Marie-Audrey Kautzmann

Cristina Sandu

Jean-Georges Lorentz

David Hicks

Anand Swaroop

Marie-Paule Felder-Schmittbuhl

Daily variation of photoreceptor specific-gene expression in the rat retina

Keywords: Rétine, photorécepteurs, Leucine zipper de la rétine neurale, rétinogenèse, transcription, horloge circadienne, Rhodopsine

Le facteur de transcription Nrl (Leucine zipper de la rétine neurale) associé à d'autres facteurs de transcription, active l'expression de gènes des bâtonnets tels que le photopigment Rhodopsine,.et est impliqué dans la Rétinite Pigmentaire. Ainsi, le gène Nrl constitue un modèle intéressant pour la compréhension des programmes contrôlant le développement et l'homéostasie des photorécepteurs.

Ce travail de thèse vise à caractériser les mécanismes régulateurs de l'expression de Nrl au cours du développement rétinien. Avec l'électroporation in vivo de vecteurs rapporteurs portant des portions distinctes du promoteur Nrl dans la rétine de souris nouveaux-nés, nous avons identifié des séquences minimales de promoteur nécessaires à une expression spécifique dans les photorécepteurs. Nous avons identifié ROR comme facteur requis pour cette expression, et montré que les facteurs de transcription 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 Nrl minimal de 0.3 kb, et montré que ce dernier est adapté à la délivrance de gène spécifiquement dans les photorécepteurs.

Nous avons également 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 Nrl peut être due à l'activation par ROR , un composant moléculaire de l'horloge circadienne. Enfin, nous avons identifié un nouveau facteur de transcription, NonO, au niveau de la région du promoteur proximal de la Rhodopsine, et démontré que ce facteur, en combinaison avec NRL et CRX, active le promoteur de la Rhodopsine. L'invalidation de NonO au cours du développement rétinien a mis en évidence son implication pour le développement et l'homéostasie des bâtonnets.

Marie-Paule, j'ai pu bénéficier de tes précieux conseils depuis le projet tutoré en M1 en cosupervision avec François Lasbenne. J'ai ensuite profité de ton sens de la pédagogie et de ton mentoring au cours de mon stage de Master 2, cependant, nous sommes allés de déception en déception quand les financements pour une thèse sous ta direction n'aboutissaient pas. C'est alors que s'est présentée l'opportunité de réaliser une thèse en co-direction avec Dr Anand Swaroop au NIH. Au final, j'ai réalisé la majeure partie de ma thèse aux Etats-Unis, mais cela ne nous a pas empêché d'être en contact par e-mail quasiment toutes les semaines pour suivre le déroulement et l'avancement de mon projet.

Tes conseils ont été les bienvenus et surtout tes encouragements tout au long de mon doctorat m'ont confortés dans la réalisation et l'achèvement de cette thèse. Merci pour ces cinq années d'encadrement entre le Master et la thèse qui se sont révélées formatrices mais aussi agréables.

I would like to sincerely thank Dr Anand Swaroop you accepted me in his lab as a graduate student at NEI. Dr Swaroop, you taught me how being meticulous while preparing figures and writing an article can be fruitful for a publication. You taught me to always aim higher ("think Nature and you will publish in PNAS"). You insisted that I be independent in my research, this last point was somehow difficult the first two years I worked with you but it slowly taught me to be autonomous, which is one of the quality required when thinking of doing a postdoc. For this matter, I appreciated your advices so I could find a good lab and a good mentor for the next step in my carreer, which is doing a postdoc.

Je tiens à remercier Dr David Hicks, qui m'a acceptée dans l'équipe «Rétine» dès mon stage de Master et dont les conseils et corrections de manuscripts ont été très appréciés. Je tiens également à te remercier pour avoir permis la collaboration avec le labo de Dr Swaroop. Ton I am grateful to all N-NRL lab members, for you advices during our meetings and for some of you, for showing me how to perform particular techniques.

Je voudrai particulièrement remercier Jérôme, mon premier contact et premier pote aux US.

Merci de m'avoir appris un peu toutes les techniques de bio mol que j'ai appliquées au cours de ma thèse, merci d'avoir eu des conseils objectifs quant à mon projet de thèse. Sur le plan perso, merci pour ton accueil avec Gaëlle. Une pensée affectueuse à Ayden et Neilan! Many thanks to my bench neighbourg, Hong, thanks for all your precious advices during my thesis and for our talks about anything. Best wishes for the future. I am thankful to Jacob "the human Wikipedia" whom knowledge about anything is impressive. Thanks for teaching me many techniques that I've applied for my project.

Merci beaucoup à Samélia ;-) Her friendship was -and still is -precious for me. Thanks for taking me out of the lab and for our discussions about everyday life. I wish you good luck for your thesis achievement and for the next step in your carreer.

Thanks to Douglas Kim for teaching me one of the main technique of my project, which is the in vivo electroproation into neonatal mouse retina. Your mentoring during the 1 st year of my thesis was really appreciable.

Briefly, I'd like to thanks Chunqiao for giving me really interesting suggestions about my project. Sharda, for being patient enough to answer so many questions of mine. Lynn, one of the nicest person I ever met, thank you for your help in and outside the lab. Winette, the other Caribbean girl of the lab, which is a remarkable person, thanks for your friendship. Suja Zhijian and Peter, thanks for the talks and help for the AAV project. Szilard and Miruna, I wish you good luck for your respective thesis achievement.

Du côté Strasbourgeois, je tiens à remercier Jean-Georges qui m'a appris la technique de Western Blot. Cristina, qui m'a permis de démystifier la technique de qPCR et dont l'aide pour les prélevements de rétine sur 24h a grandement été appréciée. Tounes, merci pour ta gentillesse tout au long de mon séjour au labo, je te souhaite bonne chance pour tes futurs projets. Madah, I wish you good luck for your thesis, hoping it will be fruitful. Daniella, good luck with the photoreceptor lipids! Claudine, merci pour ta gentillesse et je te souhaite un bon rebondissement ;-) Virginie, bonne chance pour tes manip ! Aux doctors-to-be : Ouafaa, je te souhaite beaucoup de courage pour l'achèvement de ton manuscript de thèse et bonne chance pour ta soutenance ! Catherine, je te souhaite une grande réussite pour cette dernière année de thèse, qu'elle te soit fructueuse. Cristina Saenz, merci pour ta bonne humeur et ta gentilesse, bon courage pour la suite de ton doctorat. Ibtissam et Caroline Ancel, bon courage pour la dernière ligne droite ! …Et à ceux qui ont encore quelques années devant eux avant la soutenance : Coralie, j'espère que la collaboration avec Atlanta se réalisera ! Caroline Allemann, Rajae, David et JBS, bon courage pour cette unique expérience qu'est le doctorat.

Une pensée à Julien, mon compère de thèse ! J'espère que ton choix de postdoc correspondra à tes attentes que ce soit en Europe ou aux States.

Vy, merci de t'être inquiéter régulièrement de l'évolution de la rédaction de mon manuscript. Tu as les qualités d'un bon futur doctorant, tu es dévoué à la tâche, j'espère que tu décrocheras une offre de thèse.

Un clin d'oeil à Domitille, dont la passation de bureau m'a permis de réaliser dans des conditions optimales ma fin de thèse.

Enfin, j'exprime toute ma gratitude à ma famille qui m'a encouragée et soutenue dans tout ce que j'ai entrepris. A mon significant other, dont la patience et les encouragments m'ont accompagnés tout au long de ce labeur.

Résumé en anglais

The Neural Retina Leucine zipper transcription factor (Nrl) plays a central role in rod photoreceptor development and homeostasis. Nrl, combined with other photoreceptor-specific transcription factors, activates expression of rod-specific genes such as the visual photopigment, Rhodopsin. Moreover, mutations in Nrl have been associated with Retinitis Pigmentosa. Thus, Nrl gene is an interesting model for understanding genetic programs controlling photoreceptors development and homeostasis. This thesis work aimed at characterizing regulatory mechanisms of Nrl expression during retinal development.

Using in vivo electroporation of reporter vectors carrying distinct portions of Nrl promoter into neonatal mouse retina, we identified the minimal sequences necessary to drive reporter gene expression specifically in photoreceptors layer. We identified ROR as being required for this expression and showed that OTX2, CRX and CREB transcription factors also directly bind to the defined regulatory regions.

Based on these results 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 ROR which is part of the photoreceptor circadian clock. Finally, we investigated the role of a novel Rhodopsin transcriptional regulator, NonO, identified in the Rhodopsin 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.

Retina, photoreceptors, Neural retina leucine zipper, retinogenesis, transcription, circadian clock, rhodopsin

RECURRENT ABBREVIATIONS

AAV

RÉSUMÉ DE LA THÈSE EN FRANÇAIS

RÉSUMÉ DE LA THÈSE EN FRANÇAIS

Chez les Eukaryotes, la transcription d'un gène repose sur la présence d'éléments régulateurs autour de ce gène: le promoteur basal, ainsi que des séquences situées en aval et/ou en amont du promoteur. Ces séquences sont le siège d'accrochage direct de facteurs trans-activateurs ou facteurs de transcription (FT), qui travaillent en synergie avec des co-transactivateurs, ne liant pas l'ADN directement mais agissant par le biais des FT. Ces régulateurs sont essentiels à l'accrochage de la machinerie d'initiation de la transcription sur l'ADN. Cette machinerie est constituée d'un large complexe protéique, dont l'unité principale est l'ARN polymerase II. L'intervention de FT influencera le niveau de transcription d'un gène donné, mais également déterminera l'expression ou non de ce gène en ayant des fonctions activatrice ou inhibitrice. Le mode d'action des FT est vraisemblablement de recruter des protéines co-régulatrices de la transcription qui altèrent localement la conformation de la chromatine, permettant ainsi l'accès de l'ARN polymerase II au site d'initiation de la transcription. Une autre possibilité est le recrutement par les FT de co-activateurs qui possèdent des fonctions enzymatiques affectant la chromatine. De par sa structure primaire, la chromatine est hautement condensée par l'action des histones; l'accès aux gènes nécessite donc une altération transitoire de cette structure histone-ADN. Des enzymes telles que les transférases de groupements acétyl, méthyl ou de phosphates, permettent de marquer les gènes transcriptionnellement actifs et de déstabiliser ces complexes histones-ADN pour permettre l'ouverture de la chromatine et l'accès aux séquences régulatrices du promoteur pour initier la transcription. Certains FT permettent d'activer leur gène cible de façon spatio-temporelle et confèrent aux cellules dans lesquelles ils sont exprimés une identité cellulaire. En d'autre terme, c'est la fine régulation de l'expression de gènes spécifiques qui permettra de générer des types cellulaires variés constituant un tissu donné.

La diversité des types de neurones peut être étudiée en utilisant un tissu modèle : la rétine. Son accessibilité et son répertoire cellulaire bien défini font de la rétine un tissu de choix pour l'étude des mécanismes sous-jacents au développement de types cellulaires spécifiques. La rétine est un fin tissu nerveux situé au fond de l'oeil, et est composée de six types de neurones et un type de glie organisés en couches bien distinctes. La lumière traverse l'ensemble de ces couches pour être perçue au niveau de la couche la plus externe, composée des photorécepteurs (PR). Les PR, qui possèdent des pigments visuels (opsines) sensibles aux photons, sont chargés de traduire l'influx lumineux en message nerveux et de le transmettre aux autres couches de la rétine qui relaient ce signal par le biais des axones des cellules ganglionnaires jusqu'aux centres d'intégration dans le cerveau. On distingue deux types de photorécepteurs, sur la base de leurs caractéristiques morphologiques et fonctionnelles, les cônes et les bâtonnets.

Chez l'Homme, il existe trois types de cônes dissemblables par l'opsine qu'ils expriment.

Les cônes sont responsables de la vision en forte intensité lumineuse et de la détection des couleurs, alors que les bâtonnets, qui ne possèdent qu'un type d'opsine, la Rhodopsine, fonctionnent en faible intensité lumineuse et sont responsables de la détection des mouvements et des formes. Les bâtonnets ont un rôle trophique pour les cônes [START_REF] Leveillard | Identification and characterization of rod-derived cone viability factor[END_REF]) et sont particulièrement sensibles aux altérations d'expression génique ainsi qu'à la qualité de l'environnement extracellulaire [START_REF] Parapuram | Distinct signature of altered homeostasis in aging rod photoreceptors: implications for retinal diseases[END_REF]. De ce fait, l'élucidation de la genèse et du maintien fonctionnel des photorécepteurs de type bâtonnets est nécessaire pour une meilleure élaboration de stratégies thérapeutiques pour le traitement des affections rétiniennes conduisant à la cécité.

Chez les Mammifères, la genèse des différents types neuronaux composant la rétine s'effectue selon un ordre précis [START_REF] Young | Cell differentiation in the retina of the mouse[END_REF]. L'intervention binaire de facteurs extrinsèques et intrinsèques est critique pour le développement rétinien [START_REF] Livesey | Vertebrate neural cell-fate determination: lessons from the retina[END_REF], cependant, il semblerait que les facteurs intrinsèques soient prédominants dans l'acquisition de la spécificité du type cellulaire. Parmi les facteurs intrinsèques agissant au niveau des cellules post-mitotiques, Nrl (Neural retina leucine zipper) [START_REF] Swaroop | A conserved retina-specific gene encodes a basic motif/leucine zipper domain[END_REF]), un facteur de transcription de type bZIP, joue un rôle primordial dans la détermination des précurseurs des photorécepteurs post-mitotiques à se différencier en bâtonnets plutôt qu'en cônes [START_REF] Oh | Transformation of cone precursors to functional rod photoreceptors by bZIP transcription factor NRL[END_REF]. Nrl est le facteur clé responsable de la formation de la lignée des bâtonnets, en effet, l'ablation du gène Nrl résulte en la formation d'une rétine composée uniquement de photorécepteurs de type cône [START_REF] Mears | Nrl is required for rod photoreceptor development[END_REF]. L'expression ectopique de Nrl chez des animaux Nrl -/-résulte en une rétine composée uniquement de bâtonnets [START_REF] Oh | Transformation of cone precursors to functional rod photoreceptors by bZIP transcription factor NRL[END_REF].

Ces derniers se révèlent fonctionnels d'après l'électrorétinogramme (ERG) en conditions La dissection du promoteur de Nrl a permis de mettre en évidence la fonctionnalité du cluster B (-938 à -657) en association avec la région A1 (-35 à +16) du cluster A. La combinaison de ces deux éléments de promoteur résulte en une séquence de 0.3 kb qui montre une activité transcriptionnelle similaire à celle du plus long fragment (-2734 à +119). Barre d'échelle,20 µm. scotopiques. Ces données suggèrent que Nrl est un facteur non seulement essentiel mais aussi suffisant à la genèse des photorécepteurs de type bâtonnet. De plus, Nrl est impliqué dans le maintien de l'homéostasie des bâtonnets, puisqu'il agit avec d'autres cofacteurs tels que Crx et Nr2e3 pour activer l'expression de la Rhodopsine [START_REF] Rehemtulla | The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression[END_REF]) et de plusieurs autres gènes spécifiques des bâtonnets [START_REF] Pittler | Functional analysis of the rod photoreceptor cGMP phosphodiesterase alpha-subunit gene promoter: Nrl and Crx are required for full transcriptional activity[END_REF][START_REF] Oh | Rod differentiation factor NRL activates the expression of nuclear receptor NR2E3 to suppress the development of cone photoreceptors[END_REF]. Une altération de l'expression des gènes des photorécepteurs, dont notamment NRL, résulte en une dégénérescence de ces cellules à l'origine d'un certain nombre de pathologies telles que la Rétinite Pigmentaire (Bessant, Payne et al. 1999;Kanda, Friedman et al. 2007;[START_REF] Hernan | Novel p.M96T variant of NRL and shRNA-based suppression and replacement of NRL mutants associated with autosomal dominant retinitis pigmentosa[END_REF]. Ainsi, un fin contrôle de l'expression du gène NRL semble crucial pour le développement et le fonctionnement correct des photorécepteurs de type bâtonnet. De fait, NRL constitue un bon modèle pour comprendre les programmes génétiques contrôlant le développement des photorécepteurs.

Au cours de ma thèse, je me suis attachée à caractériser les mécanismes régulant l'expression du gène Nrl. Une partie de ce travail a donné lieu à une première publication dont je suis le premier auteur (Kautzmann, Kim et al. 2011). L'analyse des régions du promoteur m'a permis de cibler trois régions principales fortement conservées chez les vertébrés (Figure 1'). J'ai exploré les propriétés de ces régions en termes de régulation transcriptionnelle, en utilisant la technique d'électroporation in vivo qui consiste à faire pénétrer des acides nucléiques dans la rétine d'animaux nouveaux nés, correspondant à la période où la prolifération des bâtonnets est maximale (Figure 2'). L'injection de vecteurs rapporteurs contenant des segments du promoteur de Nrl de différentes tailles a permis d'identifier une région de 0.3 kilobases suffisante pour une expression du rapporteur fluorescent spécifiquement dans les bâtonnets (Figure 3'). Une analyse des séquences de promoteur transcriptionnellement actives a permis de mettre en évidence l'accrochage direct de facteurs de transcription ROR, CRX, OTX2 et CREB sur des sites consensus prédits (Figure 4').

La définition d'un promoteur Nrl minimal, fonctionnel et spécifique pour l'expression dans les bâtonnets, a ouvert une perspective d'utilisation de ce promoteur en thérapie génique en utilisant des rAAV (virus recombinants adéno-associés). J'ai testé (D and E). Les oligonucléotides B3, B4 et A4 sont indiqués. B4 contient 18 nucléotides supplémentaires à l'extrémité 5' de B3, lui conférant ainsi une meilleure séquence de reconnaissance aux facteurs de transcription de transcription à homéodomaine. la fonctionnalité de ce promoteur de NRL à exprimer le gène rapporteur suivant son clonage dans le génome du vecteur viral AAV8 (dérivé de l'Adenovirus Associated Virus), et ai montré que ce promoteur pouvait être activé chez de jeunes souris (10 jours postnatal). De plus, deux types de rétines de souris ont été infectés in vivo, des rétines sauvages et des rétines de souris NRL KO composées uniquement de cônes. L'infection par les AAV8-NRLp-EGFP a montré une expression spécifique du gène rapporteur au niveau de la couche des photorécepteurs, et une activation du promoteur dans la rétine des NRL KO (Figure 5 RevErbqui régulent aussi d'autres gènes cibles, entraînant ainsi les fonctions rythmiques de la cellule. Le zeitgeber le plus puissant est le cycle jour/nuit, perçu chez les Mammifères exclusivement par la rétine. Le rôle de l'horloge rétinienne est probablement d'adapter la rétine aux changements d'intensité lumineuse subits au cours de 24 h, de l'ordre de 1x10 6 fois le niveau basal. Parmi les fonctions rythmiques de la rétine, nous pouvons citer des phénomènes d'ordre physiologique, tels que la sensibilité visuelle et le traitement de l'information lumineuse qui se manifeste au niveau des variations de l'ERG, ainsi que la régulation du pH extracellulaire caractérisés chez le lapin [START_REF] Brandenburg | Circadian changes in the response of the rabbits retina to flashes[END_REF][START_REF] Vaughan | Evidence for a circadian rhythm of susceptibility to retinal light damage[END_REF], d'un point de vue moléculaire, les voies de signalisation de l'AMPc et phospho-CREB chez le Xénope et le poulet [START_REF] Liu | Circadian regulation of nocturnin transcription by phosphorylated CREB in Xenopus retinal photoreceptor cells[END_REF][START_REF] Ivanova | Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the melatoninsynthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells[END_REF], l'expression rythmique de l'ARN messager du gène de la melanopsine chez le poulet [START_REF] Chaurasia | Molecular cloning, localization and circadian expression of chicken melanopsin (Opn4): differential regulation of expression in pineal and retinal cell types[END_REF], ou encore de gènes impliqués dans la phototransduction, tels que la transducine chez le rat (Brann and Cohen 1987) et les opsines (Bowes, van Veen et al. 1988;von Schantz, Lucas et al. 1999;[START_REF] Li | CLOCK is required for maintaining the circadian rhythms of Opsin mRNA expression in photoreceptor cells[END_REF]. La variation journalière d'opsine étant l'une des sorties de l'horloge de la rétine, le contrôle de cette fonction rythmique peut être investiguée. Nous avons vérifié que l'expression de la Rhodopsine présente effectivement un rythme journalier dans la rétine de rat et montré qu'il en va de même pour l'expression de ses FT régulateurs principaux, Nrl, Crx, Nr2e3 et RevErbα. C'est pourquoi, sur la base de la caractérisation du promoteur de Nrl que j'avais réalisée, nous avons choisi d'aller un plus loin dans la compréhension de la régulation rythmique de ce gène.

Des expériences complémentaires liées à l'observation de l'existence d'un site « RORE » (Retinoid related orphan nuclear receptor response element, séquence cible des facteurs de transcription ROR et RevEr) au niveau du promoteur Nrl m'ont permis de montrer que la fixation du facteur ROR est requise pour l'expression du gène Nrl dans les bâtonnets (Figure 7'). ROR se révèle être un candidat intéressant pour l'étude de la régulation de Nrl du point de vue développemental parce qu'il est requis pour l'induction du lignage des photorécepteurs [START_REF] Jia | Retinoid-related orphan nuclear receptor RORbeta is an early-acting factor in rod photoreceptor development[END_REF], mais également d'un point de vue circadien. En effet, ROR est aussi un constituant moléculaire de l'horloge circadienne et

FIGURES AND TABLES

FIGURE TITLE PAGE

RÉSUMÉ DE LA THÈSE EN FRANÇAIS Figure 1'

Expression de séquences du promoteur/enhancer de Nrl au cours du développement de la rétine Scheme of extensively diversified metazoan regulatory modules

Figure 2

Pathway for the pre-initiation complex assembly

Figure 3

Core promoter elements

Figure 4

Functions of TAFs

Figure 5

Current models of transcriptional activation involve multiple steps

Figure 6

Transcription factor domains

Figure 7

Suggested sequence of coactivator function in transcription initiation.

Figure 8

Model for a dimeric bZIP protein

Figure 9

Various members of the Maf family transcription factors

Figure 10

The human retina

Figure 11

The mammalian retina drawing by Cajal

Figure 12

Retinal organization

Figure 13

The mature retina

Figure 14

Rod and cone structures

Figure 15

Absoprtion spectra of visual pigments in humans

Figure 16

Development of the vertebrate eye cup

Figure 17

Retinal cell diversification

Figure 18

Schematic representation of Notch signaling

Figure 19

Regulation of retinal cell fate specification by transcription factors

Figure 20

Stages of photoreceptor development

Figure 21

The visual phototransduction cascade

Figure 22

Location of the suprachiasmatic nuclei

Figure 23

The molecular circadian clock machinery

Figure 24

Melatonin and dopamine rhythmic synthesis in the retina

Figure 25

Light-dark cycling of opsin, G, G, and 48 kDa protein mRNA levels

Figure 26

Model of transcription initiation complex at the rhodopsin promoter

Figure 27

Opsin immunohistochemistry for Nrl KO retina

Figure 28

Nrl promoter directs GFP expression to rods and pineal gland in transgenic mice

MATERIALS AND METHODS Figure 29

In vivo retinal transfection by electroporation

Table 1

List of antibodies used for immunostaining

Table 2

List of antibodies used for supershift assays

Table 3

Inventoried TaqMan gene expression assays used in the study

Table 4

Composition of buffers used for Western blot analysis

Table 5

List of antibodies used for Western blot analysis

Figure 30

Recombinant Adeno-associated virus production

RESULTS

Figure 31

Conserved regions in Nrl promoter/enhancer

Figure 32

Identification of the sequence elements within Nrl promoter/enhancer required for rod-specific expression in vivo

Figure 33

Nrl promoter/enhancer sequence showing predicted sites for trans-acting regulators

Figure 34

ROR, CRX, OTX2, and CREB bind to Nrl promoter/enhancer sequence elements.

Figure 35

Mutations in ROR binding site abolish Nrl promoter activity

Figure 36

Nrl promoter B4 sequence is a transcriptional enhancer

Figure 37

Nrl minimal and functional promoter

Figure 38

Determination of the working dilution for AAV-Nrlp-EGFP

Figure 39

Analysis of GFP reporter expression in Nrl KO mouse retina

Figure 40

Expression of Nrl in Nrl KO retina restores Rhodopsin expression

Table 6

Cosinor analysis of mRNA levels of clock output genes in whole retina and isolated photoreceptors.

Figure 41

Expression profiles of rhodopsin transcript in rat retina

Figure 42

Expression kinetics of Nrl and Crx transcripts and protein products

Table 7

Cosinor analysis of mRNA levels of clock output genes in all retina and isolated photoreceptors

Table 8

Cosinor analysis of clock output protein levels in rat whole retina.

Figure 43

Expression kinetics of Nr2e3 transcript and protein product.

Figure 44

Transcriptional effect of clock factors on Nrl promoter.

Figure 45

Expression profiles of recoverin transcript in isolated photoreceptors

Figure 46

Schematic representation of upstream regulatory elements in rhodopsin promoter, sequence alignment of RER and scheme of the isolation of bovine retina proteins binding to it.

Figure 47

Luciferase reporter assay showing transactivation of the 2.2 kb bovine rhodopsin promoter by NonO

Figure 48

NonO is required for rod photoreceptor survival. In Eukaryotes, synthesis of messenger RNA from protein encoding genes requires regulatory elements that generally include a core promoter and upstream -or downstream -activator-binding sequences necessary for anchoring the trans-acting factors. These factors work with co-activators, which may not bind directly DNA, to direct transcriptional initiation by the RNA polymerase II apparatus [START_REF] Maniatis | Regulation of inducible and tissue-specific gene expression[END_REF][START_REF] Tjian | Transcriptional activation: a complex puzzle with few easy pieces[END_REF]). The promoter is essential for efficient synthesis of a gene transcript by enabling the binding of the transcription initiation complex machinery. The core promoter corresponds to a region typically localized around 40 base pairs (bp) upstream of the transcription start site (TSS), while the proximal promoter represents a region that includes the core promoter and the further upstream and/or downstream elements (Figure 1). The proximal promoter contains DNA sequence elements that bind transcription factors, which then modulate levels of transcription, whereas the core promoter is necessary for the formation and binding of the pre-initiation complex (PIC).

