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. En effet, c’est une large partie du cerveau moyen localisée en position dorsale et qui présente la particularité de croitre de manière orientée tout au long de la vie de l’animal grâce aux cellules neuroépitheliales présentes à sa périphérie (dans la « peripheral midbrain layer », PML). De plus, les progéniteurs neuroépithéliaux, les progéniteurs lents et les cellules post-mitotiques sont localisées dans des domaines adjacents du toit, conséquence de sa croissance orientée. Chaque population cellulaire est marquée par des profils d’expression particuliers. Ainsi, une recherche dans la base de données ZFIN nous a permis d’identifier environ 50 gènes ayant une forte expression dans les cellules de la PML (progéniteurs neuroépithéliaux). De façon intéressante, beaucoup de « gènes PML » codent pour des facteurs de la biogenèse des ribosomes. L’accumulation de ce type de transcrits dans les progéniteurs lents était surprenante. Ainsi, au cours de mon doctorat, j’ai étudié le rôle spécifique des facteurs de la biogenèse des ribosomes dans le maintien des cellules neuroepithéliales de la PML. En effet, bien qu’il soit généralement admis que la biogenèse des ribosomes est un processus essentiel dans toutes les cellules, il a été récemment démontré que plusieurs facteurs nécessaires à la synthèse des ribosomes ont un rôle tissu-spécifique. Par exemple, Notchless est requis pour la survie de la masse cellulaire interne de l’embryon préimplantatoire de souris. Récemment, des expériences de knock-out conditionnel chez la souris ont montré que Notchless était nécessaire au maintien des cellules souches hématopoïétiques et intestinales, mais pas à celui des cellules différenciées. En effet, en absence de Notchless dans les cellules souches, la grosse sous-unité ribosomique (60S) ne peut pas être exportée hors du noyau et s’accumule. Au contraire, dans les cellules différenciées, où Notchless n’est pas indispensable, cette accumulation n’est pas observée. J’ai commencé une étude fonctionnelle basée sur la surexpression conditionnelle de la forme dominante-négative du gène notchless homolog 1 (nle1, homologue poisson zèbre du gène Notchless mammifère). Selon mon hypothèse, les progéniteurs lents de la PML (Slow amplifying progenitors, SAPs) pourraient avoir besoin de Notchless pour la maturation de la sous-unité 60S, contrairement aux cellules différenciées qui pourraient survivre après la délétion de ce gène. Des expériences sont encore en cours, mais nous avons déjà pu démontrer que nle1 joue un rôle crucial dans la survie des progénitéurs neuroépithéliaux de la PML. En parallèle, j’ai étudié des lignées de poisson-zèbre mutantes pour des gènes codants pour des composants du complexe de snoRNP (box C/D small nucleolar ribonucleoprotein : Fibrillarine, Nop56, Nop58). Les trois mutants présentent des phénotypes similaires, en particulier une apoptose massive et une dérégulation du cycle cellulaire dans l’ensemble du toit optique à 48 heures de développement. Étonnamment, ces résultats sont en faveur d’un arrêt du cycle cellulaire à la transition G2/M. Ainsi, cette étude pourrait permettre de mettre en évidence de nouveaux mécanismes d’arrêt du cycle cellulaire lors de défauts de biogenèse des ribosomes. L’ensemble de ces résultats montrent comment les facteurs de la biogenèse des ribosomes (tout comme le processus) contribue à la régulation fine de l’homéostasie cellulaire, et donc à la détermination de l’identité des cellules progénitrices. / 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.

Adult skeletal muscle is highly plastic and responds readily to environmental stimuli. One of the most commonly utilized methods to study skeletal muscle adaptations is immunofluorescence microscopy. By analyzing images of adult muscle cells, also known as myofibers, one can quantify changes in skeletal muscle structure and function (e.g. hypertrophy and fiber type). Skeletal muscle samples are typically cut in transverse or cross sections, and antibodies against sarcolemmal or basal lamina proteins are used to label the myofiber boundaries. The quantification of hundreds to thousands of myofibers per sample is accomplished either manually or semi-automatically using generalized pathology software, and such approaches become exceedingly tedious. In the first study, I developed MyoVision, a robust, fully automated software that is dedicated to skeletal muscle immunohistological image analysis. The software has been made freely available to muscle biologists to alleviate the burden of routine image analyses. To date, more than 60 technicians, students, postdoctoral fellows, faculty members, and others have requested this software. Using MyoVision, I was able to accurately quantify the effects of β-catenin knockout on myofiber hypertrophy. In the second study, I tested the hypothesis that myofiber hypertrophy requires β-catenin to activate c-myc transcription and promote ribosome biogenesis. Recent evidence in both mice and human suggests a close association between ribosome biogenesis and skeletal muscle hypertrophy. Using an inducible mouse model of skeletal myofiber-specific genetic knockout, I obtained evidence that β-catenin is important for myofiber hypertrophy, although its role in ribosome biogenesis appears to be dispensable for mechanical overload induced muscle growth. Instead, β-catenin may be necessary for promoting the translation of growth related genes through activation of ribosomal protein S6. Unexpectedly, we detected a novel, enhancing effect of myofiber β-catenin knockout on the resident muscle stem cells, or satellite cells. In the absence of myofiber β-catenin, satellite cells activate and proliferate earlier in response to mechanical overload. Consistent with the role of satellite cells in muscle repair, the enhanced recruitment of satellite cells led to a significantly improved regeneration response after chemical injury. The novelty of these findings resides in the fact that the genetic perturbation was extrinsic to the satellite cells, and this is even more surprising because the current literature focuses heavily on intrinsic mechanisms within satellite cells. As such, this model of myofiber β-catenin knockout may significantly contribute to better understanding of the mechanisms of satellite cell priming, with implications for regenerative medicine.

