Internalisation mechanisms of the endoderm during gastrulation in the zebrafish embryo Florence Giger To cite this version: Florence Giger. Internalisation mechanisms of the endoderm during gastrulation in the zebrafish embryo. Development Biology. Université Pierre et Marie Curie - Paris VI, 2016. English. NNT : 2016PA066203. tel-01426014 HAL Id: tel-01426014 https://tel.archives-ouvertes.fr/tel-01426014 Submitted on 4 Jan 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Thèse de Doctorat de l’Université Pierre et Marie Curie Spécialité : Biologie moléculaire et cellulaire du développement École doctorale 515 – Complexité du Vivant Présentée par Florence GIGER Internalisation Mechanisms of the Endoderm During Gastrulation in the Zebrafish Embryo Dirigée par Nicolas DAVID Présentée et soutenue le 23 septembre 2016 Devant un jury composé de : Professeur Claire FOURNIER-THIBAULT Présidente Docteur Pia AANSTAD Rapporteur Docteur Maximilian FÜRTHAUER Rapporteur Docteur Jo BEGBIE Examinateur Docteur Estelle HIRSINGER Examinateur Docteur Laurent KODJABACHIAN Examinateur Docteur Nicolas DAVID Directeur de thèse Institut de Biologie de l’École Normale Supérieure – INSERM U1024 – CNRS UMR 8197 46 rue d’Ulm, 75005 Paris 1 2 During development, cells are progressively separated into distinct territories, delimited by embryonic boundaries. The first segregation event occurs during gastrulation, when the embryo is organised in three germ-layers, the ectoderm, the mesoderm and the endoderm. The molecular and cellular mechanisms ensuring this segregation have not yet been elucidated. During my PhD thesis, I have focused on the endoderm internalisation in the zebrafish embryo. Based on in vitro results, it has been suggested that germ-layer progenitors would be segregated by a passive cell sorting. Combining cell transplantation, live confocal microscopy and functional analyses, I have shown that endodermal cell internalisation actually results from an active migration process dependent on Rac1 and its effector Arp2/3, a direct regulator of actin. Strikingly, endodermal cells are not attracted to their internal destination but rather appear to migrate out of their neighbouring cells. This process is dependent on the Wnt/PCP pathway and N-cadherin. Furthermore, N-cadherin is sufficient to trigger the internalisation of ectodermal cells, without affecting their fate. Overall, these results lead to a new model of germ-layer formation, in which endodermal cells actively migrate out of the epiblast to reach their internal position. Au cours du développement, les cellules sont progressivement séparées dans des territoires distincts délimités par des frontières embryonnaires. La première ségrégation a lieu pendant la gastrulation, quand l’embryon s’organise en trois feuillets embryonnaires, l’ectoderme, le mésoderme et l’endoderme. Les mécanismes moléculaires et cellulaires assurant cette ségrégation n’ont pas encore été élucidés. Au cours de ma thèse, je me suis focalisée sur l’internalisation de l’endoderme chez le poisson-zèbre. À partir de résultats in vitro, il a été suggéré que les progéniteurs de feuillets embryonnaires soient ségrégés par un tri cellulaire passif. En combinant des expériences de transplantation de cellules, une imagerie confocale en temps réel et des analyses fonctionnelles, j’ai montré que l’internalisation des cellules endodermiques est due en réalité à un processus de migration active dépendante de Rac1 et de son effecteur Arp2/3, un régulateur direct de l’actine. De manière surprenante, les cellules endodermiques ne sont pas attirées par leur destination interne, mais semblent plutôt migrer hors de leurs voisines. Ce processus est dépendant de la voie Wnt/PCP et de la N-cadhérine. De plus, la N-cadhérine est suffisante pour induire l’internalisation de cellules ectodermiques, sans modifier leur identité. Dans leur ensemble, ces résultats conduisent à un nouveau modèle de formation des feuillets embryonnaires dans lequel les cellules endodermiques migrent activement hors de l’épiblaste pour atteindre leur position interne dans l’embryon. 