The PIC is composed of multiple factors regulating the initiation of transcription and stabilizing the enzyme responsible for the transcription, RNA polymerase II (Pol II), on the promoter. The PIC is recruited to the core promoter at a site typically located immediately upstream (25 -30 bp) of the TSS. This site known as the TATA box

[consensus DNA sequence, TATA(A/T)A(A/T)] allows binding of the first general transcription factor TFIID through its TATA-binding protein (TBP) [START_REF] Tjian | The biochemistry of transcription in eukaryotes: a paradigm for multisubunit regulatory complexes[END_REF][START_REF] Naar | Transcriptional coactivator complexes[END_REF]) (Figure 2). Some promoters lack a TATA box element and are defined as TATA-less promoters. In this case, transcription initiation is accomplished through other DNA elements in the core promoter, these include INR (initiator), DPE

Figure 2. Pathway for the pre-initiation complex assembly

Stepwise assembling of the pre-initiation complex (PIC) occurring via the binding of TATA binding protein from TFIID at the core promoter, followed by the sequential recruitment of other general transcription factors (TFII) , completed by the stabilization of RNA polymerase II on this multi-protein complex. From Thomas and Chiang, Crit Rev Biochem Mol Biol., 2006. independent manner [START_REF] Usheva | TATA-binding protein-independent initiation: YY1, TFIIB, and RNA polymerase II direct basal transcription on supercoiled template DNA[END_REF]. The DPE is generally located from +28 to +33 relative to the INR and is classically found in TATA-less promoters (Figure 3). TFIID recognizes DPE sequence and binds to it to initiate basal machinery anchoring.

Therefore, DPE could be considered as a "downstream TATA box" for TFIID binding [START_REF] Kadonaga | The DPE, a core promoter element for transcription by RNA polymerase II[END_REF]. The DCE is a downstream regulatory element relatively frequent in TATA box containing promoters and appears to be distinct from the DPE. The XCPE1 motif is located around the TSS and acts in association with trans-acting factors such as Specificity protein 1 (Sp1) [START_REF] Tokusumi | The new core promoter element XCPE1 (X Core Promoter Element 1) directs activator-, mediator-, and TATA-binding protein-dependent but TFIIDindependent RNA polymerase II transcription from TATA-less promoters[END_REF]). Sp1 acts as a basal transcription factor on 'Sp1 sites' (GC-boxes, CACCC-boxes and basic transcription elements) that represent constitutive promoter elements supporting the regulation of expression of thousands of genes [START_REF] Tan | Sp1 phosphorylation and its regulation of gene transcription[END_REF]). XCPE1 appears to collaborate as well with sequence-specific activators on CpG islands which, when methylated by 5methylcytosine will lead to gene silencing.

1.2 Recruitment of the initiation complex TFIID is the first general transcription factor to make contact with the chromatin on the TATA box element via its TBP. TFIID serves as a foundation for the recruitment of other TFII proteins to the TATA box (Figure 2). Indeed, TFIID is stabilized by TFIIA, and then addition of TFIIB and TFIIH will complete this multi-factor structure. Finally, Pol II binds to this complex, facilitates supplemental general/basal transcription factors, TFIIE and TFIIF [START_REF] Thomas | The general transcription machinery and general cofactors[END_REF]. Besides the general transcription factors, the TBPassociated factors (TAFs) stabilize TBP on the TATA sequence, which is in general only a few base pairs long, by making contact with the INR and the DPE. TFIID is in fact a multiprotein complex composed of TBP and 12-15 distinct TAFs [START_REF] Dynlacht | Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation[END_REF][START_REF] Tanese | Coactivators for a proline-rich activator purified from the multisubunit human TFIID complex[END_REF]. TAFs are thought to interact with activators binding upstream DNA sequences, and to create a bridge between these activators and the basal machinery complex (Figure 4). Data suggest that TAF subunits can function as direct promoter-recognition factors, as coactivators capable of transducing signals from enhancer-bound activators to the basal machinery, and even as enzymatic modifiers of other proteins [START_REF] Albright | TAFs revisited: more data reveal new twists and confirm old ideas[END_REF]. Mediator is a multi-subunit co-activator complex that seems to facilitate the binding and/or function of Pol II at the core promoter [START_REF] Kim | A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II[END_REF]) (Figure 5). In metazoans, there are several Mediator-related complexes: TRAP, CRSP, ARC/DRIP, SMCC and hMed that are recruited to DNA template via interactions with sequencespecific transcriptional activators. The function of these complexes is similar to the TAFs, to create bridges between distal activators and the PIC. It is not until the carboxyterminal domain (CTD) of Pol II is phosphorylated by the Mediator complex [START_REF] Malik | The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation[END_REF] and TFIIH [START_REF] Oelgeschlager | Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control[END_REF]) that Pol II is released from the proximal promoter for an efficient elongation of the transcript (Levine 2011).

Regulation of the transcription

Role of enhancers

Promoter and enhancers appear to be similar in their functions. Indeed, despite the operational distance -enhancers can activate a promoter from a distance reaching 50kb and even when located in intronic regions -enhancers facilitate gene activation by increasing the rate of transcription through the binding of multiple regulatory proteins.

While the promoter is necessary for initiation of the transcription, enhancers can regulate the temporal and tissue-specific expression of differentially regulated genes [START_REF] Maniatis | Regulation of inducible and tissue-specific gene expression[END_REF] In some cases, the intact Mediator that consists of the core and the kinase module might be recruited at this stage. Modified from Malik and Roeder, Nat Rev Genet., 2010. recruiting repressor transcription factors to their sites to interfere with the PIC assembly or by preventing the binding of activators to their sequences [START_REF] Ogbourne | Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes[END_REF]. Silencers and enhancers are working in balance to activate or repress the transcription of specific genes in a spatial and temporal manner. The fundamental unit of chromatin is the nucleosome, which consists of 147 bp of DNA wrapped around histone proteins. As a result, some mechanisms are required for activators to access to the DNA and for the PIC to navigate through the chromatin, which appears then as a direct actor of the transcription regulation. Two types of mechanisms are responsible for chromatin decondensation; the first is the nucleosome dissociation releasing the chromatin. The second, which is better characterized, is the recruitment of proteins with enzymatic activities that will modify chromatin structure. Nucleosome remodeling complexes will destabilize histone-DNA interactions in an ATP-dependent manner, subsequently revealing chromatin segments that were masked, to allow binding of activators and the transcriptional machinery to enhancer and promoter regions. The first chromatin-remodeling complex identified is SWI/SNF in yeast and possesses a DNAstimulated ATPase activity through its Swi2/Snf2p subunit, altering nucleosome structure and facilitating transcription factors to bind to their cognate sites via the protein-protein interaction domain found in SWI/SNF [START_REF] Naar | Transcriptional coactivator complexes[END_REF]) (Figure 7). Another class of chromatin modifying enzymes is the histone acetyltransferases (HAT), whose function is to tag specific lysine residues with an acetyl group on the amino-terminal tails of histones 3 and 4 of the core of histone proteins. Acetylation will neutralize the basic charge of the lysine and destabilize the histone-DNA interaction [START_REF] Marmorstein | Histone acetyltransferases: function, structure, and catalysis[END_REF]. In general, hyperacetylation of histones is correlated with actively transcribed genes, while hypoacetylation of histones (accomplished by histone deacetylase, HDAC) is associated with repressed genes. Gcn5 was first identified as a co-activator that bridges activator proteins with basal transcription factors, but has since been found to exhibit HAT activity. Gcn5 is part of two distinct complexes, the Spt-Ada-Gcn5-acetyltransferase (SAGA) and the adaptor (ADA) co-activators. In human, hGCN5 and P/CAF (the homologs of yeast Gcn5) and SAGA have been found to interact with several TAFs but not with TFIID. It seems that TAFs are coordinating and ensuring the assembly and the integrity of multiple co-activator complexes, and function independently in transcription regulation through interaction with TFIID. Interestingly, TFIID also harbors HAT and protein kinase activities via one of its sub-unit TAF250 [START_REF] Dikstein | TAFII250 is a bipartite protein kinase that phosphorylates the base transcription factor RAP74[END_REF][START_REF] Mizzen | The TAF(II)250 subunit of TFIID has histone acetyltransferase activity[END_REF], suggesting a possible role for enzymatic modification of downstream targets upon TFIID recruitment. Two additional HATs, p300 and CBP (protein of 300 kDa and CREBbinding protein), have over 90% of sequence homology and are conserved in metazoans [START_REF] Ogryzko | The transcriptional coactivators p300 and CBP are histone acetyltransferases[END_REF]. p300 and CBP catalyze the acetylation of all four core histones, and are reported to acetylate a large number of proteins, including themselves [START_REF] Wang | Structure and chemistry of the p300/CBP and Rtt109 histone acetyltransferases: implications for histone acetyltransferase evolution and function[END_REF].

Therefore, p300 and CBP appear to be general transcriptional integrators. Other chromatin modifications during gene transcription are phosphorylation that rather serves as a covalent tag than a direct alteration of chromatin [START_REF] Banerjee | A peek into the complex realm of histone phosphorylation[END_REF] and methylation which either activates or represses gene transcription [START_REF] Kouzarides | Chromatin modifications and their function[END_REF]).

Sub-family of basic motif leucine zipper

TFs play a pivotal role in contributing to the initiation of transcription and further in regulating expression levels of specific genes. TFs can be grouped in families based on structural homology in their DNA-binding domain. In this chapter, we focused on one specific family of TFs, the Maf sub-family.

Maf stands for MusculoAponeurotic Fibrosarcoma; the structural organization and sequence homology of these proteins allowed classifying this family into the AP1 superfamily of leucine zipper containing transcription factors (Figure 8). Maf proteins are divided into two subgroups: the large Mafs, including c-Maf, MafA/L-Maf, MafB/Kreisler and Nrl proteins, comprise an acidic transactivation domain in their N terminus (Figure 9). The large Mafs are major regulators of tissue-specific gene expression and cellular differentiation in mammals; for example, mouse model studies revealed their importance for mammalian gene regulation of the retina (Nrl) [START_REF] Mears | Nrl is required for rod photoreceptor development[END_REF], the lens (c-Maf), brain (MafB), kidney, pancreas (c-Maf, MafA and MafB), bones (c-maf) and in hematopoietic cells (MafB) [START_REF] Blank | Small Maf proteins in mammalian gene control: mere dimerization partners or dynamic transcriptional regulators?[END_REF]. The small Mafs include the highly homologous MafF, MafG and MafK factors. In contrast to the large Mafs, the small Maf family members lack a recognizable transactivation domain. Maf proteins contain two specific domains; the bZIP domain common to all members of the AP1 family, the extended homology region (EHR) specific for Maf proteins, rich in glycines and containing repetition of histidine residues. Proteins with a bZIP DNA binding domain such as Jun, Fos, ATF/CREB, c/EBP, GCN4 or Maf, can bind palindromic sequences only when they are forming a dimer. The bZIP domain is constituted of two distinct subregions required for DNA binding. In the N-terminal part the bZIP contains a region rich in basic residues that are in direct contact with the DNA. The C-terminal part of the protein represents the "Leucine Zipper", which is a protein-protein domain characterized by presence of leucine residues every seven amino-acids necessary for the dimerization. Maf proteins form homodimers or heterodimers and bind to palindromic region of the DNA called MARE (Maf response element, TGCTGAC(G)TCAGCA) containing consensus sites for fixation of AP1 and CREB proteins [START_REF] Kataoka | Maf nuclear oncoprotein recognizes sequences related to an AP-1 site and forms heterodimers with both Fos and Jun[END_REF]. The specific flanking regions TGC and GCA at one or the other end of the core are crucial for recognition of the MARE by Maf proteins (Kerppola and Curran 1994).

INTRODUCTION / General mechanisms of transcriptional regulation The visual system enables organisms to perceive light and to process images that will be treated in the visual cortex in the brain. The eye is the first component of this system, and is composed of various anatomical structures working together to project images on the retina (Figure 10). The retina is a thin tissue (500 µm in humans and 200 µm in mice)

sitting on the back of the eye. It is a highly organized tissue composed of neurons and glia. The first description of the retina anatomy comes from the work of Santiago Ramon y Cajal in the late 19 th century "Structure of the Mammalian Retina" (Figure 11). In this piece of classical work, he described several types of cells in the retina and classified them according to their morphology and arborization. He used the technique of Golgi silver staining to expose the details of retinal structure. The retina was then used by Cajal to support the neuron doctrine, providing clues to identify the direction of the signal transmission at the interneuronal contacts. This tissue, given its relatively simple structure, facilitated the understanding of signal flow from photoreceptors to retinal ganglion cells (Figure 12). Hence, for Cajal the retina was a model to decipher the connections between neurons allowing transduction of signal. Nowadays, the retina is still used as a neuronal model based on its anatomical accessibility and a well defined cell repertoire (Figure 13).

Outer Nuclear and Plexiform layers

The outer nuclear layer (ONL) contains the cell bodies of the photoreceptors (PRs) rods and cones, so named based on their morphological structure. The rod nuclei are more numerous and are placed on different levels throughout the retina, while the cone nuclei are fewer and placed on the sclera side, close to the outer limiting membrane. The proportion of rods versus cones in the ONL seems to be correlated with the lifestyle of species; a diurnal animal will generally contain a higher number of cones compared to rods and vice versa for a nocturnal animal. For example, mouse and rat, two nocturnal rodents have a percentage of cones below 3% over the total PRs population, while the grass rat Arvicanthis ansorgei, a diurnal rodent has around 33% of cones [START_REF] Bobu | Photoreceptor organisation and phenotypic characterization in retinas of two diurnal rodent species: potential use as experimental animal models for human vision research[END_REF]. . This percentage is even higher in another diurnal rodent species, the sand rat Psammomys obesus [START_REF] Saidi | Diurnal rodents as animal models of human central vision: characterisation of the retina of the sand rat Psammomys obsesus[END_REF]). The cone/rod ratio of the diurnal primate human is 5-6%. The rods are in majority and are distributed on the whole surface of the retina, except in one particular region called the fovea, which is constituted exclusively of cone photoreceptors.

Rods and cones have the same basic structure with four distinct sections: the outer segment, the inner segment, the cell body and the synaptic terminal.

The outer segment is the truly receptive part of the PR as it is photosensitive. It is composed of disk membranes containing the visual pigments (cone and rod opsins) of the PRs. In rods the disks result from the internalization of the cell membrane and are isolated from the plasma, while the cone disks originate from the successive folding of the cell membrane and are in continuity and opened to the extracellular space. Outer segments of PRs are in fact highly specialized primary cilia responsible for the photosensitivity. The proteins involved in the phototransduction cascade are synthesized in the inner segment and fill up the outer segment via the connecting cilium, which constitutes a passage for the proteins traveling to and from the outer segment.

The inner segment contains mitochondria, endoplasmic reticulum, Golgi apparatus and ribosomes, structures required for proper functioning of the translational machinery and trafficking of proteins targeted to the outer segment. Opsin molecules are synthesized in the endoplasmic reticulum, glycolysated in the Golgi and docked in lipidic membranes that will form the disks of the outer segment.

The cell body is located at the transition between the inner segment and the axon.

The cell body contains almost exclusively the nucleus leaving reduced room for cytoplasm.

Both rod and cone axon terminals contain vesicles filled with an excitatory neurotransmitter, the glutamate. The rod axon terminal is called spherule, and is smaller than the cone pedicle, each of these structures form an invaginated synapse with bipolar and horizontal cells in the outer plexiform layer. The outer plexiform layer is the layer where the first synapses of the retina are made. This layer contains the axon terminals of the rods and cones that are contacted by the axon terminals of horizontal cells (for rods), by the dendrites of horizontal cells (for cones) and by dendrites of bipolar cells (for both rods and cones) [START_REF] Kolb | Synaptic connections of the interplexiform cell in the retina of the cat[END_REF][START_REF] Nelson | Cat cones have rod input: a comparison of the response properties of cones and horizontal cell bodies in the retina of the cat[END_REF]. Two types of synapses are found in the outer plexiform layer: ribbon synapses between the PRs and ON-bipolar cells or horizontal cells, and flat synapses between PRs and OFF-cone bipolar cells.

Cone photoreceptors

The cones are responsible for the bright light (photopic) and color visions (Figure 14).

They are less light sensitive than rods but allow finer detail perception. Different categories of chromatic vision emerged from evolution; hence monochromatic vision is when only one color can be perceived and polychromatic vision, two or more colors. The ability to detect colors is due to the presence of visual pigments. In vertebrates, cones can have up to four spectrally different visual pigments or opsins, forming then one type of cone depending on the opsin expressed in this cell (Figure 15). Each opsin belongs to one gene family: Short wavelength sensitive 1 (SWS1), allowing detection from ultraviolet to violet; Short wavelength sensitive 2 (SWS2), from violet to blue; Rhodopsin type 2 (RH2), middle wavelength sensitive, green range; Middle wavelength sensitive (MWS), for green, and Long wavelength (LWS) for red [START_REF] Jacobs | Evolution of colour vision in mammals[END_REF]. Human and Old

Primates have typically three types of cones named after their spectral sensitivity maxima Short (blue cone), Medium (green cone) and Long (red cone) [START_REF] Nathans | Molecular genetics of human color vision: the genes encoding blue, green, and red pigments[END_REF][START_REF] Jacobs | A perspective on color vision in platyrrhine monkeys[END_REF][START_REF] Yokoyama | Molecular evolution of vertebrate visual pigments[END_REF]. This trichromatic vision results from the split of the M/L opsin into two distinct types of pigments, the green and the red opsins [START_REF] Linberg | Comparative anatomy of major retinal pathways in the eyes of nocturnal and diurnal mammals[END_REF].

Several retinal diseases are related with cone dysfunction. These pathologies are called cone-rod dystrophies resulting in the primary loss of cone cells followed by rod cells [START_REF] Hamel | Cone rod dystrophies[END_REF]. Some cone dystrophies may be part of other syndromes such as the Bardet Biedl syndrome [START_REF] Mockel | Retinal dystrophy in Bardet-Biedl syndrome and related syndromic ciliopathies[END_REF], which is a ciliopathy causing among other symptoms retinal dystrophy (rod-cone or cone-rod). Seven BBS proteins (BBS1,2,4,[START_REF] Ravasi | An atlas of combinatorial transcriptional regulation in mouse and man[END_REF][START_REF] Manolio | Finding the missing heritability of complex diseases[END_REF]8 and 9) participate in the formation of a stable protein complex named Rod cell (brown) and cone cell (orange) bodies are located in the outer nuclear layer (ONL) and have downward processes that synapse with horizontal (yellow) and bipolar (violet) processes in the outer plexiform layer (OPL). Horizontal, bipolar, Müller (blue) and amacrine (green) cell bodies are located in the inner nuclear layer (INL). In addition to upward processes that end in the OPL, bipolar cells have downward processes that terminate with amacrine and ganglion process in the inner plexiform layer (IPL). Müller glia, which provide support functions in the retina, have upward and downward processes that terminate at the outer edge of the ONL and the inner surface of the ganglion cell layer (GCL), respectively. From Dyer and Bremmer, Nat Rev Cancer, 2005. the BBSome involved in vesicular trafficking to the ciliary membrane [START_REF] Nachury | A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis[END_REF]). Mutations in one of these genes cause basal body and/or cilium dysfunction at the origin of the syndrome. Other cone dystrophies can be non-syndromic, such as Stargardt disease caused by mutation in ABCA4 gene and affecting the phototransduction cascade [START_REF] Briggs | Mutations in ABCR (ABCA4) in patients with Stargardt macular degeneration or cone-rod degeneration[END_REF]).

Rod photoreceptors

Contrary to the cones, rod photoreceptors can function in less intense light (Figure 14). Rods are responsible for the dim light perception (scotopic vision in gray scale) and peripheral vision. One rod cell is enough to respond to one single photon, making it 100 times more sensitive than cones. Rhodopsin, the visual pigment of the rod, belongs to the G protein-coupled receptor family. This protein family consists of seven transmembrane helices connected to each other by protein loops. The chromophore of Rhodopsin is the 11-cis retinal. This latter is isomerized when a photon of light strikes the Rhodopsin protein and will trigger the phototransduction cascade.

Mutations in Rhodopsin are among the most common cause of Retinitis Pigmentosa (RP). RP, a form of rod-cone dystrophy, is a group of retinal degenerative diseases that are characterized by the loss of rod photoreceptor cells, followed by cone degeneration.

Patients with RP experience night blindness, the progressive loss of peripheral vision and eventually central vision as cone-cell viability is compromised by rod-cell death. More than 120 point mutations in Rhodopsin have been identified, some of them have been linked to recessive RP and congenital stationary night blindness, but the vast majority cause autosomal dominant RP [START_REF] Mendes | Mechanisms of cell death in rhodopsin retinitis pigmentosa: implications for therapy[END_REF]).

Inner Nuclear and Plexiform Layers

The

Amacrine cells

There is a high variety of amacrine cells in the IPL. These cells do not have an axon but can have a long axon-like projection. Amacrine cells receive inputs from bipolar cells and ganglion cells. The diversity of amacrine cell types comes from their morphology and from the range of neurotransmitters in these cells [START_REF] Pycock | Retinal neurotransmission[END_REF]. Indeed, depending on the cell subtype, amacrine cells can release glycine or GABA, representative of an inhibitory signaling. Acetylcholine, or GABA plus acetylcholine can be found in the same cell, reflecting the excitatory or inhibitory function of the cell depending on the context. Dopaminergic amacrine cells type 1 make the bridge between rod bipolar cells and ganglion cells. Finally, nitric oxide seems to be used as a neurotransmitter or neuromodulator since NADPH-diaphorase activity is found in amacrine cells that contain nitric oxide synthase (NOS) immunoreactivity [START_REF] Eldred | Real time imaging of the production and movement of nitric oxide in the retina[END_REF]).

Horizontal cells

Two types of horizontal cells are found in the mammalian retina, HI-H1 (or B type)

and HII-H2 (or A type). HI-H1 subtype makes contact with cone cells; HII-H2 branches out with cones on the dendritic part, and with rods on the axonal part. The horizontal-cone cell connection is inhibitory, horizontal cells using GABA as neurotransmitter. The role of horizontal cells is to provide an inhibitory feedback to the PRs and an inhibitory feedforward to the bipolar cells and hence to reinforce perception of contrasts.

Ganglion cell layer and Optic Nerve

The retinal ganglion cells (RGCs) are the third order of neurons carrying the visual information. The midget retinal ganglion cells, the parasol cells, the bistratified cells and the photosensitive ganglion cells are amongst the different types of RGCs found in the ganglion cell layer. This cell type classification is based on their morphologies, projections and functions. As an example, the photosensitive RGCs contain a photopigment called melanopsin, which allows the RGCs to respond directly to the light signal, even in absence of rod and cone photoreceptors. These particular cells have a function in setting and maintaining circadian rhythms [START_REF] Berson | Phototransduction by retinal ganglion cells that set the circadian clock[END_REF][START_REF] Hattar | Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity[END_REF].

RGCs collect the visual information in the IPL through their dendrites and send it to the brain via their axons that are gathering in a bundle to form the optic nerve that leaves the ocular globe. On their way to the brain, the neuronal fibers are myelinated; in primates this phenomenon occurs not until the optic nerve exits the ocular globe. The optic nerves of each eye reach the brain; the fibers from the nasal optic field partially decussate and form then the optic chiasma and then project to the lateral geniculate body; a small fraction of RGCs project to the supra-optic and hypothalamic structures, participating in non-visual processes like the pupillary reflexes and circadian rhythm regulation. glia cells is to regulate the extracellular environment of the retina by controlling the amount of potassium released by the cells following the light signal, they also uptake the glutamate from the extracellular space [START_REF] Bringmann | Muller cells in the healthy and diseased retina[END_REF]. The Müller glia cells play a fundamental role in response to a retinal injury, by proliferating and secreting some extrinsic factors that will interact directly with the surrounding cell types [START_REF] Dyer | Control of Muller glial cell proliferation and activation following retinal injury[END_REF]. In fish and birds, it is well-established that Müller glia cells can serve as a source to generate new neurons in damaged retina [START_REF] Fischer | Muller glia are a potential source of neural regeneration in the postnatal chicken retina[END_REF][START_REF] Anthony | Radial glia serve as neuronal progenitors in all regions of the central nervous system[END_REF]). Indeed, Müller cells seem to acquire the ability to de-differentiate, proliferate, and become neuronal progenitors in acutely damaged retinas. This finding presents a therapeutic potential to regenerate injured retina.

INTRODUCTION / Anatomy and development of the retina

Figure 17. Retinal cell diversification

Retinal neurogenesis proceeds in a fixed histogenetic order: Ganglion cells and horizontal cells are born first, followed by cone-photoreceptors, amacrine cells, rod-photoreceptors, bipolar cells and Müller glia cells. The prenatal (E) and postnatal days (PN) refer to the respective stages of mouse development. After [START_REF] Young | Cell differentiation in the retina of the mouse[END_REF]. (B) A set of transcription factors is initially coexpressed in mitotic retinal progenitor cells (RPCs). However, with advancing retinogenesis, their expression domains start to segregate so that finally each retinal layer expresses a unique combination of these factors (given below

Retinogenesis

Development of the eye cup in vertebrates

The retina formation begins from the neural ectoderm, the source of the entire nervous system of an individual (Figure 16). The optic vesicle formation is the second step in the process of neural retina development. In parallel, occurs the development of the lens that will exert an effect on the optic cup shape by forcing it to invaginate. The inner wall of the optic cup will define the retina, while the outer wall develops into the RPE, forming a single-layer pigmented epithelium. At this point, the RPE will not undergo further changes other than growing. By the time the optic cup is formed, the retina is already undergoing cellular differentiation, forming two nuclear layers in the inner wall of the optic cup, called inner and outer neuroblastic layers. Cells start then to divide and proliferate. Daughter cells that are not leaving the mitosis cycle continue to proliferate, whereas the ones leaving the cell cycle start their differentiation.

