La rétine est constituée de plusieurs types de neurones incluant les cellules amacrines, ganglionnaires, bipolaires et les photorécepteurs. Les photorécepteurs, qui englobent les cônes et les bâtonnets, sont des neurones sensoriels hautement spécialisés qui permettent la conversion de la lumière en signaux électriques par le mécanisme de phototransduction. Les mécanismes moléculaires par lesquels les progéniteurs rétiniens (RPCs) se différencient en différents neurones spécialisés comme les photorécepteurs sont encore peu connus. Le gène Polycomb Bmi1 appartient à la famille des gènes Polycomb qui forment des complexes multimériques impliqués dans la répression de l’expression génique via le remodelage de la chromatine. Au niveau biologique, le gène Bmi1 régule, entre autre, le contrôle de la prolifération cellulaire, le métabolisme des radicaux libres, et la réparation de l’ADN. Récemment, il a été démontré que Bmi1 joue un rôle critique dans la prolifération et l’auto-renouvellement d’un groupe de RPCs immatures. De plus, Bmi1 est essentiel au développement post-natal de la rétine. L'objectif de cette étude est d'analyser le rôle de Bmi1 dans le développement et la survie des photorécepteurs chez la souris. Nos résultats révèlent un phénotype de dégénérescence des photorécepteurs de types cônes chez notre modèle de souris déficiente pour Bmi1. Les bâtonnets sont insensibles à la mutation. De plus, Bmi1 est exprimé de façon prédominante dans les cônes. Nos expériences de culture de cellules rétiniennes suggèrent que le phénotype est cellule-autonome. Par ailleurs, la co-délétion du gène Chk2, membre de la réponse aux dommages à l'ADN, permet de ralentir la progression du phénotype. Les rétines Bmi1-/- et Bmi1-/-Chk2-/- présentent une augmentation importante des dommages oxydatifs à l'ADN. Ces résultats suggèrent que le stress oxydatif pourrait jouer un rôle important dans la survie des cônes. L'étude du rôle du gène Polycomb Bmi1 dans les photorécepteurs est importante pour une meilleure compréhension des mécanismes contribuant à la survie des cônes et pourrait mener à la découverte de nouveaux traitements des maladies dégénératives des cônes. / The retina is composed of several types of neurons such as amacrin, ganglion, bipolar and photoreceptor cells. Photoreceptors, which include cones and rods, are highly specialized neurons that convert light into electrical signals by phototransduction. The molecular mechanisms involved in differentiation of retinal progenitors (RPCs) into specialized neurons such as photoreceptors are poorly understood. The polycomb gene Bmi1 is a member of the Polycomb gene family that forms multimeric complexes involved in chromatin remodeling leading to gene repression. Biological functions of Bmi1 include regulation of cell proliferation, free radical metabolism, and DNA repair. Recently, it was shown that Bmi1 plays a critical role in the proliferation and self-renewal of a specific immature RPC group. Moreover Bmi1 is essential for post-natal retinal development. The objective of the current study is to analyze Bmi1 function in photoreceptor development and survival. Our results show that Bmi1 deficiency in mice causes degeneration of cone photoreceptors, but not of rods. Furthermore, Bmi1 is predominantly expressed in cones. Experiments using primary retinal cell cultures suggest a cell-autonomous phenotype. In addition, codeletion of Bmi1 and the critical DNA damage response protein Chk2 resulted in partial rescue and slow-down of cone degeneration. Bmi1-/- and Bmi1-/-Chk2-/- retinas also exhibit an important increase in oxidative DNA damage, suggesting that cellular redox state could play an important role in cone survival. Our studies on the role of Bmi1 in photoreceptors elucidate the mechanisms contributing to cone survival, and could lead to the development of new treatments for cone degenerative diseases.

