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Seasonal changes in the environment lead to depression-like behaviors in humans and animals. The underlying mechanisms, however, are unknown. We observed decreased sociability and increased anxiety-like behavior in medaka fish exposed to winter-like conditions. Whole brain metabolomic analysis revealed seasonal changes in 68 metabolites, including neurotransmitters and antioxidants associated with depression. Transcriptome analysis identified 3,306 differentially expressed transcripts, including inflammatory markers, melanopsins, and circadian clock genes. Further analyses revealed seasonal changes in multiple signaling pathways implicated in depression, including the nuclear factor erythroid-derived 2-like 2 (NRF2) antioxidant pathway. A broad-spectrum chemical screen revealed that celastrol (a traditional Chinese medicine) uniquely reversed winter behavior. NRF2 is a celastrol target expressed in the habenula (HB), known to play a critical role in the pathophysiology of depression. Another NRF2 chemical activator phenocopied these effects, and an NRF2 mutant showed decreased sociability. Our study provides important insights into winter depression and offers potential therapeutic targets involving NRF2.

Biological clocks have evolved as an adaptation to life on a rhythmic planet, synchronising physiological processes to the environmental light–dark cycle. Here we examine circadian clock function in Mexican blind cavefish Astyanax mexicanus and its surface counterpart. In the lab, adult surface fish show robust circadian rhythms in per1 , which are retained in cave populations, but with substantial alterations. These changes may be due to increased levels of light-inducible genes in cavefish, including clock repressor per2 . From a molecular standpoint, cavefish appear as if they experience ‘constant light’ rather than perpetual darkness. Micos River samples show similar per1 oscillations to those in the lab. However, data from Chica Cave shows complete repression of clock function, while expression of several light-responsive genes is raised, including DNA repair genes. We propose that altered expression of light-inducible genes provides a selective advantage to cavefish at the expense of a damped circadian oscillator. Body clocks modulate physiological processes to follow a day–night cycle, but whether animals exposed to constant darkness express circadian rhythms is unknown. Here the authors examine the expression of circadian genes in Mexican cavefish, and find that these resemble a pattern expected from exposure to constant daylight.

Chronic circadian disruption due to shift work or frequent travel across time zones leads to jet‐lag and an increased risk of diabetes, cardiovascular disease, and cancer. The development of new pharmaceuticals to treat circadian disorders, however, is costly and hugely time‐consuming. We therefore performed a high‐throughput chemical screen of existing drugs for circadian clock modulators in human U2OS cells, with the aim of repurposing known bioactive compounds. Approximately 5% of the drugs screened altered circadian period, including the period‐shortening compound dehydroepiandrosterone (DHEA; also known as prasterone). DHEA is one of the most abundant circulating steroid hormones in humans and is available as a dietary supplement in the USA. Dietary administration of DHEA to mice shortened free‐running circadian period and accelerated re‐entrainment to advanced light–dark (LD) cycles, thereby reducing jet‐lag. Our drug screen also revealed the involvement of tyrosine kinases, ABL1 and ABL2, and the BCR serine/threonine kinase in regulating circadian period. Thus, drug repurposing is a useful approach to identify new circadian clock modulators and potential therapies for circadian disorders. Synopsis Chronic circadian misalignment has long term consequences on our health and leads to increased risk of developing diabetes, cardiovascular disease and cancer. Using a drug repurposing approach, dehydroepiandrosterone (DHEA) was identified as an important circadian clock modulator. Approximately 5% of the screened drugs altered circadian period. DHEA shortened circadian period in cells and tissues. When fed to mice, DHEA shortened circadian period and significantly reduced jet‐lag. ABL1/2 tyrosine kinases and BCR serine/threonine kinase are involved in regulating circadian period. Drug repurposing is a useful approach to identify new circadian clock modulators. Graphical Abstract Chronic circadian misalignment has long term consequences on our health and leads to increased risk of developing diabetes, cardiovascular disease and cancer. Using a drug repurposing approach, dehydroepiandrosterone (DHEA) was identified as an important circadian clock modulator.

