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Dynamin is Required for the Maintenance of Enveloping Layer Integrity and Epiboly Progression in the Zebrafish EmbryoLepage, Stephanie E 19 June 2014 (has links)
During early development, a series of regulated cell movements is required to set up the adult body plan of an organism. Collectively referred to as gastrulation, these coordinated cell movements organize the germ layers and establish the major body axes of the embryo. One such coordinated cell movement, epiboly, describes the thinning and spreading of a multilayered cell sheet to cover the embryo during gastrulation. The zebrafish embryo has emerged as a vital model system to study the cellular and molecular mechanisms that drive epiboly. In the zebrafish, the blastoderm undergoes epiboly to engulf the yolk cell and close the blastopore at the vegetal pole. This is achieved through the coordinated movement of the deep cells, which make up the embryo proper, and two extra-embryonic tissues, the enveloping layer and yolk syncytial layer. Epiboly is essential to the development of most organisms; however, the cellular and molecular mechanisms driving epiboly are poorly understood.
Here I report the findings of two distinct projects which addressed the cellular and molecular basis for epiboly in the zebrafish. One cellular mechanism thought to be involved in driving epiboly is the removal of yolk cell membrane ahead of the advancing blastoderm margin. Using a combination of drug- and dominant-negative based approaches to inhibit Dynamin, a key component of the endocytic machinery, I demonstrated that marginal yolk cell endocytosis is dispensable for the successful completion of epiboly. Instead, I found that Dynamin primarily acts in the blastoderm where it maintains integrity of the enveloping layer (EVL) during epiboly. Dynamin maintains EVL integrity through regulation of the Ezrin/Radixin/Moesin (ERM) family of proteins and the activity of the small GTPase Rho A. With the goal of identifying genes involved in regulating epiboly, I characterized the calpain family of calcium-dependent cysteine proteases in the zebrafish and examined the developmental expression patterns of these genes. My study provided insight into the evolution of this large gene family. Furthermore, I found that most members of this family are expressed in the early embryo, suggesting that they may play a role in regulating early developmental processes such as epiboly.
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Dynamin is Required for the Maintenance of Enveloping Layer Integrity and Epiboly Progression in the Zebrafish EmbryoLepage, Stephanie E 19 June 2014 (has links)
During early development, a series of regulated cell movements is required to set up the adult body plan of an organism. Collectively referred to as gastrulation, these coordinated cell movements organize the germ layers and establish the major body axes of the embryo. One such coordinated cell movement, epiboly, describes the thinning and spreading of a multilayered cell sheet to cover the embryo during gastrulation. The zebrafish embryo has emerged as a vital model system to study the cellular and molecular mechanisms that drive epiboly. In the zebrafish, the blastoderm undergoes epiboly to engulf the yolk cell and close the blastopore at the vegetal pole. This is achieved through the coordinated movement of the deep cells, which make up the embryo proper, and two extra-embryonic tissues, the enveloping layer and yolk syncytial layer. Epiboly is essential to the development of most organisms; however, the cellular and molecular mechanisms driving epiboly are poorly understood.
Here I report the findings of two distinct projects which addressed the cellular and molecular basis for epiboly in the zebrafish. One cellular mechanism thought to be involved in driving epiboly is the removal of yolk cell membrane ahead of the advancing blastoderm margin. Using a combination of drug- and dominant-negative based approaches to inhibit Dynamin, a key component of the endocytic machinery, I demonstrated that marginal yolk cell endocytosis is dispensable for the successful completion of epiboly. Instead, I found that Dynamin primarily acts in the blastoderm where it maintains integrity of the enveloping layer (EVL) during epiboly. Dynamin maintains EVL integrity through regulation of the Ezrin/Radixin/Moesin (ERM) family of proteins and the activity of the small GTPase Rho A. With the goal of identifying genes involved in regulating epiboly, I characterized the calpain family of calcium-dependent cysteine proteases in the zebrafish and examined the developmental expression patterns of these genes. My study provided insight into the evolution of this large gene family. Furthermore, I found that most members of this family are expressed in the early embryo, suggesting that they may play a role in regulating early developmental processes such as epiboly.