The main focus of my PhD studies was aimed at understanding the general mechanism of nuclear translocation and isolating novel components of the nuclear translocation pathway in neurons. Using the Drosophila visual system as an in vivo model to study nuclear motility in developing photoreceptor cells (R-cells), I have identified a novel role for the Ser/Thr kinase Misshapen (Msn) and the endocytic trafficking pathway in regulating the nuclear translocation process. / The development of R-cells in the Drosophila eye disc is an excellent model system for the study of nuclear motility owing to its monolayer organization and the stereotypical translocation of its differentiating R-cell nuclei along the apical-basal plane. Prior to my thesis work, several laboratories had identified dynein and its associating proteins in R-cell nuclear translocation, however nothing was known about the signalling pathway that controlled their function in nuclear migration. Thus, one of my thesis goals was to elucidate the signalling mechanism controlling nuclear translocation in R-cells. / Using a combination of molecular and genetic approaches, I identified Msn as a key component of a novel signalling pathway regulating R-cell nuclear translocation. Loss of msn causes a failure of R-cell nuclei to migrate apically. Msn appears to control R-cell nuclear translocation by regulating the localization of dynein and Bicaudal-D (Bic-D). My results also show that Msn enhances Bic-D phosphorylation in cultured cells, suggesting that Msn regulates R-cell nuclear migration by modulating the phosphorylation state of Bic-D. Consistently, my results show that a Bic-D-phosphorylation-defective mutation disrupted the apical localization of both Bic-D and dynein. I propose a model in which Msn induces the phosphorylation of Bic-D, which in turn modulates the activity and/or subcellular localization of dynein leading to the apical migration of R-cell nuclei. / In addition to studying Msn, I have also searched for additional players in R-cell nuclear migration. From a gain-of-function approach, I found that the misexpression of the GTPase-activating-protein (GAP) RN-Tre caused a severe defect in R-cell nuclear migration. Since mammalian RN-Tre is involved in negatively regulating Rab protein activity, I speculated that the RN-Tre misexpression phenotype reflected a role for Rab-mediated vesicular transport in regulating R-cell nuclear migration. I systematically examined the potential role of Rab family proteins in R-cell nuclear migration and found that interfering with the function of Rab5, Rab11 or Shibire caused a similar nuclear migration phenotype. I propose that an endocytic pathway involving these GTPases is required for the targeting of determinants to specific subcellular locations, which in turn drive the apical migration of R-cell nuclei during development.

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

No description available.

La leucine zipper de la rétine neurale (Nrl) joue un rôle central dans le développement et l'homéostasie des bâtonnets en activant I'expression de gènes tels que le photopigment Rhodopsine. Nrl est aussi associé à la Rétinite Pigmentaire, faisant ainsi de ce gène un modèle intéressant pour la compréhension des programmes contrôlant le développement et I'homéostasie des photorécepteurs.Ce travail de thèse vise à caractériser les mécanismes régulateurs de I'expression de Nr/ au cours du développement rétinien. L'électroporation in vivo de vecteurs rapporteurs dans des rétines de souris en développement, a révélé des séquences minimales de promoteur Nr/ nécessaires à une expression spécifique dans les photorécepteurs. Nous avons identifié RORI3 comme facteur requis pour cette expression, et montré que les facteurs OTX2, CRX et CREB s'accrochent aussi directement à des régions régulatrices particulières du promoteur. Nous avons construit un virus adéno-associé (AAV) contenant un promoteur minimal Nrl de 0.3 kb, et montré qu'il est adapté à la délivrance de gène spécifiquement dans les photorécepteurs.Nous avons montré que NRL, CRX et NR2E3, les régulateurs principaux de la Rhodopsine, ont une expression rythmique au cours de 24 h, et que l'expression cyclique de Nr/ peut être due à l'activation par RORp, un composant l'horloge circadienne. Enfin, nous avons identifié un nouveau facteur de transcription, NonO, au niveau de la région du promoteur proximal de la Rhodopsine, qui en combinaison avec NRL et CRX, active le promoteur de la Rhodopsine. L'invalidation de NonO au cours du développement rétinien a prouvé son implication pour le développement et I'homéostasie des bâtonnets. / The Neural Retina Leucine zipper transcription factor (Nrl) plays a central role in rod photoreceptor development and homeostasis, by activating the expression of rod-specific genes such as the visual photopigment, Rhodopsin. Nrlhave been also associated with Retinitis Pigmentosa, making this gene an interesting model for understanding genetic programs controlling photoreceptors development and homeostasis.This thesis work aimed at characterizing regulatory mechanisms of Nr/ expression during retinal development. Using in vivo electroporation of reporter vectors carrying distinct portions of Nrlpromoter into neonatal mouse retina, we