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Daily biological rhythms are fundamental to retinal physiology and visual function. They are generated by a local circadian clock composed of a network of cell type/layer-specific, coupled oscillators. Animal models of retinal degeneration have been instrumental in characterizing the anatomical organization of the retinal clock. However, it is still unclear, among the multiple cell-types composing the retina, which ones are essential for proper circadian function. In this study, we used a previously well-characterized mouse model for autosomal dominant retinitis pigmentosa to examine the relationship between rod degeneration and the retinal circadian clock. This model carries the P23H mutation in rhodopsin, which induces mild rod degeneration in heterozygous and rapid loss of photoreceptors in homozygous genotypes. By measuring PER2::LUC bioluminescence rhythms, we show that the retinal clock in P23H/+ heterozygous mice displays circadian rhythms with significantly increased robustness and amplitude. By treating retinal explants with L-α aminoadipic acid, we further provide evidence that this enhanced rhythmicity might involve activation of Müller glial cells.

Light can restrict the activity of an animal to a diurnal or nocturnal niche by synchronizing its endogenous clock (entrainment) which controls the sleep wake cycle. Light can also directly change an animal's activity level (masking). In mice, high illumination levels decrease activity, i.e. negative masking occurs. To investigate the role of core circadian clock genesiPer1/iandiPer2/iin masking, we used a 5-day behavioral masking protocol consisting of 3 h pulses of light given in the night at various illuminances (4-5 lux, 20 lux and 200 lux). Mice lacking theiPer1/igene had decreased locomotion in the presence of a light pulse compared to wild-type,iPer2/iandiPer1 Per2/idouble mutant mice.iPer2/isingle mutant andiPer1 Per2/idouble mutant mice did not show significantly different masking responses compared to wild-type controls. This suggests thatiPer1/isuppresses negative masking responses in mice.

The retinal circadian system consists of a network of clocks located virtually in every retinal cell-type. Although it is established that the circadian clock regulates many rhythmic processes in the retina, the links between retinal cell-specific clocks and visual function remain to be elucidated. Bmal1 is a principal, non-redundant component of the circadian clock in mammals and is required to keep 24 h rhythms in the retinal transcriptome and in visual processing under photopic light condition. In the current study, we investigated the retinal function in mice with a rod-specific knockout of Bmal1. For this purpose, we measured whole retina PER2::Luciferase bioluminescence and the dark-adapted electroretinogram (ERG). We observed circadian day-night differences in ERG a- and b-waves in control mice carrying one allele of Bmal1 in rods, with higher amplitudes during the subjective night. These differences were abolished in rod-specific Bmal1 knockout mice, whose ERG light-responses remained constitutively low (day-like). Overall, PER2::Luciferase rhythmicity in whole retinas was not defective in these mice but was characterized by longer period and higher rhythmic power compared to retinas with wild type Bmal1 gene. Taken together, these data suggest that a circadian clock located in rods regulates visual processing in a cell autonomous manner.

Retinal photoreceptors undergo daily renewal of their distal outer segments, a process indispensable for maintaining retinal health. Photoreceptor outer segment (POS) phagocytosis occurs as a daily peak, roughly about 1 hour after light onset. However, the underlying cellular and molecular mechanisms which initiate this process are still unknown. Here we show that, under constant darkness, mice deficient for core circadian clock genes (Per1 and Per2) lack a daily peak in POS phagocytosis. By qPCR analysis, we found that core clock genes were rhythmic over 24 hours in both WT and Per1, Per2 double mutant whole retinas. More precise transcriptomics analysis of laser capture microdissected WT photoreceptors revealed no differentially expressed genes between time points preceding and during the peak of POS phagocytosis. In contrast, we found that microdissected WT retinal pigment epithelium (RPE) had a number of genes that were differentially expressed at the peak phagocytic time point compared to adjacent ones. We also found a number of differentially expressed genes in Per1, Per2 double mutant RPE compared to WT ones at the peak phagocytic time point. Finally, based on STRING analysis, we found a group of interacting genes that potentially drive POS phagocytosis in the RPE. This potential pathway consists of genes such as: Pacsin1, Syp, Camk2b, and Camk2d among others. Our findings indicate that Per1 and Per2 are necessary clock components for driving POS phagocytosis and suggest that this process is transcriptionally driven by the RPE.