3 4 Acknowledgements I cannot start my acknowledgements otherwise than by thanking my thesis director. Nicolas, what I can thank you for most is quite a difficult question. I probably won’t elaborate on your role in my entering the École Normale Supérieure as you keep saying you don’t remember the oral examination. Obviously I do. As I remember my first one-week internship that gave me the taste for research, the advice and encouragement you provided as my school tutor, until I finally joined the lab for my M2 and PhD. Thanks for the exceptional patience with which you taught me technical skills, scientific reasoning, oral expression competences... Thanks for your constant attention and the confidence you had in me. I do hope this is not the end of us working together. Because everyone needs an older sister, many thanks to Aline and Aurélie, you have both played this role wonderfully for me for five years. Thanks for your sensible advice for science and everyday life, provided over the screen of my computer or around the Luxembourg garden. Élodie, thanks for having been at my side during this time, because everyone also needs a twin sister, going through the same steps of academic life and fighting administrative obstacles set by the university. Thanks for your moral support during the not-too-good periods of my PhD, we have shared much more than lég’Ulm baskets during these years! Thanks to the students who have contributed to the young life of the lab. Julien, I forgive you for having abandoned me to the claws of Nicolas, thanks for your availability whenever I needed help when I first arrived… and even after! Thanks to Marina for her smiles and complicity, to Alex for his sarcastic humour, to Patrick for his big heart behind the metal madness, and to Gaspard for his teasing spirit. Students and postdocs are not the only contributors to a nice lab atmosphere. I thank all of the present and past members of the Rosa and Charnay labs. However I say a special thank you to Frédéric for giving me the opportunity to work in his lab, the precise scientific advice and the ironic smile when asking me about the “pourquoi du comment”; to Sylvain for being as hindered as me by France Musique’s social movements; to Pascale for her sense of humour and everyday kindness; to Patrick for the discrete attention to young members of the lab (and next-door lab) and the witty discussions after lunch; to Marika for the scientific life advice; to 5 6 Aurélie for the words of encouragement during the tea breaks; and to Firas for the care of our fish. Speaking of fish, thank you Estelle for your help when I needed embryos. I am really grateful to you as well as Stéphane and Alex at Jussieu’s fish facility. Many thanks also for your scientific advice as part of my thesis committee. Another colourful figure of my thesis committee was my “thesis godmother”; thank you Sonia for listening to my concerns and shouting the very personal optimism you have for scientific life. Let’s keep your motto in mind: « au pire, tout ce qui peut arriver, c’est que ça se passe très mal ! » Let’s now cross the Chanel and spend some time in Oxford. Thanks Jo for introducing me to the second-best model in developmental biology, and offering me my first opportunity to present my work at a meeting. Thanks Lexy and Stephen for the incomparable family atmosphere you gave to this lab. And finally, Jo, thank you so much for welcoming me repeatedly, I look forward to seeing more of you during my postdoc in London. A few words for my family, thanks for being each of you, in your own way, interested in my work, thanks for sharing my expectations, dismissing my doubts, and being always at my side. Thanks to my friends outside the lab and in particular my music partners. Finally, many thanks to the members of the jury for accepting to be part of it, and in particular to the reviewers, Pia Aanstad and Max Fürthauer. Bonne lecture ! 7 8 List of abbreviations BMP: bone morphogenetic protein CIL: contact inhibition of locomotion DIC: differential interference contrast EC: extracellular EMT: epithelial-to-mesenchymal transition EVL: enveloping layer FGF: fibroblast growth factor FGFR: fibroblast growth factor receptor GPCR: G-protein coupled receptor GPI: glycosylphosphatidylinositol hpf: hours post fertilisation Irf: interferon regulatory factor MAPK: mitogen-associated protein kinase MMP: matrix metalloproteinase MLC: myosin light chain MLCK: myosin light chain kinase MTOC: microtubule organising centre PCP: planar cell polarity PI3K: phosphoinositide 3 kinase PIP3: phosphatidylinositol-triphosphate PKC: protein kinase C PLC: phospholipase C RTK: receptor tyrosine kinase WASP: Wiskott-Aldrich syndrome protein WAVE: WASP-family verprolin homology protein YSL: yolk syncytial layer 9 10 Table of contents INTRODUCTION 17 I. Establishment of Embryonic Boundaries 21 1. Tissue Segregation during Development 21 2. Cell Sorting Based on Biophysical Properties 23 3. Cell Sorting Based on Different Repertoires of Adhesion Molecules 27 4. Cell Sorting Achieved by Contact Inhibition 29 II. Mechanisms of Active Cell Migration Segregating Cell Populations 35 1. Actin Cytoskeleton 35 2. Cell Polarity 37 3. Models of Migration in Vitro 41 4. In Vivo: Integration of the Environment 45 III. Endoderm and Mesoderm Internalisation in Model Organisms 51 1. Lessons from Sea Urchin Gastrulation: Two Ways for Cell Internalisation 51 2. Internalisation of a Coherent Layer of Cells 51 3.