identified minimal sequences required for expression photoreceptors-specific expression. We identified RORI3 as being required for this expression and showed that OTX2, CRX and CREB transcription factors also directly bind to the defined regulatory regions.We designed a novel adeno-associated virus (AAV) vector containing a minimal Nrl promoter fragment of 0.3 kb, and showed that it is well-suited for gene delivery specifically into photoreceptors.We also showed that NRL, CRX, and NR2E3, the main transcriptional regulators of Rhodopsin, display rhythmic expression over 24 h. and that Nrl might undergo cyclic activation by RORB which is part of the photoreceptor circadian clock. Finally, we investigated the role of a novel Rhodopsin transcriptional regulator, NonO, identified in theRhodopsin proximal promoter region. We demonstrated that NonO co-activates Rhodopsin promoter along with NRL and CRX. By knocking down this gene during retinal development we provided evidence for its role in rod development and homeostasis.

Circadian clocks are biological timekeepers that help maintain an organism’s behavior and physiological state optimally timed to the Earth’s day/night cycle. To do this, these internal pacemakers must accurately keep track of time. Equally importantly, they must be able to adjust their oscillations in response to external time cues to remain properly synchronized with the environment, and correctly anticipate environmental changes. When the internal clock is offset from its surrounding day/night cycle, clinically relevant disruptions develop, ranging from inconveniences such as jet-lag to more severe problems such as sleep disorders or mood disorders. In this work, I have used the fruit fly, Drosophila melanogaster, as a model organism to investigate how light and temperature can synchronize circadian systems. My initial studies centered on an intracellular photoreceptor, CRYPTOCHROME (CRY). CRY is a blue light photoreceptor previously identified as a major component of the primary light-input pathway into the Drosophila circadian clock. We used molecular techniques to show that after light-activation, CRY binds to the key circadian molecule TIMELESS (TIM). This interaction irreversibly targets TIM, but not CRY, for degradation. Further studies characterizing a newly isolated cry mutant, crym, showed that the carboxyl-terminus of CRY is not necessary for CRY’s ability to impart photic information to the molecular clock. Instead, the C-terminus appears to be necessary for normal CRY stability and protein-protein interactions. Thus, we conclude that in contrast to previous reports on CRYs of other species, where the C-terminal domain was required for transduction of photic information, the C-terminus of DrosophilaCRY has a purely modulatory function. During the second part of my dissertation work, I focused my studies on circadian thermoreception. While the effects of light in synchronization of the Drosophilaclock to environmental cycles have been extensively characterized, significantly less is known about temperature input pathways into the circadian pacemaker. I have used two approaches to look at how temperature affects the circadian system. First, I conducted a series of behavioral analyses looking at how locomotor rhythms can be phase-shifted in response to temperature cycles. By examining the behavior of genetically ablated flies, we determined that the well-characterized neurons controlling morning and evening surges of activity during light/dark cycles are also implicated in morning and evening behaviors under temperature cycles. However, we also find evidence of cells that contribute to modulating afternoon and evening behavior specifically under temperature cycles. These data contribute to a growing number of studies in the field suggesting that pacemaker cells may play different roles under various environmental conditions. Additionally, we provide data showing that intercellular communication plays an important role in regulating circadian response to temperature cycles. When the morning oscillator is absent or attenuated, the evening cells respond abnormally quickly to temperature cycles. My work thus provides information on the roles of different cell groups during temperature cycles, and suggests that beyond simply synchronizing individual oscillating cells, intercellular network activity may also have a role in modulating proper response to environmental time cues. Finally, I present some preliminary work looking at effects of temperature on known circadian molecules. Using a combination of in vivo and cell culture techniques, I have found that TIM protein levels decrease at higher temperatures. My cell culture data suggest that this is a proteasome-independent degradation event. As TIM is also a key molecule in the light-input pathway, the stability of TIM proteins may be a key point of integration for light and temperature input pathways. While additional research needs to be conducted to confirm these effects in vivoin wild-type flies, these preliminary results identify a possible avenue for further study. Taken together, my work has contributed new data on both molecular and neuronal substrates involved in processing light and temperature inputs into the Drosophila circadian clock.