Multipotent progenitors

Six early expressed transcription factors are involved in defining retinal identity: Rx1, Pax6, Six6, Six3, Tlx, and Lhx2 have been shown to stimulate retinoblast proliferation [START_REF] Zuber | Specification of the vertebrate eye by a network of eye field transcription factors[END_REF]) (Figure 17). Suppression of these TFs during the development leads to either a reduction of the retinal progenitor cell (RPC) proliferation (Pax6 and Tlx) [START_REF] Marquardt | Pax6 is required for the multipotent state of retinal progenitor cells[END_REF][START_REF] Miyawaki | Tlx, an orphan nuclear receptor, regulates cell numbers and astrocyte development in the developing retina[END_REF]), or to micropthalmia (Rx1) [START_REF] Andreazzoli | Role of Xrx1 in Xenopus eye and anterior brain development[END_REF]; whereas overexpression of some of these factors is associated with stimulation of RPC proliferation (Rx1 and Pax6) [START_REF] Casarosa | Xrx1 controls proliferation and multipotency of retinal progenitors[END_REF][START_REF] Zaghloul | Alterations of rx1 and pax6 expression levels at neural plate stages differentially affect the production of retinal cell types and maintenance of retinal stem cell qualities[END_REF] and giant eye development (Six6) [START_REF] Zuber | Giant eyes in Xenopus laevis by overexpression of XOptx2[END_REF] or induction of Müller glia cell differentiation (Rax) [START_REF] Furukawa | rax, Hes1, and notch1 promote the formation of Muller glia by postnatal retinal progenitor cells[END_REF]. The differentiation of certain retinal cell types generated from RPCs may be influenced by the action of secreted factors. Among these are the Ciliary Neurotrophic Factor (CNTF) and Leukemia Inhibitory Factor (LIF), both involved in changing the fate of rod precursors into bipolar-like cells, resulting in the loss of rod markers in these precursor cells [START_REF] Ezzeddine | Postmitotic cells fated to become rod photoreceptors can be respecified by CNTF treatment of the retina[END_REF]). The Epidermal Growth Factor (EGF) and

Transforming Growth factor- (TGF-) are both binding to the EGF receptor (EGFR) and were found to have mitogenic effects on the RPCs [START_REF] Anchan | EGF and TGF-alpha stimulate retinal neuroepithelial cell proliferation in vitro[END_REF]. Retinoic acid is highly produced in the developing retina, likely by both RPCs and postmitotic cells, and promotes rod photoreceptor development [START_REF] Kelley | Retinoic acid promotes differentiation of photoreceptors in vitro[END_REF]. Another

factor supporting photoreceptor differentiation, is the Fibroblast Growth Factor (FGF), which was shown to have an effect in stimulating cells to express opsins, and therefore to differentiate into photoreceptors [START_REF] Hicks | Fibroblast growth factor stimulates photoreceptor differentiation in vitro[END_REF]).

b. The Notch pathway

There is a high contribution of cell-cell interactions for cell fate specification. This is explained by the presence of cell surface proteins, such as receptors and membranebound ligands that are working together to transduce the signal pathway to modulate retinal development. The Delta-Notch pathway has been extensively characterized for its role in retinogenesis [START_REF] Perron | Determination of vertebrate retinal progenitor cell fate by the Notch pathway and basic helix-loop-helix transcription factors[END_REF]. Notch1 (Neurogenic locus notch homolog protein 1) is a transmembrane receptor activated by one of its ligands, Delta (Figure 18).

Both elements are widely expressed in the developing retina and are associated with retinal cell differentiation. It is suggested that the Notch pathway inhibits neuronal differentiation, and rather supports RPC proliferation as well as gliogenesis [START_REF] Furukawa | rax, Hes1, and notch1 promote the formation of Muller glia by postnatal retinal progenitor cells[END_REF]. There is a balance between Notch and Delta expression in adjacent cells that will make them become neurons or keep them in proliferation state.

This phenomenon is called lateral inhibition, in which a pool of cells expressing more Delta on their surfacewill inhibit the neighboring cells from exiting the mitogenic cycle to become neurons, by enhancing Notch expression in those cells. A high expression of Notch in cells will contribute to glia development [START_REF] Louvi | Notch signalling in vertebrate neural development[END_REF].

Importantly, isolated embryonic retinal cells cultured with postnatal retinal cells do not preferentially adopt the cell fate of these postnatal RPCs. Therefore, RPC differentiation is influenced by other mechanisms than extrinsic signaling to adopt diverse cell type identity during retinogenesis.

Contribution of intrinsic cues in cell fate determination

 Basic helix-loop-helix transcription factors Notch signaling activates the expression of Hes1 and Hes5 (E(spl)/hairy homologue) (Figure 18), two basic helix-loop-helix (bHLH) transcription factors that will in turn modulate the expression of downstream targets [START_REF] Ohtsuka | Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation[END_REF]. Some bHLH factors like Neurogenin and Mash1 act on keeping the proliferation of RPCs, whereas others are expressed in postmitotic neurons or differentiated neurons like the neurogenic differentiation factor 1 (NeuroD) (Figure 19). NeuroD plays a role in neuron and glia cell fate determination, and regulates amacrine cell type development over bipolar cell differentiation [START_REF] Morrow | NeuroD regulates multiple functions in the developing neural retina in rodent[END_REF]). The achaete-scute homologue 1 (Ascl1; also known as Mash1) factor is expressed in amacrine cells, rod photoreceptors and late-born retinal cells and later is restricted to the INL [START_REF] Cepko | The roles of intrinsic and extrinsic cues and bHLH genes in the determination of retinal cell fates[END_REF].

Math5 (also called Atoh7) is required for retinal ganglion cell development [START_REF] Brown | Math5 encodes a murine basic helix-loop-helix transcription factor expressed during early stages of retinal neurogenesis[END_REF]) and regulates the transcription factor Brn3b, which in turn is necessary for the terminal differentiation of retinal ganglion cells [START_REF] Liu | The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development[END_REF]).

Immature retinal cell specification

There is a highly conserved histogenic order in retinal neurogenesis (Figure 17). In mice, the RPC genesis starts around embryonic day 9,5. There is an overlap of births with retinal ganglion cells produced first, followed by horizontal cells, cone photoreceptors, amacrine cells, rod photoreceptors, bipolar cells and finally Müller glia cells [START_REF] Young | Cell differentiation in the retina of the mouse[END_REF][START_REF] Livesey | Vertebrate neural cell-fate determination: lessons from the retina[END_REF]. Upon becoming postmitotic, newly generated neurons have to express various post-mitotical TFs to induce their terminal differentiation; however, these TFs must also be expressed throughout the whole life of the cell for a normal function of the mature neuron. Hence, post-mitotical TFs appear essential for both maturation and maintenance of homeostasis of the cells.

Photoreceptor fate commitment

Studies of gene ablation brought to light the complex mechanisms accountable for a particular cell type development. TFs are typically responsible for regulating cell fate specification; however, it is rather combinations of multiple TFs than action of a single one that leads to the maturation of a particular cell type (Figure 20). However, some exception can be made, for example, in the rod versus cone cell fate specification, the basic-leucine zipper Nrl (Neural retina leucine zipper) is a transcription factor preferentially expressed in precursor cells that will become rod photoreceptors [START_REF] Swaroop | A conserved retina-specific gene encodes a basic motif/leucine zipper domain[END_REF]. Deletion of Nrl induces the loss of rod photoreceptors that are converted into S-cones [START_REF] Mears | Nrl is required for rod photoreceptor development[END_REF]) (Nrl transcription factor will be further detailed in chapter D). In retinogenesis, the role of Nrl is to activate the rod-pathway and to repress the cone-specific genes in association with other TFs, such as the nuclear receptor subfamily 2, group E, member 3 (Nr2e3) [START_REF] Oh | Rod differentiation factor NRL activates the expression of nuclear receptor NR2E3 to suppress the development of cone photoreceptors[END_REF], and the cone rod homeobox (Crx) (Mitton, Swain et al. 2000). Nr2e3 (formerly Photoreceptor-specific Nuclear Receptor, PNR) [START_REF] Kobayashi | Identification of a photoreceptor cell-specific nuclear receptor[END_REF]) supports the rodphotoreceptor lineage by inhibiting the expression of cone genes (Cheng, Khanna et al. 2004;Peng, Ahmad et al. 2005;Cheng, Aleman et al. 2006), and Nrl and Crx, activating rod genes [START_REF] Chen | The rod photoreceptor-specific nuclear receptor Nr2e3 represses transcription of multiple cone-specific genes[END_REF]. Mutations reported in Nr2e3 are associated with enhanced S-cones syndrome (ESCS), characterized in patients by an increased number of the S-cone subtype. Electroretinographies of patients show an increased S-cone mediated sensitivity and extinction in rod response. Moreover sensitivity of L/M cones is reduced in variable degrees [START_REF] Haider | Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate[END_REF]. These data suggest that the precursors of photoreceptors develop into an S-cone default pathway unless additional factors influence the pluripotent cells to adopt a particular photoreceptor-subtype fate.

Another transcription factor involved in the photoreceptor pathway is the homeobox gene Crx. Crx is a transcription factor contributing to the photoreceptor maturation and homeostasis [START_REF] Furukawa | Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation[END_REF][START_REF] Hennig | Regulation of photoreceptor gene expression by Crxassociated transcription factor network[END_REF]. In Crx deficient mice, the ONL is formed of photoreceptors that lack outer segments and whose neuronal activity is impaired [START_REF] Furukawa | Retinopathy and attenuated circadian entrainment in Crxdeficient mice[END_REF]. This implies that Crx is critical for photoreceptor maturation although it does not specify the photoreceptor cell fate.

Another homeobox gene necessary for the photoreceptor lineage is the paired-type homeodomain transcription factor Otx2. Otx2 is early expressed in the transcriptional cascade leading to photoreceptor cell fate (Swaroop, Kim et al. 2010). Otx2

transactivates Crx and is a key regulator of photoreceptor development; indeed, deletion of Otx2 leads to a retina deprived of photoreceptors, and an increase of amacrine-like cells, suggesting that the precursors for photoreceptors changed their fate in absence of Otx2 to become amacrine cells [START_REF] Nishida | Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development[END_REF]. ROR, an orphan nuclear receptor, acts for both rod and cone differenciation. Lack of RORb in developing mouse retina results in the absence of rods and features an excess of primitive S cone-like nphotoreceptors that are not functional. In RORb -/-mice, Nrl is not expressed, suggesting that RORb lies upstream of Nrl in the photoreceptor cell fate lineage [START_REF] Jia | Retinoid-related orphan nuclear receptor RORbeta is an early-acting factor in rod photoreceptor development[END_REF]).

Cone-photoreceptor lineage is highly influenced by the expression of thyroid hormone receptor. Indeed, in the Thyroid hormone receptor 2 (TR2)-deficient mice, while the rod photoreceptors seem normal, the only cone subtype found in the retina of these animals, is the S-cone subtype. No M-cone immunoreactivity is found, suggesting that showed an increase in both amacrine and horizontal cells [START_REF] Inoue | Math3 and NeuroD regulate amacrine cell fate specification in the retina[END_REF].

Deletion of Foxn4 results in a total disappearance of horizontal cells, likely due to the fact that Foxn4 is expressed early in the retinogenesis and likely affects Math3 expression [START_REF] Li | Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors[END_REF]).

One critical regulator of the RGCs development is the bHLH factor Math5. Deletion of this factor results in an almost total disappearance of RGCs that are converted into other cell types [START_REF] Wang | Requirement for math5 in the development of retinal ganglion cells[END_REF][START_REF] Le | Math5 is required for both early retinal neuron differentiation and cell cycle progression[END_REF]). Downstream of Math5, expression of Brn3b is likely required for formation of RGC axons and axon path-finding and is essential for early retinal ganglion cell differentiation [START_REF] Gan | POU domain factor Brn-3b is essential for retinal ganglion cell differentiation and survival but not for initial cell fate specification[END_REF][START_REF] Erkman | A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system[END_REF].

Mature and functional photoreceptors

After specification of the cell type, photoreceptors will start to express specific genes that are involved in their functioning, such as genes for the phototransduction cascade and genes that will confer them their typical morphology. Then, axonal growth and synapse formation between photoreceptors and bipolar and horizontal cells will establish the connections necessary for the signal transduction. Finally, outer segment development will intricate their structure within the RPE for establishment of the phototransduction (Swaroop, Kim et al. 2010;[START_REF] Hao | Transcriptional Regulation of Rod Photoreceptor Homeostasis Revealed by In Vivo NRL Targetome Analysis[END_REF].

INTRODUCTION / Anatomy and development of the retina

Figure 22. Location of the suprachiasmatic nuclei

The master clock regulating the circadian cycles is located in two small structures in the brain, at the base of the hypothalamus, called the suprachiasmatic nuclei. From http://thebrain.mcgill.ca

The retina and generation of rhythmic functions

The circadian system

Circadian rhythms in physiology and behavior exist in virtually all living organisms and allow adaptation to and anticipation of daily changes induced by earth rotation. These rhythms are controlled by circadian clocks, endogenous time-keeping systems able to generate ~24 hour rhythms even in the absence of external time-giving cues or zeitgebers. The most powerful zeitgeber is the light/dark cycle, which in mammals is perceived exclusively by the retina. In addition to the photoreceptors, the classical photosensitive cells of the retina, there is another class of cells sensitive to light intensity variations: a small group of retinal ganglion cells expressing the photopigment melanopsin [START_REF] Hattar | Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity[END_REF]. Axons of these cells, called intrinsically photosensitive ganglion cells (ipRGC), project via the optic nerve and optic chiasm to the suprachiasmatic nuclei (SCN), which is considered as the "master clock" of the entire organism [START_REF] Hannibal | Melanopsin: a novel photopigment involved in the photoentrainment of the brain's biological clock?[END_REF]. The SCN are a paired structure of the hypothalamus, composed of distinct groups of neurons receiving and interpreting information on environmental light and dark conditions and day length, both important for the entrainment of the master clock [START_REF] Ralph | A mutation of the circadian system in golden hamsters[END_REF][START_REF] Ralph | Transplanted suprachiasmatic nucleus determines circadian period[END_REF]) (Figure 22). SCN coordinate brain and peripheral secondary clocks by various output signals such as hormonal cues or neuronal efferences. In particular, they direct the pineal gland to secrete the hormone melatonin which is considered one of the main molecular outputs of the clock. This hormone is secreted by the pineal gland, with a higher expression during the night, and its expression is related to the length of the night, which varies depending on the season (short in summer and long in winter).

Although the pineal gland was found to be the principal site to produce melatonin, the retina produced as well this horamone rhtymically, giving to this tissue an importance in rhythm generation. The melatonin in the retina has a neuromodulatory role in acting on several aspects of retinal physiology. For instance, melatonin is suggested to be a chemical mediator for dark-adaptation in the retinal network [START_REF] Besharse | Regulation of rhythmic photoreceptor metabolism: A role for post-receptoral neurons[END_REF]; in non-primate vertebrates, melatonin is responsible for the dark-adaptive migration of the photopigment in the OS [START_REF] Cheze | Role of the epiphysis in the migration of retinal epithelial pigment during unilateral photic stimulation in Salmo irideus][END_REF]. Moreover, this hormone is involved in the rod OS disc shedding in Xenopus and rat retina [START_REF] Besharse | Regulation and possible role of serotonin Nacetyltransferase in the retina[END_REF][START_REF] White | Effects of exogenous melatonin on circadian disc shedding in the albino rat retina[END_REF].

At the molecular level, the clock machinery relies on interactions of specific clock genes, working together in positive and negative regulatory loops during a period close to 24 hours, and this mechanism is quite similar between SCN and the secondary clocks

The retinal clock

Almost 30 years ago, the first demonstration that the vertebrate retina contains an endogenous oscillator was made in Xenopus. In this initial study, the authors showed in vivo that the retina displays oscillations in N-acetyltransferase activity in constant conditions, substantiating that the retina is a peripheral clock controlling the rhythms of local functions and can work independently of the master clock located in the SCN [START_REF] Besharse | Circadian clock in Xenopus eye controlling retinal serotonin N-acetyltransferase[END_REF]. Existence of an oscillator in the retina is likely to prepare the retina to the > 1000 000 fold-change in light intensity encountered throughout the light/dark cycle. Several rhythms have been described in the vertebrate retina; some of them will be described further in the next paragraphs. Among the rhythmic functions of the retina, we can cite the visual sensitivity and ERG response [START_REF] Brandenburg | Circadian changes in the response of the rabbits retina to flashes[END_REF]) and extracellular pH [START_REF] Dmitriev | Circadian clock regulation of pH in the rabbit retina[END_REF] both characterized in rabbit;

sensitivity to light-induced photoreceptor degeneration in rat [START_REF] Vaughan | Evidence for a circadian rhythm of susceptibility to retinal light damage[END_REF]; melatonin biosynthesis in photoreceptor cells in chicken [START_REF] Hamm | Retinal rhythms in chicks: circadian variation in melantonin and serotonin N-acetyltransferase activity[END_REF], dopamine content and release from amacrine and interplexiform cells in fish (Ribelayga, [START_REF] Ribelayga | Dopamine mediates circadian clock regulation of rod and cone input to fish retinal horizontal cells[END_REF]; rod-cone dominance in quail [START_REF] Manglapus | Circadian rhythms of rod-cone dominance in the Japanese quail retina[END_REF]; rod-cone coupling in goldfish and mouse retina (Ribelayga, Cao et al. 2008); cAMP [START_REF] Ivanova | Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the melatoninsynthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells[END_REF] and pCREB [START_REF] Liu | Circadian regulation of nocturnin transcription by phosphorylated CREB in Xenopus retinal photoreceptor cells[END_REF] signaling pathways in photoreceptors of chicken and Xenopus, respectively; transducin mRNA in rat (Brann and Cohen 1987); iodopsin mRNA [START_REF] Pierce | Circadian regulation of iodopsin gene expression in embryonic photoreceptors in retinal cell culture[END_REF]) and melanopsin mRNA [START_REF] Chaurasia | Molecular cloning, localization and circadian expression of chicken melanopsin (Opn4): differential regulation of expression in pineal and retinal cell types[END_REF], both studies in chicken; nocturnin mRNA, characterized in Xenopus and mouse [START_REF] Green | Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin[END_REF][START_REF] Wang | Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse[END_REF]) (Iuvone, Tosini et al. 2005). Bmal1 function within the retina is required to generate circadian rhythms for the inner retinal visual processing. Deletion of Bmal1 affects rhythmicity of genes normally rhythmic in LD, suggesting that Bmal1 plays an important role in light-dependent gene regulation (Storch, Paz et al. 2007). Virtually all the layers oscillate in an independent manner (C. Jaeger, unpublished data) and express the same clock genes considered to be part of the core clockwork found in the SCN (Tosini and Fukuhara 2002).

Rhythmic functions have been particularly characterized in photoreceptors, supporting existence of a circadian clock in the ONL. Experiments that led to this conclusion are described below.

Melatonin and Dopamine rhythms

The initial characterization of a circadian clock in the photoreceptors was made with

Xenopus isolated photoreceptors kept in culture. Although these cells were disconnected from the SNC, they showed sustained circadian oscillations of melatonin release for many days in vitro. Moreover, addition of dopamine in the culture reset the melatonin rhythm, meaning that the photoreceptors cells possess the receptors, identified as the D2/D4-like receptors, to respond to the dopamine release and this symbolizes a setting for the entrainment of the clock [START_REF] Cahill | Circadian clock functions localized in xenopus retinal photoreceptors[END_REF][START_REF] Tosini | Dopamine inhibits melatonin release in the mammalian retina: in vitro evidence[END_REF]. In addition to that, AANAT (the enzyme catalyzing the rate limiting step in melatonin synthesis) is more or less regulated by a circadian clock, likely located in photoreceptors, further strengthening melatonin rhythmicity. Thus, melatonin is secreted by the photoreceptors at night, whereas the dopamine, produced by the amacrine/interplexiform cells, is found during the daytime in the retina (Figure 24).

There are inhibitory effects of the melatonin on the dopamine release through the action on MT2-like melatonin receptors [START_REF] Ribelayga | A circadian clock in the fish retina regulates dopamine release via activation of melatonin receptors[END_REF]). Thus, there is a cellular feedback loop formed by the photoreceptors releasing melatonin and the amacrine/interplexiform cells, to regulate circadian retinal physiology (Iuvone, Tosini et al. 2005;[START_REF] Tosini | The circadian clock system in the mammalian retina[END_REF]).

Disc shedding

Another aspect of the retinal circadian functions found in photoreceptors, is the phagocytosis of the outer segment of these cells by the RPE. Both rod and cone outer segments are constantly renewed. The degradation of visual pigment-containing discs implies the formation of new discs that are pushing the old ones at the tip of the OS.

Next, the disc detaches from the OS and is phagocytosed by the adjacent RPE; this phenomenon is materialized by the formation of phagosomes in the RPE cells. The quantification of the number of phagosomes in rod photorecetors during a circadian cycle shows a rhythmic apparition of these structures, with a peak of disk shedding at the beginning of the light period in a light/dark cycle (LaVail 1976;Bobu and Hicks 2009). In chicken retina, the peak of disk shedding in cones is observed at the beginning of the dark period [START_REF] Young | The daily rhythm of shedding and degradation of rod and cone outer segment membranes in the chick retina[END_REF]. Disc shedding persists in constant darkness, supporting the fact that this output is driven by a circadian oscillator. to an open reading frame of 237 amino acids. Hence, the first exon is untranslated and may be required for the recruitment of a complex participating to the initiation of transcription. Analysis of the 5'-untranslated region helped identifying some of the critical regulatory elements that might play a role in the transcription of Nrl. Among these elements, a non-canonical TATA box, a CAAT box, two E boxes and putative transcription factor binding sites for octamer and AP2 proteins were identified [START_REF] Farjo | Molecular characterization of the murine neural retina leucine zipper gene, Nrl[END_REF].

Circadian expression

NRL can form homodimers and heterodimers with c-Maf, Fos and Jun, and binds to an AP-1 like sequence TGCN 6-8 GCA designated as NRL Response Element (NRE) (Kerppola and Curran 1994;Kerppola and Curran 1994). The work of Rehemtulla and collaborators was the first to show the functional role of NRL as being part of a retinal protein complex able to specifically transactivate the promoter of rhodopsin gene [START_REF] Rehemtulla | The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression[END_REF]. NRL was also shown to interact with the TATA-Binding protein (TBP) through 35 amino acid residues identified in NRL protein as the minimal transactivation domain [START_REF] Friedman | The minimal transactivation domain of the basic motifleucine zipper transcription factor NRL interacts with TATA-binding protein[END_REF]).

These data suggest that NRL could play an active role in the formation or stabilization of the transcription initiation complex at the promoter of target genes. NRL protein undergoes post-translational modifications (PTMs), such as phosphorylation, which result in the detection of several isoforms in retinal immunoblots (Swain, Hicks et al. 2001). The role of PTMs is thought to modulate the activity of transcription factors. Thus, alterations in NRL-mediated transactivation were observed with NRL proteins carrying mutated residues p.S50 and p.P51. These residues are important sites for NRL phosphorylation: it was shown that mutations at these residues -identified in patients with autosomal dominant retinitis pigmentosa (Bessant, Payne et al. 1999;Kanda, Friedman et al. 2007) -result in reduced number of NRL isoforms by immunoblotting.

Moreover, p.S50 and p.P51 mutants displayed a significantly higher capacity to transactivate rhodopsin promoter as compared to the WT protein, conferring them a gain-of function (Kanda, Friedman et al. 2007). Interestingly, p.S50 and p.P51 residues are located within NRL minimal transactivation domain, which is important for the interaction of NRL with TBP [see above, [START_REF] Friedman | The minimal transactivation domain of the basic motifleucine zipper transcription factor NRL interacts with TATA-binding protein[END_REF])], suggesting that for SUMO sites exhibit lesser transactivation properties on the target promoter of Nr2e3 (Roger, Nellissery et al. 2010). Hence, it appears that PTMs modulate the activity of NRL to activate promoters of specific target genes that contribute to rod development and function.

Transcriptional regulation of the rod-photopigment Rhodopsin

Multi-factorial complex formation for Rhodopsin expression

Rhodopsin is one of the most critical genes for the rod-photoreceptor. Fine-tuning its expression is a critical step for proper development and functioning of the rods.

Mutations in Rhodopsin are associated with autosomal dominant or recessive Retinitis Pigmentosa, an inherited retinal dystrophy leading progressively to vision loss [START_REF] Humphries | Retinitis pigmentosa: genetic mapping in X-linked and autosomal forms of the disease[END_REF][START_REF] Olsson | Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa[END_REF]. In mice, disruption of Rhodopsin gene leads to a decline with age of the total number of photoreceptors, ultimately resulting in a thinning of the ONL. In these mice, rod-outer segments appear shorther and no immunoreactivity for Rhodopsin is found. Moreover, electroretinograms (ERG) display a reduced cone response, but no rod response is detected (Humphries, Rancourt et al. 1997). These data put emphasis on the importance of an accurate regulation of Rhodopsin for rod cell homeostasis, as proven also by using transgenic mouse strains overexpressing Rhodopsin (Tan et al, IOVS 2001). Among the classical factors known to positively regulate Rhodopsin, NRL is the first transcription factor identified in a retinal protein complex that binds to a sequence spanning the NRE found in Rhodopsin promoter and to stimulate a Rhodopsin promoter-reporter gene construct in retinal cell cultures [START_REF] Kumar | The bZIP transcription factor Nrl stimulates rhodopsin promoter activity in primary retinal cell cultures[END_REF][START_REF] Rehemtulla | The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression[END_REF]. The leucine zipper domain of NRL is important for protein-protein interaction; it is via this domain that NRL binds to CRX homeodomain (Mitton, Swain et al. 2000). CRX binds three distinct regions in Rhodopsin promoter and regulates other rod-specific gene promoters [START_REF] Chen | Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes[END_REF][START_REF] Lerner | Nrl and Sp nuclear proteins mediate transcription of rodspecific cGMP-phosphodiesterase beta-subunit gene: involvement of multiple response elements[END_REF][START_REF] Pittler | Functional analysis of the rod photoreceptor cGMP phosphodiesterase alpha-subunit gene promoter: Nrl and Crx are required for full transcriptional activity[END_REF]). Therefore, NRL in synergy with CRX is required for elevated expression of Beside the classical cis-regulators Nrl, Crx and Nr2e3 involved in rhodopsin promoter activation, other signaling pathways/transcription factors have been identified to modulate rhodopsin expression. For example, QRX in bovine [START_REF] Wang | QRX, a novel homeobox gene, modulates photoreceptor gene expression[END_REF]),

Fiz1 in bovine [START_REF] Mitton | Interaction of retinal bZIP transcription factor NRL with Flt3interacting zinc-finger protein Fiz1: possible role of Fiz1 as a transcriptional repressor[END_REF] and cAMP in chicken [START_REF] Voisin | Cyclic AMP-dependent activation of rhodopsin gene transcription in cultured retinal precursor cells of chicken embryo[END_REF] were all found to regulate rhodopsin expression levels.

In summary, regulation of Rhodopsin expression throughout development and life requires multiple factors working in synergy for sustained transcription level (Figure 26).

Control of Nrl expression

3.1 NRL-GFP mice, a tool to study rod development A considerable progress in the study of rod development has been made with the generation of transgenic mice expressing a reporter gene specifically in rods. Akimoto and his collaborators studied 2,5 kb of mouse Nrl 5'-unstranslated region (from -2408 to +115) and aligned it with its human homolog. This syntenic analysis highlighted four regions in the promoter, called clusters I-IV, which are highly conserved sequences.

Cloning of this 2,5 kb Nrl promoter with a reporter gene, in this case EGFP, allowed to generate transgenic mice. The characteristic of these mice is to express EGFP only in rod photoreceptors and interestingly, into the pineal gland as well (Figure 28). It is then easy to follow expression of these EGFP-expressing cells during retinal development, and to determine when the reporter gene is detected (and explicitly transcribed) in these cells.