Retinal photoreceptors undergo daily renewal of their distal outer segments, a process indispensable for maintaining retinal health. Photoreceptor Outer Segment (POS) phagocytosis occurs as a daily peak, roughly about one hour after light onset. However, the underlying cellular and molecular mechanisms which initiate this process are still unknown. Here we show that, under constant darkness, mice deficient for core circadian clock genes (Per1 and Per2), lack a daily peak in POS phagocytosis. By qPCR analysis we found that core clock genes were rhythmic over 24h in both WT and Per1, Per2 double mutant whole retinas. More precise transcriptomics analysis of laser capture microdissected WT photoreceptors revealed no differentially, expressed genes between time-points preceding and during the peak of POS phagocytosis. By contrast, we found that microdissected WT retinal pigment epithelium (RPE) had a number of genes that were differentially expressed at the peak phagocytic time-point compared to adjacent ones. We also found a number of differentially expressed genes in Per1, Per2 double mutant RPE compared to WT ones at the peak phagocytic time-point. Finally, based on STRING analysis we found a group of interacting genes which potentially drive POS phagocytosis in the RPE. This potential pathway consists of genes such as: Pacsin1, Syp, Camk2b and Camk2d among others. Our findings indicate that Per1 and Per2 are necessary clock components for driving POS phagocytosis and suggest that this process is transcriptionally driven by the RPE.DeclarationsFundingThis project has been funded with support from the NeuroTime Erasmus+ grant (European Union), Rotterdamse Stichting Blindenbelangen (Netherlands), Nelly Reef fund (Netherlands), Stichting voor Ooglijders (Netherlands), Stichting tot Verbetering van het Lot der Blinden (Netherlands) and Retina France (France).Conflicts of interest/Competing interestsThe authors declare no competing interests.Availability of data and materialData supporting the conclusions of this article are included within the article and are available from the corresponding authors on reasonable request.Code availabilityThe R code for analysis is available from the corresponding authors on reasonable request.Ethics approvalAll experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement on Use of Animals in Ophthalmic and Vision Research, as well as with the European Union Directive (2010/63/EU).Consent to participateNot applicableConsent for publicationAll authors read and approve of the contents of this manuscript.Author contributionsN.M. performed experiments, analysis, prepared figures, wrote the manuscript and obtained funding. O.A.-H.H. performed experiments, data analysis, prepared figures and obtained funding. P.D.M. and A.J. performed bioinformatics analysis and edited the manuscript. U.B., J.B.t.B. and C.S. provided technical assistance, performed experiments, prepared figures and edited the manuscript. D.H., A.A.B. and M.-P.F.-S. conceptualized and directed the project, obtained funding, provided resources, performed analysis and edited the manuscript.

Multiple circadian clocks dynamically regulate mammalian physiology. In retina, rhythmic gene expression serves to align vision and tissue homeostasis with daily light changes. Photic input is relayed to the brain to entrain the master circadian clock, the suprachiasmatic nucleus, which matches behaviour to environmental changes. Circadian organization of the mouse retina involves coordinated, layer-specific oscillators, but so far little is known about the cone photoreceptor clock and its role in the circadian system. Using the cone-only Nrl-/- mouse model we show that cones contain a functional self-sustained molecular clockwork. By bioluminescence-combined imaging, we also show that cones provide substantial input to the retinal clock network. Furthermore, we found that light entrainment and negative masking in cone-only mice are subtly altered and that constant light displayed profound effects on their central clock. Thus, our study demonstrates the contribution of cones to retinal circadian organisation and their role in finely tuning behaviour to environmental conditions.