Most organisms, from cyanobacteria to humans are equipped with circadian clocks. These endogenous and self-sustained pacemakers allow organisms to adapt their physiology and behavior to daily environmental variations, and to anticipate them. The circadian clock is synchronized by environmental cues (i.e. light and temperature fluctuations). The fruit fly, Drosophila melanogaster, is well established as a model for the study of circadian rhythms. Molecular mechanisms of the Drosophilacircadian clock are conserved in mammals. Using genetic screens, several essential clock proteins (PER, TIM, CLK, CYC, DBT, SGG and CK-II) were identified in flies. Homologs of most of these proteins are also involved in generating mammalian circadian rhythms. In addition, there are only six neuronal groups in the adult fly brain (comprising about 75 pairs of cells) that express high levels of clock genes. The simplicity of this system is ideal for the study of the neural circuitry underlying behavior. The first half of this dissertation focuses on a genetic screen designed to identify novel genes involved in the circadian light input pathway. The screen was based on previous observations that a mutation in the circadian photoreceptor CRYPTOCHROME (CRY) allows flies to remain rhythmic in constant light (LL), while wild type flies are usually arrhythmic under this condition. 2000 genes were overexpressed and those that showed a rhythmic behavior in LL (like crymutants) were isolated. The candidate genes isolated in the screen present a wide variety of biological functions. These include genes involved in protein degradation, signaling pathways, regulation of transcription, and even a pacemaker gene. In this dissertation, I describe work done in order to validate and characterize such candidates. The second part of this dissertation focuses on identifying the pacemaker neurons that drive circadian rhythms in constant light (LL) when the pacemaker gene period is overexpressed. We found that a subset of pacemaker neurons, the DN1s, is responsible for driving rhythms in constant light. This attractive finding reveals a novel role for the DN1s in driving behavioral rhythms under constant conditions and suggests a mechanism for seasonal adaptation in Drosophila.

A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.

Cell polarity is an essential property of adult neurons, which rely on asymmetric distribution of receptors and transmitters for proper signal propagation and cell function. In the retina, loss of photoreceptor (PR) polarity can lead to retinal dystrophies such as Leber Congenital Amaurosis, but the molecular mechanisms involved in regulating PR polarity remain unclear. A highly conserved protein complex involved in the establishment of cell polarity from C. elegans to mammals is the Par complex. Localized at the subapical region of polarized cells, it is composed of the “partitioning defective” PDZ domain-containing proteins Par3/Par6 and the atypical protein kinase C (aPKC). Although extensively studied in epithelial cells, the role of the Par complex in mammalian neurons remains poorly understood. Our unpublished results indicate that conditional inactivation (cKO) of Par3 in the developing retina interferes with the polarized growth of the photosensitive cilium at the apical tip of PR cells, eventually leading to PR degeneration. To uncover how Par3 might regulate ciliogenesis in PR cells, we immunoprecipitated Par3 from mouse retinal extracts and carried out mass spectrometry analysis. We found a cluster of calcium/calmodulin-dependent protein kinase II (CaMKII) proteins as potential Par3-interacting partners in the retina. CaMKII is one of the most abundant proteins found in the central nervous system, where it constitutes 1-2% of total proteins. While extensive studies have demonstrated the importance of CaMKII in long-term potentiation (LTP), long term depression (LTD) and dendrite