EGFP positive cells are detectable in the retina as early as embryonic day E12 (Rhodopsin starts to be detected at post natal day P4) and subsequently increase in abundance over time. Expression profile of these EGFP-positive cells is central-to-peripheral, consisting with spatial and temporal rod genesis [START_REF] Akimoto | Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors[END_REF]. These data are the first to use an early-expressed factor specific to rod cells to trace their fate, from retinal precursor cell to mature photoreceptor. These mice have also proven highly useful in Putative RA response elements have been identified in the 2,5 kb mouse Nrl promoter, and revealed a positive binding of proteins from bovine retinal nuclear extracts [START_REF] Khanna | Retinoic acid regulates the expression of photoreceptor transcription factor NRL[END_REF]. In summary, this study was the first to investigate potential candidates for Nrl regulation.

Identification of factors controlling Nrl expression will deepen our knowledge on the transcriptional hierarchy influencing retinal precursor cells to commit to one cell fate and to maintain this cellular identity.

MATERIALS AND METHODS

A. Bioinformatic analysis

Promoter/enhancer sequences from mouse, rat, human, orangutan and dog Nrl genes were downloaded from the July 2007 (mm9) mouse genome assembly [START_REF] Karolchik | The UCSC Genome Browser[END_REF]) and submitted to syntenic alignment using CLUSTALW program [START_REF] Chenna | Multiple sequence alignment with the Clustal series of programs[END_REF]) in order to identify conserved sequence clusters. To predict the transcription factor binding sites on the promoter region, we analyzed the mouse genomic sequence with TF search program [START_REF] Heinemeyer | Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL[END_REF],

MultiTF tool and Mulan program [START_REF] Matys | TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes[END_REF] found in the TRANSFAC database [START_REF] Matys | TRANSFAC: transcriptional regulation, from patterns to profiles[END_REF].

B. Animals

For the developmental part of our study, we purchased untimed E17-

C. In vivo electroporation

In vivo transfection of the retina by electroporation was performed as previously described [START_REF] Matsuda | Analysis of gene function in the retina[END_REF]. Briefly, neonatal animals were placed on ice several seconds for anaesthesia. Only the right eye was injected. At birth, the eyelid of the animal is still closed; thus, to expose the eyeball for injection, the future edge of the eyelid was cleaned with 70% ethanol and cut with the tip of a sharp 30-gauge needle.

Once the eyeball was exposed, a pre-hole was made in the sclera near the cornea using the tip of a sharp 30-gauge needle. Through that same pre-hole an injection needle (Hamilton Syringe, 701SN, 10 ul, Special: Gauge 33, length 0.5 in, point style 3, blunt end)

was inserted until feeling a resistance (the inserted needle is visible through the lens).

DNA solution (1 µl) containing a dye (1% Fast green) was injected into the subretinal space. After the injection, the DNA was submitted to electroporation (ECM 830, BTX-Harvard Apparatus, Square Wave Electroporator). Tweezer-type electrodes were soaked in PBS and squeezed around the head of the animal. For a subretinal injection, the positive electrode was placed at the DNA-injected side, to allow the DNA to migrate towards the retinal cell progenitors (Figure 29). Five 80 volts square pulses of 50-ms duration and 950-ms intervals were applied using a pulse generator. After the procedure, the animals were placed on a heating pad until total recovery and then returned to their mother.

D. Tissue preparation and immunohistochemistry

Animals were sacrificed at post-natal day 14 (P14) and electroporated eyeballs were collected in Hank's Balanced Salt Solution (HBSS) medium. Before dissection, eyeballs positive for fluorescent reporter gene were sorted out and only positive eyeballs were kept for dissection. Two different techniques of dissection were used. First technique:

under a dissection microscope, in HBSS medium, eyeballs were cut along the ora serrata, removing the cornea and the sclera, leaving only the retina still attached to the lens.

Retina (plus lens) was immerged in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT) to allow the retina to keep its "cup-shape" for further processes. After this pre-fixation, the lens and vitreous humor were removed in HBSS. The retina was placed back into the 4% PFA bath for an additional 20 min. After fixation, retinas were rinsed in HBSS and placed in 30% sucrose /PBS for several hours or overnight for cryoprotection. Finally, retinas were embedded in OCT (Sakura Finetek) and processed for cryosectioning (10-12 µm). Second technique of dissection: eyeballs were harvested from the animals and directly immerged in 4% PFA for 15 min at RT. After this prefixation, eyeballs were placed in HBSS and cut along the ora serrata to remove the cornea, lens and vitreous humor. An additional fixation in 4% PFA was performed for 20 min and then dissected eyeballs were rinsed in HBSS and immerged in 30% sucrose/PBS and processed as described previously.

Immunohistochemistry: Retinal sections stained only with DAPI (1 mg/ml in PBS) were rinsed several times with washing buffer (0.1 % Triton-X-100 in PBS). Then DAPI solution was applied and incubated 10 min at RT. Finally, the slide was rinsed several times in washing buffer before applying mounting medium. For antibody use in immunohistochemistry, retinal sections were first rinsed with washing buffer, then incubated for 2h in blocking buffer (DMEM, 10% FBS, Penicillin/Streptomycin, 0.1% Triton X100 and 4% goat serum). Primary antibodies were diluted in blocking buffer and applied overnight at 4°C. After rinsing the slides with washing buffer, we applied the secondary antibody diluted in blocking buffer. DAPI was applied right after the secondary antibody incubation and retinal sections were proceeded as described above for the mounting. deoxyribonucleoside triphosphate (dNTPs) to produce amplicons. Amplified DNA fragments were purified, enzymatically digested at the new restriction sites, and then ligated into pEGFP-N1 (Clontech) and SV40-mCherry-IRES-alkaline phosphatase plasmid vectors [START_REF] Kim | A core paired-type and POU homeodomain-containing transcription factor program drives retinal bipolar cell gene expression[END_REF]. Individual clones were analyzed by restriction endonuclease digestion or PCR screening, and sequenced before being tested in functional assay.

Electrophoretic Mobility Shift Assays (EMSAs)

Based on our bioinformatic analysis, we designed oligonucleotide sequences (30 -40 nucleotides) spanning the predicted transcription factor binding sites. Corresponding mutant oligonucleotides were obtained by substituting up to 5 nucleotides in the putative binding sites. Wild type (WT) oligonucleotides were annealed and radiolabeled as described below. Nuclear protein extracts from HEK293 cells transfected with mammalian expression constructs or from adult or neonatal (P1-2) mouse retinas were incubated with 1 µg poly (dI-dC) and 1 µg salmon sperm at 4°C for 15 minutes in binding buffer (12 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.9; 60 mM KCl; 4 mM MgCl 2 ; 1 mM EDTA [ethylenediaminetetraacetic acid]; 12% glycerol; 1 mM dithiothreitol; supplemented with protease and phosphatase inhibitors). Then 30 and the reaction was incubated at 4°C for 20 minutes. In competition studies, nuclear extracts were preincubated with 25 or 100 ng unlabeled probes at room temperature for 15 minutes and then incubated with labeled probe at 4°C for 20 minutes. In order to immunologically identify protein components in the protein-DNA complexes, nuclear extracts were incubated with appropriate antibody at 4°C for 15 minutes followed by the addition of labeled probe and a further incubation at room temperature for 20 minutes.

The reaction mixtures were electrophoresed on 8% non-denaturating polyacrylamide gels at 80 volts for 2 hours and subjected to autoradiography.

Site directed mutagenesis

The DNA construct containing a mutated retinoid-related orphan nuclear receptor response element (RORE) was generated from a Nrl conserved region (-938 to -119) by sequential PCR using the following primers: 5'-GCTGAAAATGTATGGCACACCCCAGCC-3' and 5'-GGCTGGGGTGTGCCATACATTTTCAGC-3'. The mutations consisted in substituting two oligonucleotides of the consensus core motif AAAATGTAGGTCA. Effect of the mutation was assessed by in vivo tranfection by electroporation of neonatal animals.

Analysis of transcript level variation during 24 h cycle 4.1 Extraction of total RNA and reverse transcription

Extraction of total RNA (from whole retina and isolated photoreceptors of 6-8 week old rats) and reverse transcription were described in (Sandu, Hicks et al. 2011). 300 ng of total RNA was reverse transcribed into first strand cDNA and stored at -80ºC. We used inventoried TaqMan Gene Expression Assays for Rho, Nrl and Crx, designed to specifically amplify mRNA (Applied Biosystems, Table1), and custom designed assays for Nr2e3 based on the predicted RefSeq XM_002727061.1 sequence (forward 5'GGCTGCAGCGGCTTCTT3', reverse 5'CCCCTACCTGGCACCTGTAG3', probe 5'AGGAGTGTGAGACGGAG3'; amplicon size, 64bp).

Table 3: Inventoried TaqMan gene expression assays used in the study

Reference (Applied Biosystems), GenBank accession number (RefSeq), location of the assay in the gene (nucleotide number indicates position of the probe in the RefSeq sequence), size (bp, base pairs) of the amplicons are given for all assays. Gene expression assays used in this study are designed across the exon-exon junction indicated in the table.

Prior to qPCR total RNA was extracted from isolated photoreceptors by using the Absolutely RNA Microprep kit (Stratagene, La Jolla, CA, USA), and from whole retina using the Absolutely RNA Miniprep kit (Stratagene) according to the manufacturer's instructions. Samples were homogenized using a 1-mL syringe and a 27-gauge needle.

On-column digestion with DNase was performed to ensure removal of possible genomic DNA contamination. Total RNA was eluted with elution buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA) using 14 and 30 µL for photoreceptor layers and whole retina, for 1 min. The acquisition of fluorescence data was performed at the end of the elongation step using the 7300 System Sequence Detection Software V 1.3.1 (Applied Biosystems). Each PCR reaction was done in duplicate, and the coefficient of variation among duplicates was 0.18% on average. A dilution curve of the pool of all cDNA samples was used to calculate the amplification efficiency for each assay. No-template control reactions were performed as negative controls for each assay. One 96-well plate corresponded to the analysis of one gene within one type of sample (retina or photoreceptors) and one experimental condition (LD or DD cycles). Transcript levels were normalized using -actin and Pde6b [START_REF] Kamphuis | Circadian expression of clock genes and clock-controlled genes in the rat retina[END_REF]) that showed constant expression in their mRNA levels in both LD and DD during the 24-h cycle, in photoreceptor layers as well as in the whole retina (data not shown). qPCR data analysis was done using the qBase software (free v1.3.5; [START_REF] Hellemans | qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data[END_REF])) for the management and automated analysis of qPCR data. Expression of target genes was quantified based on the Cq method modified to take into account gene-specific amplification efficiencies and multiple reference genes. Transcript levels were calculated relative to the sample showing lowest expression, and which was rescaled to one.

F. Cell culture

HEK293 human embryonic kidney cell line has high transfection efficiency and rapid growth in conventional cell culture environment, and therefore was used for transfection experiments. Frozen aliquot of HEK293 was rapidly thawn and transferred to a 75 cm 2 flask containing 10 ml of Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12

(DMEM/F-12; Gibco, Invitrogen) pre-warmed at 37°C. Cultures were kept at 37°C in a 5% CO2 atmosphere. Medium was renewed two times a week. Cells at 80% confluence were split and seeded in 24-well plates (40000 cells/well). After 24 h cells were transiently transfected using XtremeGENE 9 (Roche Applied Science) according to the manufacturer's protocol.

HEK293T cell co-transfections were performed using 0.5 μg of the reporter plasmid expressing firefly luciferase under the control of mouse Nrl promoter (-938 to +119) (Kautzmann et al., 2011)

G. Luciferase assays

At 80% confluence, cells were split counted and seeded at 40 000 cells per well into 12-well plates. 24h after splitting, cells were transfected with Fugene6 or XtremGene9 (Roche) with 1 µg of total DNA, and further incubated 48h. Cultures were then processed with Dual-Luciferase Reporter Assay kit according to the manufacturer's instructions. 20 µl of cell lysate was loaded into each well in a 96 well-plate. Activity of firefly and renilla was measured sequentially using GloMax Luminometer (Promega) with Dual Auto-Injector.

H. Western blot

Retinas were flash-frozen on dry ice and maintained in -80°C until protein extraction.

Retinas were mechanically dissociated using a sonicator

Secondary Antibody

Goat anti rabbithorseradish peroxidase 1:10 000/1:80 000 Fermentas Goat anti mousehorseradish peroxidase 1:10 000 Fermentas

Figure 30. Recombinant Adeno-associated virus production

Co-transfection of rAAV vector and AAV packaging plasmid followed by infection with helper adenovirus, results in the generation of rAAV in 293 cells. Modified from Büeler, Biol Chem., 1999.

Vectors titration by quantitative PCR

I. AAV production and purification.

1. Vector design AAV2 ITRs (Inverted Terminal Repeats) were used in all AAV vector constructs.

pCMV-EGFP DNA [START_REF] Park | Intravitreal delivery of AAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the Rs1-KO mouse[END_REF] rAAV particles were produced and purified as decribed in [START_REF] Grimm | Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy[END_REF].

Briefly, vented cap roller bottles (850 cm2; Corning, USA) were seeded with 3 × 10 7 using primer and probe sets specific for EGFP cDNA.

Animals and vector delivery

Postnatal day (P)10 wild-type mice were used for promoter expression study and P4, P10 and 1 month-old Nrl KO mice were used for rescue experiments (n=4 per DNA vector tested). Animals were anesthetized with intraperitoneal injection of ketamine (90 mg/kg)/xylazine (9 mg/kg). Pupils were dilated with topical application of cyclopentolate and phenylephrine hydrochloride. Under binocular microscope, a small incision was made through the cornea adjacent to the limbus with an 18-gauge needle. A 33-gauge blunt needle injection syringe (Hamilton) was inserted through the incision, with care taken to avoid the lens, and was pushed through the retina. All injections were made subretinally. P4 and P10 animals received 0.5 µL of AAV-mNrlp-EGFP at 0.6, 2 or 6 e12

vector genomes per milliliter (vg/mL), 0.5 µL of AAV-hRKp-EGFP at 2 e12 vg/mL for promoter characterization study, and 0.5 µL of AAV-CMV-NRL at 1 e12 vg/mL for rescue experiments. Adult animals received 1 µL of either AAV-CMV-NRL or AAV-hRKp-EGFP at 0.5 e12 vg/mL. Visualization during injection was aided by addition of fluorescein (100 mg/mL AK-Fluor; Alcon, Inc., USA) to the vector suspensions at 0.1% V/V. NRL is a key factor for rod photoreceptor differentiation and homeostasis. Deletion of Nrl in mice leads to a cone-only retina and mutations in this gene have been correlated

RESULTS

RESULTS

RESULTS

I. Characterization of mouse

in humans with Retinitis Pigmentosa (Bessant, Payne et al. 1999;[START_REF] Martinez-Gimeno | Mutations P51U and G122E in retinal transcription factor NRL associated with autosomal dominant and sporadic retinitis pigmentosa[END_REF]Kanda, Friedman et al. 2007;[START_REF] Hernan | Novel p.M96T variant of NRL and shRNA-based suppression and replacement of NRL mutants associated with autosomal dominant retinitis pigmentosa[END_REF]

II. Identification of a minimal Nrl promoter as a tool for specific photoreceptor expression using AAV

The anatomical accessibility of the retina makes it a tissue of choice for gene therapy experiments targeting degenerating photoreceptors. Hereditary diseases such as Retinitis Pigmentosa or Leber Congenital Amaurosis primarily affect photoreceptor survival and lead ultimately to blindness. During the last decades, use of adenoassociated virus (AAV)-based vectors showed increasing promise in gene therapy due to their relative safety and long-term gene expression [START_REF] High | AAV-mediated gene transfer for hemophilia[END_REF]. Various promoters specific for photoreceptor expression have been successfully used for foreign gene delivery in photoreceptors [START_REF] Chen | The human blue opsin promoter directs transgene expression in short-wave cones and bipolar cells in the mouse retina[END_REF][START_REF] Glushakova | Human blue-opsin promoter preferentially targets reporter gene expression to rat s-cone photoreceptors[END_REF][START_REF] Khani | AAV-mediated expression targeting of rod and cone photoreceptors with a human rhodopsin kinase promoter[END_REF]). However, most promoters used for this type of study are shorter versions of opsins' promoters. Utilization of these promoters could rather be a disadvantage as opsin expression is a tightly controlled function. Opsin promoter produces abundant amounts of transcripts and may generate a potential toxicity for the cell by overproducing the protein. Identification of small promoter, for optimal expression of photoreceptor-specific genes, presents a clinical interest. Thus, primary characterization of regulatory elements in such a promoter would allow modulation of its expression. A. Further dissection of Nrl promoter elements required for the transcription We knew from previous experiments that full length cluster B (-938 to -657 bp from the transcriptional start site) cloned upstream of the minimal SV40 promoter was unable to activate transcription. Subsequently, we hypothesized that B4 would also be unable to stimulate transcription of the reporter gene. For that reason, we cloned B4 sequence upstream of a rod-specific promoter (mouse rhodopsin proximal promoter, -250 to +1)

In vivo analysis of

and compared the reporter gene expression in P0 electroporated retinas in presence or not of B4 sequence.

Addition of B4 sequence to mRhodopsin promoter triggered an apparent increase in rhodopsin promoter activity compared to the single rhodopsin promoter (Figure 36A).

Relative pixel intensity ratio was measured using ImageJ software (National Institutes of Health, USA) to quantify the effect of B4 cloning. Differences of expression level between the two DNA constructs were checked using the one way ANOVA analysis of variance.

Results showed a significant variation in mCherry expression between the two groups (n= 20; P =0.002) with the highest pixel intensity when B4 was cloned with rhodopsin promoter (Figure 36B). These data suggest that B4 sequence is an enhancer element for the transcription and strategies for cloning of Nrl upstream region should include this B4 sequence for correct gene activation.

RESULTS / Identification of a minimal Nrl promoter as a tool for specific photoreceptor expression…

Nrl promoter drives robust and specific transgene expression in retinal photoreceptors

In this study, we examined the promoter of the mouse Neural retina leucine zipper (Nrl) and tested its ability to drive gene expression in photoreceptors mediated by AAV vector. To determine the promoter activity of minimal Nrl promoter (A1+B, 0.3 kb)

sequence in vivo, we placed an EGFP reporter gene under its transcriptional control and packaged the resultant expression construct into an AAV8 vector to generate AAV-Nrlp-EGFP. We first determined the appropriate working dilution of this construct by injecting P10 wild type (C57Bl6) retinas with a range (0.3 e12 -3 e12 vector genome/milliliter) of AAV-Nrlp-EGFP in subretinal space. For comparison, we looked at human Rhodopsin kinase promoter activity in AAV (AAV-RKp-EGFP) previously reported to be functional and robust in both rod and cone photoreceptors [START_REF] Khani | AAV-mediated expression targeting of rod and cone photoreceptors with a human rhodopsin kinase promoter[END_REF]). Three weeks after injection reporter gene analysis revealed a high difference of promoter transactivation between the two promoters tested. While AAV-RKp-EGFP showed a strong reporter gene expression at the concentration tested (1 e12 vg/mL), cells were only able to express strong EGFP reporter gene with the highest AAV-Nrlp-EGFP concentration (3 e12 vg/mL). A barely detectable EGFP expression was seen with injection of AAV-Nrlp-EGFP at 1 e12 vg/mL and no expression was detected with the lowest concentration 0.3 e12 vg/mL (Figure 38). Neither RPE nor inner retina expressed the reporter gene, confirming the photoreceptor specificity of the two promoters. Both promoters appeared similarly effective in driving uniform expression in photoreceptors although AAV-RKp-EGFP showed a stronger promoter response for a lower working dilution, meaning that Rhodopsin kinase expression is actively stimulated in photoreceptors. These data prove that cloning of Nrl 5'-upstream sequence in AAV is a working tool for photoreceptor-specific expression and brings us some knowledge about the behavior of this promoter in AAV system as this experimental design has never been tested before.

Figure 39. Analysis of GFP reporter expression in Nrl KO mouse retina

Representative retinal sections from wild type (C57Bl6; a-b') and Nrl KO mice (c-d'). In parallel groups, animals were injected at P10 with 1 µL of AAV-Nrlp-EGFP concentrated at 3 e12 vg/mL (a, a', c, c') or with AAV-RKp-EGFP concentrated at 1 e12 vg/mL. 3 weeks postinjection, retinas were harvested for EGFP analysis. Green, EGFP; blue, DAPI. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar 50 µm

AAV-Nrlp-EGFP is functional in cone-like cells

We next asked whether AAV -Nrlp-EGFP could also drive EGFP reporter expression in photoreceptors other than rods. To answer this question, we used recombinant mice deleted of Nrl. In these animals, the photoreceptor layer is composed of cone-like cells and does not contain rods. In Nrl -/-animals, rods are replaced by photoreceptors similar to S-cones [START_REF] Mears | Nrl is required for rod photoreceptor development[END_REF]. It was previously reported that AAV-RKp-EGFP injection in Nrl KO retina showed positive expression of the reporter gene [START_REF] Khani | AAV-mediated expression targeting of rod and cone photoreceptors with a human rhodopsin kinase promoter[END_REF]; therefore, we used this construct as positive control. P10 Nrl KO or wild type retinas were injected with AAV-Nrlp-EGFP (3 e12 vg/mL) or AAV-RKp-EGP(1 e12 vg/mL).

Retinas were harvested 3 weeks post-injection for reporter gene analysis. We found EGFP expression in the photoreceptor layer of wild type animals as reported previously (Figure 39a-b') and in the outer nuclear layer of Nrl KO retina (Figure 39c-d').Although EGFP was detected in the transgenic context, cells displayed an apparent diminished promoter activity as compared to the wild type background. These data suggest that Nrl promoter can be activated in cone-like cells.

Figure 40. Expression of Nrl in Nrl KO retina restores rhodopsin expression

Representative retinal sections of Nrl KO retina injected at P4, P10 and 1 month-old mice with AAV-CMV-Nrl at 0.5 e12 vg/mL or with AAV-RKp-EGFP at 1 e12 vg/mL. On these retinas, assessment of rhodopsin expression was done using immunohistochemistry with antirhodopsin antibody (RHO). We detected specific expression of rhodopsin in the ONL for all the developmental time points tested. AAV-RKp-EGFP (far right column) was used as negative control, and did not show positive cells expressing rhodopsin. ONL, outer nuclear layer; INL, inner nuclear layer, GCL, ganglion cell layer. Scale bars, 20 µm.

Ectopic expression of Nrl in Nrl KO retina induces rhodopsin expression

NRL transcription factor is a major contributor to the rod cell fate specification by directing retinal progenitors to develop into rod photoreceptors. Indeed, ectopic expression of Nrl in immature photoreceptors leads to loss of cone genes expression and transformation into rods. Moreover, NRL activates several rod-specific genes for rod proper functioning, such as rhodopsin and cGMP-phodphodiesterase -subunit [START_REF] Rehemtulla | The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression[END_REF]Mitton, Swain et al. 2000;[START_REF] Lerner | Nrl and Sp nuclear proteins mediate transcription of rodspecific cGMP-phosphodiesterase beta-subunit gene: involvement of multiple response elements[END_REF]). To evaluate the cellular plasticity of post-mitotic retinal neurons, we drove the expression of Nrl coding sequence not only in developing (P4 and P10) and but also in adult Nrl KO retina. We evaluated how the action of this transcription factor modulates retinal development by ectopically expressing Nrl open reading frame under the control of cytomegalovirus promoter in cone-like cells. Nrl KO retinas were injected with AAV-CMV-Nrl (made by S. Hiriyanna) (0.5 e12 vg/mL). For negative control experiment, we used AAV-RKp-EGFP (1 e12 vg/mL), and as output of this experimental design, we performed immunohistochemistry with rhodopsin antibody for injected retina (Figure 40). We observed a positive rhodopsin staining for P4 and P10 Nrl KO retina injected with AAV-CMV-Nrl, and interestingly for adult retina injected with AAV-CMV-Nrl as well. Injection of AAV-RKp-EGFP did not show any rhodopsin-labeled cells. Hence, these data give additional weight to the major contributing role of Nrl in rod cell fate specification.

Discussion

Definition of a minimal, functional and yet specific promoter for photoreceptor expression is a major interest for gene therapy targeting retinal degenerative disorders.

In this study, we identified by in vivo electroporation cis-regulatory sequences of the Neural retina leucine zipper gene essential for its transcription. Among the identified elements, B4 enhancer showed elevated promoter activation when linked to the mouse rhodopsin proximal promoter. When placed in Nrl genomic context, B4 enhancer linked to the basal promoter A1 previously described (Kautzmann, Kim et al. 2011), could not transcribe efficiently the reporter gene, resulting in weak expression of mCherry in some cells in the ONL. In this situation, likely the combination of A1+B4 sequence (50 bp and 51 bp, respectively), similar to the B2+A1 combination, was too short for correct promoter transactivation, explaining the decline in reporter gene expression. Our results

suggest that the sequences and potential interacting factors corresponding to B4 region are sufficient to drive expression specifically in the ONL, but likely other factors are missing to fully activate the promoter. Therefore, based on the information provided by in vivo retinal transfections, we chose Nrl promoter A1+B that has the characteristics to be small in size (0.3 kb) and has relatively high transactivation abilities. The specificity of expression of this promoter was partially demonstrated using in vivo electroporation, where we observed location of mCherry reporter gene in the ONL. The technique of in vivo electroporation has several advantages; one of them is the possibility to quickly evaluate expression of a gene of interest as compared to the generation of transgenic mice. Another advantage of this technique which could be perceived in some cases by a disadvantage is the limitation of cell types targeted during neonatal stages, time of the injection. Indeed, in vivo electroporation is performed at P0-2 in mouse retina; at this time of the development, there is a peak of rod proliferation that will thus be preferentially targeted for the ectopic gene expression. Only dividing cells will be able to express the foreign DNA as long as these cells possess appropriate transcription factors for promoter activation. Based on these data, we presumed in our study that Nrlp-mCherry expression observed in the ONL was due to rod-expression only but not to cones, as previously shown for the -938 to +119 construct (chapter I of results).

However, confirmation of this statement would require further co-labeling with cone specific markers like cone-arrestin.

Adeno-associated virus (AAV)-based vectors showed increasing promise in gene therapy because of to their relative safety and long-term gene expression [START_REF] High | AAV-mediated gene transfer for hemophilia[END_REF].