Multiple retinal cells harbor a circadian oscillator, including retinal pigment epithelial cells (RPE). However, little is known about the functions that are regulated by the RPE clock. The aim of this study was to investigate whether the circadian clock in the RPE regulates the transport of glucose and its glycolytic metabolic by-product - lactate. To that end, we first characterized the mRNA expression profile of glucose and monocarboxylate transporters in ARPE-19 cells. We found that SLC2A1 and SLC16A1 were, respectively, the most abundantly expressed glucose and lactate (monocarboxylate) transporters. We further observed that the protein products of SLC2A1 (encoding GLUT1) and SLC16A1 (encoding MCT1) localize on the apical membrane of ARPE-19 monolayers. In a subsequent time-course experiment, we found that SLC2A1 and SLC16A1 mRNA oscillated in ARPE-19 monolayers, but not in dispersed cells, suggesting that monolayer cellular organization is necessary for rhythmic regulation of these transporters. In these monolayers, we found that MCT1 proteins varied over time, in contrast to GLUT1 proteins which did not vary over time. Spectrophotometric measurements of supernatants sampled from ARPE-19 monolayer cultures revealed that glucose concentrations did not significantly differ between apical (Api) supernatants and basolateral (BL) ones. In addition, we did not find rhythms in Api or BL glucose concentrations. Conversely, we found higher lactate concentrations in Api supernatants than BL ones. Further, we found that Api lactate concentrations were rhythmic. Pearson's r revealed that the concentration gradients (Api - BL) of glucose and lactate correlated with the gene expression of respective SLC2A1 and SLC16A1 transporters. Incubation with photoreceptor outer segments (POS) affected the mRNA expression of SLC16A1 and SLC2A1 in ARPE-19 monolayers in a time-dependent manner, thus suggesting that the retina might modulate the RPE clock-controlled expression of transporters via interactions with POS. In conclusion, this work provides evidence that the transport of lactate is regulated by the circadian clock in the RPE.

The retinas from Period 1 (Per1) and Period 2 (Per2) double-mutant mice (Per1−/− Per2Brdm1) display abnormal blue-cone distribution associated with a reduction in cone opsin mRNA and protein levels, up to 1 year of age. To reveal the molecular mechanisms by which Per1 and Per2 control retina development, we analyzed genome-wide gene expression differences between wild-type (WT) and Per1−/− Per2Brdm1 mice across ocular developmental stages (E15, E18 and P3). All clock genes displayed changes in transcript levels along with normal eye development. RNA-Seq data show major gene expression changes between WT and mutant eyes, with the number of differentially expressed genes (DEG) increasing with developmental age. Functional annotation of the genes showed that the most significant changes in expression levels in mutant mice involve molecular pathways relating to circadian rhythm signaling at E15 and E18. At P3, the visual cascade and the cell cycle were respectively higher and lower expressed compared to WT eyes. Overall, our study provides new insights into signaling pathways -phototransduction and cell cycle- controlled by the circadian clock in the eye during development.

For many years, the suprachiasmatic nucleus (SCN) was considered as the unique circadian pacemaker in the mammalian brain. Currently, it is known that other brain areas are able to oscillate in a circadian manner. However, many of them are dependent on, or synchronized by, the SCN. The Habenula (Hb), localized in the epithalamus, is a key nucleus for the regulation of monoamine activity (dopamine, serotonin) and presents circadian features; nonetheless, the clock properties of the Hb are not fully described. Here, we report, first, circadian expression of clock genes in the lateral habenula (LHb) under constant darkness (DD) condition in wild-type mice which is disturbed in double Per1−/−-Per2Brdm1 clock-mutant mice. Second, using Per2::luciferase transgenic mice, we observed a self-sustained oscillatory ability (PER2::LUCIFERASE bioluminescence rhythmicity) in the rostral and caudal part of the Hb of arrhythmic SCN-ablated animals. Finally, in Per2::luciferase mice exposed to different lighting conditions (light-dark, constant darkness or constant light), the period or amplitude of PER2 oscillations, in both the rostral and caudal Hb, were similar. However, under DD condition or from SCN-lesioned mice, these two Hb regions were out of phase, suggesting an uncoupling of two putative Hb oscillators. Altogether, these results suggest that an autonomous clock in the rostral and caudal part of the Hb requires integrity of circadian genes to tick, and light information or SCN innervation to keep synchrony. The relevance of the Hb timing might reside in the regulation of circadian functions linked to motivational (reward) and emotional (mood) processes.

Vision is a highly rhythmic function adapted to the extensive changes in light intensity occurring over the 24-hour day. This adaptation relies on rhythms in cellular and molecular processes, which are orchestrated by a network of circadian clocks located within the retina and in the eye, synchronized to the day/night cycle and which, together, fine-tune detection and processing of light information over the 24-hour period and ensure retinal homeostasis. Systematic or high throughput studies revealed a series of genes rhythmically expressed in the retina, pointing at specific functions or pathways under circadian control. Conversely, knockout studies demonstrated that the circadian clock regulates retinal processing of light information. In addition, recent data revealed that it also plays a role in development as well as in aging of the retina. Regarding synchronization by the light/dark cycle, the retina displays the unique property of bringing together light sensitivity, clock machinery, and a wide range of rhythmic outputs. Melatonin and dopamine play a particular role in this system, being both outputs and inputs for clocks. The retinal cellular complexity suggests that mechanisms of regulation by light are diverse and intricate. In the context of the whole eye, the retina looks like a major determinant of phase resetting for other tissues such as the retinal pigmented epithelium or cornea. Understanding the pathways linking the cell-specific molecular machineries to their cognate outputs will be one of the major challenges for the future.

