Functional study of the role played by nucleolar proteins in the control of neural progenitor homeostasis using zebrafish as a model Alessandro Brombin To cite this version: Alessandro Brombin. Functional study of the role played by nucleolar proteins in the control of neural progenitor homeostasis using zebrafish as a model. Molecular biology. Université Paris Sud -Paris XI, 2015. English. NNT : 2015PA112237. tel-01599238 HAL Id: tel-01599238 https://tel.archives-ouvertes.fr/tel-01599238 Submitted on 2 Oct 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. 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UNIVERSITÉ PARIS-SUD ÉCOLE DOCTORALE 426 : GÈNES GÉNOMES CELLULES Laboratoire : CNRS, UMR 9197, Institut des Neurosciences Paris‐Saclay THÈSE DE DOCTORAT SCIENCES DE LA VIE ET DE LA SANTÉ par Alessandro BROMBIN Functional study of the role played by nucleolar proteins in the control of neural progenitor homeostasis using zebrafish as a model Date de soutenance : 29/09/2015 Composition du jury : Directeur de thèse : Jean-Stéphane JOLY DR1 (INRA) Rapporteurs : Francesco ARGENTON Professeur (Università degli Studi di Padova) Pierre-Emmanuel GLEIZES Professeur (Université de Toulouse – Paul Sabatier) Examinateurs : Pierre CAPY Professeur (Université de Paris Sud) Frédéric CATEZ CR1 (CNRS) Morgane LOCKER MCF (Université de Paris Sud) Membres invités : Françoise JAMEN MCF (Université de Paris Sud) Acknowledgements Because so many people helped me so much during these years, I think I will never be able to express all my gratitude well enough towards everybody and I apologize in advance to people I may have forgotten to mention. To begin, I would like to especially thank Dr. Jean-Stéphane Joly and Dr. Françoise Jamen without whom I would have never settled down in France and I would have possibly never started a thesis after all. Six years ago, you took the decision to bet on a non-French-speaker Italian student and you brought me until here. Thank you for all the support during all these years: for all the meetings, discussions, advice, preparations for presentations, searching for the best strategy to make experiments work and helping me with my written works. Thanks to both of you for never letting go. I must express my special thanks to all the members of my examination committee who readily agreed to participate in the evaluation and defense of my thesis: Prof. Francesco Argenton, Prof. Pierre Capy, Dr. Frédéric Catez, Prof. Pierre-Emmanuel Gleizes, Dr. Morgane Locker. Thank you very much to Francesco Argenton and Pierre-Emmanuel Gleizes who accepted the heavy task of being a “rapporteur” without hesitation. During these years all my colleagues turned into friends and I wish to thank all of them for this. First of all I wish to thank my past and present bureau: we shared so much all together and I consider you as my family. Aurélie, Emilie, Mathilde, Matthieu, Rosaria, Stéphanie and Tibi (in strict alphabetical order) we had never ending discussion about science, thesis, postdoc, fashion, films, series, food and everything. It was a joy to come every day at work. Emilie, thanks because you have been “ma postdoc” and my Timon. Aurélie, thanks for all the things that we shared in the lab and outside (I will miss our gossip dance with the chairs). Matthieu, thanks for being our conscience. Thanks also to my “pote” Joshua. Thanks Rosaria and Stéphanie because you went through my writing period without punching me. Stéphanie and Aurélie, you are my “padawans”: hold on, girls! Thanks to all of you because you forced me to stop being “sentimentalement coincé” (you know that I would have used another term) and made me write these acknowledgements. How to forget the members of the “extended bureau”? I wish to thank Jean-Michel for his precious support, long discussions amazing dinners and wine. Thanks also to Franck for all the scientific discussions. Thanks to Johanna for being always so positive and for laughing as much loud as me (I am still in debt with you), to Pierre for the amazing breaks and for making me feel like a professional chef. I Thanks Lucie for making me feel like a genius, to Barbara for our car discussions and to Maryline for her advices both on my experiments and recipes. Thanks to all the members of the “couloir des fous” (Arnim, Elodie, Laurie, Sylvie, Stéphane, Victor, Adeline, …): I always found help, support and friendship. Thanks to the past and present members of the Amagen unit for technical help, but also for being what you are. I wish to thank Sosthène for being exactly like me, Joanne for our “good morning meetings” before 8 a.m., Naïma for her smile, Sylvia for always remembering that Italy is an amazing place, Zlatko for being my example (I