RTEL1 (Regulator of telomere length1) DNA helicase has been demonstrated to be vital for the maintenance of telomere length and genomic stability. However, its biological role during development is unknown. Our recent finding that RTEL1 is selectively expressed in several types of adult stem cells, suggests that RTEL1 could play an essential role in the maintenance of these cells. Depending on the function of RTEL1 in the maintenance of genomic stability, we hypothesize that RTEL1 could be required for protecting adult stem cells from genomic instability, whose dysfunction may not only impair tissue homeostasis/regeneration, but also could transform these cells to form tumors. In this study, we have used mouse intestinal stem/progenitor cells model to address this hypothesis. With a transgenic lineage tracing assay, we demonstrated that RTEL1-expressing cells in intestinal crypts can self renew and differentiate to the progeny cells required for intestinal homeostasis. Using a conditional knockout approach, we also showed that loss of RTEL1 function could induce genomic instability in intestinal stem/progenitor cells, which significantly affected the survival of intestinal stem cells and intestinal regeneration. Finally, in this study, we also observed intestinal hyperplasia in our RTEL1 conditional knockout mice, indicating that loss of RTEL1 function may initiate intestinal tumorigenesis. All of these findings strongly support that RTEL1 could be one the key molecules necessary for the maintenance of intestinal stem/progenitor cells and this function could be important for preventing intestinal tumorigenesis.

Wolbachia is a vertically transmitted, obligate intracellular bacterium infecting ~40% of all known species of arthropods, as well as filarial nematodes. The nature of Wolbachia-host interactions ranges from reproductive parasitism to increased fecundity and pathogen protection. Wolbachia reduces the ability of mosquitoes to transmit human pathogens, which is being explored as a novel method for the control of vector-borne diseases like Dengue and Zika. The mechanisms of Wolbachia blocking the transmission of these diseases are not fully understood. There are studies indicating that Wolbachia-induced changes in the insect immunity could block the virus, however there is no consensus in the literature. A necessary step in the transmission of these diseases is the viral entry into the insect vector. This occurs trough the gut epithelium, highlighting the importance of understanding the interaction of this tissue with microorganisms. We have recently shown that Wolbachia colonizes the Drosophila gut epithelium and affects the gut microbiome composition. Wolbachia’s presence did not affect the gene expression of immune effector molecules from the main regulators of gut immunity, Imd and ROS pathways. Our understanding of the mechanisms of Wolbachia’s colonization of the gut epithelium and modulation of gut microbiome are still very limited. This work characterizes Wolbachia’s kinetics of colonization in Drosophila midguts. Imaging analysis revealed that Wolbachia colonizes adult and larval midguts in different patterns. We have also characterized a preferential colonization in specific adult midgut sub-regions. We observed that Wolbachia patches are confined to specific midgut subregions, in a pattern similar to the arrangement of intestinal stem cell (ISC) clones. These results led us to hypothesize that Wolbachia colonizes Drosophila midguts by infecting intestinal progenitor cells and spreading vertically to their progeny with limited lateral transmission between neighboring cells. We provide evidence to support this hypothesis by showing that Wolbachia is present in intestinal progenitor cells in all stages of the fly’s life cycle as well as by analyzing the infection status of ISC clones and differentiated cells surrounding ISCs. Finally, we found that ISC proliferation is reduced by the intracellular presence of Wolbachia, which also decreases ISC tumor incidence triggered by the downregulation of Notch signaling specifically in ISCs. These findings will aid in our understanding of Wolbachia tropisms and its phenotypic consequences. It has been shown that in the Wolbachia wMelPop strain excessive growth of intracellular bacteria leads to damage to the host cell, suggesting a mechanism of controlling intracellular growth in other strains. To better understand the molecular mechanisms behind Wolbachia-Drosophila interactions, we turned to the gonads, as Wolbachia colonization of these tissues has been well characterized. We chose to investigate the interplay between Reactive Oxygen Species (ROS) and Wolbachia, as intracellular ROS could regulate bacterial density but also be affected by Wolbachia and play a role in symbiont-related phenotypes. Using direct and indirect measurements of ROS, we show that the pathogenic strain wMelPop increases ROS in the germarium, while the symbiotic strains wMel and wMelCS reduce ROS in the terminal filaments. None of the Wolbachia strains tested affected ROS levels in the testes. In addition, genetically altering ROS levels in the germline or systemically in the fly did not affect Wolbachia levels in the ovaries. We conclude that ROS does not significantly affect Wolbachia in the fruit fly gonads.

