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  1. Evaluation Summary:

    Light and light perception are important factors that modulate several aspects of behavior and physiology in all animals, including humans. More specifically, the paper examines circadian cycling of phototransduction regulators in diurnal zebrafish and nocturnal mice, and links them to function at the level of ERGs. Interestingly, the transcriptional cycling is shifted between zebrafish and mice. This work is of relevance to vision researchers, but also of interest to a broader audience of behavioral (neuro)scientists and chronobiologists.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

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  2. Reviewer #1 (Public Review):

    Zang and colleagues present an intriguing manuscript on circadian oscillations in retinal gene expression and function in zebrafish and mice. They find that many key regulators of phototransduction-shutoff cascade show marked oscillations that persist in darkness, and that are shared across adult and larval zebrafish. Moreover, the same set of regulators oscillate in antiphase in mice, which unlike zebrafish are nocturnal. Using ERGs, the authors then go on to show that zebrafish photo-recovery is modulated in a circadian manner, and this continues to occur in constant darkness, indicating that it is indeed intrinsic oscillations (rather than light entrainment alone). This also appears to be mirrored in flicker fusion rates and perhaps at the level of some behaviours.

    Overall, the manuscript is very interesting and usefully adds to our understanding of retinal/ photoreceptor function in vertebrates in the context of circadian rhythms. Moreover, the manuscript is well presented and written.

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  3. Reviewer #2 (Public Review):

    Circadian clock function is an intrinsic property of most cells and tissues. The retina is no exception with many lines of evidence pointing to retinal physiology being under the control of a local circadian clock thereby matching the very different physiological demands of the retina at different time of day. In zebrafish, previous studies have documented response threshold, photoreceptor retinomotor movement and cone synaptic ribbon assembly to be under clock control. This manuscript takes some important steps forward in our understanding of the links between the circadian clock and retinal physiology in zebrafish. Rhythms of photoresponse recovery are visualised convincingly by electroretinography and these are shown to be mirrored consistently by rhythms in optokinetic and optomotor responses. The authors also present clear data revealing that this correlates with circadian clock-regulated rhythmic mRNA and protein expression of key regulators of the visual transduction cascade, specifically those involved in so-called shut-off kinetics. What is particularly elegant is that the phase of the equivalent gene expression rhythms in the mouse, a nocturnal species, are 12 hours phase-shifted with respect to the diurnal zebrafish, probably reflecting the different timing of visual function in these two species with respect to the day-night cycle.

    The main weak point of this work in my opinion concerns the many questions that are raised, but not convincingly answered, regarding how the clock is coupled to these retinal outputs. Attempts to identify enhancer elements in the promoter region of the rhythmically expressed visual transduction cascade genes which are targeted by the core circadian clock machinery (one assumes, E-boxes) were apparently not conclusive (Lines 23 24, page 21 and lines 1 and 2, page 22: "Although we identified some conserved binding sites of core clock proteins in our analyzed genes, neither of them was conserved in mammalian genomes (....), suggesting that the regulatory pattern of circadian regulation is more complex."). Although this statement is based on bioinformatic inspection, rather than unbiased promoter analysis, this leaves much uncertainty as to precisely how, or indeed if the core clock machinery within the cone photoreceptors cells directly regulates these gene targets. If not direct regulation by core clock transcription factors, which mechanism might operate? One possibly related issue is that in the experiments examining retinal gene expression in larvae raised under constant darkness, rhythmicity was observed where previous studies might have anticipated clock desynchronisation. The authors extrapolate from these findings with the following prediction: (line 7-9, page 22, "We did not observe this phenomenon in our study of visual transduction genes in the retina, suggesting the existence of an inheritable maternal clock in the eye (Delaunay et al., 2000)"). The data presented in this manuscript is insufficient to test this hypothesis and indeed many other studies have been unable to provide any evidence for maternal inheritance of the clock in this species. However, the persistence of rhythmicity in larvae raised in DD conditions is a result that might provide some clues as to how the circadian clock mechanism is operating here.

    So, in summary, while making some important contributions to the circadian clock and retinal physiology field, the work tends to raise more questions than it answers.

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  4. Reviewer #3 (Public Review):

    The authors investigate the putative molecular mechanisms underlying circadian changes in visual behavior using the diurnal zebrafish as model. They focus on arrestins, G-protein receptor kinases, Regulator of G protein signallin 9, and recoverins, which are all regulators of the termination of the visual transduction cascade, hence modulators of visual temporal resolution. Despite two exceptions (i.e. rcv2a which was not fluctuating and rcv1b which was not expressed in the larval retina) the authors find that the expression levels of all tested genes show time dependent fluctuations over the 24 hours in both larvae's and adult eyes. To support a circadian regulation of the tested genes, the observed fluctuations are maintained in constant darkness (where circadian rhythms should be self-sustained) and disrupted in constant light (where circadian rhythms should be lost). The authors also test for fluctuations in the expression levels of the proteins encoded by 2 of the tested genes, namely GRK7a and ARR3.These might also be present in the eyes of adult fish at different amounts over the 24 hours. The fluctuations in the expression levels of the regulators of the visual transduction cascade correlate with changes in visual behavior and physiology. In particular:

    • the photoreceptors take longer to recover from a flash of light in the evening compared to the morning, and the difference is lost when the animals are reared in constant light

    • the animals are less able to resolve flicker frequencies in the evening

    • startle responses following lights-off and lights-on are more intense in the morning or evening, respectively

    • optokinetic responses (i.e. velocity of eye movement in response to a contrast stimulus) change over the day being lower in the evening compared to noon.