International audience; The cerebellum contains a circadian clock, generating internal temporal signals. The daily oscillations of cerebellar proteins were investigated in mice using a large-scale two-dimensional difference in gel electrophoresis (2D-DIGE). Analysis of 2D-DIGE gels highlighted the rhythmic variation in the intensity of 27/588 protein spots (5%) over 24 h based on cosinor regression. Notably, the rhythmic expression of most abundant cerebellar proteins was clustered in two main phases (i.e., midday and midnight), leading to bimodal distribution. Only six proteins identified here to be rhythmic in the cerebellum are also known to oscillate in the suprachiasmatic nuclei, including two proteins involved in the synapse activity (Synapsin 2 [SYN2] and vesicle-fusing ATPase [NSF]), two others participating in carbohydrate metabolism (triosephosphate isomerase (TPI1] and alpha-enolase [ENO1]), Glutamine synthetase (GLUL), as well as Tubulin alpha (TUBA4A). Most oscillating cerebellar proteins were not previously identified in circadian proteomic analyses of any tissue. Strikingly, the daily accumulation of mitochondrial proteins was clustered to the mid-resting phase, as previously observed for distinct mitochondrial proteins in the liver. Moreover, a number of rhythmic proteins, such as SYN2, NSF and TPI1, were associated with non-rhythmic mRNAs, indicating widespread post-transcriptional control in cerebellar oscillations. Thus, this study highlights extensive rhythmic aspects of the cerebellar proteome.

Retinal photoreceptor outer segments (POS) are renewed daily through phagocytosis by the adjacent retinal pigment epithelial (RPE) monolayer. Phagocytosis is mainly driven by the RPE circadian clock but the underlying molecular mechanisms remain elusive. Using ARPE-19 (human RPE cell-line) dispersed and monolayer cell cultures, we investigated the influence of cellular organization on the RPE clock and phagocytosis genes. PCR analysis revealed rhythmic expression of clock and phagocytosis genes in all ARPE-19 cultures. Monolayers had a tendency for higher amplitudes of clock gene oscillations. In all conditions ARNTL, CRY1, PER1-2, REV-ERBα, ITGB5, LAMP1 and PROS1 were rhythmically expressed with REV-ERBα being among the clock genes whose expression showed most robust rhythms in ARPE-19 cells. Using RPE-choroid explant preparations of the mPer2Luc knock-in mice we found that Rev-Erbα deficiency induced significantly longer periods and earlier phases of PER2-bioluminescence oscillations. Furthermore, early phagocytosis factors β5-Integrin and FAK and the lysosomal marker LAMP1 protein levels are rhythmic. Finally, POS incubation affects clock and clock-controlled phagocytosis gene expression in RPE monolayers in a time-dependent manner suggesting that POS can reset the RPE clock. These results shed some light on the complex interplay between POS, the RPE clock and clock-controlled phagocytosis machinery which is modulated by Rev-Erbα.

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the main circadian clock, synchronized by the light-dark cycle, which generates behavioral rhythms like feeding, drinking and activity. Notwithstanding, the main role of the SCN clock on the control of all circadian rhythms has been questioned due to the presence of clock activity in many brain areas, including those implicated in the regulation of feeding and reward. Moreover, whether circadian rhythms of particular motivated behaviors exist is unknown. Here, we evaluated the spontaneous daily and circadian behavior of consumption of a sweet caloric solution (5-10% sucrose), and the effects of sucrose intake on the expression of clock genes in the mouse brain. Mice showed a daily (in a light-dark cycle) and a circadian (in constant darkness conditions) rhythm in the intake and sucrose preference with a rise for both parameters at night (or subjective night). In addition, we observed changes in the circadian day-night expression of the clock gene Per2 in the SCN, cortex and striatum of animals ingesting sucrose compared to control mice on pure water. Finally, daily rhythms of sucrose intake and preference were abolished in Per2Brdm1- and double Per1-/-Per2Brdm1-mutant animals. These data indicate that the expression of circadian rhythms of hedonic feeding behaviors may be controlled by brain circadian clocks and Per gene expression.

