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Glutamate is the major excitatory amino acid in the vertebrate brain. Glutamatergic signaling is involved in most of the central nervous system functions. Its main components, namely receptors, ion channels, and transporters, are tightly regulated at the transcriptional, translational, and post-translational levels through a diverse array of extracellular signals, such as food, light, and neuroactive molecules. An exquisite and well-coordinated glial/neuronal bidirectional communication is required for proper excitatory amino acid signal transactions. Biochemical shuttles such as the glutamate/glutamine and the astrocyte-neuronal lactate represent the fundamental involvement of glial cells in glutamatergic transmission. In fact, the disruption of any of these coordinated biochemical intercellular cascades leads to an excitotoxic insult that underlies some aspects of most of the neurodegenerative diseases characterized thus far. In this contribution, we provide a comprehensive summary of the involvement of the Aryl hydrocarbon receptor, a ligand-dependent transcription factor in the gene expression regulation of glial glutamate transporters. These receptors might serve as potential targets for the development of novel strategies for the treatment of neurodegenerative diseases.

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.

Circadian clocks are cell-autonomous, molecular pacemakers regulating a wide variety of behavioural and physiological processes in accordance with the 24 ​h light/dark cycle. The retina contains a complex network of cell-specific clocks orchestrating many biochemical and cellular parameters to adapt retinal biology and visual function to daily changes in light intensity. The gene regulatory networks controlling proliferation, specification and differentiation of retinal precursors into the diverse retinal cell types are evolutionary conserved among vertebrates. However, how these mechanisms are interconnected with circadian clocks is not well-characterized. Here we explore the existing evidence for the regulation of retinal development by circadian clock-related pathways, throughout vertebrates. We provide evidence for the influence of clock genes, from early to final differentiation steps. In addition, we report that the clock, integrating environmental cues, modulates a number of pathological processes. We highlight its potential role in retinal diseases and its instructive function on eye growth and related disorders.

International audience; Melatonin, a major signal of the circadian system, is also involved in brain functions such as learning and memory. Chronic melatonin treatment is known to improve memory performances, but the respective contribution of its central receptors, MT1 and MT2, is still unclear. Here, we used new single receptor deficient MT1-/and MT2-/mice to investigate the contribution of each receptor in the positive effect of chronic melatonin treatment on long-term recognition memory. The lack of MT2 receptor precluded memory-enhancing effect of melatonin in the object recognition task and to a lesser extent in the object location task, whereas the lack of MT1 receptor mitigated its effect in the object location task only. Our findings support a key role of MT2 in mediating melatonin's beneficial action on long-term object recognition memory, whereas MT1 may contribute to the effect on object location memory.

Melatonin (MLT), secreted during the night by the pineal gland, is an efferent hormonal signal of the master circadian clock located in the suprachiasmatic nucleus (SCN). Consequently, it is a reliable phase marker of the SCN clock. If one defines as "chronobiotic," a drug able to influence the phase and/or the period of the circadian clock, MLT is a very potent one. The most convincing data obtained so far come from studies on totally blind individuals. Exogenous MLT administered daily entrains the sleep-wake cycle of these individuals to a 24-h cycle. MLT, however, is not essential to sleep. In nocturnally, active mammals, MLT is released during the night concomitantly with the daily period of wakefulness. Therefore, MLT cannot be simply considered as a sleep hormone, but rather as a signal of darkness. Its role in the circadian system is to reinforce nighttime physiology, including timing of the sleep-wake cycle and other circadian rhythms. MLT exerts its effects on the sleep cycle especially by a direct action on the master circadian clock. The sleep-wake cycle is depending not only on the circadian clock but also on an orchestrated network of different centers in the brain. Thus, the control of sleep-wake rhythm might be explained by a parallel and concomitant action of MLT on the master clock (chronobiotic effect) and on sleep-related structures within the brain. MLT acts through two high-affinity membrane receptors (MT1 and MT2) with striking differences in their distribution pattern. MLT is a powerful synchronizer of human circadian rhythms, thus justifying the use of MLT and MLT agonists in clinical medicine as pharmacological tools to manipulate the sleep-wake cycle, and to treat sleep disorders and other circadian disorders. Available MLT analogs/drugs are all nonspecific MT1/MT2 agonists. The development of new ligands which are highly selectivity for each subtype is clearly a new challenge for the field and will be at the root of new therapeutic agents for curing specific pathologies, including sleep disorders.

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.