think about you) and Noémie for the many advices and for our beers. Thanks to Fred, Laurent, Ben and Céline. Thanks to all the past and present members of DEPSN/NED/NeuroPSI (yes, I have been part of the three units): Yan and Hélène (my zombie friends), Julia (always helping me), Alex, Olivier, Hélène, Karine, Karen, Raphaël, Jean-Baptiste, Yannick, Ingrid, Audrey, Gaëlle, Yvette. All of you made me feel like home. Now, I must also express my special gratitude to Ludo who welcomed me in his apartment. We fought and we laughed, we cooked and we discussed. For this I thank you! (you know that I will be always ready to start the “coloc 2.0”). Thanks to all my “Parisian” friends: Vale and Andi (always caring about me, even if I disappeared), Marco and Clèm (for having brought a little bit of “Colli” here), Micol, Simone, Elisa, Ale and Anna. Thanks as well to the “non-Parisians” who are in Italy (I always think about you). Finally, I wish to thank my family with a “grazie” : mi avete sempre supportato dall’Italia e non sapete quanto sia stato importante sapervi sempre vicini (finalmente riuscirete anche voi a godervi Parigi come ho fatto io in questi anni). II ABSTRACT In neural stem cells (NSCs) and neural progenitors (NPs), as in other cell types, cell identity is characterized by specific molecular signatures that depend on the environment provided by neighboring cells. Thus, it is important to study progenitor cells in vivo. The zebrafish optic tectum (OT) is a suitable model for that purpose. Indeed, this large structure of the dorsal midbrain displays life-long oriented growth supported by neuroepithelial cells present at its periphery (in the peripheral midbrain layer, PML). Moreover, neuroepithelial progenitors, fast-amplifying progenitors and post-mitotic cells are found in adjacent domains of the OT, as a consequence of its oriented growth. Each cell population is marked by concentric gene expression patterns. Interestingly, a datamining of the ZFIN gene expression database allowed us to identify around 50 genes displaying biased expression in PML cells (neuroepithelial progenitors). Interestingly, many “PML genes” code for ribosome biogenesis factors. The accumulation of transcripts for such ubiquitously expressed genes in SAPs was very surprising so during my thesis I examined whether ribosome biogenesis may have specific roles in these neuroepithelial cells, while improving our knowledge. Indeed, although it is generally admitted that ribosome biogenesis is essential in all cells, it has been shown quite recently that several components of the ribosome biogenesis have tissue restricted roles. For example, Notchless is required for the survival of the inner cell mass in the preimplantation mouse embryo. More recently, conditional knock-out experiments in mice showed that Notchless is necessary for the maintenance of hematopoietic stem cells and intestinal stem cells, but not for committed progenitors and differentiated cells. Indeed in the absence of Notchless in stem cells, the immature 60S subunit cannot be exported from the nucleus and accumulates. This does not happen in differentiated cells where Notchless is dispensable. I started a functional study based on the conditional overexpression of a dominant-negative form of the gene notchless homolog 1 (nle1, the zebrafish homolog of the mammalian gene Notchless). My hypothesis was that the PML slow-amplifying progenitors (SAPs) may require Notchless for the maturation of the 60S subunit, but not the differentiated cells which could survive also after the deletion of this gene. Experiments are still underway. So far we could demonstrate that nle1 has a crucial role in SAPs. I studied zebrafish mutants for genes coding for the components of the box C/D small nucleolar ribonucleoprotein (snoRNP) complex (Fibrillarin, Nop56, Nop58). Mutants displayed a similar phenotype with massive apoptosis and a deregulation of the cell cycle in the whole tectum at 48hpf. Our data suggest a cell cycle arrest at the G2/M transition, highlighting novel possible mechanisms of cell cycle arrest upon impaired ribosome biogenesis. All together, these data highlight how ribosome biogenesis factors and the whole ribosome biogenesis contribute to the fine regulation of cell homeostasis thereby contributing to the determination of progenitor cell identity. III RESUME L’identité des cellules souches et des progéniteurs neuraux, comme celle de tout type cellulaire, est caractérisée par des signatures moléculaires spécifiques qui dépendent de l’environnement dans lesquelles les cellules se trouvent. Ainsi, il est primordial d’étudier ces cellules dans un contexte in vivo. Le toit optique du poisson zèbre est un modèle idéal pour ce type d’étude.