International audience; The circadian clock is thought to adjust retinal sensitivity to ambient light levels, yet the involvement of specific clock genes is poorly understood. We explored the potential role of the nuclear receptor subfamily 1, group D, member 1 (REV-ERB; or NR1D1) in this respect. In light-evoked behavioral tests, compared with wild-type littermates, Rev-Erb(-/-) mice showed enhanced negative masking at low light levels (0.1 lx). Rev-Erb(-/-) mouse retinas displayed significantly higher numbers of intrinsically photosensitive retinal ganglion cells (ipRGCs; 62% more compared with wild-type) and more intense melanopsin immunostaining of individual ipRGCs. In agreement with a pivotal role for melanopsin, negative masking at low light intensities was abolished in Rev-Erb(-/-)Opn4(-/-) (melanopsin gene) double-null mice. Rev-Erb(-/-) mice showed shortened latencies of both a and b electroretinogram waves, modified scotopic and photopic b-wave and scotopic threshold responses, and increased pupillary constriction, all of which suggested increased light sensitivity. However, wild-type and Rev-Erb(-/-) mice displayed no detectable differences by in vivo fundus imaging, retinal histology, or expression of cell type-specific markers for major retinal cell populations. We conclude that REV-ERB plays a major role in retinal information processing, and we speculate that REV-ERB and melanopsin set sensitivity levels of the rod-mediated ipRGC pathway to coordinate activity with ambient light.Ait-Hmyed Hakkari, O., Acar, N., Savier, E., Spinnhirny, P., Bennis, M., Felder-Schmittbuhl, M.-P., Mendoza, J., Hicks, D. Rev-Erb modulates retinal visual processing and behavioral responses to light.

Skin acts as a barrier between the environment and internal organs and performs functions that are critical for the preservation of body homeostasis. In mammals, a complex network of circadian clocks and oscillators adapts physiology and behavior to environmental changes by generating circadian rhythms. These rhythms are induced in the central pacemaker and peripheral tissues by similar transcriptional-translational feedback loops involving clock genes. In this work, we investigated the presence of functional oscillators in the human skin by studying kinetics of clock gene expression in epidermal and dermal cells originating from the same donor and compared their characteristics. Primary cultures of fibroblasts, keratinocytes, and melanocytes were established from an abdominal biopsy and expression of clock genes following dexamethasone synchronization was assessed by qPCR. An original mathematical method was developed to analyze simultaneously up to nine clock genes. By fitting the oscillations to a common period, the phase relationships of the genes could be determined accurately. We thereby show the presence of functional circadian machinery in each cell type. These clockworks display specific periods and phase relationships between clock genes, suggesting regulatory mechanisms that are particular to each cell type. Taken together, our data demonstrate that skin has a complex circadian organization. Oscillators are present not only in fibroblasts but also in epidermal keratinocytes and melanocytes and are likely to act in coordination to drive rhythmic functions within the skin.

Mammalian retina harbours a self-sustained circadian clock able to synchronize to the light : dark (LD) cycle and to drive cyclic outputs such as night-time melatonin synthesis. Clock genes are expressed in distinct parts of the tissue, and it is presently assumed that the retina contains several circadian oscillators. However, molecular organization of cell type-specific clockworks has been poorly investigated. Here, we questioned the presence of a circadian clock in rat photoreceptors by studying 24-h kinetics of clock and clock output gene expression in whole photoreceptor layers isolated by vibratome sectioning. To address the importance of light stimulation towards photoreceptor clock properties, animals were exposed to 12 : 12 h LD cycle or 36 h constant darkness. Clock, Bmal1, Per1, Per2, Cry1, Cry2, RevErbα and Rorβ clock genes were all found to be expressed in photoreceptors and to display rhythmic transcription in LD cycle. Clock genes in whole retinas, used as a reference, also showed rhythmic expression with marked similarity to the profiles in pure photoreceptors. In contrast, clock gene oscillations were no longer detectable in photoreceptor layers after 36 h darkness, with the exception of Cry2 and Rorβ. Importantly, transcripts from two well-characterized clock output genes, Aanat (arylalkylamine N-acetyltransferase) and c-fos, retained sustained rhythmicity. We conclude that rat photoreceptors contain the core machinery of a circadian oscillator likely to be operative and to drive rhythmic outputs under exposure to a 24-h LD cycle. Constant darkness dramatically alters the photoreceptor clockwork and circadian functions might then rely on inputs from extra-photoreceptor oscillators.