Zebrafish tissues and cells have the unusual feature of not only containing a circadian clock, but also being directly light-responsive. Several zebrafish genes are induced by light, but little is known about their role in clock resetting or the mechanism by which this might occur. Here we show that Cryptochrome 1a (Cry1a) plays a key role in light entrainment of the zebrafish clock. Intensity and phase response curves reveal a strong correlation between light induction of Cry1a and clock resetting. Overexpression studies show that Cry1a acts as a potent repressor of clock function and mimics the effect of constant light to \"stop\" the circadian oscillator. Yeast two-hybrid analysis demonstrates that the Cry1a protein interacts directly with specific regions of core clock components, CLOCK and BMAL, blocking their ability to fully dimerize and transactivate downstream targets, providing a likely mechanism for clock resetting. A comparison of entrainment of zebrafish cells to complete versus skeleton photoperiods reveals that clock phase is identical under these two conditions. However, the amplitude of the core clock oscillation is much higher on a complete photoperiod, as are the levels of light-induced Cry1a. We believe that Cry1a acts on the core clock machinery in both a continuous and discrete fashion, leading not only to entrainment, but also to the establishment of a high-amplitude rhythm and even stopping of the clock under long photoperiods.

Melanopsin (OPN4) is an opsin photopigment that, in mammals, confers photosensitivity to retinal ganglion cells and regulates circadian entrainment and pupil constriction. In non-mammalian species, two forms of opn4 exist, and are classified into mammalian-like ( m ) and non-mammalian-like ( x ) clades. However, far less is understood of the function of this photopigment family. Here we identify in zebrafish five melanopsins ( opn4m - 1, opn4m - 2, opn4m - 3, opn4x - 1 and opn4x - 2) , each encoding a full-length opsin G protein. All five genes are expressed in the adult retina in a largely non-overlapping pattern, as revealed by RNA in situ hybridisation and immunocytochemistry, with at least one melanopsin form present in all neuronal cell types, including cone photoreceptors. This raises the possibility that the teleost retina is globally light sensitive. Electrophysiological and spectrophotometric studies demonstrate that all five zebrafish melanopsins encode a functional photopigment with peak spectral sensitivities that range from 470 to 484 nm, with opn4m-1 and opn4m-3 displaying invertebrate-like bistability, where the retinal chromophore interchanges between cis - and trans -isomers in a light-dependent manner and remains within the opsin binding pocket. In contrast, opn4m-2, opn4x-1 and opn4x-2 are monostable and function more like classical vertebrate-like photopigments, where the chromophore is converted from 11- cis to all- trans retinal upon absorption of a photon, hydrolysed and exits from the binding pocket of the opsin. It is thought that all melanopsins exhibit an invertebrate-like bistability biochemistry. Our novel findings, however, reveal the presence of both invertebrate-like and vertebrate-like forms of melanopsin in the teleost retina, and indicate that photopigment bistability is not a universal property of the melanopsin family. The functional diversity of these teleost melanopsins, together with their widespread expression pattern within the retina, suggests that melanopsins confer global photosensitivity to the teleost retina and might allow for direct “fine-tuning” of retinal circuitry and physiology in the dynamic light environments found in aquatic habitats.

Specific stages of the cell cycle are often restricted to particular times of day because of regulation by the circadian clock. In zebrafish, both mitosis (M phase) and DNA synthesis (S phase) are clock-controlled in cell lines and during embryo development. Despite the ubiquitousness of this phenomenon, relatively little is known about the underlying mechanism linking the clock to the cell cycle. In this study, we describe an evolutionarily conserved cell-cycle regulator, cyclin-dependent kinase inhibitor 1d (20 kDa protein, p20), which along with p21, is a strongly rhythmic gene and directly clock-controlled. Both p20 and p21 regulate the G1/S transition of the cell cycle. However, their expression patterns differ, with p20 predominant in developing brain and peak expression occurring 6 h earlier than p21 . p20 expression is also p53-independent in contrast to p21 regulation. Such differences provide a unique mechanism whereby S phase is set to different times of day in a tissue-specific manner, depending on the balance of these two inhibitors.

The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell types and fluorescent markers.

The methyl cycle is a universal metabolic pathway providing methyl groups for the methylation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deficiencies. Fustin et al. reveal the evolutionarily conserved link between methyl metabolism and biological clocks. This study suggests the possibility of translating fundamental understanding of methylation deficiencies to clinical applications.

The circadian clock ensures that behavioral and physiological processes occur at appropriate times during the 24-hour day/night cycle, and is regulated at both the cellular and organismal levels. To identify pathways acting on intact animals, we performed a small molecule screen using a luminescent reporter of molecular circadian rhythms in zebrafish larvae. We identified both known and novel pathways that affect circadian period, amplitude and phase. Several drugs identified in the screen did not affect circadian rhythms in cultured cells derived from luminescent reporter embryos or in established zebrafish and mammalian cell lines, suggesting they act via mechanisms absent in cell culture. Strikingly, using drugs that promote or inhibit inflammation, as well as a mutant that lacks microglia, we found that inflammatory state affects circadian amplitude. These results demonstrate a benefit of performing drug screens using intact animals and provide novel targets for treating circadian rhythm disorders.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.