    These results suggest that circadian modulation of visual behavior might depend on the cyclic expression of the genes involved in the visual transduction cascade. These conclusions are well supported by the molecular and behavioral data presented, especially by the fact that time dependent fluctuations are observed in several aspects of visual behavior. A potential weakness is that only one visual behavior is tested under constant darkness or constant light, namely the cone photo response recovery. Testing a second visual behavior (e.g. visual motor response) under these conditions would probably strengthens the correlation between changes in the expression levels of genes of the visual transduction cascade and behavioral rhythms. In addition, while it seems quite clear that transcripts level are changing over time in a circadian manner, this cannot be stated for sure with the statistical analysis performed. Indeed, the One Way ANOVA allows to test for an effect of time, but can not provide proof for circadian oscillation. This is even more important in case of GRK7a and ARR3, where the effect of time on protein expression. The authors investigate the putative molecular mechanisms underlying circadian changes in visual behavior using the diurnal zebrafish as model. They focus on arrestins, G-protein receptor kinases, Regulator of G protein signallin 9, and recoverins, which are all regulators of the termination of the visual transduction cascade, hence modulators of visual temporal resolution. Despite two exceptions (i.e. rcv2a which was not fluctuating and rcv1b which was not expressed in the larval retina) the authors find that the expression levels of all tested genes show time dependent fluctuations over the 24 hours in both larvae's and adult eyes. To support a circadian regulation of the tested genes, the observed fluctuations are maintained in constant darkness (where circadian rhythms should be self-sustained) and disrupted in constant light (where circadian rhythms should be lost). The authors also test for fluctuations in the expression levels of the proteins encoded by 2 of the tested genes, namely GRK7a and ARR3.These might also be present in the eyes of adult fish at different amounts over the 24 hours. The fluctuations in the expression levels of the regulators of the visual transduction cascade correlate with changes in visual behavior and physiology. In particular:

    • the photoreceptors take longer to recover from a flash of light in the evening compared to the morning, and the difference is lost when the animals are reared in constant light

    • the animals are less able to resolve flicker frequencies in the evening

    • startle responses following lights-off and lights-on are more intense in the morning or evening, respectively

    • optokinetic responses (i.e. velocity of eye movement in response to a contrast stimulus) change over the day being lower in the evening compared to noon.

    These results suggest that circadian modulation of visual behavior might depend on the cyclic expression of the genes involved in the visual transduction cascade. These conclusions are well supported by the molecular and behavioral data presented, especially by the fact that time dependent fluctuations are observed in several aspects of visual behavior. A potential weakness is that only one visual behavior is tested under constant darkness or constant light, namely the cone photo response recovery. Testing a second visual behavior (e.g. visual motor response) under these conditions would probably strengthens the correlation between changes in the expression levels of genes of the visual transduction cascade and behavioral rhythms. In addition, while it seems quite clear that transcripts level are changing over time in a circadian manner, this cannot be stated for sure with the statistical analysis performed. Indeed, the One Way ANOVA allows to test for an effect of time, but can not provide proof for circadian oscillation. This is even more important in case of GRK7a and ARR3, where the effect of time on protein expression seems to be somewhat milder. To better test for circadian oscillation, the authors could perhaps try to use RAIN, which allows to detect rhythms in time series independently of the wave form.

    One very interesting finding of this study is that the expression profiles of genes involved in the visual transduction cascade is antiphasic when comparing diurnal fish to nocturnal mice. Nevertheless, zebrafish and mouse are quite distant species.Ti generalize these findings it would be necessary to discuss data also from diurnal rodents or nocturnal fish.

    In general, the study, and its conclusions, are straightforward and provide new insights into the mechanisms underlying circadian regulation of visual behavior.
    n expression seems to be somewhat milder. To better test for circadian oscillation, the authors could perhaps try to use RAIN, which allows to detect rhythms in time series independently of the wave form.

    One very interesting finding of this study is that the expression profiles of genes involved in the visual transduction cascade is antiphasic when comparing diurnal fish to nocturnal mice. Nevertheless, zebrafish and mouse are quite distant species.Ti generalize these findings it would be necessary to discuss data also from diurnal rodents or nocturnal fish.

    In general, the study, and its conclusions, are straightforward and provide new insights into the mechanisms underlying circadian regulation of visual behavior.

    Was this evaluation helpful?