Melatonin, a major signal of the circadian system, is also involved in brain functions such as learning and memory. Chronic melatonin treatment is known to improve memory performances, but the respective contribution of its central receptors, MT

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.

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.

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.

The presence of a circadian clock in the retinal pigment epithelium (RPE) was discovered recently. However, little is known about mechanisms or processes regulated by the RPE clock. We cultured ARPE-19 monolayers in a transwell culture system, and we found rhythmic mRNA expression of the sodium-potassium-chloride co-transporter

International audience; Melatonin (MLT) exerts its physiological effects principally through two high-affinity membrane receptors MT1 and MT2. Understanding the exact mechanism of MLT action necessitates the use of highly selective agonists/antagonists to stimulate/inhibit a given MLT receptor. The respective distribution of MT1 and MT2 within the CNS and elsewhere is controversial, and here we used a "knock-in" strategy replacing MT1 or MT2 coding sequences with a LacZ reporter. The data show striking differences in the distribution of MT1 and MT2 receptors in the mouse brain: whereas the MT1 subtype was expressed in very few structures (notably including the suprachiasmatic nucleus and pars tuberalis), MT2 subtype receptors were identified within numerous brain regions including the olfactory bulb, forebrain, hippocampus, amygdala and superior colliculus. Co-expression of the two subtypes was observed in very few structures, and even within these areas they were rarely present in the same individual cell. In conclusion, the expression and distribution of MT2 receptors are much more widespread than previously thought, and there is virtually no correspondence between MT1 and MT2 cellular expression. The precise phenotyping of cells/neurons containing MT1 or MT2 receptor subtypes opens new perspectives for the characterization of links between MLT brain targets, MLT actions and specific MLT receptor subtypes.

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.

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.

In mice, mutations or targeted disruptions of the core circadian gene Bmal1 have been implicated in early onset of ocular pathologies, including premature/congenital cataract development. The aim of the present study was to analyze probands of consanguineous Pakistani cataract families to identify the novel pathogenic variants in the BMAL1 gene. We have studied 21 congenital cataract families. Ophthalmic examination was performed for the probands and available family members. Genomic DNA was isolated from peripheral blood. PCR and Sanger sequencing was performed for the entire coding region of the BMAL1 gene. Targeted Sanger sequencing of BMAL1 revealed a heterozygous variant c.41A>T; p.(Asp14Val) in one proband, but it did not co-segregate with the disease phenotype in the family. In addition, a nonsynonymous variant (rs2290037) was identified in five probands. Our study is the first one to analyze the role of BMAL1 gene mutations in humans for their association with congenital cataract. Although we were unable to find the variants associated with congenital cataract families from Pakistan, more studies from other populations will be informative to further prove the role of BMAL1 with the disease.

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.

Development and homeostasis require stringent spatiotemporal control of gene expression patterns that are established, to a large extent, by combinatorial action of transcription regulatory proteins. The bZIP transcription factor NRL (neural retina leucine zipper) is critical for rod versus cone photoreceptor cell fate choice during retinal development and acts as a molecular switch to produce rods from postmitotic precursors. Loss of Nrl in mouse leads to a cone-only retina, whereas ectopic expression of Nrl in photoreceptor precursors generates rods. To decipher the transcriptional regulatory mechanisms upstream of Nrl, we identified putative cis-control elements in the Nrl promoter/enhancer region by examining cross-species sequence conservation. Using in vivo transfection of promoter-reporter constructs into the mouse retina, we show that a 0.9-kb sequence upstream of the Nrl transcription initiation site is sufficient to drive reporter gene expression in photoreceptors. We further define a 0.3-kb sequence including a proximal promoter (cluster A1) and an enhancer (cluster B) that can direct rod-specific expression in vivo. Electrophoretic mobility shift assays using mouse retinal nuclear extracts, in combination with specific antibodies, demonstrate the binding of retinoid-related orphan nuclear receptor β (RORβ), cone rod homeobox, orthodenticle homolog 2, and cyclic AMP response element-binding protein to predicted consensus elements within clusters A and B. Our studies demonstrate Nrl as a direct transcriptional target of RORβ and suggest that combinatorial action of multiple regulatory factors modulates the expression of Nrl in developing and mature retina.

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.