Le cancer colorectal représente la deuxième cause de mortalité par cancer en France. Dans l’intestin, l’initiation et la progression tumorale sont corrélées à l’accumulation séquentielle de mutations génétiques et épigénétiques au niveau du compartiment de renouvellement de l’épithélium. Ces altérations ont pour conséquence une croissance incontrôlée de l’épithélium et, à terme, la formation de lésions cancéreuses. En tenant compte du nombre croissant de cas de cancers colorectaux, la découverte de facteurs de prédisposition de cette pathologie reste d’un intérêt majeur. Les données de séquençage du génome humain ne suffisant pas à expliquer la prévalence de la maladie à l’échelle de la population, nous nous intéressons aux mécanismes permettant le contrôle de l’expression de certains gènes : les mécanismes épigénétiques. Dans notre équipe nous disposons de modèles animaux génétiquement modifiés nous permettant d’étudier, dans des conditions proches de la pathologie humaine, les phases précoces de l’initiation tumorale. Ces souris, bien que génétiquement identiques, développeront pourtant un nombre de tumeurs intestinales très variable. En comparant les profils moléculaires de souris développant peu ou beaucoup de tumeurs intestinales, nous souhaitons mettre en évidence les facteurs épigénétiques mis en jeu pour expliquer cette différence de susceptibilité à la tumorigenèse. Pour ce faire, nous avons mis en place une stratégie visant à prélever un échantillon intestinal, par chirurgie, sur de jeunes souris qui ont ensuite été suivies jusqu’à l’âge adulte et à l’apparition des tumeurs. Cette stratégie innovante nous a permis de corréler les profils d'expression et de methylation d'ADN d’intestins développant peu ou beaucoup de tumeurs, ouvrant la possibilité de disposer de nouveaux marqueurs prédictifs quant aux chances de développer un cancer. Ces données sont complétées par une étude sur les conséquences de la perte du gène suppresseur de tumeur Apc, un gène couramment muté dans les cancers colorectaux humain. A l’aide de modèles de souris d’invalidation inductible, nous avons déterminé les conséquences de la perte d’Apc sur les profils de méthylation de l'ADN des cellules souches intestinales, et leur capacité à initier une tumeur. L’ensemble de ces différentes projets développés dans le cadre de ma thèse nous ont permis de mieux comprendre les mécanismes cellulaires liés à la prédisposition et à l’initiation tumorale et de proposer des nouvelles stratégies diagnostiques et d’évaluation du risque individuel. / Cancer initiation and progression represent the outcome of the progressive accumulation of genetic and epigenetic alterations. Global changes in the epigenome are now considered as a common hallmark of malignancies. However, most of our present knowledge represents the result of the comparison between fully established malignancies and their surrounding healthy tissue. Such comparison is not informative about the epigenetic contribution to the very early steps of cancer onset. By performing DNA methylation and gene expression profiling of the intestinal epithelium of relevant in vivo models we aim at shedding light on the correlation between the interindividual epigenetic polymorphisms within the population and the relative risk to develop malignancies, and establish the existence of a molecular signature associated with an increased susceptibility to develop intestinal cancer. Our results confirm that a considerable degree in the variability associated to cancer susceptibility cannot be ascribed to major genetic changes and that such heterogeneity seems to correlate with distinct molecular profiles associated to classes of poorly or highly susceptible isogenic animals.We also investigated in vivo the timing at which the remodeling occur at the epigenomic scale by analyzing the alterations in the DNA methylation and gene expression profiles of intestinal stem cells upon the loss of the Apc gene, the most common genetic lesion associated with human colorectal cancer initiation. We found that the loss of function of Apc in the Lgr5-positive intestinal stem cell compartment is rapidly accompanied by a reprogramming of the DNA methylation profiles resulting in altered gene expression and impaired fate determination in those cells. The results show that part of the phenotype resulting from the constitutive activation of the Wnt pathway upon Apc loss is acquired via differential epigenetic regulation of key biological processes controlling the balance between self-renewal and differentiation. By using conditional genetic ex vivo models we found part of these oncogenic effects to be reversible via the modulation of the machinery responsible for de novo methylation of the DNA.Overall, this work confirms that the epigenetic remodeling is an early event in tumorigenesis that might even precede actual cell transformation. The functional impact of our findings on cancer initiation is currently under investigation.