As a peripheral tissue localized at the interface between internal and external environments, skin performs functions which are critical for the preservation of body homeostasis, in coordination with environmental changes. Some of these functions undergo daily variations, such as temperature or water loss, suggesting the presence of time-keeping mechanisms. Rhythmic functions are controlled by a network of circadian oscillators present virtually in every cell and coordinated by the central clock located in the suprachiasmatic nuclei. At the molecular level, circadian rhythms are generated by conserved transcriptional–translational feedback loops involving several clock genes, among which Per1 and Per2 play a central role. Here we characterize clock activity in skin of the transgenic Per1-luciferase rat during postnatal development and adulthood, by real-time recording of bioluminescence in explants and primary dermal fibroblasts, and report marked transformation in circadian properties, from early life to aging. Using primary dermal fibroblast cultures we provide evidence that melatonin treatment phase dependently increases the amplitude of circadian oscillations and that ambient temperature impacts on their period, with slight overcompensation. Together, these findings demonstrate that skin contains a self-sustained circadian clock undergoing age-dependent changes. Dermal fibroblasts, one of the major skin cell types, also exhibit robust, yet specific, circadian rhythmicity which can be fine-tuned by both internal (melatonin) and external (temperature) factors.

Many aspects of retinal physiology are modulated by circadian clocks, but it is unclear whether clock malfunction impinges directly on photoreceptor survival, differentiation or function. Eyes from wild-type (WT) and Period1 (Per1) and Period2 (Per2) mutant mice (Per1(Brdm1) Per2(Brdm1) ) were examined for structural (histology, in vivo imaging), phenotypical (RNA expression, immunohistochemistry) and functional characteristics. Transcriptional levels of selected cone genes [red/green opsin (Opn1mw), blue cone opsin (Opn1sw) and cone arrestin (Arr3)] and one circadian clock gene (RORb) were quantified by real-time polymerase chain reaction. Although there were no changes in general retinal histology or visual responses (electroretinograms) between WT and Per1(Brdm1) Per2(Brdm1) mice, compared with age-matched controls, Per1(Brdm1) Per2(Brdm1) mice showed scattered retinal deformations by fundus inspection. Also, mRNA expression levels and immunostaining of blue cone opsin were significantly reduced in mutant mice. Especially, there was an alteration in the dorsal-ventral patterning of blue cones. Decreased blue cone opsin immunoreactivity was present by early postnatal stages, and remained throughout maturation. General photoreceptor differentiation was retarded in young mutant mice. In conclusion, deletion of both Per1 and Per2 clock genes leads to multiple discrete changes in retina, notably patchy tissue disorganization, reductions in cone opsin mRNA and protein levels, and altered distribution. These data represent the first direct link between Per1 and Per2 clock genes, and cone photoreceptor differentiation and function.

The cerebellum participates in motor coordination as well as in numerous cerebral processes, including temporal discrimination. Animals can predict daily timing of food availability, as manifested by food-anticipatory activity under restricted feeding. By studyingex vivoclock gene expression byin situhybridization and recordingin vitro Per1-luciferasebioluminescence, we report that the cerebellum contains a circadian oscillator sensitive to feeding cues (i.e., whose clock gene oscillations are shifted in response to restricted feeding). Food-anticipatory activity was markedly reduced in mice injected intracerebroventricularly with an immunotoxin that depletes Purkinje cells (i.e., OX7-saporin). Mice bearing the hotfoot mutation (i.e.,Grid2ho/ho) have impaired cerebellar circuitry and mild ataxic phenotype.Grid2ho/homice fedad libitumshowed regular behavioral rhythms and day–night variations of clock gene expression in the hypothalamus and cerebellum. When challenged with restricted feeding, however,Grid2ho/homice did not show any food-anticipatory rhythms, nor timed feeding-induced changes in cerebellar clock gene expression. In hypothalamic arcuate and dorsomedial nuclei, however, shifts inPer1expression in response to restricted feeding were similar in cerebellar mutant and wild-type mice. Furthermore, plasma corticosterone and metabolites before mealtime did not differ between cerebellar mutant and wild-type mice. Together, these data define a role for the cerebellum in the circadian timing network and indicate that the cerebellar oscillator is required for anticipation of mealtime.

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