The Maf oncoproteins are b-Zip transcription factors of the AP-1 superfamily. They are involved in developmental, metabolic, and tumorigenic processes. Maf proteins are overexpressed in about 50% of human multiple myelomas. Here, we show that Maf-transforming activity is controlled by GSK-3-dependent phosphorylation and that phosphorylation by GSK-3 can increase the oncogenic activity of a protein. Using microarray analysis, we identify a gene-expression subprogram regulated by GSK-3-mediated Maf phosphorylation involved in extracellular matrix remodeling and relevant to cancer progression. We also demonstrate that GSK-3 triggers MafA sequential phosphorylation on residues S61, T57, T53, and S49, inducing its ubiquitination and degradation. Paradoxically, this phosphorylation increases MafA-transcriptional activity through the recruitment of the coactivator P/CAF. We further demonstrate that P/CAF protects MafA from ubiquitination and degradation, suggesting that, upon the release of the coactivator complex, MafA becomes polyubiquitinated and degraded to allow the response to terminate.

Maf proteins are basic-leucine zipper transcription factors belonging to the AP1 superfamily. Several developmental processes require Maf proteins yet, the redundancy or complementarity of their respective roles in common processes has been only partially investigated. We present for the first time a complete comparative analysis of maf gene expression patterns in vertebrates. Expression of c-maf, mafB/kreisler, mafA/L-maf, mafF, mafG and mafK was analyzed by whole-mount in situ hybridization within chick embryos and their extraembryonic tissues ranging from embryonic day (E) 1 to 7. We carefully examined the extent of overlap between distinct maf genes and report that the developing lens, kidney, pancreas and apoptotic zones of limb buds show sustained co-expression of large maf genes. Small maf genes also exhibit overlap, for example in the dermomyotome. We also describe so far unidentified sites of maf gene expression. mafA is found in the developing neural tube and dorsal root ganglia. c-maf hybridization is detected in the neuroretina, the notochord and the endothelium of extraembryonic blood vessels.

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.

In the endocrine pancreas, alpha-cell-specific expression of the glucagon gene is mediated by DNA-binding proteins that interact with the G1 proximal promoter element. Among these proteins, the paired domain transcription factor Pax-6 has been shown to bind to G1 and to transactivate glucagon gene expression. Close to the Pax-6-binding site, we observed the presence of a binding site for a basic leucine zipper transcription factor of the Maf family. In the present study, we demonstrate the presence of Maf family members in the endocrine pancreas that bind to G1 and transactivate glucagon promoter expression. In transient transfection experiments, we found that the transactivating effect on the glucagon promoter was greatly enhanced by the simultaneous expression of Maf transcription factors and Pax-6. This enhancement on glucagon transactivation could be correlated with the ability of these proteins to interact together but does not require binding of Maf proteins to the G1 element. Furthermore, we found that Maf enhanced the Pax-6 DNA binding capacity. Our data indicate that Maf transcription factors may contribute to glucagon gene expression in the pancreas.

Ras-induced cell transformation is mediated through distinct downstream signaling pathways, including Raf, Ral-GEFs-, and phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathways. In some cell types, strong activation of the Ras–Raf–MEK–extracellular signal-regulated kinase (ERK) cascade leads to cell cycle arrest rather than cell division. We previously reported that constitutive activation of this pathway induces sustained proliferation of primary cultures of postmitotic chicken neuroretina (NR) cells. We used this model system to investigate the respective contributions of Ras downstream signaling pathways in Ras-induced cell proliferation. Three RasV12 mutants (S35, G37, and C40) which differ by their ability to bind to Ras effectors (Raf, Ral-GEFs, and the p110 subunit of PI 3-kinase, respectively) were able to induce sustained NR cell proliferation, although none of these mutants was reported to transform NIH 3T3 cells. Furthermore, they all repressed the promoter of QR1, a neuroretina growth arrest-specific gene. Overexpression of B-Raf or activated versions of Ras effectors Rlf-CAAX and p110-CAAX also induced NR cell division. The mitogenic effect of the RasC40–PI 3-kinase pathway appears to involve Rac and RhoA GTPases but not the antiapoptotic Akt (protein kinase B) signaling. Division induced by RasG37-Rlf appears to be independent of Ral GTPase activation and presumably requires an unidentified mechanism. Activation of either Ras downstream pathway resulted in ERK activation, and coexpression of a dominant negative MEK mutant or mKsr-1 kinase domain strongly inhibited proliferation induced by the three Ras mutants or by their effectors. Similar effects were observed with dominant negative mutants of Rac and Rho. Thus, both the Raf-MEK-ERK and Rac-Rho pathways are absolutely required for Ras-induced NR cell division. Activation of these two pathways by the three distinct Ras downstream effectors possibly relies on an autocrine or paracrine loop, implicating endogenous Ras, since the mitogenic effect of each Ras effector mutant was inhibited by RasN17.