Various genes' promoters specific for photoreceptor expression have been successfully used for foreign gene delivery in photoreceptors [START_REF] Chen | The human blue opsin promoter directs transgene expression in short-wave cones and bipolar cells in the mouse retina[END_REF][START_REF] Glushakova | Human blue-opsin promoter preferentially targets reporter gene expression to rat s-cone photoreceptors[END_REF][START_REF] Khani | AAV-mediated expression targeting of rod and cone photoreceptors with a human rhodopsin kinase promoter[END_REF]). In addition, they have arisen as convenient tool in research, to deliver ectopic genes within the whole outer retina/RPE, even in the adult animal. We generated AAV vectors containing Nrl promoter; the cloning size limitation in AAV pushed us to preferentially use the minimal Nrl A1+ B promoter. We first checked the functionality of this promoter in AAV genome by generating AAV-Nrlp-EGFP construct and injecting it into wild type mouse retina. We tried different dilutions of the vector genomes to give us an overview of this construct expression. The best results of EGFP expression was observed with the highest concentration of AAV-Nrlp-EGFP (3 e12 vg/mL), which showed a strong and specific expression of the gene reporter all over the ONL, is in the range of currently used virus dilutions and did not show any toxicity. Thus AAV-Nrlp-EGFP appears of potential convenient use for photoreceptor gene delivery in the retina. Once the correct AAV working dilution was determined, we assessed promoter specificity by injecting AAV-Nrlp-EGFP in Nrl KO retina, which have the specificity to contain only cone like cells in the ONL. Although EGFP expression was weaker in Nrl KO retina in comparison to the wild type background, we showed positive expression of GFP in the ONL. These data suggest that in this particular context, photoreceptors other than rods are able to activate Nrl promoter. However, we cannot precisely explain th expression in these particular conelike cells is due to limited sequence elements found in the minimal Nrl promoter that may be active also in cone-like cells, or whether the characteristic molecular environment, found in the photoreceptors of the Nrl KO retina, is permissive for Nrl promoter activation. Nonetheless, we can correlate our data to previous work reporting GFP expression under the control of Nrl (2.5 kb) promoter in Nrl KO retina [START_REF] Akimoto | Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors[END_REF]. In this study, the authors crossed Nrl-GFP mice with Nrl KO mice and analyzed expression of GFP in this Nrl deleted background. They observed strong GFP expression in the cone-like cells and proposed that early in development, retinal progenitors have not committed their cell fate specification yet and remain plastic. It is not until action of key transcription factors such as Nrl that the retinal progenitors will restrict their cellular competence to give rise to one type of neuron and restrict the panel of genes able to be expressed. Our results show that this permissive state of this cone-like cells (or maybe of cones in general) still exists in the P10 retinas and extends over 1 month, age at which retinas were harvested.

III. Nrl and co-activators of rhodopsin expression show daily variations in the rat retina during the 24 h cycle Introduction

Night/day transitions are major events to which living organisms have to adapt their physiology and behaviour. Such daily changes rely on circadian rhythmicity at cellular and molecular levels. In mammals, these rhythms are generated by a hierarchical network of oscillators, comprising a central clock located in the suprachiasmatic nuclei (SCN) which is synchronized with daily environmental light cues through the retina, and a series of peripheral oscillators receptive to synchronizing information produced by the SCN (Stratmann and Schibler 2006). Circadian rhythmicity is a cell autonomous process based on the interconnection of transcriptional/post-translational regulatory feedback loops These molecular phenomena involve clock genes such as Bmal1, Clock, Per1-2, Cry1-2, RevErbα and Rorα or Rorβ, which have the property to entrain the expression of "clock-controlled genes" and thereby to drive rhythmic gene expression programs (Guillaumond, Dardente et al. 2005;Ko and Takahashi 2006).

Retinas have evolved endogenous timekeeping systems ensuring the adjustment of their function to the daily changes in light intensity (Iuvone, Tosini et al. 2005). Rhythms have been described in most retina layers. For instance, phospholipid synthesis is rhythmic in avian ganglion cells (Garbarino-Pico, Carpentieri et al. 2004) as is melanopsin expression in rat intrinsically sensitive ganglion cells (Hannibal, Georg et al. 2005). Rhythmic synthesis and release of dopamine from the inner nuclear layer (INL) has been documented in many species (Wirz-Justice, Da Prada et al. 1984;Pozdeyev, Doroshenko et al. 2000;Doyle, McIvor et al. 2002). In photoreceptors, numerous processes, such as synthesis and release of melatonin (Tosini and Menaker 1996), rod-cone coupling (Ribelayga, Cao et al. 2008) or phagocytosis of rod outer segments (LaVail 1976;Bobu and Hicks 2009) follow a daily schedule and are regulated by a circadian clock. Expression of genes encoding elements of the phototransduction cascade is rhythmic as well, notably in rodents. In the mouse, expression of UV-opsin and rhodopsin genes were shown to cycle under constant darkness with maximum mRNA amounts found around the transition between subjective day and night (von Schantz, Lucas et al. 1999), as is also expression of rhodopsin in the zebrafish retina (Yu, Gao et al. 2007). Daily changes in elements of the phototransduction cascade were more frequently reported under lightdark cycles: in these conditions, mRNAs for the α-subunit of transducin (Brann and Cohen 1987) and for rhodopsin (Bowes, van Veen et al. 1988), with both peaks around the night to day transition.

Rhythmic control of retina function proceeds from complex regulations involving a local internal clock in the retina, capable to be entrained by light and to maintain rhythmic synthesis of melatonin in culture (Tosini and Menaker 1996;Ruan, Allen et al. 2008). In addition, Bmal1 gene was shown to be indispensable in the eye to generate appropriate rhythms in gene expression and to process light information (Storch, Paz et al. 2007).

Interestingly, a large part of the transcriptome found cyclic under LD conditions, was also altered upon ablation of the Bmal1 gene, confirming the central role that the circadian clock plays in the synchronization of the retina visual function with the day/night cycle (Storch, Paz et al. 2007). Photoreceptors were shown to harbor a functional circadian clock machinery [START_REF] Tosini | Localization of a circadian clock in mammalian photoreceptors[END_REF]Sandu, Hicks et al. 2011). So far, clockcontrolled genes directly activated by the CLOCK/BMAL1 complex have been identified in photoreceptors, such as AC1 (Adenylyl cyclase 1) (Fukuhara, Liu et al. 2004) and Aanat which encodes the rate-limiting enzyme in melatonin synthesis (Tosini and Fukuhara 2002). It has been suggested that E-box activation through CLOCK/BMAL1 does not account for all the genes undergoing rhythmic expression. Other clock factors or tissuespecific/clock-controlled transcription factors are likely to provide activating signals at phases of the 24 h cycle that do not coincide with CLOCK/BMAL1 peak (Doherty and Kay 2010). Thus in photoreceptors, mechanisms underlying rhythmic expression of other genes remain to be identified, although the cAMP/protein kinase A (PKA)/cAMP response element binding (CREB) signaling pathway has been proposed to provide strong contribution (Iuvone, Tosini et al. 2005). In the present study, we investigated potential mechanisms for the control of rhodopsin rhythmic expression in the rat retina. We show that Crx and Nrl, as well as Nr2e3 photoreceptor-specific transcription factors responsible for high level of rhodopsin expression in rod-photoreceptor, display rhythmic expression in LD, with peaks occurring around the night to day transition. We also provide evidence that Nrl is not a direct target of CLOCK/BMAL1 dimers but rather controlled by the REVERBα and RORβ clock factors.

Materials and methods

Animals and tissue preparation

Experiments were performed on 6-9 week old male Wistar rats (Charles River Laboratories, L'Arbresle, France) bred and housed at the Chronobiotron UMS3415 animal facility (Strasbourg) in a 12 h light/12 h dark cycle (LD). Rats had access to water and food ad libitum and were handled according to the European Communities Council Directive of 24 November 1986 (86/609/EEC) and the Animal Use and Care Committee from Strasbourg (CREMEAS). Animals were euthanized with CO 2 (20% in an air tight box).

A group of animals kept in LD was euthanized starting at ZT0 (light onset; ZT12 dark onset) every 4 hours through a period of 24 h. A second group of animals was exposed to constant dark (DD, dark/dark), for 36 hours prior to eye sampling (starting at projected ZT0, every 4 hours through a 24 h period).

Whole retina sampling. After enucleation of the eye, a small incision was performed on the cornea with a sterile blade. The lens and vitreous body were discarded and the retina was directly collected with sterile forceps, immediately frozen on dry ice and stored at -80°C for further total RNA and protein extractions.

Isolation of photoreceptor layer. Photoreceptor layer was isolated from the retina by tangential sectioning using a vibratome-based procedure (Sandu, Hicks et al. 2011).

Briefly, retina was dissected from the eye, flattened carefully with four radial cuts and mounted with photoreceptor surface down on a 20% gelatin block. To obtain the photoreceptor layer, a first vibratome cut of 170 µm depth from the vitreal surface was performed. The remaining photoreceptor layer was isolated with a single further slice (100 µm). The preparation was permanently maintained on ice cold medium and the photoreceptor samples were immediately frozen on dry ice and stored at -80°C.

Extraction of total RNA and reverse transcription

Extraction of total RNA (from whole retina and isolated photoreceptors of 6-8 week old rats) and reverse transcription were described in (Sandu, Hicks et al. 2011). 300 ng of total RNA was reverse transcribed into first strand cDNA and stored at -80ºC. Table 1 Inventoried TaqMan gene expression assays used in the study.

Reference (Applied Biosystems), GenBank accession number (RefSeq), location of the assay in the gene (nucleotide number indicates position of the probe in the RefSeq sequence), size (bp, base pairs) of the amplicons are given for all assays. Gene expression assays used in this study are designed across the exonexon junction indicated in the table.

As described in (Sandu, Hicks et al. 2011) (Bradford 1976).

Retinal proteins (3µg, denaturated in 2X Laemmli buffer) were separated by 10 % SDSpolyacrylamide gels electrophoresis and transferred on PVDF (polyvinylidene fluoride:

Millipore, Molsheim, France) membranes. Membranes were blocked in TBST buffer (Tris buffered saline containing 0,2% Tween 20) supplemented with 5% fat-free milk and then incubated overnight at 4°C in primary antibodies diluted in blocking buffer. Membranes were washed in TBST buffer and incubated with secondary antibody coupled to horseradish peroxidase diluted in TBST supplemented with 5% fat-free milk. Membranes were further processes using the chemoluminescence Immobilion reagent (Millipore), exposed to autoradiographic films. For loading control analysis, immunoblots were stripped and probed with -tubulin or -actin primary antibody solution.

Protein band densities were determined by scanning the blots on a professional scanner (Epson 4990). Each image was subjected to quantification using ImageJ software (version 1.45s, National Institutes of Health, USA). Plotted data represent the ratio of normalized pixel band values obtained for the studied protein with respect to those measured for the loading control protein.

Antibodies

The following antibodies were used: anti-NRL polyclonal antibody, a gift from Dr A.

Swaroop (Swain, Hicks et al. 2001); anti-CRX polyclonal antibody (La Spada, Fu et al. 2001) and anti-NR2E3-p183 polyclonal antibody (Peng, Ahmad et al. 2005), gifts from Dr S. Cheng; anti--actin (Sigma, L'Isle d'Abeau Chesnes, France); anti--tubulin (Abcam, Paris, France); anti-rabbit and anti-mouse light chain specific horseradish peroxidaseconjugated anti-IgG antibodies (Beckmann-Coulter, Nyon, Switzerland).

Cell culture and transfection HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Saint Aubin, France) containing 10% fetal bovine serum, 100 units/ml penicillin G and 100 μg/ml streptomycin and 2 mM L-glutamine. Cells at 80% confluence were split and seeded in 24-well plates (40000 cells/well). After 24 h cells were transiently transfected using XtremeGENE 9 (Roche Applied Science) according to the manufacturer's protocol.

HEK293T cell co-transfections were performed using 0.5 μg of the reporter plasmid expressing firefly luciferase under the control of mouse Nrl promoter (-938 to +119) (Kautzmann, Kim et al. 2011)

Dual luciferase assays

Firefly and Renilla luciferase activities were determined using the Dual-Luciferase reporter assay system (Promega) and measured with Glomax luminometer (Turner BioSystems, Sunnyvale, CA). Renilla luciferase activity was used as internal control for transfection efficiency. All the transfections were done in triplicate.

Statistical analysis

Results are presented as mean ± SEM.

Differences among groups were analysed using the one-way ANOVA analysis of variance (P < 0.05 was considered significant) (STATISTICA 8.0, StatSoft Inc., Tulsa, OK, USA) and post-hoc analysis (Tukey's HSD test).

Data were further analysed by the cosinor method (Sigmaplot V 10.0, Systat Software Inc., San Jose, CA, USA): rhythmicity in gene expression was assessed by fitting the 24 h data to a cosine curve f=a+ (b*cos(2*3.1416*(x-c)/ 24)) (Nelson, Tong et al. 1979)

Nrl and Crx expression in rat photoreceptor layers

Nrl and Crx are photoreceptor-specific genes encoding the major transcriptional regulators of rhodopsin promoter in the adult retina by acting on neighbouring enhancer sequences (Mitton, Swain et al. 2000;Cheng, Khanna et al. 2004). 24 h expression kinetics of Nrl and Crx transcripts in LD conditions (n = 5 per time point, total n = 30)

showed strong daily variations (Figure 2A) by one-way ANOVA analysis: Nrl F 5,24 = 2.78, P = 0.04; Crx F 5,24 = 12.07, P = 0.0001. Expression of these genes showed circadian rhythmicity with acrophases around ZT1 for Nrl ZT20 for Crx (Table 2, cosinor analysis P < 0.05). In DD conditions (n = 3-8 per time point, total n = 33) the amplitude of Nrl and Crx profiles was strongly reduced with respect to LD condition and no significant temporal variation could be revealed by one-way ANOVA analysis (Figure 2A) (Nrl F 5,27 = 1.14, P = 0.37; Crx F 5,27 = 2.14, P = 0.09).

To correlate gene transcript variation with protein expression, we harvested retinas of animals sacrificed every 4 hours over a 24 h period (ZT0-20) (n = 3-4 animals per time point, total n = 22). We extracted whole retina proteins and performed immunoblotting.

Figure 2B shows the daily variations of NRL and CRX with their respective immunoblots.

NRL shows rhythmic protein expression, according to one-way ANOVA analysis (F(5,16) = 2.8618, P = 0.04948), with acrophase around ZT7-8 (Table 3, cosinor analysis P < 0.05).

CRX expression also revealed significant changes over 24 h (F(5,16) = 10.911, P = 0.0001) but no rhythmic profile (Table 3). Table 2 Cosinor analysis of mRNA levels of clock output genes in whole retina and isolated photoreceptors.

Nr2e3 expression in whole rat retina

Photoreceptor specific transcription factor NR2E3 was also reported to regulate rhodopsin expression (Cheng, Khanna et al. 2004;Cheng, Aleman et al. 2006). Nr2e3

transcript levels show a strong tendency to vary with time (F(5,14) = 2.8780, P = 0.054) (Figure 3A). These data were confirmed by cosinor analysis (Table 2, P < 0.05). Figure 3B shows the daily variation of NR2E3 protein with its respective immunoblot. NR2E3

protein expression profile shows strong variation over 24 h as evidenced by one-way ANOVA analysis (F(5,16) = 22.454, P < 0.0001) and peaks around ZT7 ( Table 3 Cosinor analysis of clock output protein levels in rat whole retina.

Effects of clock transcription factors on mNrl promoter

We tested the abilities of clock factors to transactivate luciferase reporter activity driven by mouse Nrl promoter fragment (-938 to +119), previously reported to be sufficient and necessary for rod-specific expression (Kautzmann, Kim et al. 2011) and to carry E-box (-74) and RORE (Retinoic acid-related Orphan Receptor binding Element; -783) sequences .

We performed luciferase assays to compare the transactivation abilities of different factors reported to be involved in the molecular clock machinery. Co-expressed CLOCK and BMAL1 did not lead to any increase in Nrl promoter activity (Figure 4), although they strongly stimulated the promoter of the Per1 clock gene in parallel assays (data not shown). In contrast, REVERB and ROR triggered statistically significant increase of Nrl promoter activity as compared to its basal level. These data suggest that the daily regulation of Nrl may be controlled by secondary clock factors rather than by the direct action of the core clock elements CLOCK and BMAL1.

Discussion

Photoreceptor properties are synchronized to the light/dark cycle allowing organisms to optimize visual function to each photic situation. Although molecular mechanisms regulating outputs have not been yet characterized, it is clear that a circadian clock plays a central role in the temporal distribution of retina specific functions over the 24 h cycle (Storch, Paz et al. 2007). Altered expression of rhodopsin gene induces photoreceptor degeneration, suggesting that a strict control of its synthesis is required for retina homeostasis (Humphries, Rancourt et al. 1997;Tan, Wang et al. 2001). In the present study, we investigated 24 h expression profiles of rhodopsin and of its principal regulators NRL, CRX and NR2E3 and show that all of them display rhythmic expression in LD, with peaks occurring by the end of the night or beginning of the day. None of these genes was found rhythmic in DD, although mRNA profiles look rather similar to the ones in LD, probably owing to decreased amplitude of circadian oscillations previously observed in photoreceptors in DD, for clock genes as well as for outputs (Sandu, Hicks et al. 2011). We also provide evidence that Nrl transcription might be controlled by clock factors REVERBα and RORβ.

By performing qPCR analysis with whole retina, we show that in Wistar rats, rhodopsin expression is rhythmic in LD with maximal transcript amount by the end of the night. This result is in agreement with what was reported in mice (Bowes, van Veen et al. 1988) but was found different in regard to data obtained previously in Fisher rats (Sakamoto, Liu et al. 2006) and likely reflects strain differences. Interestingly, levels of α-transducin mRNA also peak by the night/day transition, indicating that expression of some phototransduction cascade elements cluster around that moment (Brann and Cohen 1987). To assess this hypothesis we analyzed 24 h expression profiles of recoverin, another gene associated with phototransduction which product inhibits rhodopsin kinase and sustains light effects on rhodopsin. Since recoverin was shown to be expressed in the inner retina (McGinnis, Stepanik et al. 1999), we performed the qPCR analysis on vibratome isolated photoreceptor layers and found that this gene also displays sustained rhythmicity in LD with peak expression around ZT21 and a large dampening in DD (Supplemental data, Figure S1). Thus, several key players of the phototransducing function appear to be maximally co-expressed around the transition between night and day in the Wistar rat. While we do not have any clear explanation for the occurrence of these peak expressions around that time of the cycle, it is possible that it reflects similar regulatory mechanisms allowing coincidence between synthetic pathways and catabolic processes such as outer segment disk phagocytosis which takes place just after lights on (Bobu and Hicks 2009).

Our results show that expression of three photoreceptor-specific transcription factors varies along the day/night cycle, both at the level of their mRNA and gene products which levels increase from the beginning of the light phase and peak 7 to 11 hours after their respective transcript. Interestingly, mRNA profiles also display maxima by the end of the night/beginning of the day, as was also reported for Crx and Nr2e3 upon microarray analysis of the circadian eye transcriptome: peak expression was found at ZT20.5 and ZT4.5 respectively (Storch, Paz et al. 2007). However, Crx rhythmicity was not observed in a previous Northern blot study performed in whole retinas from Wistar rats (Sakamoto, Oishi et al. 1999) possibly because of the lower dynamic range of the technique. Because Nrl has been reported to be a "master gene" for rod differentiation and the principal activator of rhodopsin promoter, we further investigated its daily regulation. Transcriptional activity of NRL, notably toward rhodopsin promoter, is regulated by post-translational mechanisms involving phosphorylation (Bessant, Payne et al. 1999;Kanda, Friedman et al. 2007) and sumoylation (Roger, Nellissery et al. 2010).

We did not detect any variation in NRL apparent mobility in western blot, which remained at the highest apparent molecular weights (3 bands around 30-35kDa) (Figure 2B) described to correspond to phosphorylated proteins (Swain, Hicks et al. 2001) indicating that the daily regulation of Nrl principally takes place at the transcriptional level. We recently mapped the minimal promoter of Nrl required for rod-specific expression in the postnatal retina and described the presence of both RORE and E-box sequences therein (Kautzmann, Kim et al. 2011). Here we investigated whether Nrl promoter could be a target for clock factors binding to these elements and found that it is not transactivated by the CLOCK/BMAL1 dimer but shows significant stimulation by two clock components that are also involved in photoreceptor development (Swaroop, Kim et al. 2010;Mollema, Yuan et al. 2011;Forrest and Swaroop 2012): RORβ and, to a lesser extent but still significant, by REVERBα (Figure 4). We previously reported that RORE site is bound by RORβ and that this factor is required for expression of Nrl in rods (Kautzmann, Kim et al. 2011). In addition, Rorβ expression shows robust rhythmicity in photoreceptors in LD, with maximum occurring at ZT20 (Sandu, Hicks et al. 2011), a few hours before the peak of Nrl transcript. Thus, it is possible that RORβ takes part in the rhythmic regulation of Nrl gene. While REVERBα was essentially described as a transcriptional inhibitor, we found a mild activating effect on Nrl promoter. Similar positive effect was reported on the promoter of rhodopsin, yet in the context of a transcriptional complex containing other transcription factors and in which REVERBα might play an unusual role. Since Reverbα also cycles in photoreceptors with peak expression around ZT22, further analysis is required to definitely understand the role of this factor in Nrl regulation and notably the competitive effect it might display towards RORβ effect.

Taken together, our results suggest that synchronization of rhythmic expression patterns by the LD cycle in photoreceptors likely proceeds from a complex interplay of transcription factors. We provide evidence that NRL expression is regulated by the clock factor RORβ. One could hypothesize that NRL, together with CRX and NR2E3 which likewise peak around the middle of the day, contribute to rhythmic expression of rhodopsin gene. Interestingly, REVERBα which is also part of the photoreceptor clock is recruited by direct interaction with NR2E3 into this complex and also provides activating effect (Cheng, Khanna et al. 2004). Respective contribution of each of these factors will need additional studies. While our study, together with results from other groups, put forward the expression of rhodopsin, recoverin, Nrl, Crx and Nr2e3 around the end of the night, it did not explore the possible contribution of the cAMP/PKA/CREB pathway which is maximally activated by the end of the dark phase as well and was shown to regulate other clock outputs in photoreceptors Effects of cyclic AMP regarding the studied genes will require further experiments. Finally, alternative post-transcriptional regulatory mechanisms involving mRNA decay, deadenylation of polyA tails or microRNAs (Tan, Wang et al. 2001;Staiger and Koster 2010) should not be forgotten in order to get a complete picture of the molecular pathways linking the photoreceptor clock to its outputs.

IV. A novel transcription factor, NonO, appears as a coactivator of rhodopsin expression and is necessary for rod-photoreceptor survival.

Introduction

Complex biological processes, including development and homeostasis, require quantitatively precise expression of genes in specific spatiotemporal patterns (Swaroop, Kim et al. 2010). Regulatory information necessary for the transcription of a gene is generally confined to the proximal promoter region, upstream of the transcription start site (TSS); however, distal sequence elements (called "enhancers") are frequently required for cell type-specific expression (Bulger and Groudine 2010). Enhancer elements are not directionally selective and can exert their influence over long distances [START_REF] Banerji | Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences[END_REF]. Most enhancers contain binding sites for multiple proteins; some of these can associate with transcriptional co-activators, relocate into physical proximity of TSS through a looping mechanism, and enhance RNA polymerase II-mediated gene expression [START_REF] Visel | Genomic views of distant-acting enhancers[END_REF]). The combinatorial interaction of specific factors that bind to promoter and/or enhancer elements together determines the activation or repression of a gene [START_REF] Ravasi | An atlas of combinatorial transcriptional regulation in mouse and man[END_REF]. The evolution of enhancers in developmentally regulated genes is believed to exert a major drive for animal morphology [START_REF] Boffelli | Comparative genomics at the vertebrate extremes[END_REF]. Recent genome wide studies have revealed that a majority of variations associated with complex multifactorial human diseases are present in non-coding regions, and many of these may be within potential enhancer sequence elements [START_REF] Manolio | Finding the missing heritability of complex diseases[END_REF]).

Retinal and macular neurodegenerative diseases are a major cause of incurable blindness (Neitz and Neitz 2011), with photoreceptor death being the primary cause of visual dysfunction (Malanson and Lem 2009). Rod photoreceptors constitute ~70% of the retinal cells in most mammals and contain the visual pigment, rhodopsin [START_REF] Curcio | Human photoreceptor topography[END_REF]. Rhodopsin represents as much as 90% of the total protein in rod outer segments (ROS) that capture photons to initiate visual transduction (Deretic 2006). The renewal of almost 10% of ROS discs, subsequent to their daily circadian clock-regulated shedding requires quantitatively precise expression and transport of rhodopsin and other phototransduction proteins (Young 1967;Winkler 2008). Indeed, abnormal expression and/or trafficking of rhodopsin are associated with dysfunction or death of rod photoreceptors (Malanson and Lem 2009).

RESULTS / A novel transcription factor, NonO, appears as a co-activator of rhodopsin expression… 123

The expression of rhodopsin is primarily regulated at the level of transcription. Two distinct sequence elements have been defined upstream of rhodopsin TSS: Rhodopsin Proximal Promoter Region (RPPR) and Rhodopsin Enhancer Region (RER) (Kumar, Chen et al. 1996;[START_REF] Nie | RER, an evolutionarily conserved sequence upstream of the rhodopsin gene, has enhancer activity[END_REF]. RPPR includes about 200 bp sequence, immediately upstream of TSS, that can direct rod-specific expression of a reporter gene in the transgenic mice [START_REF] Zack | Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas[END_REF]. Electrophoretic mobility shift assays and promoter activity analyses have delineated the binding sites for multiple transcription factors in RPPR; these include basic motif leucine zipper transcription factor NRL, cone rod homeobox CRX, and orphan nuclear receptor NR2E3 (Chen and Zack 1996;[START_REF] Rehemtulla | The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression[END_REF]. Loss of Nrl results in a cone-only retina with no rhodopsin expression [START_REF] Mears | Nrl is required for rod photoreceptor development[END_REF], whereas loss of Crx leads to rods but no outer segments and minimal rhodopsin expression [START_REF] Furukawa | Retinopathy and attenuated circadian entrainment in Crxdeficient mice[END_REF],

establishing their synergistic and pivotal role in regulating rhodopsin expression (Mitton, Swain et al. 2000;[START_REF] Hao | Transcriptional Regulation of Rod Photoreceptor Homeostasis Revealed by In Vivo NRL Targetome Analysis[END_REF].

Transgenic mouse studies with murine or bovine RPPR revealed somewhat leaky and lower level of reporter gene expression [START_REF] Zack | Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas[END_REF]; however, a larger approximately 5 kb upstream fragment demonstrated more precise and high expression levels [START_REF] Zack | Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas[END_REF], suggesting the importance of sequences upstream of RPPR in determining rhodopsin expression in vivo. Footprinting of bovine rhodopsin promoter identified a highly conserved sequence RER, approximately 2 kb upstream of TSS [START_REF] Nie | RER, an evolutionarily conserved sequence upstream of the rhodopsin gene, has enhancer activity[END_REF]. More recently, RER was shown to contact physically with RPPR and within the rhodopsin coding region by intrachromosomal loop formation in rod photoreceptors while maintaining a linear configuration in other cell types (Peng and Chen 2011). Despite its demonstrated significance, RER binding proteins have not been identified as yet.