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Towards an Understanding of Zebrafish Epiboly: The Characterization of the Epiboly Initiation Mutant Eomesodermin ADu, Susan 31 December 2010 (has links)
How cell movements are coordinated during
morphogenesis is not well understood. We focus on epiboly, which describes the thinning and spreading of a multilayered cell sheet. The first phase of epiboly involves the doming of the yolk
cell up into the overlying blastoderm. We previously showed that over-expression of a dominant– negative eomesodermin a construct inhibits doming. Here I report my analysis of embryos lacking both maternal and zygotic Eomesodermin A (MZeomesa). eomesafh105 mutant embryos (1) exhibit a doming delay, (2) have defective yolk cell microtubules, (3) have tightly packed deep cells with more bleb – like protrusions and (4) express early endoderm markers abnormally.
Despite these phenotypes, the majority of MZeomesa embryos are able to complete epiboly and form endodermal derivatives. In both Xenopus and mice, Eomesodermin has also been implicated in the regulation of gastrulation movements and cell fate specification, suggesting a
conserved role for Eomesodermin throughout vertebrate development.
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Towards an Understanding of Zebrafish Epiboly: The Characterization of the Epiboly Initiation Mutant Eomesodermin ADu, Susan 31 December 2010 (has links)
How cell movements are coordinated during
morphogenesis is not well understood. We focus on epiboly, which describes the thinning and spreading of a multilayered cell sheet. The first phase of epiboly involves the doming of the yolk
cell up into the overlying blastoderm. We previously showed that over-expression of a dominant– negative eomesodermin a construct inhibits doming. Here I report my analysis of embryos lacking both maternal and zygotic Eomesodermin A (MZeomesa). eomesafh105 mutant embryos (1) exhibit a doming delay, (2) have defective yolk cell microtubules, (3) have tightly packed deep cells with more bleb – like protrusions and (4) express early endoderm markers abnormally.
Despite these phenotypes, the majority of MZeomesa embryos are able to complete epiboly and form endodermal derivatives. In both Xenopus and mice, Eomesodermin has also been implicated in the regulation of gastrulation movements and cell fate specification, suggesting a
conserved role for Eomesodermin throughout vertebrate development.
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Evidence for partial epithelial-to-mesenchymal transition and recruitment of motile blastoderm edge cells during avian epibolyFutterman, Matthew 06 June 2011 (has links)
Embryonic epiboly has become an important developmental model for studying the mechanisms underlying collective movements of epithelial cells. In the last couple of decades, most studies of epiboly have utilized Xenopus or zebrafish as genetically tractable model organisms, while the avian epiboly model has received virtually no attention. Here, we re-visit epiboly in quail embryos and characterize several molecular markers of epithelial-to-mesenchymal transition (EMT) in the inner zone of the extraembryonic Area Opaca and at the blastoderm edge. Our results show that the intermediate filament vimentin, a widely-used marker of the mesenchymal phenotype, is strongly expressed in the edge cells compared to the cells in the inner zone, and that epiboly is inhibited when embryos are treated with Withaferin-A, a vimentin-targeting drug. Laminin, an extracellular matrix protein that is a major structural and adhesive component of the epiblast basement membrane, is notably absent from the blastoderm edge, and shows three distinct morphological regions approaching the leading edge. While these expression profiles are consistent with a mesenchymal phenotype, several other epithelial markers, including cytokeratin, β-catenin, and E-cadherin, were present in the blastoderm edge cells. Moreover, the results of a BrDU proliferation assay suggest that expansion of the edge cell population is primarily due to recruitment of cells from the inner zone, and not proliferation. Taken together, our data suggest that the edge cells of the avian blastoderm have characteristics of both epithelial and mesenchymal cells, and could serve as an in-vivo model for cancer and wound healing studies.