arborisation, its role in cell polarity remains unknown. Using tagged versions of Par3 and CaMKIID, we validated their interaction in vivo and in vitro by co-immunoprecipitation. Interestingly, we found that CaMKIID localizes to the ciliary region of PRs, suggesting that Par3 might recruit CaMKIID at the apical membrane of PR cells, where it could be involved in ciliogenesis. To explore this hypothesis, we investigated whether dominant-negative or constitutively active forms of CaMKIID could impact cilia formation in PRs. Interestingly, overexpression of both mutant forms of CaMKIID during PR development resulted in shortening of the photosensitive cilia (outer segments), similar to what we observed in Par3 cKO retinas. This study suggests that a CaMKIID/Par3 protein complex regulates the establishment of PR cell polarity, raising the possibility that this complex may be generally involved in controlling neuronal polarity throughout the nervous system. / Le traitement et la propagation de l’information nerveuse repose sur une distribution asymétrique de récepteurs et d’émetteurs à la surface de chaque neurone. Ce cloisonnement en domaines sous-cellulaires distincts est également appelé polarité cellulaire. Dans la rétine, la perte de polarité des photorécepteurs peut entraîner des dystrophies rétiniennes telle que l'amaurose congénitale de Leber, mais les mécanismes moléculaires impliqués restent flous. Un complexe protéique impliqué dans l'établissement de la polarité cellulaire, hautement conservé de C. elegans aux mammifères, est le complexe PAR. Localisé au niveau de la région sous-apicale des cellules polarisées, le coeur de ce complexe est constitué des protéines de la famille partitioning defective Par3 / Par6 et de la protéine kinase C atypique aPKC. Bien que largement étudié dans les cellules épithéliales, le rôle du complexe Par dans les neurones de mammifères reste mal compris. Nos résultats indiquent que l'inactivation conditionnelle (cKO) de Par3 dans la rétine de souris en développement interfère avec la croissance polarisée du cil photosensible à la pointe apicale des cellules photoréceptrices (PR), conduisant finalement à une dégénérescence des PRs. Pour découvrir comment Par3 pourrait réguler la ciliogenèse des PRs, nous avons immunoprécipité Par3 à partir d'extraits rétiniens de souris et effectué une analyse par spectrométrie de masse. Nous avons trouvé un ensemble de protéines appartenant à la famille des calcium-calmoduline-dépendantes de la protéine kinase II (CaMKII) comme partenaires potentiels de Par3 dans la rétine. Les CaMKII figurent parmi les protéines les plus abondantes du système nerveux central où elles constituent 1 à 2% des protéines totales. Alors que des études approfondies ont démontré l'importance de CaMKII dans la potentialisation et la dépression à long terme (LTP et LTD), et l'arborisation des dendrites, son rôle dans la polarité cellulaire reste inconnu. En utilisant des versions étiquetées de Par3 et CaMKIID, nous avons validé leur interaction in vivo et in vitro par co-immunoprécipitation. Nous avons mis en évidence une localisation de CaMKIID dans la région ciliaire des PR, suggérant que Par3 pourrait recruter CaMKIID à la membrane apicale des cellules PR, où il pourrait être impliqué dans la ciliogenèse. Pour explorer cette hypothèse, nous avons étudié si les formes dominantes négatives ou constitutivement actives de CaMKIID pouvaient avoir un impact sur la formation des cils des PRs. vii La surexpression des deux formes mutantes au cours du développement des PRs a entrainé un raccourcissement des segments externes, semblable à ce que nous avons observé dans les rétines Par3 cKO. Cette étude montre qu'un complexe de protéines CaMKIID / Par3 pourrait réguler l’établissement et le maintien de polarité des PRs, suggérant l’implication ce complexe dans le contrôle de la polarité neuronale de l’ensemble du système nerveux central.