Purpose Circadian rhythms are central to vision and retinal physiology. A circadian clock located within the retina controls various rhythmic processes including melatonin synthesis in photoreceptors. In the present study, we evaluated the rhythmic expression of clock genes and clock output genes in retinal explants maintained for several days in darkness. Methods Retinas were dissected from Wistar rats, either wild-type or from the Per1-luciferase transgenic line housed under a daily 12 h:12 h light-dark cycle (LD12/12), and put in culture at zeitgeber time (ZT) 12 on semipermeable membranes. Explants from wild-type rats were collected every 4 h over 3 days, and total RNA was extracted, quantified, and reverse transcribed. Gene expression was assessed with quantitative PCR, and the periodicity of the relative mRNA amounts was assessed with nonlinear least squares fitting to sine wave functions. Bioluminescence in explants from Per1-luciferase rats was monitored for several days under three different culture protocols. Results Rhythmic expression was found for all studied clock genes and for clock downstream targets such as c-fos and arylalkylamine N-acetyltransferase (Aanat) genes. Clock and output genes cycled with relatively similar periods and acrophases (peaks of expression during subjective night, except c-fos, which peaked around the end of the subjective day). Data for Per1 were confirmed with bioluminescence monitoring, which also permitted culture conditions to be optimized to study the retina clock. Conclusions Our work shows the free-running expression profile of multiple clock genes and potential clock targets in mammalian retinal explants. This research further strengthens the notion that the retina contains a self-sustained oscillator that can be functionally characterized in organotypic culture.

We reported previously that post-mitotic chicken embryonic neuroretina (NR) cells are induced to proliferate following in vitro infection with RAV-1, a retrovirus that does not carry an oncogene. NR cell multiplication results from the frequent activation and subsequent retroviral transduction of two related serine/threonine protein kinases, the c-mil/c-raf or c-Rmil/B-raf genes. We also showed that a very early event in the activation of these proto-oncogenes is the synthesis of chimeric mRNAs containing viral and cellular sequences joined by a splicing mechanism. In the current study, we have examined the ability of RAV-1 to induce proliferation of quail NR cells. By using the reverse transcription-polymerase chain reaction technique, we identified, in several proliferating quail NR cultures infected with RAV-1, a chimeric mRNA containing cellular sequences joined to the RAV-1 splice donor site. These cellular sequences are derived from a gene designated R10, which is expressed through a 1.9-kilobase (kb) mRNA detected in several embryonic tissues. A second transcript of 2.3 kb is specifically expressed in the NR, where both transcripts are developmentally regulated. The R10 cDNA encodes a 251-amino acid polypeptide that contains a leucine zipper motif. It exhibits significant similarity with the putative D52/N8L protein, encoded by an mRNA reported previously to be overexpressed in human breast and lung carcinomas. By using polyclonal antibodies specific for its amino-terminal and leucine zipper-containing regions, we identified the R10 gene product as a cytoplasmic protein of 23 kDa in cultured avian fibroblasts. A second protein of 30 kDa is detected in post-mitotic NR cells that express the 2.3-kb transcript. We also show, by in vitro transcription/translation and immunoprecipitation, that the R10 protein can readily form homodimers, presumably through its leucine zipper motif.

Purpose Prolonged periods of constant lighting are known to perturb circadian clock function at the molecular, physiological, and behavioral levels. However, the effects of ambient lighting regimes on clock gene expression and clock outputs in retinal photoreceptors—rods, cones and intrinsically photosensitive retinal ganglion cells—are only poorly understood. Methods Cone-rich diurnal rodents (Muridae: Arvicanthis ansorgei) were maintained under and entrained to a 12 h:12 h light-dark cycle (LD; light: ~300 lux). Three groups were then examined: control (continued maintenance on LD); animals exposed to a 36 h dark period before sampling over an additional 24 h period of darkness (DD); and animals exposed to a 36 h light period before sampling over an additional 24 h period of light (~300 lux, LL). Animals were killed every 3 or 4 h over 24 h, their retinas dissected, and RNA extracted. Oligonucleotide primers were designed for the Arvicanthis clock genes Per1, Per2, Cry1, Cry2, and Bmal1, and for transcripts specific for rods (rhodopsin), cones (short- and mid-wavelength sensitive cone opsin, cone arrestin, arylalkylamine N-acetyltransferase) and intrinsically photosensitive retinal ganglion cells (melanopsin). Gene expression was analyzed by real-time PCR. Results In LD, expression of all genes except cone arrestin was rhythmic and coordinated, with acrophases of most genes at or shortly following the time of lights on (defined as zeitgeber time 0). Arylalkylamine N-acetyltransferase showed maximal expression at zeitgeber time 20. In DD conditions the respective profiles showed similar phase profiles, but were mostly attenuated in amplitude, or in the case of melanopsin, did not retain rhythmic expression. In LL, however, the expression profiles of all clock genes and most putative output genes were greatly altered, with either abolition of daily variation (mid-wavelength cone opsin) or peak expression shifted by 4–10 h. Conclusions These data are the first to provide detailed measures of retinal clock gene and putative clock output gene expression in a diurnal mammal, and show the highly disruptive effects of inappropriate (nocturnal) lighting on circadian and photoreceptor gene regulation.