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Global Analysis Of Transcriptional Control Driving Zebrafish GastrulationSimon Wilkins Unknown Date (has links)
Gastrulation, literally “formation of the gut” is ultimately an inadequate term to describe one of the most dynamic periods during vertebrate developmental biology. During gastrulation coordinated cell movements reshape the non-descript blastula into the structured gastrula and simultaneously specify the three germ layers: endoderm, mesoderm and ectoderm. The morphogenetic movements of gastrulation are highly conserved between species, but the links between their genetic and biomechanical regulation are poorly understood. The zebrafish embryo – externally hatched, optically clear and amenable to genetic manipulation – is an ideal vertebrate model in which to study both morphogenetic movements and their genetic control. This thesis provides a detailed analysis of the zebrafish Mix-type homeobox transcription factor, Mtx2, both in terms of its role in gastrulation and the molecular mechanisms regulated by Mtx2. This approach involved detailed examination of the Mtx2 loss-of-function phenotype and, subsequently, use of this phenotype as the basis for a microarray screen to identify and investigate Mtx2-dependent genes. One specific Mtx2-dependent gene, katanin-like 1 was investigated further by loss-of-function studies. Prior to this study the mtx2 gene was identified by homology, within its homeodomain, to other Mix-family transcription factors, but both its function and transcriptional targets remained unknown. Using a morpholino knockdown approach, this thesis demonstrates that Mtx2 is essential for vegetal movement (epiboly), but not specification, of the embryonic germ layers and extra-embryonic tissues during zebrafish gastrulation. The recruitment of filamentous actin (F-actin) to a punctate band at the blastoderm margin, was previously shown to be responsible for progression of epiboly. However, formation of this structure is demonstrated to be Mtx2-dependent. Microarray expression profiling of the Mtx2 loss-of-function phenotype was performed to screen for novel genes with roles in gastrulation. This approach identified Mtx2-dependent genes with roles in cytoskeletal dynamics, cell-cell adhesion and endocytosis and vesicular trafficking – processes known to be involved in morphogenetic movements. Many Mtx2-dependent genes are co-expressed with mtx2 in the extra-embryonic yolk syncytial layer (YSL), the teleost functional equivalent of mammalian visceral endoderm. The subset of Mtx2-dependent genes co-expressed with mtx2 and that contain Mtx2-binding sites within their 2kb proximal promoter represent the genes with the greatest likelihood of being direct Mtx2 transcriptional targets. A novel homologue of the microtubule severing protein Katanin, known as katanin-like 1 (katnal1) met all these conditions. Morpholino knockdown of Katnal1 demonstrates that like Mtx2, Katnal1 is essential for gastrulation in zebrafish. A cloned Katnal1mCherry fusion construct was observed to associate with microtubules, and demonstrated bi-directional trafficking around transfected mammalian cells. Analysis of the microtubule network in wild-type and morpholino injected zebrafish embryos demonstrated that remodelling of the extensive microtubule network found in the YSL and yolk cytoplasmic layer (YCL) is Katnal1-dependent. Nuclear division within the YSL and F-actin recruitment to the blastoderm margin are also Katnal1-dependent. This thesis therefore demonstrates, for the first time directly, the multiple, specific roles played by the microtubule network of the YSL/YCL. Katnal1 is highly conserved from Drosophila to mammals and is dynamically expressed during mouse gastrulation. The Mtx2 binding motif in the katnal1 2kb proximal promoter can be bound by both Mtx2 and its putative mouse homologue Mixl1. This suggests that katnal1 may also be a direct target of Mtx2. At the technical level, these results demonstrate the validity of screening for novel developmentally important genes using a zebrafish microarray-based approach, the potential of such an approach to, ab initio, identify a candidate list of transcription factor targets and confirm the utility of the zebrafish as a developmental model. At the biological level, this work collectively suggests that Mtx2 is a central regulator of the morphogenetic movement of epiboly and that Katnal1-dependent microtubule remodelling drives multiple aspects of gastrulation, potentially from Drosophila through to humans.