In this study, we took a proteomic approach to identify protein, which bind to and might be involved in bringing RER in close proximity to RPPR in order to achieve quantitatively precise expression of rhodopsin in rod photoreceptors. We hypothesize that being a highly transcribed gene in rod photoreceptors, the splicing of rhodopsin pre-messenger RNA might be coupled to transcription of this gene by looping of RER on RPPR. By mass spectrum analysis we identified several candidate proteins bound to RER and selected the non-POU domain containing octamer-binding protein (NonO/p54 nrb ) for downstream analysis since it showed the maximum number of peptides. Here we report that NonO can activate NRL and CRX transcriptional activity on the rhodopsin promoter. Finally, we looked at the effect of the NonO gene transcript knockdown in neonatal murine retina and evaluated the survival of rod photoreceptors.

Results

Identification of proteins binding to RER of bovine rhodopsin promoter.

To identify factors binding to RER, we first analyzed the bovine rhodopsin RER genomic sequence using MacVector (version 11.11.1). Phylogenetic comparison of the -2155 to -2027 region relative to TSS revealed the presence of several evolutionarily conserved elements (Figure 1A). RER contains predicted binding sites (Transfac software AliBaba2. 1C). Five protein bands present in the RER but absent in the scrambled oligonucleotide elute lane (Figure 1C, asterisks) were selected and excised out from the gel for subsequent mass spectrometry analysis. Proteins for which two or more unique peptides were obtained from the mass spectra analysis, were considered significant (Table 1). Gene ontology analysis of the identified proteins revealed that they were mostly involved in splicing, transcription, cell cycle regulation and signal transduction. A few were protein kinases and phosphatases modulating the activity of a wide range of regulatory proteins. Several candidate coactivators bound to RER were identified, such as NonO, PPP1CA, BUB3, CCAR1, 14-3-3, SAP180, HNRPM and P. Some of these proteins are known to be part of transcriptional complexes involved in the regulation of nuclear receptor gene promoters. We selected for further investigation the non-POU domain containing octamer-binding protein (NonO/p54 nrb ) that returned the highest number of unique peptides in our mass spectrometry analysis. NonO protein binding specificity to RER oligonucleotide as compared to scrambled oligonucleotide was confirmed by Western blot analysis of eluates (Figure 1D).

In summary, we identified several candidate proteins that bind to rhodopsin RER in rod photoreceptors and identified a candidate protein, NonO, which may be involved in rhodopsin regulation.

NonO physically interacts with RER and acts in synergy with NRL and CRX to activate rhodopsin promoter.

To investigate the functional correlation between NRL, CRX and NonO, we performed reporter gene assays using a 2.2 kb bovine rhodopsin promoter-firefly luciferase reporter containing both RPPR and RER cis regulatory regions (Figure 2A). Pol II antibodies, but not with anti-Brn3b and control rabbit IgG (Figure 2C). However, immunoprecipitation of NonO with NRL and CRX was not observed in the absence of cross-linking from bovine retina. These results suggest that NonO-containing transcriptional complexes are in close proximity to the NRL-and CRX-containing complexes on the rhodopsin promoter. However, we did not get a direct interaction of these proteins in bovine retina. Gene ontology analysis of these identified proteins revealed that they were mostly involved in splicing, transcription, cell cycle regulation and signal transduction. We propose that expression of the rhodopsin in rod photoreceptors might be cotranscriptionally coupled to splicing to cope with the timing of ROS biogenesis during daily rhythmic shedding as well as during terminal maturation of the rod cells during retinal development.

It is well established that ROS undergo disc shedding during early morning and need more membrane lipids and proteins particularly rhodopsin which constitutes about 90% of total ROS protein. Presence of several splicing proteins on the rhodopsin RER suggests that its splicing may be coupled with transcription to facilitate high expression in rod photoreceptors. Finding of several splicing proteins such as NonO, PPP1Ca and HNRPM on RER further supports the hypothesis of rhodopsin co-transcriptional coupling. Our data appear to be consistent with the ''recruitment'' model involved in co-transcriptional coupling of splicing to transcription (Munoz, de la Mata et al.), in the sense of a recruitment of splicing regulators at the promoter, or of their interaction with transcriptional regulators associated with the nascent mRNA. However, it is also possible that splicing factors like NonO may be playing a dual role in the expression of the highly transcribed genes such as rhodopsin in the retinal rod photoreceptors by altering the kinetics of transcription along with splicing.

We showed that NonO activates rhodopsin expression in in vitro cell cultures by interacting with NRL and CRX and increases the recruitment of RNA Pol II on rhodopsin promoter. In depth analyses using rhodopsin promoter luciferase reporter analysis and knockdown of NonO by in vivo electroporation of the shRNA against it revealed a functional significance of its role in rhodopsin expression in rod photoreceptors.

Remarkably, NonO shRNA knockdown displayed increased death of rod photoreceptors in the retina very early. Therefore these genetic and biochemical lines of evidence suggest that Nono is involved in the co-transcriptional coupling of the rhodopsin transcription with its splicing.

Notably, two proteins identified by our RER binding/mass spectrometry screening, serine/threonine-protein phosphatase 1 alpha catalytic subunit (Ppp1ca) and heterogeneous nuclear ribonucleoprotein M (Hnrpm), are known to interact with NonO [START_REF] Marko | hnRNP M interacts with PSF and p54(nrb) and co-localizes within defined nuclear structures[END_REF][START_REF] Liu | Consensus PP1 binding motifs regulate transcriptional corepression and alternative RNA splicing activities of the steroid receptor coregulators, p54nrb and PSF[END_REF]. Furthermore, Ppp1ca modulates the transcriptional activity of several transcription factors including NonO and PSF as well RNA Pol II by changing their phosphorylation status. We hypothesize that NonO forms a regulatory complex with Ppp1ca and Hnrnp on rhodopsin RER that in turn loops on RPPR and interacts with the transcriptional complexes containing NRL and CRX in order to enhance the recruitment of RNA Pol II on rhodopsin promoter and increase its expression in rod photoreceptors particularly in early morning when its higher amount of synthesis needed (Figure 4). Similarly, recent reports have shown that NonO is involved in regulating transcription of several genes such as steroid hormones estrogen, androgen as well as progesterone receptors, RXR and Cyp17 [START_REF] Yang | NonO, a non-POU-domain-containing, octamer-binding protein, is the mammalian homolog of Drosophila nonAdiss[END_REF][START_REF] Sewer | Transcriptional activation of human CYP17 in H295R adrenocortical cells depends on complex formation among p54(nrb)/NonO, proteinassociated splicing factor, and SF-1, a complex that also participates in repression of transcription[END_REF].

Among other proteins identified in our screening are several co-activators such as BUB3, CCAR1, 14-3-3, CREAP1, Sap180 and ERBP. Presence of these proteins on rhodopsin RER was not reported earlier and based on their known function it is possible that they might be playing an important role in rhodopsin regulation. Recently, yeast two hybrid screening studies have identified retinoic X receptor (RXR) chain of the nuclear hormone receptor superfamily as a NonO interacting protein [START_REF] Yang | NonO, a non-POU-domain-containing, octamer-binding protein, is the mammalian homolog of Drosophila nonAdiss[END_REF]. RXR is a nuclear receptor activated by 9-cis retinoic acid (RA). The role of RA in eye development is well established and interaction of NONO with the RA receptor (RAR) may have indirect effect on the transactivation abilities of NRL and CRX on rhodopsin promoter. To evaluate the effect of NonO knockdown on rhodopsin level we used P7 retina electroporated with either NonO shRNA or control shRNA and looked for the endogenous rhodopsin expression in GFP positive dissociated retinal cells by staining with anti-Rho.. Role of the retinoic acid in activation of NRL expression in Y79 cells has been recently reported [START_REF] Khanna | Retinoic acid regulates the expression of photoreceptor transcription factor NRL[END_REF]. Since NonO interacts with RXR receptor and RER has a predicted binding site for RXR, it may be possible that this interaction might be helping in increasing the rhodopsin promoter-luciferase reporter activity. The recent evidence that ERR beta is involved in rod differentiation and photoreceptor survival [START_REF] Onishi | The orphan nuclear hormone receptor ERRbeta controls rod photoreceptor survival[END_REF]) might be another line of evidence that orphan nuclear hormone receptors such as ERR beta and RXR play an important role in rhodopsin gene regulation.

In conclusion, we identified several candidate proteins binding to RER and studied the role of NonO/p54 nrb in rhodopsin gene regulation. NonO enhances rhodopsin gene transcription in the presence of NRL and CRX by a mechanism that may involve looping of RER on to RPPR on rhodopsin promoter and might be co-transcriptionally coupled to splicing (Figure 4). Finally, knockdown of NonO leads to rod photoreceptor cell death by apoptosis in the post-natal mouse retina. membranes were incubated overnight at 4 °C with primary antibody. After three washes with TBST, membranes were incubated with secondary antibody coupled to horseradish peroxidase for 1 h in TBST. Blots were washed twice in TBST and protein bands were visualized by enhanced chemiluminescence plus (Thermo Scientific, Rockford, IL).

In vivo electroporation. Neonatal CD-1 mice (Charles River Laboratories, Wilmington, MA) were used for in vivo transfection by electroporation, as described (Matsuda and Cepko 2004). For cotransfection studies, equimolar amounts of plasmids were used at a concentration of 10 μg/μL. Injection volume was 0.2 μL. Transfected eyeballs were harvested at P5, P10 and P21 for immunohistochemical analysis.

Immunohistochemistry. Cryosections were probed with specific antibodies as described (Matsuda and Cepko 2004) and visualized using Leica SP5 confocal laser scanning unit (Leica Microsystem Inc., Buffalo Grove, IL).

In this thesis work we focused on the transcriptional regulation of the major rod differentiation factor, Neural retina leucine zipper, Nrl. Our first goal, which constituted the main part of the thesis, was to understand the regulation of Nrl expression during development. To do so, we focused on three main evolutionarily conserved elements found in Nrl 5'-upstream sequence, A, B and C. We made different promoter bashings and cloned these different promoter fragments upstream of fluorescent reporter genes.

The technique of in vivo electroporation of neonatal mice retina, which allows investigating expression of DNA plasmidic vectors under the control of particular genes, led to the demonstration that the -938 to +119 fragment of the promoter (containing A+B but not intermediate sequences) was sufficient for driving strong expression in the ONL, whereas A fragment alone was not. Although we did not further focus on the whole promoter fragment (-2734 to +119 containing A, B and C) it should be underlined that this full-length 2.8 kb fragment was also efficient in driving reporter expression in the ONL, a result that is in agreement with the observation of Akimoto et al. by using 2,5kb

Nrl promoter to drive GFP expression in transgenic mice. Our results (data not shown) even suggested that 2.8 kb fragment expression was more restricted compared to the shorter fragment (-938 to +119). This result indicates that there are likely additional regulatory elements upstream of region B, to support other functions such as expression level regulation in rods and/or inhibition of expression in non-photoreceptor cell types.

We then focused on cluster B (-938 to -657) and cluster A (-304 to +119) and the observation that A+B but not A alone was working in ONL upon electroporation led to the conclusion that B is the required fragment for ONL expression. reporter labeling was increased four-fold, demonstrating its enhancer properties.

However, this element triggered very limited expression in the ONL when combined with A1. The same observation was done with B2 sequence-containing B4. Thus, 0.3 kb A1+B is so far the promoter sequence that most truly reflects Nrl promoter properties and should be used as a tool to further dissect the developmental mechanisms inducing Nrl expression.

One of the main advantages of the in vivo electroporation technique is to rapidly evaluate gene expression as compared to the generation of transgenic mice. However, this technique does not allow investigating early developmental points in the retina.

Knowing the histological order of the retinal development, we hypothesize that cone photoreceptors (peak of postmitotic cells at E16 in the mouse) would not be transfected in our neonatal electroporations. Therefore, the specificity of the tested promoter was not fully proven by using only this technique.

To tentatively answer this question, we produced EGFP constructs under the control of Nrl promoter A1+B sequence cloned in recombinant adeno-associated virus (AAV).

Generation of such viral vectors represent several advantages. First the specificity of promoter can be tested as mentioned above, since viruses are infecting cells independently of their cell type (although some AAV serotypes were proven to display higher affinity and better gene delivery for certain cell types [START_REF] Pang | Comparative analysis of in vivo and in vitro AAV vector transduction in the neonatal mouse retina: effects of serotype and site of administration[END_REF]). Second, use of a minimal promoter is a clear benefit for AAV use, since the cloning size limitation is < 4.7 kb and should include the coding sequence (CDS) to test, as well as the regulatory untranslated regions plus the two inverted terminal repeat sequences essential for AAV transcription. Hence, the importance of reducing as much as possible the size of foreign DNA to integrate into AAV genome is evident. In gene therapy targeting retinal disease, the idea is to replace the deficient gene by its wild type version carried by AAV. Uses of AAV vectors show a biological interest since they are nonpathogenic and provoke a mild immune response. In some cases, replacement genes can be particularly long and it is necessary to diminish the CDS with a risk of translating truncated protein that will have an unrewarding effect for gene therapy. To avoid this problem, it is better to focus on the promoter region by minimizing as much as possible its size. Defining a minimal Nrl promoter region for specific photoreceptor expression was among our goals. We showed expression of AAV-Nrlp-EGFP in wild type retina specifically in the photoreceptor layer; however complementary immunostaining with cone or rod-markers would have informed us on the precise identity of photoreceptorcell types that are expressing EGFP. We took a different approach to answer this question; by expressing AAV-Nrlp-EGFP in Nrl-/-animals we could evaluate whether Nrl promoter could be activated or not in cones. We found EGFP expression in the ONL of injected Nrl-/-retinas, showing that Nrl promoter can indeed be activated in the default S-cones typical of Nrl KO. These data corroborate the previous finding of our laboratory showing expression of the GFP transgene placed under the control of the 2.5 kb Nrl promoter in Nrl -/-retina [START_REF] Akimoto | Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors[END_REF]). In our case, we showed the promoter activation from P10, while Akimoto and his collaborators assessed the expression earlier in retinal development by generating transgenic mice. Nonetheless, it is complicated based only on these observations to affirm that cones are able to activate Nrl since the biological outcome of this activation would be transformation of cones in rods as proven by Oh and collaborators [START_REF] Oh | Rod differentiation factor NRL activates the expression of nuclear receptor NR2E3 to suppress the development of cone photoreceptors[END_REF]. One piece of element we can bring forward is the fact that there are more likely further 5'-upstream regulatory elements in Nrl promoter (even above 2.5 kb) that are not taken in consideration with artificial Nrl promoter constructions. Therefore, further promoter characterization is required for better understanding of the regulatory mechanisms implicated in the control of Nrl expression that will give rise to mature and functional retina.

Nrl regulation found its interest also in the investigation of rhythmic function of the retina. Previous studies showed a daily rhythm in rhodopsin transcription (Bowes, van Veen et al. 1988), as is also the case for other genes involved in the phototransduction cascade (Iuvone 2005). Given the central role played by rhodopsin in photoreceptor [START_REF] Tosini | Localization of a circadian clock in mammalian photoreceptors[END_REF]Sandu, Hicks et al. 2011) and this probably drive rhythmic expression of rod and cone functions by entraining more or less directly expression of key transcription factors and their target genes. Based on our partial Nrl promoter characterization, we can relate the control of its regulation with the action of ROR as transcriptional regulator. ROR is a molecular component of the circadian clock and was shown to display robust rhythmic expression in photoreceptors (Sandu, Hicks et al. 2011). Control of Nrl promoter by RORtranscription factor may explain in part the daily variations of Nrl; these data are corroborated by luciferase expression showing Nrl promoter activation by ROR. The overall picture we can draw from this study is that rhodopsin expression proceeds from complex mechanisms, which ensure expression of this gene during rod photoreceptor development and throughout adulthood, by making sure that rhodopsin is expressed at the right time of the day. Our data suggest that rhodopsin is highly expressed at the end of the night, in phase with expression of other elements of the phototransduction cascade and also with the rhythmic shedding of photopigment-filled outer segments. The concerted regulation of these processes probably contributes to the fine regulation of an over wise highly active outer segment renewal that is fundamental for photoreceptor survival.

Finally, we investigated the transcriptional relevance of a novel trans-acting factor NonO in rhodopsin regulation. NonO was identified on rhodopsin proximal promoter and was shown to highly regulate reporter gene expression in combination with NRL and CRX. To characterize NonO function in vivo, we knocked-down this factor in the developing retina using shRNA targeted against NONO transcript. We showed early degeneration of rod-photoreceptors mediated by apoptosis probably due to rhodopsin down-regulation, a data which puts further emphasis on the fact that abnormal expression levels of rhodopsin strongly impair photoreceptor homeostasis. These data present NonO as an original transcription factor required for rhodopsin activation. From this observation, it is legitimate to wonder whether NonO could play a role in rhodopsin daily variation. It was previously reported that NonO interacts with Period1 protein in mammalian cells and this interaction was suggested to be essential for normal circadian rhythmicity in mammals and drosophila [START_REF] Kyriacou | Circadian rhythm mutations in Drosophila melanogaster affect short-term fluctuations in the male's courtship song[END_REF]. It would be interesting to investigate the profile of expression of Nono to determine whether this gene is also rhythmic in the retina.

  amitié avec Anand a certainement positivement influencée mon recrutement dans son labo, donc merci sincèrement! Je remercie Valérie Simmoneaux pour l'accueil au sein du Département de Neurobiologie des rythmes à l'INCI.

Figure 1 '

 1 Figure 1'. Expression de séquences du promoteur/enhancer de Nrl au cours du développement de la rétine

Figure 2 Figure 3 '

 23 Figure 2'. Ordre de naissance des cellules de la rétine de souris La neurogenèse rétinienne suit un ordre histogénique précis. Les cellules ganglionnaires et horizontales sont les premières à se différencier, suivies des phases de naissance juxtaposées des photorécepteurs de type cône, des cellules amacrines, des photorécepteurs de type bâtonnets, des cellules bipolaires et enfin des cellules gliales de Müller. Chaque courbe reflète la proportion relative de chaque type cellulaire produit, plutôt qu'un nombre absolu de cellules nées. (D'aprèsMarquardt et al. 2002)

Figure 4 '

 4 Figure 4'. ROR, CRX, OTX2 et CREB s'accrochent aux éléments de séquence du promoteur /enhancer de Nrl Autoradiogrammes de gels pour des oligonucléotides radiomarqués avec du 32 P et retardés avec des protéines nucléaires extraites de rétines de souris adultes (A et D) et nouveaux-nés P1-2 (B et E), ou extraites de cellules HEK293 transfectées avec les constructions d'ADN CMV-RORβ (A, lignes 10 et 12), CMV-CRX ou CMV-OTX2 (C). L'ajout de 25 ou 100 ng d'oligonucléotides spéciques non-marqués entre en compétition avec l'oligonucléotide marqué et réduit son signal. L'oligonucléotide non-marqué mutant (m), comportant cinq substitutions de nucléotides dans la séquence consensus d'accrochage, n'entre pas en compétition. Des supershifts ont été réalisés avec des anticorps contre OTX2, CRX, CREB, RORβ, et ded IgG non dirigés contre ces facteurs (A and B), avec des anticorps anti-CRX ou -OTX2 (C), et avec des anticorps contre CREB, NRL, c-Fos, et d'IgG normaux (D and E). Les oligonucléotides B3, B4 et A4 sont indiqués. B4 contient 18 nucléotides supplémentaires à l'extrémité 5' de B3, lui conférant ainsi une meilleure séquence de reconnaissance aux facteurs de transcription de transcription à homéodomaine.

  photorécepteurs se différencient à partir d'un pool commun de précurseurs et que ces cellules expriment donc des facteurs de transcription communs aux deux types de photorécepteurs. Sur le même registre de thérapie génique, nous avons cherché à mettre en évidence la plasticité des photorécepteurs post-mitotiques dans leur capacité à changer leur spécificité cellulaire. Pour cela, nous avons utilisé des AAV pour réintroduire NRL sous le contrôle du promoteur ubiquitaire CMV dans la rétine de souris NRL KO. Trois âges distincts du développement de la rétine ont été choisis, P4, P10 et adulte. Par des expériences d'immunomarquage, nous avons démontré l'expression de la Rhodopsine pour chaque âge testé (Figure 6'); ces résultats nous indiquent donc que NRL est un facteur de transcription qui a la capacité de moduler la spécificité cellulaire aussi bien dans des cellules post-mitotiques en cours de développement que dans des cellules déjà matures. L'utilisation du promoteur NRL avec des rAAV ouvre une base d'étude pour le développement de futurs vecteurs viraux ciblant spécifiquement les photorécepteurs.

Figure 5 '

 5 Figure 5'. Expression de la GFP dans des rétines de souris infectées avec des virus recombinants adéno-associés Des rétines de souris de type sauvage (C57Bl6; A-B') et transgénique (Nrl KO; C-D') ont été injectées à P10 avec 1µL de constructions virales AAV8-Nrlp-EGFP (A, A', C, C'; concentré à 3 e12 particules virales/mL ; Nrl promoteur murin de 0.3 kilobases) et AAV-RKp-EGFP (B, B', D, D'; concentré à 1 e12 particules virales/mL ; Rhodopsin kinase promoteur humain de 1.2 kb). Trois semaines après l'injection, les rétines montrent une expression de la GFP uniformément sur la couche des photorécepteurs (couche nucléaire externe, ONL). Aucune expression de la GFP n'a été détectée dans la couche nucléaire interne (INL) ou dans la couche des cellules ganglionnaires (GCL). Barre d'échelle, 50 µm.

Figure 6 '

 6 Figure 6'. L'expression de la Rhodopsine est rétablie suite à la ré-introduction de NRL dans des rétines Nrl KO Des rétines de souris transgéniques Nrl KO ont été injectées à P4, P10 et 1 mois avec 1µL de constructions virales AAV8-CMV-NRL et AAV-RKp-EGFP. L'évaluation de l'expression de la Rhodopsine et de la GFP s'est effectuée trois semaines après l'injection. L'immunomarquage anti-Rhodopsine (RHO) sur des rétines Nrl KO injectées avec AAV8-CMV-NRL a permis de détecter la présence de cette protéine spécifiquement dans la couche des photorécepteurs (ONL) à tous les âges testés. L'injection de AAV-RKp-EGFP n'a pas montré de marquage positif, par contre nous observons une immunoréactivité des vaisseaux sanguins due à l'anticorps souris RHO employé. ONL, couche nucléaire externe ; INL, couche nucléaire interne, GCL, couche des cellules ganglionnaires. Barre d'échelle, 20 µm.

Figure 7 '

 7 Figure 7'. ROR est nécessaire à l'expression de Nrl au cours du développement La transfection in vivo de promoteur Nrl WT montre une forte expression du gène rapporteur mCherry dans la couche des photorécepteurs. La mutagenèse dirigée contre le site RORE (substitution de deux nucléotides dans la séquence wild type, AAAATGTAGGTCA) abolit l'expression de mCherry. Vert, Green Flurorescent Protein ; rouge, fluorescence du mCherry ; bleu, DAPI. Barre d'échelle, 20 µm.

Figure 8 'Figure 9 '

 89 Figure 8'. Profiles d'expression d'ARNm de ROR et RevErb dans la couche des photorécepteurs de rat Analyse en qPCR des profiles d'expression de gènes horloge sous des conditions lumière/obscurité (barres blanche et noire respectivement) de la couche de photorécepteurs isolée par coupe au vibratome. Les valeurs représentent la moyenne ± l'erreur type (SEM ; n = 5). Les variations significatives (P < 0.05) au cours du temps sont indiquées. Les données pour ZT0 et ZT24 sont dupliquées. (D'aprèsSandu et al. 2011, EJN)

  III.Nrl and co-activators of rhodopsin expression show daily variations in the rat retina during the 24 h cycle ............................................................................................................................................... IV. A novel transcription factor, NonO, appears as a co-activator of rhodopsin expression and is necessary for rod-photoreceptor survival. .......................................................................................... GENERAL DISCUSSION ......................................................................................................................... GENERAL DICUSSION ........................................................................................................................... REFERENCES ........................................................................................................................................ REFERENCES ........................................................................................................................................ ABSTRACT IN ENGLISH .........................................................................................................................

Figure 2 'Figure 3 'Figure 4 'Figure 5 'Figure 6 'Figure 7 'Figure 8 'Figure 9 '

 23456789 Figure 2'Ordre de naissance des cellules de la rétine de souris Figure 3' Identification d'une région minimale du promoteur de Nrl transcriptionnellement active Figure 4' ROR, CRX, OTX2 et CREB s'accrochent aux éléments de séquence du promoteur /enhancer de Nrl Figure 5' Expression de la GFP dans des rétines de souris infectées avec des virus recombinants adéno-associés Figure 6' Expression de la Rhodopsine est rétablit suite à la ré-introduction de NRL dans des rétines Nrl KO Figure 7' ROR est nécessaire à l'expression de Nrl au cours du développement

Figure 49

 49 Figure 49Schematic representation of the RPPR and RER bound transcriptional complexes on rhodopsin promoter

(Figure 1 .

 1 Figure 1. Scheme of extensively diversified metazoan regulatory modulesA complex arrangement of multiple clustered enhancer modules interspersed with silencer and insulator elements which can be located 10-50 kb either upstream or downstream of a composite core promoter containing TATA box (TATA), Initiator sequences (INR), and downstream promoter elements (DPE). Modified fromLevine and Tjian, Nature, 2003.

Figure 3 .

 3 Figure 3. Core promoter elementsThis diagram shows com-mon core promoter ele-ments with their con-sensus se-quences and relative position to the transcription start site (+1). The core promoter can show considerable variability as there are no universal elements. Each of the motifs is found in only a fraction of core promoters with different combinations. The TATA box, Inr, DPE, and DCE are recognition sites for binding of TFIID, whereas BREu and BREd interact with TFIIB. From Baumann et al.,Mol. Biotechnol., 2010.

Figure 4 .

 4 Figure 4. Functions of TAFs Various functions of individual TBD-associated factors (TAFs) subunits in facilitating the process of transcriptional activation through the subunit TBP of TFIID. Modified from Näär et al, Annu Rev Biochem, 2001.

Figure 5 .

 5 Figure 5. Current models of transcriptional activation involve multiple steps (a)Transcriptionally inert chromatin, in which DNA is tightly packaged with nucleosomes. (b)Transcriptional activators bind to their cognate sites in the regulatory region of the gene and recruit series of chromatin co-activators that can covalently modify nucleosomes (c)Chromatin is characterized by distinct covalent modifications, such as acetylation (Ac) and methylation (Me), and by a relative dearth of nucleosomes. The activators then recruit Mediator.In some cases, the intact Mediator that consists of the core and the kinase module might be recruited at this stage. Modified fromMalik and Roeder, Nat Rev Genet., 2010.