La vision est considérée comme un des sens les plus importants, prenant en charge environ 80% des perceptions que nous recevons dans notre vie quotidienne. Les photorécepteurs de type cônes sont responsables de la vision centrale de haute résolution et en couleurs, et leur dégénérescence est souvent la cause de la perte de vision dans les maladies dégénératives rétiniennes (RDs). Les RDs sont un groupe hétérogène de maladies affectant des millions de personnes dans le monde, qui pour le moment sont pour la plupart sans aucune option thérapeutique. Les modèles animaux sont extrêmement utiles pour étudier le développement ou la dégénérescence de la rétine, ainsi que pour comprendre les mécanismes moléculaires des maladies génétiques héréditaires affectant les photorécepteurs. La modélisation des maladies dégénératives et du développement peut être particulièrement difficile, spécialement dans le cas de maladies humaines rares et complexes pour lesquelles aucun modèle animal exhaustif n'est disponible. De nos jours, la génération et le maintien de modèles de maladies humaines permettant une analyse approfondie du mécanisme moléculaire représente un grand défis . La technologie des cellules souches possède un grand potentiel dans la modélisation des maladies et représente un outil puissant pour générer des modèles évolutifs, sans l’utilisation d’animaux qui peuvent illustrer plus précisément les phénotypes cliniques de maladies humaines complexes. Nous avons développé un protocole pour différencier les cellules souches pluripotentes (PSCs) en feuillets rétiniens (RSs), qui sont des tissus polarisés et multicouches contenant des photorécepteurs cône et exprimant les marqueurs spécifiques du segment externe (OS), du cilium connecteur (CC) et du noyau. En utilisant à la fois des modèles de souris et des modèles humanisés à base de cellules souches, nous avons étudié le rôle de BMI1 dans les photorécepteurs matures. La protéine du groupe Polycomb Bmi1 est connue pour ses fonctions neuroprotectrices en contrôlant la sénescence et l'apoptose, et est exprimée à la fois dans le progéniteur rétinien et les neurones, mais on en sait peu sur son rôle spécifique dans la rétine adulte. Elle a été récemment associée à des troubles neurodégénératifs d'apparition tardive, et elle pourrait avoir un rôle dans la pathologie des RDs d'apparition tardive, comme la dégénérescence maculaire liée à l'âge (DMLA). Nous avons montré que les photorécepteurs cône et les neurones bipolaires sont générés normalement mais subissent ensuite une dégénérescence rapide chez les souris Bmi1-/- par nécroptose associée à Rip3. La dégénérescence était associée à des anomalies de compactage de la chromatine, à l'activation des répétitions en tandem et au stress oxydatif. De plus, nous montrons que BMI1 est préférentiellement exprimé dans les cônes au niveau des foyers hétérochromatiques dans la rétine humaine. Son inactivation dans les cellules souches embryonnaires humaines (hESCs) a altéré la différenciation terminale du cône et a entraîné des anomalies de compactage de la chromatine, l'activation des répétitions en tandem et l'induction de P53. Ces résultats fournissent un mécanisme expliquant comment une carence en Bmi1 conduit à la dégénérescence des cônes et révèlent des fonctions biologiques conservées et des différences pour Bmi1 dans la biologie des photorécepteurs entre la souris et l'homme. En utilisant un modèle humain basé sur les cellules souches pluripotentes induites (iPSCs), nous avons ensuite étudié le processus dégénératif chez les patients atteints de ciliopathies, un groupe de maladies génétiques hétérogènes affectant les protéines impliquées dans la structure et la fonction du cil primaire, qui sont fréquemment accompagnée d'une dégénérescence rétinienne. Nous générons des feuillets rétiniens dérivés d'iPSCs à partir de patients atteints de deux ciliopathies, les syndromes de Meckel-Gruber (MKS) et de Bardet-Biedl (BBS). Les photorécepteurs ciliopathiques présentaient des altérations communes significatives dans l'expression de centaines de gènes de développement. De plus, ils ont montré plusieurs anomalies dans la formation et le maintien du cilium interne, le positionnement du centriole mère, l'activation d'une réponse au stress aux protéines mal repliées, instabilités génomiques et l'accumulation de dommages à l'ADN. Cette étude révèle comment la combinaison des technologies de reprogrammation cellulaire et d'organogenèse avec le séquençage de nouvelle génération permet d'élucider les mécanismes moléculaires et cellulaires impliqués dans les troubles dégénératifs et développementaux de la rétine humaine. La même