanding cell migration during development is highly relevant to a number of human physiological processes. Abnormal cell migration during early development can lead to congenital defects, with improper cell migration during adult life potentially leading to the invasion and metastasis of cancer. By studying cell migration events, in vivo, new insights are to be found to both the function and malfunction of key embryonic and postembryonic migratory events. The non-canonical Wnt pathway has been identified as an evolutionarily conserved signaling pathway, regulating C&E cell movements during vertebrate gastrulation. With the absence of the non-canonical Wnts (ncWnts), Wnt5 and Wnt11, during zebrafish development leading to a shorter and broader body axis with defects in elongation during segmentation resulting in undulation of the notochord. While it is clear ncWnts are necessary for C&E, many of the downstream effectors regulating these cell movements have not been defined. Previous research has shown that activation of ncWnt signaling through Wnt5 or Wnt11 results in an increase in intracellular Ca2+ during zebrafish gastrulation. To determine if the Ca2+/Calmodulin-dependent protein kinase, CaMK-II, is a potential downstream target of the Ca2+ increases during ncWnt activation, CaMK-II’s role in C&E was assessed. This study identifies camk2b1 and camk2g1 as being necessary for C&E movements, and outlines the phenotype of the overall embryo as well as individual cells of camk2b1 and camk2g1 morphants. The defects of CaMK-II morphants are specifically linked to alterations in C&E cell movements, while cell fate and proliferation are unaffected. An increase in CaMK-II activation during gastrulation produces similar C&E defects, demonstrating the specificity of CaMK-II’s activation in facilitating these highly coordinated cellular movements. We show that CaMK-II is working downstream Wnt 11 and in parallel to JNK signaling during gastrulation C&E. Overall, these data identify CaMK-II as a required component of C&E movements during zebrafish development, downstream ncWnt signaling, and altering cell migration through changes in cell shape