L'épithélium intestinal adulte des mammifères est un tissu hautement organisé entièrement renouvelé tous les 5 à 7 jours, grâce à la présence de cellules souches intestinales (CSI). Localisées à la base des cryptes, les CSI sont capables de s'auto-renouveler et de générer l'ensemble des types cellulaires de ce tissu. Afin de préserver l'équilibre entre leur auto-renouvellement et leur différenciation, véritable garant de l'homéostasie de l'épithélium intestinal, les CSI résident dans un microenvironnement finement régulé, « la niche », leur procurant l'ensemble des signaux nécessaires à leurs fonctions. La Nétrine-1, molécule sécrétée et apparentée à la famille des Laminines, est exprimée dans l'environnement des cryptes intestinales, mais également au cours du développement intestinal. Initialement découverte pour son rôle dans le guidage axonal, cette protéine est à ce jour considérée comme une molécule pléïotropique impliquée dans divers processus physiologiques tels que la morphogénèse, la migration, l'adhésion cellulaire, la prolifération mais également pathologiques comme la tumorigénèse. Considérant ces observations nous nous sommes donc intéressés au rôle potentiel de la Nétrine-1 dans la régulation du compartiment souche intestinal adulte, ainsi que lors de l'ontogénèse intestinale. Dans une première partie, nous montrons qu'ex vivo la Nétrine-1 promeut la croissance des entéroïdes et régule l'expression génique de certains marqueurs spécifiques des CSI. Dans une seconde partie, nous montrons, grâce à la génération et caractérisation de nouveaux modèles murins, que la Nétrine-1 est impliquée dans le développement de l'épithélium intestinal grêle et que sa délétion conduit à un retard d'émergence des villi / The adult intestinal epithelium is a highly organized tissue, which is completely self-renewed every 5 to 7 days, due to a pool of multipotent intestinal stem cells (ISC). Located at the base of intestinal crypts, ISC have the ability to self- renew and to give rise to all epithelial intestinal cell types. To preserve the balance between their self-renewal and their differentiation, and therefore to maintain the epithelial tissue homeostasis, ISC reside in a tightly regulated microenvironment - called “niche”- that provides them all factors required for their functions. Netrin-1, a laminin-related secreted protein, is expressed in the microenvironment of the crypt, and is also expressed during intestinal development. Initially described as an axonal guidance cue, Netrin-1 is now considered as a pleiotropic molecule involved in many different processes such as morphogenesis, cell migration, cell adhesion, proliferation and also tumorigenesis. Based on these observations, we hypothesized that Netrin-1 could play a role in the maintenance of the adult intestinal stem cell compartment, and also in the intestinal ontogenesis. In a first part, we showed that Netrin-1 promotes the growth of enteroids ex vivo while regulating gene expression of specific intestinal stem cell markers. In the second part, we demonstrated, by using two novel genetically engineered mouse models, that Netrin-1 is involved in the embryonic development of the intestinal epithelium and that its deletion leads to a delay in villi emergence

Vascular Endothelial Growth Factor (VEGF) is a highly conserved ligand that is involved in the regulation of angiogenesis and vasculogenesis, however, alternative roles of the ligand have been emerging. Organisms such as jellyfish and Drosophila contain VEGF homologs, yet they do not possess endothelial cells or a vascular system indicating that VEGF might have other primitive roles. In this current study we investigated how VEGF affects the post-natal development of the intestinal epithelial by either overexpressing VEGF or by reducing the bioavailability of VEGF with the overexpression of soluble VEGF receptor (sFLT-1) within the gastrointestinal tract. After three weeks of VEGF overexpression, mutant mice displayed an increase in villus height and proliferation in the transit-amplifying zone with the decrease of crypts per measured length and Lgr5 expression. On the other hand, sFLT-1 overexpressing mice had an increase in crypt depth with a decrease in villus height, proliferation in the transit-amplifying zone, crypts per measured length and reduced expression of Dll1 and Bmp4. Overall the availability of VEGF has the ability to alter the proliferation of progenitor cells in the crypt by either a direct or indirect signals. These studies reveal that by some means VEGF is altering the developing post-natal intestinal epithelium and proliferation. Largely, elucidating the interaction between VEGF and intestinal stem cells in intestinal development and differentiation may help to advance intestinal stem cell therapies in intestinal dysfunction or disease

The canonical Wnt signaling pathway is one of the most important pathways involved in cell proliferation and differentiation. It is highly conserved in evolution and participates not only in embryonic development but also in adult tissue homeostasis. In the intestine, Wnt signaling is closely connected to maintenance of intestinal stem cells and renewal of the epithelia. Conversely, aberrant activation of the Wnt signaling pathway underlies different types of human diseases. Its constitutive activation results in neoplasia and specifically in development of colorectal cancer, which is the third most common malignancy in western world. The aim of this thesis was to uncover various aspects of the regulatory mechanisms of the Wnt/β-catenin signaling cascade. Furthermore, I headed to find novel Wnt pathway modulators and confirm their function in vivo. The results are presented in four publications. The first study examines murine Wnt proteins processing and the sequential order of Wnt post-translational modifications which are required for the secretion and signaling activity of the ligands. Next publication focuses on the gene Troy, which we identified as negative regulator of Wnt signaling. TROY was discovered as a Wnt target gene during DNA microarray profiling of human colorectal cancer cells....