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Evolutionary implication of mechanotransduction in developmentBouclet, Adrien 17 June 2014 (has links) (PDF)
In this thesis, I first focused on the testing of the hypothesis of the mechanotransductive activation of the apical accumulation of Myosin-II (Myo-II) that leads to Drosophila embryos mesoderm invagination, in response to the active cell apex pulsations preceding gastrulation in the mesoderm. This hypothesis was proposed on the basis of previous experiments realized in my host lab, having consisted in the rescue of mesoderm invagination in pulsation and invagination defective mutants, in response to a simple mechanical indent of the mesoderm. Here I demonstrated quantitatively the plausibility of such mechanical trigger of the active apical accumulation of Myo-II leading to subsequent mesoderm invagination, in response to the mechanical strains developed by the endogenous pulsative movements of mesoderm cell apexes, in silico. In a second part, I tested experimentally the role of the mechanical strains developed by the very first morphogenetic movements of zebrafish (Danio rerio) and Drosophila embryos, in the early specification of mesoderm cells identity. Specifically, to test this hypothesis, I developed magnetic biophysical tools to mimic the epiboly morphogenetic movements in epiboly defective zebrafish embryos. We found the beta-catenin (B-cat) Y667 phosphorylation as the common mechano-transductive pathway involved in earliest mesoderm genes expression notail and twist respectively, in response to the very first morphogenetic movements of embryogenesis in both species, epiboly and mesoderm invagination, respectively. This allowed to suggest such mechanotransduction pathway as conserved from the last common ancestor of both species, namely the last common ancestor of bilaterians, therefore possibly involved in the origins of mesoderm emergence in the ancestor, which represents a currently important opened question of evo-devo. In a third part, I developed experiments of mechanical indent of Drosophila embryos germ cells, and demonstrated the production of generational heritable developmental defects induced on at least 3 generations. These experiments suggest accidental mechanical perturbation of germ cells as a putative new motor mode of heritable modulations in the genetic developmental program of embryogenesis, with the molecular mechanism underlying such transmission being currently in progress.
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Evolutionary implication of mechanotransduction in development / Implication d'un point de vue évolutif de la méchanotransduction dans le développementBouclet, Adrien 17 June 2014 (has links)
Durant ma thèse je me suis intéressé à trois sujets différents connectés par la méchanotransduction au cours du développement. Mon intérêt principal étant porté sur les impacts évolutifs apportés par la méchanotransduction. Mais qu’est-ce que la méchanotransduction ? C’est la conversion d’un stimulus mécanique en une activité biochimique. Ces mécanismes sont présents dans bien des domaines : motilité des cellules, différentiation cellulaire, création de structures. Durant ma thèse je me suis principalement intéressé aux mécanismes de méchanotransduction liés à l’invagination du mésoderme chez la drosophile. Ce mouvement est l’un des tous premiers mouvements morphogénétique et fait partie d’une étape clé du développement qu’est la gastrulation mécanisme commun à toutes les espèces animales. Dans un premier temps je me suis focalisé cette invagination du mésoderme chez la Drosophile. Cette invagination est générée par des accumulations apicales de myosine qui vont entrainer une constriction apicale des cellules. Mon premier travail a été de comprendre et de modéliser ce mouvement en me basant sur le travail effectué dans le laboratoire. En effet il a été prouvé que les contraintes mécaniques entrainent directement l’invagination du mésoderme : par une indentation sur le mésoderme d’embryons incapables de réaliser une invagination on arrive à restaurer cette dernière. La modélisation que j’ai réalisée permet de montrer la plausibilité de réaliser un couplage bio-mécanique afin d’expliquer la formation de cette invagination. Le modèle est composé d’une chaine de cellules couplées mécaniquement entre elles par des jonctions adhérentes. Les cellules sont excitées individuellement par une force sinusoïdale leur taille va donc être