approche, combinant la différenciation en RSs avec des techniques de séquençage du génome à large spectre, pourrait être appliquée pour modéliser de nombreuses maladies génétiques, développementales et dégénératives affectant les photorécepteurs. Il peut également aider à élucider les mécanismes moléculaires sous-jacents à ces maladies, au criblage de médicaments de composés ayant des effets thérapeutiques potentiels et à prédire les effets secondaires des médicaments. / Vision is considered the most important sense, taking on about 80% of the perceptions we receive in our everyday life. Cone-photoreceptors are responsible for high-resolution central vision and color discrimination, and their degeneration is frequently the cause of vision loss in retinal degenerative diseases (RDs). RDs are a heterogeneous group of diseases affecting millions of people worldwide, which at the moment are mostly without any therapeutic option. Animal models are extremely useful in studying the retina's development or degeneration and understanding the molecular mechanisms in inherited genetic disease affecting photoreceptors. Modeling human developmental and degenerative diseases can be particularly challenging, especially in the case of rare and complex diseases where no exhaustive animal models are available. Generation of sustainable human disease models that allow in-depth analysis of the molecular mechanism is one of the big challenges nowadays. Stem cell technology holds great potential in disease modeling and represents a new powerful tool for generating scalable and animal-free models that can more accurately illustrate clinical phenotypes of complex human diseases. We developed a protocol to differentiate pluripotent stem cells (PSCs) into retinal sheets (RSs), which are polarized, multi-layered tissues containing cone photoreceptors and expressing outer segment (OS), connecting cilium (CC), and nuclear specific markers. Using both mouse and stem cells-based humanized models, we first investigate the role of BMI1 in mature photoreceptors. The Polycomb group protein Bmi1 is known for its neuroprotective functions by controlling senescence and apoptosis and is expressed in both retinal progenitor and neurons, but little is known about its specific role in the adult retina. It has been recently linked to late-onset neurodegenerative disorders, and it could have a role in the pathology of late-onset RDs, such as Age-related Macular Degeneration (AMD). We showed that cone photoreceptors and bipolar neurons are generated normally but then undergo rapid degeneration in Bmi1-/- mice through Rip3-associated necroptosis. Degeneration was associated with chromatin compaction anomalies, activation of tandem-repeats, and oxidative stress. Furthermore, we show that BMI1 is preferentially expressed in cones at heterochromatic foci in the human retina. Its inactivation in human embryonic stem cells (hESCs) impaired cone terminal differentiation and resulted in chromatin compaction anomalies, activation of tandem-repeats, and P53 induction. These findings provide a mechanism explaining how Bmi1 deficiency leads to cone degeneration and reveal conserved biological functions and differences for Bmi1 in photoreceptor biology between mouse and man. Using an induced Pluripotent Stem Cells (iPSCs) based human model, we then investigate the degenerative process in patients with ciliopathies, a group of heterogeneous genetic diseases affecting proteins involved in primary cilium structure and function frequently accompanied by retinal degeneration. We generate iPSC-derived retinal sheets from patients affected by two ciliopathies, Meckel-Gruber (MKS) and Bardet-Biedl syndromes (BBS). Ciliopathic photoreceptors displayed significant common alterations in the expression of hundreds of developmental genes. Moreover, they showed several anomalies in the formation and maintenance of cilia, the mother centriole's positioning, the activation of a stress response to misfolded proteins, genomic instabilities, and DNA damage accumulation. This study reveals how combining cell reprogramming and organogenesis technologies with next-generation sequencing enables the elucidation of molecular and cellular mechanisms involved in human retinal degenerative and developmental disorders. The same approach, combining photoreceptor sheet differentiation and wide-genome expression profile, could be applied to model many genetic, developmental, and degenerative diseases affecting photoreceptors. It may help elucidate the molecular mechanisms underlining these diseases, drug screening of compounds with potential therapeutic effects, and predict drug side effects.