Ribosomes are the cellular structures responsible for the synthesis of protein in all branches of life. All ribosomes are made from a large and small subunit that in turn are composed of protein and RNA. The synthesis of eukaryotic ribosomes is a complex process involving more than 200 factors and spans three cellular compartments: the nucleolus, the nucloplasm, and the cytoplasm. The precise function of most of these ribosome biogenesis factors remains unknown. The RNA component of ribosomes is in part processed from a large RNA transcript that yields most of the RNA present in mature ribosomes. Part of the maturation process involves modification of this ribosomal RNA as processing is carried out. Recent work constructing protein interaction networks in Saccharomyces cerevisiae suggested the methyltransferase Bud23 was involved in ribosome biogenesis (1). This thesis describes my work to characterize Bud23 and place it within the ribosome biogenesis pathway. Bud23 is a SAM methyltransferase important for the proper biogenesis of the small ribosomal subunit. Here I will demonstrate that Bud23 methylates G1575 of the small subunit ribosomal RNA (SSU rRNA), and its absence delays export of the SSU rRNA from the nucleolas, and the nucleus, and results in the delayed maturation of the SSU rRNA. Finally, I will show that Bud23 function is connected to small subunit processome factor Utp14 through identification of a Utp14 mutant that suppresses the bud23[Delta] deletion phenotype. / text

No description available.

Les cellules souches (CS) se démarquent des progéniteurs et cellules différenciées à de nombreux égards. Notamment, les CS présentent des caractéristiques particulières dans des processus cellulaires fondamentaux, et il a été récemment proposé que la biogenèse des ribosomes (BiRi) participe à la régulation des CS. Pendant ma thèse, j’ai utilisé diverses approches pour étudier le rôle et la régulation de la BiRi dans des populations de CS, in vivo et ex vivo dans des modèles murins.Grâce à un modèle d’inactivation génétique du facteur de BiRi Notchless (Nle), j’ai participé à l’étude de son rôle dans le lignage hématopoïétique et l’épithélium intestinal adultes, et cours du développement embryonnaire précoce. In vivo, la perte constitutive de Nle entraîne une létalité embryonnaire, et j’ai montré ex vivo que l’inactivation de Nle dans des CS embryonnaires induit une réponse au stress ribosomique médiée par le suppresseur de tumeur p53, et des défauts de prolifération/survie. L’induction de la perte de Nle chez l’adulte active également p53 dans les CS hématopoïétiques et intestinale, entraînant leur rapide élimination.En parallèle, j’ai utilisé plusieurs méthodes pour mesurer l’activité de BiRi des progéniteurs immatures et CS hématopoïétiques (CSH) à l’homéostasie, in vivo chez la souris adulte. J’ai ainsi mis en évidence des variations de l’activité de BiRi dans ces populations, révélant notamment une activité de BiRi des CSH jusqu’ici insoupçonnée du fait de leur quiescence.Dans l’ensemble, mon travail renforce l’idée d’un rôle de la BiRi dans la régulation des CS, et apporte une meilleure compréhension de la régulation de ce processus dans le lignage hématopoïétique. / Stem cells (SCs) differ from progenitors and differentiated cells on many aspects. Notably, SCs display particular characteristics in fundamental cellular processes, and ribosome biogenesis (RiBi) has recently been proposed to play an important role in the regulation of SCs. During my thesis, I have used various approaches to study the role and regulation of RiBi in SC populations, using in vivo and ex vivo mouse models.Using genetic inactivation of the RiBi factor Notchless (Nle), I have participated to the analysis of its role in the adult hematopoietic system and intestinal epithelium, and in the establishment of the first cell lineages during early embryogenesis. In vivo, constitutive Nle deficiency causes early embryonic lethality, and I showed ex vivo that Nle inactivation in embryonic SCs induces a ribosomal stress response mediated by the tumor suppressor p53, and proliferation/survival defects. Conditional Nle inactivation in the adult mouse also induces activation of p53 in hematopoietic and intestinal SCs in vivo, leading to their rapid elimination.In parallel, I have used different methods to analyze the RiBi activity of hematopoietic SCs (HSCs) and immature progenitors at homeostasis, in vivo in the adult mouse. Thus, I have unraveled variations in the RiBi activity of these populations, and notably uncovered previously unsuspected RiBi activity in HSCs despite their quiescent state.Altogether, my work supports the hypothesis of a role for RiBi in the regulation of SCs and provides better understanding of the activity of this process during hematopoietic differentiation.