modifiée. Grace au couplage certains comportements collectifs vont apparaitre. Pour les cellules associées à certaines conditions génétiques une fois que la taille seuil sera dépassée, la cellule va générer une force de constriction. Comme la cellule se constricte elle va déformer les cellules voisines qui seront plus à même de franchir la taille pallier. Une invagination globale va s’en suivre. Ce modèle reproduit quantitativement la dynamique de constriction des apex et permet de vérifier la possibilité d’obtenir une invagination à partir d’interactions mécaniques entre les cellules. Il permet met aussi en évidence l’importance de l’étude des comportements collectifs qui permettent par différents couplages. Dans une seconde partie j’ai effectué le parallèle entre l’invagination du mésoderme de la drosophile et l’initiation de l’épibolie du poisson zèbre en se focalisant sur le rôle de contraintes mécaniques développées par ces premiers mouvements morpho-génétiques. Ces premiers mouvements vont déterminer la création des différents feuillets et notamment la différentiation des cellules du mésoderme. Ces deux espèces montrent des mécanismes de méchanotransduction communs avec la phosphorylation de la beta-catenin (b-cat) Y667. Cette phosphorylation entraine l’expression de gènes twist (Drosophile) et notail (Danio rerio) nécessaire aux mouvements morphogénétiques. Les expériences réalisées consistent à bloquer les mouvements par l’intermédiaire de drogues (pour le poisson zèbre) ou de mutations (pour la Drosophile) et exercer une déformation mécanique sur les embryons. En l’absence de contraintes mécaniques la betacatenin n’est plus phosphorylée, de ce fait on elle n’est plus présente dans les noyaux ce qui entraine une perte d’expression des gènes twi ou notail. Avec une déformation mécanique alors que les mouvements sont bloqués nous arrivons à réactiver la phosphorylation de la βcat et ré induire l’expression des gènes. Ma contribution majeure pour ces expériences a été la mise en place d’un système magnétique permettant de mimer les mouvements de l’épibolie. (...) / In this thesis, I first focused on the testing of the hypothesis of the mechanotransductive activation of the apical accumulation of Myosin-II (Myo-II) that leads to Drosophila embryos mesoderm invagination, in response to the active cell apex pulsations preceding gastrulation in the mesoderm. This hypothesis was proposed on the basis of previous experiments realized in my host lab, having consisted in the rescue of mesoderm invagination in pulsation and invagination defective mutants, in response to a simple mechanical indent of the mesoderm. Here I demonstrated quantitatively the plausibility of such mechanical trigger of the active apical accumulation of Myo-II leading to subsequent mesoderm invagination, in response to the mechanical strains developed by the endogenous pulsative movements of mesoderm cell apexes, in silico. In a second part, I tested experimentally the role of the mechanical strains developed by the very first morphogenetic movements of zebrafish (Danio rerio) and Drosophila embryos, in the early specification of mesoderm cells identity. Specifically, to test this hypothesis, I developed magnetic biophysical tools to mimic the epiboly morphogenetic movements in epiboly defective zebrafish embryos. We found the beta-catenin (B-cat) Y667 phosphorylation as the common mechano-transductive pathway involved in earliest mesoderm genes expression notail and twist respectively, in response to the very first morphogenetic movements of embryogenesis in both species, epiboly and mesoderm invagination, respectively. This allowed to suggest such mechanotransduction pathway as conserved from the last common ancestor of both species, namely the last common ancestor of bilaterians, therefore possibly involved in the origins of mesoderm emergence in the ancestor, which represents a currently important opened question of evo-devo. In a third part, I developed experiments of mechanical indent of Drosophila embryos germ cells, and demonstrated the production of generational heritable developmental defects induced on at least 3 generations. These experiments suggest accidental mechanical perturbation of germ cells as a putative new motor mode of heritable modulations in the genetic developmental program of embryogenesis, with the molecular mechanism underlying such transmission being currently in progress.
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