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.

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.

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.

Pineal melatonin is synthesized with daily and seasonal rhythms following the hypothalamic clock-driven release of norepinephrine (NE). The pineal gland of rats and mice, like the biological clock, expresses a number of clock genes. However, the role of pineal clock elements in pineal physiology is still unknown. We examined the expression and regulation of several clock genes (Per1, Cry2, Bmal1 and Rev-erb alpha) under different lighting conditions or following adrenergic treatments in the Syrian hamster, a seasonal rodent. We found that Per1 and Cry2 genes were similarly regulated by the nocturnal release of NE: levels of Per1 and Cry2 mRNA displayed a nocturnal increase that was maintained after 2 days in constant darkness (DD) but abolished after 2 days under constant light (LL), a condition that suppresses endogenous NE release, or after an early night administration of the adrenergic antagonist propranolol. In contrast, Bmal1 and Rev-erb alpha exhibited a different pattern of expression and regulation. mRNA levels of both clock genes displayed a marked daily variation, maintained in DD, with higher values at midday for Bmal1 and at day/night transition for Rev-erb alpha. Remarkably, the daily variation of both Bmal1 and Rev-erb alpha mRNA was maintained in LL conditions and was not affected by propranolol. This study confirms the daily regulation of Per1 and Cry2 gene expression by NE in the pineal gland of rodents and shows for the first time that a second set of clock genes, Bmal1 and Rev-erb alpha are expressed with a circadian rhythm independent of the hypothalamic clock-driven noradrenergic signal.

Maf b-Zip transcription factors are involved in both terminal differentiation and oncogenesis. To investigate this apparent contradiction, we used two different primary cell types and performed an extensive analysis of transformation parameters induced by Maf proteins. We show that MafA and c-Maf are potent oncogenes in chicken embryo fibroblasts, while MafB appears weaker. We also provide the first evidence that MafA can confer growth factor independence and promote cell division at low density. Moreover, using MafA as a model, we identified several parameters that are critical for Maf transforming activities. Indeed, MafA ability to induce anchorage-independent cell growth was sensitive to culture conditions. In addition, the transforming activity of MafA was dependent on its phosphorylation state, since mutation on Ser65 impaired its ability to induce growth at low density and anchorage-independent growth. We next examined transforming activity of large Maf proteins in embryonic neuroretina cells, where they are known to induce differentiation. Unlike v-Jun, MafA, MafB and c-Maf did not show oncogenic activity in these cells. Moreover, they counteracted transformation induced by constitutive activation of the Ras/Raf/MEK pathway. Taken together, our results show that Maf proteins could display antagonistic functions in oncogenesis depending on the cellular context, and support a dual role for Maf as both oncogenes and tumor suppressor-like proteins.

Basic-leucine zipper transcription factors of the Maf family are key regulators of various developmental and differentiation processes. We previously reported that the phosphorylation status of MafA is a critical determinant of its biological functions. Using Western blot and mass spectrometry analysis, we now show that MafA is phosphorylated by p38 MAP kinase and identify three phosphoacceptor sites: threonine 113 and threonine 57, evolutionarily conserved residues located in the transcription activating domain, and serine 272. Mutation of these residues severely impaired MafA biological activity. Furthermore, we show that p38 also phosphorylates MafB and c-Maf. Together, these findings suggest that the p38 MAP kinase pathway is a novel regulator of large Maf transcription factors.