The mammalian mitochondria contain their own genome which encodes thirteen polypeptide components of the oxidative phosphorylation (OxPhos) system, and the mitochondrial (mt-) rRNAs and tRNAs required for their translation. The maturation of the mitochondrial ribosome requires both mt-rRNAs to undergo post-transcriptional chemical modifications, folding of the rRNA and assembly of the protein components assisted by numerous biogenesis factors. The post-transcriptional modifications of the mt-rRNAs include base methylations, 2’-O-ribose methylations and pseudouridylation. However, the exact function of these modifications is unknown. Many mitoribosome biogenesis factors still remain to be identified and characterised. This work aims to broaden our understanding of two proteins involved in mitoribosome biogenesis through the study of the function of an rRNA methyltransferase and a novel biogenesis factor. Firstly, we characterised MRM1 (mitochondrial rRNA methyltransferase 1), a highly conserved 2’-O-ribose methyltransferase. We confirmed that MRM1 modifies a guanine in the peptidyl (P) transferase region of the 16S mt-rRNA that specifically interacts with the 3’ end of the tRNA at the ribosomal P-site. In bacteria, the modification is dispensable for ribosomal biogenesis and cell viability under standard conditions. However, in yeast mitochondria, Mrm1p is vital for ribosomal assembly and function. We generated knockout cells lines using programmable nuclease technology, and characterised the possible effects of MRM1 depletion on mitochondrial translation and mitoribosome biogenesis. We demonstrated that neither the enzyme nor the modification is required for human mitoribosomal assembly and translation in our experimental setup. Secondly, we identified a novel mitochondrially-targeted putative RNA endonuclease, YbeY. Using YbeY knockout cell lines, we showed that depletion of YbeY leads to loss of cell viability and OxPhos function as a consequence of a severe decrease in mitochondrial translation. Northern blotting and transcriptomic analysis using next generation RNA-Seq revealed transcript-specific changes to steady state levels. This analysis identified mt-tRNASer as a potential target of YbeY. We investigated the effect of YbeY deficiency on mitoribosomal assembly by quantitative sucrose gradient fractionation and mass spectrometry. This analysis showed that the mt-SSU is depleted in YbeY knockout cells. Further, immunoaffinity purification identified MRPS11 as a key interactor of YbeY. We propose that YbeY is a multifunctional protein that performs endonucleolytic functions in the mitochondria and also acts as a mitochondrial ribosome biogenesis factor, assisting small subunit assembly through its interaction with MRPS11.

La RNase E chez Escherichia coli est une endoribonucléase essentielle qui joue un rôle important dans la maturation des ARN stables, dans le contrôle qualité des ribosomes, ainsi que dans la dégradation constitutive et régulée des ARN messagers. La séquence de ciblage à la membrane (MTS pour Membrane Targeting Sequence), qui forme une hélice α-amphipatique, ancre la RNase E à la membrane cytoplasmique interne des cellules. La conservation absolue du MTS chez l'ensemble des -protéobactéries suggère un rôle important de la localisation membranaire RNase E dans le métabolisme de l'ARN. Pour élucider la fonction cellulaire de l'association membranaire de la RNase E, nous avons caractérisé la souche rne∆MTS qui exprime une RNase E cytoplasmique. Les résultats de cette étude nous amènent à proposer que l'association membranaire de la RNase E est nécessaire à la stabilité de la RNase E, est impliquée dans des interactions fonctionnelles avec des régulateurs associés à la membrane et protège les transcrits présents dans le nucléoïde en évitant des interactions prématurées avec la RNase E. En particulier, garder la RNase E à la membrane est critique pour la spécificité de la RNase E dans le contrôle qualité des ribosomes. Cette association membranaire est une nouvelle couche de régulation qui permet d’expliquer comment la RNase E, une enzyme avec peu de spécificité de séquence et avec beaucoup de substrat, peut remplir les fonctions de «maturase» et de «dégradase». / RNase E in Escherichia coli is an essential endoribonuclease with important roles in stable RNA maturation, in ribosome quality control and in constitutive and regulated mRNA degradation. The Membrane Targeting Sequence (MTS), which forms an amphipathic α-helix, anchors RNase E on the inner cytoplasmic membrane. The absolute conservation of the MTS among -Proteobacteria suggests an important role for RNase E membrane association in RNA metabolism. To elucidate the cellular function of the membrane association of RNase E, we characterized the rne∆MTS strain expressing cytoplasmic RNase E. The results of this study lead us to propose that RNase E membrane association is necessary for RNase E stability, for functional interactions with membrane-associated regulatory factors and for protecting nascent transcripts in the nucleoid from premature interactions with RNase E. In particular, keeping RNase E to the membrane is critical for the specificity of RNase E in ribosome quality control. Membrane association is a new layer of regulation that can explain how RNase E, an enzyme with little sequence specificity and many substrates, can fulfill both ‘maturase’ and ‘degradase’ functions.