2. 2

 2 Transcription factors and chromatin remodeling 2.2.1 Mode of action Transcription factors (TFs) are proteins that bind to specific DNA sequences found in enhancer or promoter region. TFs are generally composed of three major domains: the DNA-binding domain, responsible for recognition of specific DNA sequences. The second domain is the trans-activating domain accountable for the activation or repression of the targeted gene transcription and in certain cases can be necessary for the proteinsinteraction, however this function can be done by the protein-protein interaction domain, which allows TFs to interact with TAFs or other regulators to modulate the transcription (Figure6). TFs can recruit co-regulator proteins that are altering locally chromatin conformation and thus enabling Pol II to access to the initiation site, or else, TFs are interacting with co-activators that are themselves enzymes altering chromatin structure. As a matter of fact, chromatin is the highest condensed structure of the DNA.

Figure 6 .

 6 Figure 6. Transcription factor domainsScheme of the amino acid sequence of a prototypical transcription factor that contains a DNA-binding domain (DBD), a protein-protein interaction domain (PPD) and a transactivation domain (TAD). The order of placement and the number of domains may differ in various types of transcription factors.

Figure 7 .

 7 Figure 7. Suggested sequence of coactivator function in transcription initiation.Nucleosome (large green circle) positioned over the TSS (subtended arrow), masking the TATA box (T). (a) Once bound to an enhancer, activator(s) may recruit a kinase (Ki) that phosphorylates histone H3 at Ser 10 (circled P). (b) HAT coactivator complexes bridge the activator AD and phospho-Ser-10. Followed by acetylation of lysines in histone (circled Ac), weakening contacts between DNA and histones. Acetylated chromatin allows activator to interact with chromatin remodeling complexes such as SWI/SNF (c), which make contacts to activator, and to acetylated chromatin. ATP-dependent remodeling affects the path of DNA around the histone octamer, thus exposing the adjacent TATA box. (d) TBP/TFIID binds the TATA box and induces a bend in DNA. This bending provokes repositioning of the nucleosome in a manner also dependent on histone acetylation. Nucleosome repositioning exposes the transcriptional start site and allows access to RNA pol II and GTFs. In some instances, recruitment of Mediator (M) by activator precedes and facilitates pol II entry. This latter event can also come under the control of signaling pathways linked to the cell cycle. Modified fromFeatherstone, Curr Opin Genet Dev., 2002.

Figure 8 .

 8 Figure 8. Model for a dimeric bZIP protein Two bZIP polypeptides join via a Leucine zipper to form a Y-shaped molecule in which the stem of the Y corresponds to a coiled pair of -helices held by the leucine zipper. The arms of the Y are the respective basic regions of each polypeptide; they act as a linked set of DNA contact surfaces. Source http://web.virginia.edu

Figure 9 .

 9 Figure 9. Various members of the Maf family transcription factors Overview of the Maf transcription factor family. H: Histidine repeat; G: Glycine repeat.

Figure 10 .Figure 11 .

 1011 Figure 10. The human retinaA horizontal cross section of the human eye, showing the major parts of the eye. From Encyclopaedia Britannica, Inc, http://www.britannica.com/

Figure 12 .

 12 Figure 12. Retinal organization Rod and cone photoreceptors form an outer nuclear layer that contacts bipolar cells and horizontal cells. Bipolar cells relay information from the outer retina to the inner retina where they contact retinal ganglion cells and amacrine cells. The cell bodies and synaptic connections between the various retinal cell types are organized into distinct layers. Ph: photoreceptors. OPL/IPL: Outer/Inner Plexiform Layers. INL: Inner Nuclear Layer. GCL: Ganglion Cell Layer. From Rachel Wong laboratory, Josh Morgan.

Figure 13 .

 13 Figure 13. The mature retina

  inner nuclear layer (INL) is the layer of the interneurons that mediate and modulate the flow of information between the photoreceptors and the ganglion cells. INL contains the cell bodies of bipolar cells, amacrine cells, horizontal cells, interplexiform cells and Müller glial cells. Nuclei distribution within the INL is the following: bipolar cells are located in the middle of the INL; most of the amacrine cells are found in the innermost

Figure 14 .Figure 15 .

 1415 Figure 14. Rod and cone structuresBoth types of photoreceptors are composed of an outer segment, an inner segment a cell body, an axon and a synaptic terminal characteristic for each type of photoreceptor. From http://thebrain.mcgill.ca

Figure 16 .

 16 Figure 16. Development of the vertebrate eye cup (a) The neural plate is the starting point for the development of the vertebrate eye cup. (b)The neural plate folds upwards and inwards. (c) The optic grooves evaginate. (d) The lips of the neural folds approach each other and the optic vesicles bulge outwards. (e) After the lips have sealed the neural tube is pinched off. At this stage the forebrain grows upwards and the optic vesicles continue to balloon outwards: they contact the surface ectoderm and induce the lens placode. (f) The optic vesicle now invaginates, so that the future retina is apposed to the future retinal pigment epithelium (RPE), and the ventricular space that was between them disappears. Developing retinal ganglion cells send axons out across the retinal surface. The surface ectoderm at the lens placode begins to form the lens pit. This section is midline in the right eye, through the choroid fissure, so only the upper region of the retina and the RPE are visible. g | The eye cup grows circumferentially, eventually sealing over the choroidal fissure and enclosing the axons of the optic nerve (as well as the hyaloid/retinal vessels; not shown). The ectodermal tissue continues to differentiate and eventually forms the lens. FromLamb et al., Nat Rev Neurosci., 2007.

Figure 18 .

 18 Figure 18. Schematic representation of Notch signaling. Activation of Notch by its ligand Delta leads to the cleavage and translocation of the intracellular domain (ICD) of Notch. Notch ICD binds to the RBP-J complex and activates expression of Hes genes. Hes proteins interact with the corepressor Groucho and repress neural bHLH genes such as Mash1 and Math3, which are required for neuronal cell fate specification in the developing retina. From Ohsawa and Kageyame, Brain Res., 2008.

Figure 19 .

 19 Figure 19. Regulation of retinal cell fate specification by transcription factors Combinations of multiple transcription factors, such as bHLH-type and homeobox-type factors, are required for proper specification of retinal cell types. From Ohsawa and Kageyame, Brain Res., 2008.

Figure 20 .

 20 Figure 20. Stages of photoreceptor developmentMultipotent retinal progenitor cells (RPCs) divide and produce additional multipotent progenitors (thick circular arrow) or progenitor cells that become restricted in their competence to generate various cell types (thin circular arrow). Some of these proliferating cells become restricted to a lineage that will give rise to at least one photoreceptor cell and possibly to non-photoreceptor cells. After cell cycle exit, postmitotic precursors can remain plastic. During cell type specification of photoreceptors, precursors are directed to become cones or rods that eventually express photopigments (M opsin and S opsin in cones, and rhodopsin in rods), and form outer segments and synapses. FromSwaroop et al., Nat Rev Neurosci. 2010.

  TR2 has a dual role in cone pathway development, by stimulating M-cone development and repressing S-cone generation[START_REF] Ng | A thyroid hormone receptor that is required for the development of green cone photoreceptors[END_REF][START_REF] Ng | Developmental expression of thyroid hormone receptor beta2 protein in cone photoreceptors in the mouse[END_REF][START_REF] Ng | Two transcription factors can direct three photoreceptor outcomes from rod precursor cells in mouse retinal development[END_REF]).2.4.2 Other retinal cell fate commitmentBoth the homeobox gene Chx10 and two bHLH, Mash1 and Math3, seem to play a major role in the bipolar cell-type specification. Mutations in Chx10 result in a proliferation of RPCs and a bipolar-less retina[START_REF] Burmeister | Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation[END_REF]. The double knock-out animals for Mash1 and Math3 genes present a total absence of bipolar cells and an increase of Müller glia cells, suggesting that Mash1 and Math3 are necessary for bipolar cell development and inhibition of gliogenesis[START_REF] Tomita | Mammalian achaete-scute and atonal homologs regulate neuronal versus glial fate determination in the central nervous system[END_REF][START_REF] Hatakeyama | Roles of homeobox and bHLH genes in specification of a retinal cell type[END_REF].Amacrine cell development seems to be induced by the expression of a set of bHLH factors such as Math3, NeuroD, Pax6 and Six3, shown to promote amacrine cell genesis[START_REF] Inoue | Math3 and NeuroD regulate amacrine cell fate specification in the retina[END_REF]. In the transcriptional cascade leading to the amacrine cell specification, Sox2 is found upstream of Pax6 expression. Sox2 transcription induces Pax6 expression, which will in turn activate NeuroD, and both factors will promote the amacrine cell fate[START_REF] Lin | Sox2 plays a role in the induction of amacrine and Muller glial cells in mouse retinal progenitor cells[END_REF].Horizontal cells and amacrine cells seem to have linked development, since both cell types are sharing common transcription factors. Misexpression of Math3 and Pax6

Figure 21 .

 21 Figure 21. The visual phototransduction cascadeIn the dark, opsin is bound to 11-cis-retinal to form inactive rhodopsin (R) in the disc membranes. Basal activity of the guanylyl cyclase (GC) keeps cGMP levels high. The binding of Ca 2+ -bound calmodulin (CaM) confers high affinity for cGMP to cGMP-gated channels in the plasma membrane, allowing these channels to remain open. Both Na + and Ca 2+ enter the channels resulting in high Ca 2+ levels and Ca 2+ -bound guanylate cyclase-activating protein (GCAP). Light (H) results in photoisomerization of 11-cis-retinal to all-trans-retinal, forming activated rhodopsin (R + ), which binds and activates the heterotrimeric G protein, transducin (). The GTP-bound transducin α subunit activates cGMP phosphodiesterase (PDE), which hydrolyzes cGMP to GMP, reducing the cGMP concentration and the binding of cGMP to the cGMP-gated channels. The probability of channel closing increases proportionally to light intensity, reducing Ca 2+ influx. Intracellular Ca 2+ is further depleted by activity of the Na + -Ca 2+ , K + exchanger. Low intracellular Ca 2+ leads to active GCAP, which in turn activates GC to synthesize cGMP from GTP supplied by the guanine nucleotide cycle. This comprises guanylate kinase (GK) and nucleoside diphosphate kinase (NDPK). Release of Ca 2+ from CaM leads to its dissociation from the cGMP-gated channels conferring a lower affinity for cGMP and further closure of the channels. From http://mutagenetix.utsouthwestern.edu

Figure 23 .

 23 Figure 23. The molecular circadian clock machineryThis diagram depicts the positive (CLOCK:BMAL1) and negative (PER:CRY) limbs of the circadian clock gene feedback loop that is present in virtually all cells throughout the body. REV-ERBα and RORα represent components of a secondary feedback loop that either activate (solid line with arrow) or inhibit (dashed line) Bmal1 activity. The CLOCK:BMAL1 transcription complex has the ability to directly regulate the activity of genes (clock controlled genes) critically involved in energy metabolism, including Dbp and Pparα. In turn, PPARα can directly bind to Bmal1 and influence activation of the positive limb of the feedback loop. BMAL1, brain muscle arnt like factor; CLOCK, circadian locomotor output cycles kaput; CRY, cryptochrome; DBP, albumin D-element binding protein; PER, period; PPARα, peroxisome proliferator-activated factor alpha; REV-ERBα, reverse erythroblastosis virus alpha; RORα, retinoic acid receptor-related orphan receptor alpha; RXRα, retinoid X receptor alpha. FromLaposky et al., FEBS Lett., 2008.

(

  Figure 23). The positive loop is constituted of CLOCK (Circadian Locomotor Output Cycles Kaput) and BMAL1 (ARNTL aryl hydrocarbon receptor nuclear translocator-like), two bHLH-PAS factors forming a heterodimer binding to E-box sequence (core consensus sequence, 5'-CANNTG-3') found in the promoter of target genes. Among the genes regulated by CLOCK/BMAL1, there are Dec1-2, RevErbα-β and Rorα-γ plus the main factors involved in the negative loop, Per1-3 and Cry1-2. PER (Period) and CRY (cryptochrome) proteins are forming dimers in the cytosol and enter the nucleus to inhibit CLOCK/BMAL1 dimer and hence to repress their own expression. PER and CRY also undergo proteasome-mediated degradation. Thus, when their levels are low enough, their activation by CLOCK/BMAL1 can re-start and expression of PER and CRY proteins rises again.

Figure 24 .

 24 Figure 24. Melatonin and dopamine rhythmic synthesis in the retinaMelatonin and dopamine play opposing roles in the regulation of retinal adaptive physiology Dopamine functions as a humoral signal for light, producing light-adaptive physiology, while melatonin has dark-adaptive effects. The synthesis and release of both melatonin and dopamine are under circadian control, with melatonin released at night and dopamine during the daytime. Melatonin inhibits the release of dopamine through an action on melatonin receptors and dopamine inhibits the synthesis and release of melatonin from photoreceptor cells by acting on dopamine receptors present on photoreceptors. Thus, the melatonin synthesizing photoreceptors and dopamine secreting inner retinal neurons form a cellular feedback loop that regulates circadian retinal physiology. FromTosini et al., Bioessays, 2008.

Figure 25 .

 25 Figure 25. Light-dark cycling of opsin, G, G, and 48-kDa protein mRNA levels Graphic representation of total retinal RNA isolated from WT mouse and labeled with specific cDNA probes for opsin, G, G, and 48-kDa protein. Area of each slot is marked on the ordinate. The isolation times are indicated on the abscissa. Highest levels of opsin (black box), G(+) and G(white box) mRNAs and lowest levels of 48-kDA protein (black triangle) mRNA occur 1.5h before lights on (3.30 a.m. on day 1 and 3.30 a.m. on day 2, arrows).Modified fromBowes et al., Exp. Eye Res., 1988.

  photoreceptors in light/dark cycle showed a significant variation in the transcript amount for Clock, Per1, Per3 and Casein kinase I, whereas Bmal1 mRNA variation was not significant. In dark/dark conditions only Clock and Per3 remained significantly variable. These data are placed in correlation with analysis of the clock gene expression in the whole retina, where in LD, all the genes cited above showed significant variability during the cycle, while in DD, the variability remained in Clock, Bmal1, Per2 and Per3. During 24 h, changes in mRNA levels of clock genes in photoreceptors indicate that these cells potentially contain a circadian clock working independently to other retinal layers[START_REF] Schneider | Unique clockwork in photoreceptor of rat[END_REF]. A recent study used another technique to isolate photoreceptors, performing vibratome-dissection; the authors isolated rat photoreceptors and quantified mRNA amounts of clock genes during LD and DD conditions. They showed significant variations of transcript for Clock, Bmal1, Per1, Per2, Cry1, Cry2, Ror and RevErb genes in LD condition, whereas in DD, none of the genes' transcript remained significant except for Ror and RevErb. The clock outputs Aanat and c-Fos remained strongly rhythmic in LD and DD conditions(Sandu, Hicks et al. 2011).Thus in spite of some differences, the data of this and previous studies are globally in agreement. They suggest that the photoreceptors have the components of a functional clock in LD; however, lack of rhythmicity of genes' expression in DD suggests that the

  phosphorylation status affects NRL ability to bind TBP and/or other components of the general transcription machinery. Recently, another type of PTM affecting NRL protein has been demonstrated: the sumoylation of NRL with two small ubiquitin-like modifier (SUMO) molecules attached to the Lys-20 residue of this protein. Mutant NRL proteins

Rhodopsin.Figure 26 .

 26 Figure 26. Model of transcription initiation complex at the rhodopsin promoterIn the dark cis-regulatory elements upstream of the transcription start site in the rhodopsin promoter include Eopsin-1, Ret-1, BAT-1, NRE, Ret-4 and TATA-box. NRL is believed to bind to NRE as a homodimer. It interacts with TATA-binding protein (TBP) (unpublished data). NRL and CRX physically interact[START_REF] Chen | Ret 4, a positive acting rhodopsin regulatory element identified using a bovine retina in vitro transcription system[END_REF]. CRX can bind to Ret-4 and BAT-1 elements. BAF and FIZ-1 function as repressors of CRX and NRL, respectively. The homeodomain proteins, RX, ERX, CRX and QRX, can bind to the Ret-1/PCE-1 element in vitro. It is unclear which of these occupies this element in vivo. QRX interacts with CRX but not with NRL (57). In this report, we show that NR2E3 and NR1D1 also participate in the transcriptional activation of the rhodopsin promoter. Combinatorial and synergistic actions of various regulatory proteins recruit and stabilize the initiation complex and facilitate the transcription by RNA polymerase II (RNAP II). FromCheng et al., Hum. Mol. Genet., 2004.

1. 2 Figure 27 .

 227 Figure 27. Opsin immunohistochemistry for Nrl KO retina Immunostaining of retinal sections from 5-wk wildtype, Nrl +/-and Nrl -/-mice with antibodies. a,b, Wildtype and Nrl +/-retinae show a normal distribution of rhodopsin antibody (indicated by light brown color). c, Rhodopsin is absent in the shortened outer segment layer of the Nrl -/-retina. d,e, Wildtype and Nrl +/-retinae show normal distribution of S-opsin antibody. f, Nrl -/-retina shows S-opsin staining of the entire outer segment layer. g,h,i, M-opsin antibody shows normal distribution in all retinae. From Mearset al., Nat Genet., 2001.

  photoreceptors in mice[START_REF] Maclaren | Retinal repair by transplantation of photoreceptor precursors[END_REF]

Figure 29 .

 29 Figure 29. In vivo retinal transfection by electroporation Upper panel, DNA solution is injected in the subretinal space of neonatal animals and then submitted to series of electrical pulses for DNA transfection into retinal progenitor cells. Lower panel, electrodes for in vivo electroporation. Tweezer-type electrodes are placed to hold the head of neonatal rat or mouse. From http://genepath.med.harvard.edu

4. 2

 2 Real-time quantitative PCR Gene expression over 24 h LD and DD cycles was analysed by real-time PCR for Rho and Nr2e3 in the whole retina, and Nrl and Crx in photoreceptor layers. Real-time quantitative PCR was performed using the hydrolysed probe-based TaqMan chemistry.

  and the following expression plasmids: i. pSV-CLOCK and CMV-BMAL1 (Travnickova-Bendova et al., 2002) (0.1 µg each); ii. FLAG-tagged CMV-RORβ (Science Applications International Corporation, Frederick, MD) (0.3 µg); iii. pcDNA4c-RevErbα(Cheng et al., 2004) (0.3 µg). Co-transfection mixtures contained also 0.001 μg of the second reporter plasmid, CMV-Renilla (Promega, Madison, WI), and the empty pcDNA4c (Life Technologies,) vector to adjust the total amount of transfected DNA to 1 µg. Cells were harvested 48 h after transfection and lysed in 100 μl of passive lysis buffer (Promega).

Tween 20 (

 20 TBS-T) buffer, and then incubated in TBS-T supplemented with 5% fat-free milk for 1h incubation at RT. Later, the membrane was incubated in the primary antibody solution diluted in TBST overnight at 4°C under agitation. Membrane was washed thoroughly with TBS-T and then incubated with secondary antibody coupled to horseradish peroxidase diluted in TBS-T supplemented with milk. immunoreactive bands were revealed by chemoluminescence (Immobilon, Millipore) and detected by exposure to an auroradiographic film. Apparent molecular weights were estimated by comparison to pre-stained molecular size markers. Protein band densities were determined by scanning the blots on a professional scanner. Each image was subjected to quantification using ImageJ software (version 1.43u, National Institues of Health, USA).

  was digested with NotI and SacII to remove CMV promoter. 0.3 kb mouse Nrl promoter (composed of A1+B; -34 to +16 and -968 to -657 respectively) was PCR amplified using the following primers: A1, forward and B, forward and reverse primers, 5′-ATTTGCGGCCGCGGGAATACCCTTTA-3′ and 5′-ACGGGGTACCACCACCACACTTCTGT-3′. The PCR products were digested with NotI, KpnI and SacII, and then inserted into the AAV8 vector plasmid containing enhanced green fluorescent protein. For rescue experiments, mouse Nrl coding sequence was inserted via BamHI and XhoI sites into AAV8-vector plasmid containing CMV promoter 2. rAAV Vector production.

4 .

 4 HEK293 cells in 300 mL Dulbecco's modified Eagle medium (DMEM, Gibco) containing 9% fetal calf serum. The cultures were incubated at 37°C with 10% CO2 and rotated at 0.2 rounds per minute for 3 days before transfection. For each roller bottle, 18 mL of 300 mM CaCl2 containing 150 µg each of vector plasmid and rep/cap vector (encoding proteins required for virus replication and encapsidation) as well as adenovirus helper was rapidly mixed with 10 mL of 2 × HBS (HEPES-buffered saline; 50 mM HEPES [N-2hydroxyethylpiperazine-N′-2-ethanesulfonic acid], 280 mM NaCl, 1.5 mM NaH 2 PO 4 , pH 7.1) and was added to the roller bottle. The next morning, medium was changed and replaced with 100 mL serum-free DMEM supplemented with antibiotics. Cultures were then incubated for 72 hours. Vector-containing cells were dislodged from the sides of the roller bottles by vigorous swirling and collected by centrifugation at 1000g for 15 minutes 4°C.3. rAAV vector purificationVirus production is summarized in Figure30. The collected HEK293 cells were dispersed in TSM buffer (50 mM Tris-Cl, 150 mM NaCl, 2 mM MgCl 2 , pH 8.0, 50 mL per roller bottle) before disruption by microfluidization(3 passes, model HC 2000; Microfluidics Corporation, Newton, MA). Tissue debris were removed by centrifugation (4150 rpm, 1.30 hour total, 4°C), and the supernatant was adjusted to 25 mM CaCl 2 by adding the appropriate amount of a 1 M stock solution. After 1-hour incubation at 4°C, the resultant precipitate was removed by centrifugation (4150 rpm, 1 hour, 4°C), and the supernatant was digested with Benzonase (250 U/mL, Novagen) for 1 hour at 37°C. Vector particles were then precipitated for 2 hours at 0°C with 8% polyethylene glycol 8000 (in 650 mM NaCl) and collected by centrifugation (4000 rpm, 30 minutes, 4°C). The pellet was completely dissolved in 50 mM HEPES, 150 mM NaCl, 20 mM EDTA (ethylenediaminetetraacetic acid), 1% Sarcosyl, pH 8.0 containing 10 μg/mL RNase A (2mL per roller bottle) and was applied to a CsCl step gradient consisting of 5 mL of 1.5 g CsCl/mL for the bottom layer, 8 mL of 1.3 g CsCl/mL for the middle layer, then a 22-25 mL sample (in 25 × 89 mm polyallomer tubes, spun in an SW32 rotor at 28 000 rpm for 18 hours at 20°C). The lower full capsid band was identified visually and collected with an 18-gauge hypodermic needle and a 10-mL syringe through the side of the tube. The vector-containing solution was again centrifuged on a linear CsCl gradient (14 × 95-mm polyallomer tubes, spun in an SW40 rotor at 38 000 rpm for 72 hours at 20°C). The full capsid band was collected with a hypodermic needle, diafiltered against Tris-buffered saline (TBS, 10 mM Tris-Cl, 180 mM NaCl, pH 7.4) and stored frozen at -80°C. Titration of viral particles.Vector genome titers were established by taking the average of 3 quantitative realtime PCR (Q-PCR) determinations. All samples were DNaseI treated before measurement and were subjected to 45 cycles of amplification (2 steps per cycle: 62°C for 60 seconds, 95°C for 15 seconds) in a 7700 Q-PCR machine(Applied Biosystems, Foster City, CA)

  -injection, eyes were enucleated, placed in 4% paraformaldehyde/PBS for 15 min, and their anterior segments and lens were removed. Fixation continued in fixative for additional 20 min. The fixed tissues were soaked in 30% sucrose/PBS for 3 hours or overnight, embedded in OCT (Sakura Finetek),shock frozen and sectioned along the superior-inferior meridian at 12 µm thickness.. For immunolabeling retinal sections were washed three times x 5 min with PBS. Non-specific binding sites were blocked with DMEM supplemented with antibiotics (penicillin and streptomycin) containing 10% FBS, 4% goat serum, and 0.1% Triton X-100 for 1 h at room temperature. Rhodopsin monoclonal antibody, Rho4D2 (Dr. R. Molday, University of British Columbia, Vancouver, British Columbia, Canada) was used as primary antibody, diluted at 1:500 in blocking solution and incubated at room temperature overnight at 4°C. The next day, sections were washed three times in PBS-T (1X PBS, 0.1 % Triton) and then were incubated for 1 h with anti-mouse antibody conjugated with Alexa Fluor 568 (Molecular Probes, Invitrogen) diluted at 1:1000 in blocking buffer and (1 mg/mL in PBS) diamidino-phenylindole (DAPI). After three washes with PBS-T, sections were mounted with gel mounting medium. Immunostaining was visualized using an Olympus FluoView FV1000 confocal laser scanning unit.

  photoreceptor survival.

  , a hereditary disease leading progressively to blindness. Despite the developmental and clinical importance of this gene, little is known about its regulation. The main goal of my project was to decipher the mechanisms responsible for the transcriptional regulation of Nrl during retinal development. Previous studies had partially characterized promoter elements of Nrl that could play an active role in the positive regulation of this gene. A 2.5 kb promoter region containing conserved elements was shown to drive specifically reporter gene in rod photoreceptors[START_REF] Akimoto | Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors[END_REF]. Retinoic acid response elements were among the putative binding sequences identified in Nrl promoter; gel shift and transactivation assays demonstrated the positive role of retinoic acid for Nrl regulation.[START_REF] Khanna | Retinoic acid regulates the expression of photoreceptor transcription factor NRL[END_REF]. During my thesis, we aimed to extend Nrl promoter characterization using biochemical approaches and in vivo electroporation of neonatal mouse retina. We defined a minimal region of 0.3 kb that could strongly drive reporter gene expression specifically in rod photoreceptors. This fragment of promoter is composed of a proximal promoter (A1) and a promoter/enhancer element (B) that are both required for Nrl expression. Bioinformatic analyses of these two 5'-untranslated sequences predicted various transcription factors binding sites; further analysis of Nrl promoter elements evidenced the binding of ROR, CREB, CRX and OTX2 for developing and mature retina. Discovery of these trans-acting factors implied in Nrl regulation gave insights into the transcriptional protein complexes at the origin of rod photoreceptor differentiation.

Figure 36 .

 36 photoreceptor expression, and that is notably limited in size for use in Adeno-Associated

Figure 37 .