We have performed an exhaustive characterization of the large Maf family of basic leucine zipper transcription factors in vertebrates using the genome data available, and studied the embryonic expression patterns of the four paralogous genes thus identified in Xenopus tropicalis. Our phylogenetic analysis shows that, in osteichthyans, the large Maf family contains four orthology classes, MafA, MafB, c-Maf and Nrl, which have emerged in vertebrates prior to the split between actinopterygians and sarcopterygians. It leads to the unambiguous assignment of the Xenopus laevis XLmaf gene, previously considered a MafA orthologue, to the Nrl class, the identification of the amphibian MafA and c-Maf orthologues and the identification of the zebrafish Nrl gene. The four X. tropicalis paralogues display partially redundant but nevertheless distinct expression patterns in the somites, developing hindbrain, pronephros, ventral blood island and lens. Comparisons with the data available in the mouse, chick and zebrafish show that these large Maf expression territories are highly conserved among osteichthyans but also highlight a number of differences in the timing of large Maf gene expression, the precise extent of some labelled territories and the combinations of paralogues transcribed in some organs. In particular, the availability of robust phylogenies leads to a reinterpretation of previous expression pattern comparisons, suggesting an important part for function shuffling within the gene family in the developing lens. These data highlight the importance of exhaustive characterizations of gene families for comparative analyses of the genetic mechanisms, which control developmental processes in vertebrates.

We previously described the identification of quail MafA, a novel transcription factor of the Maf bZIP (basic region leucine zipper) family, expressed in the differentiating neuroretina (NR). In the present study, we provide the first evidence that MafA is phosphorylated and that its biological properties strongly rely upon phosphorylation of serines 14 and 65, two residues located in the transcriptional activating domain within a consensus for phosphorylation by mitogen-activated protein kinases and which are conserved among Maf proteins. These residues are phosphorylated by ERK2 but not by p38, JNK, and ERK5 in vitro. However, the contribution of the MEK/ERK pathway to MafA phosphorylation in vivo appears to be moderate, implicating another kinase. The integrity of serine 14 and serine 65 residues is required for transcriptional activity, since their mutation into alanine severely impairs MafA capacity to activate transcription. Furthermore, we show that the MafA S14A/S65A mutant displays reduced capacity to induce expression of QR1, an NR-specific target of Maf proteins. Likewise, the integrity of serines 14 and 65 is essential for the MafA ability to stimulate expression of crystallin genes in NR cells and to induce NR-to-lens transdifferentiation. Thus, the MafA capacity to induce differentiation programs is dependent on its phosphorylation.

The neuroretina is a functional unit of the central nervous system which arises through successive steps of division, growth arrest and differentiation of neuroectodermal precursors. Postmitotic quail neuroretina (QNR) cells are conditionally induced to divide upon infection with temperature sensitive mutants of Rous sarcoma virus (RSV), since QNR cell division can be arrested by either inactivating p60v-Src at the nonpermissive temperature (41 degrees C) or by serum deprivation at 37 degrees C. We are studying the transcriptional control of QR1, a neuroretina specific gene, whose expression is down-regulated in proliferating cells at 37 degrees C and is fully restored when these cells are made quiescent. We previously showed that this quiescence specific upregulation implicates a promoter region named A box, which binds Maf transcription factors. We report the identification of the C box, a second promoter sequence that activates QR1 transcription in non dividing cells. This sequence is able to form two DNA-protein complexes, one of which (C4) is predominantly detected in growth arrested NR cells. We identified the DNA binding site for C4 and described mutations that abolish both C4 binding and promoter activity in quiescent cells. Moreover, we show that a multimerized C box is able to stimulate a heterologous promoter in non dividing cells and constitutes, therefore, a novel quiescence responsive enhancer. Finally, we report that QR1 transcriptional response to cell quiescence requires cooperation between the C box and A box.

Transcription factors of the Maf proto-oncogene family have been shown to participate in the regulation of several differentiation specific genes. We previously reported that a member(s) of this family is involved in the regulation of the neuroretina specific gene, QR1, through a promoter region, designated the A box, that is closely related to the Maf recognition element (MARE). We undertook an identification of Maf family genes expressed in the quail neuroretina (QNR) and we report the isolation of mafA, a gene encoding a novel member of the large Maf proteins subgroup. Expression of this gene is developmentally regulated in the neuroretina. MafA is able to bind to MARE sequence and to heterodimerize with v-Maf, MafB, Jun and Fos, but not with the small MafF and MafK proteins. Accordingly, it is able to transactivate the QR1 promoter A box. We also show that increased expression of mafA induces sustained proliferation of postmitotic QNR cells.