Alors que la biogénèse des ribosomes a étéconsidérée comme un mécanisme ubiquiste, lesétapes de ce processus ont récemment étédémontrées comme étant tissu-spécifiques. Letoit optique (OT) du poisson-zèbre est un modèleapproprié pour étudier la prolifération cellulairepuisque les cellules à différents états dedifférenciation se trouvent dans des domainesséparés.Au cours de mon doctorat, j'ai examiné si lesgènes de la biogenèse des ribosomes peuventavoir des rôles spécifiques dans les cellulesprogénitrices neuroépithéliales (CPNe). Profitantd'une analyse transcriptomique antérieure, j'aid'abord examiné les nouveaux candidatsaccumulés dans les CPNe. J'ai décidé de meconcentrer sur proliferation-associated 2G4(pa2G4/ebp1) qui est exprimé de manièrepréférentielle dans les CPNe.Ce gène favorise ou réprime la proliférationcellulaire dans des organismes normaux oupendant la tumorigénèse. J'ai conçu une stratégiepour l'expression inductible et cellule-spécifiquede ce gène.Fibrillarin (Fbl), une méthyltranférasenucléolaire est également préférentiellementexprimée dans CPNe. Ce gène joue un rôleimportant dans le cancer. J'ai montré que lesmutants fbl présentaient des défauts OTspécifiques,en lien avec une apoptose massive etune absence de différenciation neurale. J'aiégalement démontré une diminution de l'activitéde traduction des ribosomes. En outre, lesmutants fbl montrent une progression de la phaseS altérée. Nos données suggèrent que fbl estessentiel à la prolifération des progéniteursneuronaux du poisson-zèbre. / While ribosome biogenesis has been consideredas an ubiquitous mechanism, steps of thisprocess have recently been shown to be tissuespecific. Zebrafish optic tectum (OT) is asuitable model to study cell proliferation sincecells at different differentiation states arespatially partitioned.During my PhD, I examined whether ribosomebiogenesis genes may have specific roles inneuroepithelial progenitor cells (NePCs).Taking advantage of a previous transcriptomicanalysis, I first screened for new candidatesaccumulated in NePCs. I decided to focus onproliferation-associated 2G4 (pa2g4/ebp1),which was expressed preferentially in NePCs.This gene promotes or represses cellproliferation in normal organisms or duringtumorigenesis. I designed a strategy for theinducible expression and cell specificexpression of this gene.Fibrillarin (Fbl), a small nucleolarmethyltransferase is also preferentiallyexpressed in NePCs. It plays an important rolein cancer. I showed that fbl mutants displayedspecific OT defects linked to a massiveapoptosis and an absence of neuraldifferentiation. I also demonstrated deficienciesin the ribosome translational activity.Additionally, fbl mutants showed impaired Sphaseprogression. Our data suggest that fbl isessential for the proliferation of zebrafishneuronal progenitors.

No description available.

Erythroid terminal differentiation couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. I used Epor−/− mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. I found that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. Specifically, I found that high levels of EpoR signaling increase the size and shorten the cycle of early erythroblasts, which are amongst the fastest cycling somatic cells. I confirmed the effect of erythropoietin (Epo) on red cell size in human volunteers, whose mean corpuscular volume (MCV) increases following Epo administration. Our work shows that EpoR signaling alters the expected inverse relationship between cell cycle length and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress. The ability of EpoR signaling to increase cell size in rapidly cycling early erythroblasts suggests that these cells have exceptionally efficient EpoR-driven mechanisms for growth. I found evidence for this in ongoing work, where Epor−/− and Stat5−/− single-cell transcriptomes show dysregulated expression of ribosomal proteins and rRNA transcription and processing genes. Global rates of ribosomal rRNA transcription and protein synthesis increase in an EpoR dependent manner during a narrow developmental window in early ETD, coincident with the time of cell cycle shortening. Our work therefore suggests EpoR-driven regulation of ribosome biogenesis and translation orchestrating rapid cycling and cell growth during early ETD.