 37 Figure 37. Nrl minimal and functional promoter Representative sections from neonatal mouse retinas transfected in vivo with different lengths of mouse Nrl promoter/enhancer regions. (a') Conserved clusters combination A (-304 to +119 from the TSS) and B (-938 to -657) display a strong mCherry reporter gene expression specifically in the ONL. (b') Minimal A1 sequence (-34 to +16) in cluster A behaves as a basal promoter for Nrl expression when combined to cluster B. A1+B sequence shows a sustained expression of mCherry in the ONL compared to A1+B2 (B2, -814 to -657) or to A+B4 (B4, -814 to -763) sequences (C' and D' respectively). Ubiquitin-GFP was used to verify the transfection efficiency (a-d). Merged images (a"-d"). Red, mCherry; Green, GFP; blue, DAPI. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar 20 µm.

Figure 38 .

 38 Figure 38. Determination of the working dilution for AAV-Nrlp-EGFP Retinal sections from wild type (C57Bl6) P10 injected animals with AAV-Nrlp-EGFP and AAV-RKp-EGFP. Retinas were harvested 3 weeks post-injection for EGFP analysis. AAV-RKp-EGFP (far right column) was used as positive control. Green, EGFP; blue, DAPI. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar 20 µm

  Real-time quantitative PCR Gene expression over 24 h LD and DD cycles was analysed by real-time PCR for Rho and Nr2e3 in the whole retina, and Nrl and Crx in photoreceptor layers. Real-time quantitative PCR was performed using the hydrolysed probe-based TaqMan chemistry.We used inventoried TaqMan Gene Expression Assays for Rho, Nrl and Crx, designed to specifically amplify mRNA (Applied Biosystems, Table1), and custom designed assays for Nr2e3 based on the predicted RefSeq XM_002727061.1

  and the following expression plasmids: i. pSV-CLOCK and CMV-BMAL1 (Travnickova-Bendova, Cermakian et al. 2002) (0.1 µg each); ii. FLAG-tagged CMV-RORβ (Science Applications International Corporation, Frederick, MD) (0.3 µg); iii.pcDNA4c-RevErbα(Cheng, Khanna et al. 2004) (0.3 µg). Co-transfection mixtures contained also 0.001 μg of the second reporter plasmid, CMV-Renilla (Promega, Madison, WI), and the empty pcDNA4c (Life Technologies,) vector to adjust the total amount of transfected DNA to 1 µg. Cells were harvested 48 h after transfection and lysed in 100 μl of passive lysis buffer (Promega).

  , with x indicating the time (h), a indicating the mean value of the cosine curve (mesor), b indicating the amplitude of the curve (half of the sinusoid) and c indicating the acrophase (h).

Figure 2 Expression

 2 Figure 2

Figure 3

 3 Figure 3 Expression kinetics of Nr2e3 transcript and protein product. A. 24 h expression profile of Nr2e3 transcript in the rat retina under LD condition was obtained by qPCR; B. Daily profiles of NR2E3 protein levels in the retina of rats maintained in LD. Protein levels were analyzed in total extracts and quantified by Western blots. Left panel, representative immunoblots of LD samples pooled per time point (ZT). Respective molecular weights (kDa) are reported Right panel, values are shown as relative amount of protein after normalization with β-actin. Data are expressed as mean ± SEM. Significant temporal variations (P < 0.05) are indicated. (White bar, light period, black bar, dark period). Data for ZT0 are double-plotted at ZT24. Here Nr2e3 transcript levels were normalized with respect to Pde6b only.

Figure 4 Transcriptional

 4 Figure 4 Transcriptional effect of clock factors on Nrl promoter. HEK293T cells were co-transfected with a construct of mNrl (-938 to +119) promoter region linked to firefly luciferase reporter gene and a combination of CLOCK plus BMAL1 expression vectors (0.1 µg each), or with of RevErbα alone (0.3 μg), or RORβ alone (0.3 µg). Fold-changes are relative to the mock expression of pcDNA4c control vector. Data (ratio of firefly to Renilla luciferase activity) are expressed as relative to the mock expression. Each bar represents the mean ± SEM (n = 3). Asterisks indicate P < 0.05.

Figure 1 Schematic

 1 Figure 1 Schematic representation showing upstream regulatory elements in rhodopsin promoter, sequence alignment of RER and scheme of the isolation of bovine retina proteins binding to it . Panel (A) shows the proximal and distal elements in bovine rhodopsin promoter and sequence alignment of distal enhancer region (RER) in different mammalian species. Shaded regions represent the conserved sequences. Panel (B) shows the flow chart of the isolation scheme for the control and RER oligonucleotide-binding proteins from the bovine retinal nuclear extract. Panel (C) silver stained SDS PAGE gel showing the different eluted proteins bound to rhodopsin RER and control oligonucleotide from bovine retina. Lanes: 1-Input nuclear fraction, 2-RER-and 3-Control oligonucleotide eluted proteins. Asterisks indicate the protein bands used for mass spectra analysis. Panel (D) shows the western immunoblot with NonO antibody of the RER and control oligonucleotide eluted fractions. Arrow indicates the NonO band.

  A plasmid containingCMV promoter-driving renilla luciferase was used as control. HEK 293 cells were used to examine promoter activity. Transfection of NonO alone in HEK 293 cells resulted in minimal dosage-dependent increase of rhodopsin promoter-luciferase reporter activity.Either NRL or CRX alone transactivated the Rhodopsin promoter two to three times more than NonO. However, co-transfection of NonO with NRL and CRX resulted in ~ 80-fold increase over background in rhodopsin-luciferase reporter activity. In contrast, only minimal effect was observed when the luciferase reporter driven by the 130 bp bovine rhodopsin promoter lacking RER, was co-transfected with NonO, NRL and CRX (Figure2B). This suggests that NonO specifically acts on RER and enhances the transactivation activities of NRL and CRX on rhodopsin promoter. To test whether bovine NonO interacts directly with NRL or CRX, we performed immunoprecipitation from bovine retinal extract with anti-NonO, anti-NRL, anti-CRX anti-bodies. Immunoprecipitation with anti-RNA Polymerase II (Pol II) antibody was used as positive control and anti-Brn3b was used as negative control. Blots were immunoreacted with anti-NonO antibody. Upon crosslinking, NonO was immunoprecipitated from bovine retinal extract using NRL, CRX and

Figure 2 (

 2 Figure 2 (A) Luciferase reporter assay showing transactivation of the 2.2 kb bovine rhodopsin promoter by NonO. HEK293 cells were co-transfected with 2.2 kb bovine rhodopsin promoter driving firefly luciferase reporter (0.2 µg) and increasing concentrations (0.1-0.6 μg) of NonO alone or in association with 0.1 μg of either NRL or CRX or both. Fold change is relative to the mock expression vector control. All the experiments were done in triplicate. T test was performed using Prism software version 5 and P values <0.05 were considered significant. P values <0.05 to 0.01 were given two asterisks and <0.01 were given three asterisks . (B) HEK293 cells were co-transfected with 130 bp bovine rhodopsin promoter driving firefly luciferase reporter (0.2 µg) and increasing concentrations (0.1-0.6 μg) of NonO alone or in association with, 0.1 μg of NRL and CRX together. Fold change is relative to the mock expression vector control. All the experiments were done in triplicate. T test was performed using Prism software version 5 and P values <0.05 were considered significant.

Figure 3 NonO

 3 Figure 3 NonO is required for rod photoreceptor survival. (A) Neonatal mouse retinas were coelectroporated with 2.2 kb bovine rhodopsin promoter driving DsRed (bRhoDsRed) and either control or NonO-shRNA constructs. Fluorescent reporter gene expression was analyzed at P10 and P21. Cells expressing either control or NonO-shRNA are green, cells expressing bovine rhodopsin promoterdriven DsRed are Red. Nuclei were stained with DAPI. Left panel (a and c) represents control shRNA and right panel (b and d) represents NonO-shRNA electroporated retina at P0 and harvested at P10 and P21. Green GFP, red 2.2 kb rhodopsin promoter driven DsRed. White Arrow heads indicate the GFP and DsRed double positive cells. White line indicates the 20 µm length of the section. (B) Depletion of NonO by shRNA leads to rod cell death by apoptosis. Neonatal mouse retinas were electroporated with either control or NonO-shRNA constructs. Caspase-3 staining was performed at P7 to find out the rod cell death in NonO-shRNA electroporated retina as compared to control. (e) NonO-shRNA, (f) Control shRNA. Green GFP, Red caspase-3 and nuclei were stained with DAPI.

Figure 4 Schematic

 4 Figure 4Schematic representation of the RPPR and RER bound transcriptional complexes on rhodopsin promoter. A schematic representation of rhodopsin promoter showing the RPPR and RER with bound known representative proteins. Location and binding to rhodopsin promoter is shown for different proteins. Based on our data and previous studies we propose a model in which RER bound transcriptional complexes containing NonO along with other proteins identified in this study loops to RPPR to further enhance the recruitment of the general transcription machinery and RNA Pol II to the rhodopsin promoter in order to regulate its quantitative precise expression in rod photoreceptors.

  B sequence contains numerous putative binding elements and by performing gel shift experiments we indeed showed specific binding of ROR, CRX, OTX2 and CREB. In addition, site directed mutagenesis against ROR response element abolished expression of the reporter gene upon in vivo electroporation of mouse pup retinas, demonstrating the relevance of ROR in Nrl regulation. These data support the previous work of[START_REF] Jia | Retinoid-related orphan nuclear receptor RORbeta is an early-acting factor in rod photoreceptor development[END_REF] showing the lack of Nrl expression in Rorb -/-animals. These data, together with the transactivation of Nrl promoter-reporter construct by RORβ (Results, chapter III) bring further insight into the transcriptional networks leading to rod development and maturation, by showing that in the sequential intervention of specific transcription factors leading to retinogenesis, Nrl probably lies downstream of RORβ. However, since OTX2 and CRX also interact with Nrl promoter, we can hypothesize that these factors (and likely others) which are expressed earlier in photoreceptor development, also contribute to activate Nrl expression at the right place and moment.To further characterize the conserved Nrl promoter elements, we used different cluster combinations and tested them by in vivo electroporation; we dissected out clusters B and A to reveal regions within these fragments essential for promoter activation in the ONL. We identified a minimal Nrl promoter sequence constituted of cluster B and a sub-part of cluster A (-34 to +16) that we called basal promoter A1. A1 roughly contains the TATA-like box sequence and a GC rich potential binding site for Sp1 factor that probably contributes to basal constitutive expression[START_REF] Tan | Sp1 phosphorylation and its regulation of gene transcription[END_REF]. Combination of B and A1 yielded relatively strong reporter expression in the ONL by in vivo retinal transfection. Based on the main transcription factor binding region defined by gel shift experiments within cluster B, we identified the 51 bp B4 sequence as a potential crucial regulator of Nrl expression. Indeed, when this sequence was combined with rhodopsin promoter in retinal in vivo electroporation experiments,

  photoreceptors[START_REF] Tosini | Localization of a circadian clock in mammalian photoreceptors[END_REF] Sandu, Hicks et al. 2011) and this

  Real-time quantitative PCR ............................................................................................... F. Cell culture ................................................................................................................................ Western blot ............................................................................................................................. I. AAV production and purification. .............................................................................................. 1. Vector design ......................................................................................................................... 2. rAAV Vector production. ....................................................................................................... 3. rAAV vector purification ........................................................................................................ 4. Titration of viral particles. ..................................................................................................... 5. Animals and vector delivery .................................................................................................. 6. Immunofluorescence analysis ...............................................................................................

	TABLE DES MATIÈRES
	4.2

G. Luciferase assays....................................................................................................................... H. RESULTS ................................................................................................................................................. I. Characterization of mouse Nrl promoter regulatory sequences. ................................................. II. Identification of a minimal Nrl promoter as a tool for specific photoreceptor expression using AAV 83 A. Further dissection of Nrl promoter elements required for the transcription ........................... B. AAV-mediated expression of Nrl promoter or cDNA to further decipher the developmentall properties of Nrl gene .......................................................................................................................

Table 1 : List of antibodies used for immunostaining

 1

	Antigen	Host	Dilution	References
	Primary Antibody
	Cone arrestin	Rabbit	1 :2000	Millipore
	Rhodopsin	Mouse	1:500	(Hicks and Molday 1986)
	Secondary Antibody
	Goat anti mouse-Alexa 568	Goat	1 :1000	Invitrogen
	Goat anti rabbit-Alexa 568	Goat	1 :1000	Invitrogen

a Bacterial Artificial Chromosome containing Nrl gene that was used as template and mixed with high fidelity DNA polymerase (Pfu High Fidelity and NEB Phusion) and

Table 2 : List of antibodies used for supershift assays

 2

	Antigen	Host	Type	References
	Primary Antibody
	Otx2		Polyclonal	Chemicon
	Crx		Polyclonal	(Cheng, Khanna et al. 2004)
	Ror		Polyclonal	Diagenode
	Creb		Monoclonal	Cell signaling
	Nrl		Polyclonal	(Roger, Nellissery et al. 2010)
	c-Fos		Polyclonal	Calbiochem
	Normal Immunoglobulin G		Polyclonal	Rockland

Table 4 : Composition of buffers used for Western blot analysis

 4

	Composition

Table 5 : List of antibodies used for Western blot analysis

 5

	Antigen	Host	Dilution	References
	Primary Antibody
				(Roger, Nellissery et al.
	Nrl	Rabbit	1:5000	2010)
	Crx	Rabbit	1:2000	(La Spada, Fu et al. 2001)
	Nr2e3	Rabbit	1:3000	(Peng, Ahmad et al. 2005)
	-Actin	Mouse	1:40 000	Sigma
	-Tubulin	Mouse	1:10 000	Abcam

  CV) among duplicates was 0.18% on average. A dilution curve of the pool of all cDNA samples was used to calculate the amplification efficiency for each assay (values were between 1.8 and 2 for all assays). No-template control (NTC) reactions were performed as negative controls for each assay. One 96-well plate corresponded to the analysis of one gene within one type of samples (retina or photoreceptors) and one experimental condition (LD or DD). Transcript levels were normalized using -actin and

		of variation (Pde6b.
		Western blot analysis
		Whole retinas, sampled at ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20, were dissociated in
		extraction buffer (20 mM Tris pH 7.6, 150 mM NaCl, 1.5% Triton-X-100, 1 mM EDTA,
	Gene 0.2% SDS supplemented with protease inhibitors (Roche Applied Science, Germany), TaqMan assay RefSeq Exon Assay Amplicon
		reference phosphatase inhibitors (Sigma, L'Isle d'Abeau Chesnes, France) and 1 mM DTT) using a boundary location length (bp)
	Rhodopsin sonicator (Vibra-Cell 75186, Sonics & Materials, Newtown, CT, USA) for 15 s; 30/10 pulse Rn00583728_m1 NM_033441.1 1-2 446 73
	Nrl	Rn01481925_m1 and 50% amplitude. Lysates were centrifuged at 13,000 rpm (4°C, 20 min ) and XM_224189.2 4-5 512 103
	Crx	Rn00573116_m1 supernatants kept at -80°C until further use. Concentrations of total soluble proteins in NM_021855.1 1-2 99 64
		the extracts were determined using the Bradford method
		, PCR conditions were: 1 x TaqMan Universal
		PCR Master Mix, No AMPErase UNG (Applied Biosystems), 1 x Gene Expression Assay mix
		(containing forward and reverse primers and cognate probe; Applied Biosystems) and 1
		µl of cDNA in a total volume of 20 µl. The PCR program was as follows: 10 minutes at
		95ºC, followed by 40 cycles of denaturation at 95ºC for 15 seconds and annealing-
		elongation at 60ºC for 1 minute. The acquisition of fluorescence data was performed at
		the end of the elongation step using the 7300 System Sequence Detection Software V
		1.3.1 (Applied Biosystems). Each PCR reaction was done in duplicate and the coefficient

Table 3

 3

	, cosinor

ACKNOWLEDGEMENTS

Je remercie grandement les docteurs Muriel Perron, Pierre Voisin et Hélène Dollfus d'avoir accepté d'évaluer mon travail de thèse. I would like to thanks Dr David Klein and Dr Douglas Forrest for giving me precious advices for my thesis project as members of my thesis advisory committee. Mes remerciements s'adressent à mes directeurs de thèse, Dr Marie-Paule Felder-Schmittbuhl et Dr Anand Swaroop.

Acknowledgements

We are grateful to Garry G. Borisy for the generous gift of shRNA vector pG-Super, Norimoto Gotoh and Linn Gieser for rod photoreceptor cDNAs, and Tiziana Cogliati for assistance in preparing the manuscript. We acknowledge Jerome Roger, Shobi Veleri, James Friedman and other members of N-NRL for advice and discussions. This work was supported by intramural research program of the National Eye Institute.

According to these findings therapeutic strategies aiming at suppressing and/or replacing NRL mutant gene, appear valuable in the case of Retinitis Pigmentosa due to NRL mutations, as it has been done previously with two genes involved in this disease, Rds-peripherin and Rhodopsin [START_REF] Palfi | RNAi-based suppression and replacement of rds-peripherin in retinal organotypic culture[END_REF][START_REF] O'reilly | RNA interference-mediated suppression and replacement of human rhodopsin in vivo[END_REF]).

NRL is a key factor for rod-photoreceptor development

During retinogenesis, NRL plays a pivotal role in the determination of a precursor cell to become a rod. Deletion of Nrl in mice results in the complete loss of rods, which are converted into S cones [START_REF] Mears | Nrl is required for rod photoreceptor development[END_REF]) (Figure 27). In Nrl -/-animals, S cone proliferation is not only explained by NRL loss but also by the failure to express NR2E3. Indeed, it was shown that NRL induces the expression of NR2E3; therefore NR2E3, which main function is to inhibit expression of cones gene, is defective in Nrl-null mice, resulting in the cone-only-retina observed in these animals [START_REF] Oh | Rod differentiation factor NRL activates the expression of nuclear receptor NR2E3 to suppress the development of cone photoreceptors[END_REF].

These data suggest that NR2E3 is downstream of NRL in the transcriptional hierarchy controlling retinal development. Transgenic expression of Nr2e3 in Nrl-/-animals results in the complete suppression of cone differentiation and development of rod-like cells, which are, however, non-functional (Cheng, Aleman et al. 2006). Per contra, ectopic expression of Nrl in the null background induces transformation of all photoreceptors into functional rods as attested by the scotopic ERG showing a profile similar to the WT [START_REF] Oh | Transformation of cone precursors to functional rod photoreceptors by bZIP transcription factor NRL[END_REF]). Therefore, these data suggest that contrary to NR2E3, NRL is not only essential, but is also sufficient for rod genesis. Induction of rod-specific gene expression relies on the action of multiple transcription factors. Interaction of NRL with several other transcription factors confers cell specificity in activating target genes. NRL largely contributes to the rod lineage specification and to the homeostasis of these cells by regulating genes involved in phototransduction on one hand and in cell maintenance on the other hand, such as Lman1 and Wisp1 [START_REF] Hao | Transcriptional Regulation of Rod Photoreceptor Homeostasis Revealed by In Vivo NRL Targetome Analysis[END_REF]. Therefore control of Nrl expression should be tightly regulated to generate a mature and functional retina.

Abstract

Rhythmic physiology is central to visual function and allows adaptation of the retinal response to the day/night cycle. Several photoreceptor-specific processes such as melatonin synthesis and shedding of rod outer segments are controlled by a circadian clock located within the retina and self-entraining to the light/dark cycle. Rat photoreceptors harbour a functional clock machinery but the mechanisms by which it regulates their rhythmic functions has been poorly characterized. In the present study we confirm that rhodopsin transcript displays daily rhythmicity and show that its direct regulatory transcription factors, NRL, CRX and NR2E3 are also regulated on a daily basis.

All studied mRNAs display maximal expression around the transition between night and day but don't show any significant rhythmicity in constant darkness. We provide evidence that transcription of Nrl is regulated by REVERBα and RORβ clock factors and suggest a novel link between the circadian clock and its outputs.

Results

Rhodopsin gene expression in whole rat retina

Profiles of rhodopsin were analysed over 24 h in whole rat retinas under LD and DD conditions. In LD (n = 3-5 per time point, total n = 24) a statistically significant temporal variation was found for rhodopsin transcript by one-way ANOVA analysis (Figure 1) (rhodopsin F 5,18 = 8.9419, P = 0.0002). Cosinor analysis confirmed a daily rhythmic pattern for rhodopsin (P < 0.01, Table 2), with a peak at ZT23.

In DD conditions (n = 3-6 per time point, total n= 32), rhodopsin transcript (Figure 1) did not show any significant temporal variation (Table 2).

Supplemental data

Figure S1

Expression profiles of recoverin transcript in isolated photoreceptors under LD and DD conditions during 24 h analysed by qPCR (inventoried TaqMan assays Rn00590194_m1, Applied Biosystems) (white bar, light period; black bars, dark period). Significant temporal variation (P < 0.05) is indicated: LD, F 5,24 = 5.08, P = 0.003; DD, F 5,27 = 2.41, P = 0.06. Rhythmicity in LD conditions was confirmed by cosinor analysis (mesor, 2.05  0.1; amplitude 0.62  0.14; acrophase 21.18  0.87 h, F 2.,27 =9.6181, P = 0.0007) and the tendency to be rhythmic in DD conditions was shown (mesor, 1.98  0.08; amplitude 0.31  0.12; acrophase 21.09  0.87 h, F 2.,30 =3.2393, P = 0.0532). Data for ZT0 are double-plotted at ZT24. Values represent mean ± SEM.

BIOLOGICAL SCIENCES Biochemistry

The

Manuscript in preparation

Abstract

Rhodopsin is the visual pigment in outer segments of rod photoreceptors that constitute around 70% of cells in the retina of most mammals. The daily renewal of almost 10% of outer segment discs requires a stringent homeostatic control to maintain high levels of rhodopsin expression. The expression of rhodopsin is controlled by two cis-regulatory sequences upstream of the transcription start site -a proximal promoter region (RPPR)

and an enhancer region (RER). RPPR contains binding sites for the two key transcription regulatory factors -neural retina leucine zipper (NRL) and cone rod homeobox (CRX).

However, a larger genomic region, including RER, is essential for precise high-level rhodopsin expression in vivo. Here, we report the identification of RER binding proteins from bovine retinal nuclear extract by mass-spectrometric analysis. We detected the largest number of unique peptides for the non-POU domain containing octamer-binding protein (NonO/p54 nrb ). We show that NonO activates the expression from a rhodopsin

Discussion

Regulation of transcription is mainly accomplished through activity of gene promoter and enhancer regions. Although proximal regions of promoters are sufficient for minimal expression of the majority of the genes,those genes which are highly transcribed need additional regulation, which is largely accomplished by enhancers. Enhancers contain multiple binding sites for a variety of transcription factors and act independently of their location, distance or orientation with respect to the gene [START_REF] Banerji | Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences[END_REF].

In some cases, they can even activate transcription of genes located in a different chromosome [START_REF] Geyer | Tissue-specific transcriptional enhancers may act in trans on the gene located in the homologous chromosome: the molecular basis of transvection in Drosophila[END_REF][START_REF] Lomvardas | Interchromosomal interactions and olfactory receptor choice[END_REF]. Clusters of DNA sequences in enhancers can bind combinations of transcription factors and then interact with components of the mediator complex or transcription factor II D (TFIID) to help recruit RNA polymerase II (RNAPII) [START_REF] Maston | Transcriptional regulatory elements in the human genome[END_REF]) by looping out the intervening sequences on gene promoters [START_REF] Schoenfelder | The transcriptional interactome: gene expression in 3D[END_REF]. Non-coding intergenic sequences and most likely enhancers contain the majority of the genetic variations associated with complex human diseases.

Rhodopsin is the major structural protein present in the ROS and required for the outer segment integrity and phototransduction. Mutations in its coding region are associated with rod photoreceptor degeneration. Information about the genetic variations in rhodopsin regulatory region is not well characterized but there are evidences that mutations in the proteins binding to RPPR region cause underexpression of rhodopsin, which ultimately leads to death of rod cells by apoptosis. Indeed rhodopsin null mice have no ROS and undergo degeneration after a month. Due to daily circadian-clock driven shedding of ROS discs a high level of rhodopsin expression is required to maintain the rod photoreceptor homeostasis.

Rhodopsin promoter contains two cis-regulatory elements: a RPPR which binds the transcription factors NRL, CRX, NR2E3 and Fiz-1 and recruits RNA Pol II, gives basal level of expression and a second RER region located ~2 kb upstream from the TSS that is required for restricted expression in rods and is bound and regulated by yet unknown factors. Here we show that several cis-regulatory proteins bind to distal enhancer region and may be required for quantitative and precise expression of rhodopsin in rod cells.

GENERAL DISCUSSION

GENERAL DICUSSION

ABSTRACT IN ENGLISH

PROMOTER DISSECTION OF THE ROD-SPECIFIC NRL TRANSCRIPTION FACTOR: DEVELOPMENTAL AND DAILY REGULATION

The vertebrate retina consists of seven major cell types generated from multipotent progenitors by a hierarchical and stepwise process controlled by both extrinsic factors and intrinsic genetic programs. Photoreceptors (PRs) account for up to 60% of all cells in the adult neural retina and contain generally a majority of rods responsible for night vision. PRs are highly metabolically active cells undergoing intensive outer segments renewal on a daily rhythmic basis. Mutations in rod photoreceptor-genes are linked to retinal dystrophy such as Retinitis Pigmentosa (RP), leading ultimately to blindness. Cell transplantation and gene therapy present feasible strategies to restore visual function; given the central role played by rods in these pathologies, it is meaningful to understand molecular mechanisms controlling their development and maintenance.

The Neural Retina Leucine zipper transcription factor (Nrl) plays a central role in rod photoreceptor development and homeostasis. Nrl, combined with other photoreceptor-specific transcription factors activates expression of rod-specific genes such as the visual photopigment, Rhodopsin. Moreover, Nrl is both essential and sufficient for rod cell fate specification as shown by using transgenesis. Finally, mutations in Nrl have been associated with RP. Thus, Nrl gene is an interesting model for understanding genetic programs controlling PRs development and homeostasis.

This thesis work aimed at characterizing regulatory mechanisms of Nrl expression. It mainly focused on transcriptional control during retinal development. By using in vivo electroporation of reporter vectors carrying distinct portions of Nrl promoter into neonatal mouse retina, we identified the minimal sequences necessary to drive reporter gene expression specifically in PR layer. We identified ROR as being required for this expression and showed that OTX2, CRX and CREB transcription factors also directly bind to the defined regulatory regions.

Based on these results 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 PRs.

Nrl promoter analysis also proved to be useful to get new insights into the mechanisms controlling daily physiological rhythms in PRs. We showed that NRL, CRX, and NR2E3, the main transcriptional regulators of Rhodopsin gene, display rhythmic expression over 24 h. and that Nrl might undergo cyclic activation by RORwhich is part of the PR circadian clock. Finally, we investigated the role of a novel Rhodopsin transcriptional regulator, NonO, identified in the Rhodopsin 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. Key words: Retina, photoreceptors, Neural retina leucine zipper, retinogenesis, transcription, gene regulation, circadian clock, Rhodopsin