Light-Dependent Regulation of Circadian Clocks in Vertebrates

Abstract

Circadian clocks are intrinsic time-tracking systems that endow organisms with a survival advantage. The core of the circadian clock mechanism is a cell-autonomous and self-sustained oscillator called a cellular clock, which operates via a transcription-/translation-based negative feedback loop. Under natural conditions, circadian clocks are entrained to a 24-hour day by environmental time cues, most commonly light. In mammals, circadian clocks are regulated by cellular clocks located in the central nervous system, such as the suprachiasmatic nucleus (SCN), and in other peripheral tissues. Importantly, mammals have no photoreceptors in the peripheral tissues; therefore the effect of light on peripheral clocks is indirect. By striking contrast, zebrafish peripheral cellular clocks are directly light responsive. This characteristic of the zebrafish cellular clock has contributed to the identification of molecules and signaling pathways that are involved in the light-dependent regulation of the cellular clock. Here, selected light-dependent regulatory mechanisms of circadian clocks in mammals and zebrafish are described.

1. Introduction

Circadian clocks constitute ubiquitous processes that regulate various biochemical and physiological events that occur with 24-hour periodicity, even in the absence of external cues [1]. The exact timing of this rhythm is established by cell-autonomous mechanisms, called cellular clocks, which are controlled by a transcription-/translation-based negative feedback loop [2, 3]. In both vertebrates and invertebrates, cellular clocks are scattered throughout their bodies; thus, the circadian system comprises both central and peripheral oscillators [4].

To guarantee that an organism’s behavior remains tied to the rhythms of its environment, the circadian clock must respond to environmental stimuli to be reset [5]. The main cue for animals is light, which is provided by the day-night cycle. It has been proposed that in mammals the light-induced resetting of the circadian clock is dependent on transcription activation in the suprachiasmatic nucleus (SCN), where the central clock is located [6]. The mammalian route for the regulation of the circadian clock by light uses the retinohypothalamic tract (RHT), which connects directly to the central clock located in the SCN [7]. This makes it difficult to understand the mechanisms underlying light regulation of the circadian clock at a cellular level. Thus, although changes in gene expression have been implicated in the light-induced phase shift of the circadian clock [6, 8], the induction of the expression of clock genes by light and the exact mechanism by which these gene products work remain to be elucidated at the cellular level.

Zebrafish peripheral clocks display a striking characteristic in that they are directly light responsive [9, 10]. Light induces the expression of clock genes and the circadian expression of several clock-related genes in zebrafish peripheral cells [11]. In addition, zebrafish embryonic cell lines can recapitulate the light-response characteristics of a vertebrate clock. In these cell lines, the oscillations of clock gene expression can be entrained to a new light-dark cycle, showing that cultured zebrafish cells have the clock components required for light-induced circadian clock resetting, and the cultured cell system thus provides a valuable tool for studying the light-dependent regulation of the circadian clock at a cellular level [12, 13]. Zebrafish cellular clocks can be studied in cultured cells, which facilitate the study of the photic responses of clock genes encoding cellular-clock regulators, and have revealed cellular signaling pathways that are involved in the light-dependent regulation of the cellular clock [14, 15, 16, 17, 18]. Additionally, an increased understanding of light-dependent cellular-clock regulation in zebrafish has suggested intriguing associations among the circadian clock, DNA repair, and cell cycle control [19, 20, 21, 22, 23].

Here we describe selected light-dependent regulatory aspects of vertebrate circadian machinery.

2. Cellular-clock regulation in mammals

In mammals, the cellular clock comprises the CLOCK, NPAS2, BMAL1, BMAL2, PER1, PER2, CRY1, and CRY2 proteins [1, 24]. These cellular clock components are called clock proteins. CLOCK or NPAS2 proteins heterodimerize with BMALs to form an active transcription complex that transactivates clock-controlled genes, including Crys and Pers. Once the CRY and PER proteins have been translated, they are translocated to the nucleus, where they inhibit CLOCK (NPAS2):BMAL-mediated transcription through a direct protein-protein interaction, setting up the rhythmic gene expression that drives the circadian clock. The CLOCK(NPAS2):BMAL complex also stimulates expression of the clock-controlled genes (Ccgs) to regulate various elements of physiology. This accounts in part for the presence of circadian rhythms in a variety of physiological processes [25]. Although the relatively straightforward mechanism of positive and negative feedback loops is necessary to establish and maintain circadian clocks, cellular clocks have further levels of complexity, including posttranscriptional regulation, posttranslational modification, chromatin remodeling, availability and stability of clock proteins, and regulation of intracellular localization. These regulatory mechanisms provide an interface that can be used as an entry point for stimuli that can reset or control the clock. In addition, genetic studies of genes encoding cellular-clock regulators have revealed distinct roles for clock proteins in regulating circadian clocks, as well as direct links between the circadian clock and various pathologies [26, 27, 28].

3. Photoreceptors for circadian-clock regulation in mammals

Circadian clocks regulate various biochemical, physiological, and behavioral processes with a periodicity of approximately 24 hours. Under natural conditions, circadian rhythms are entrained to this 24-hour day by environmental time cues, with light level being the most important [5]. The eye is the principal mediator of light input to the central clock in mammals. Rods and cones receive visual information within the retina [29, 30] (Figure 1). These cells, however, are dispensable for photoreception of circadian clocks. Indeed, rodents that lack classical visual responses are still capable of circadian photoentrainment [31]. Retrograde tracing experiments have identified retinal cells projecting to the SCN through the RHT, but not to the visual centers of the brain [32]. These cells constitute a small subset of retinal ganglion cells (RGCs) localized in the ganglion cell layer (GCL), and they have been shown to display intrinsic phototransduction abilities, with photic properties matching those of clock entrainment [33]. The main candidate for the circadian photoreceptor is melanopsin, which is an opsin found in the eye and other photoreceptive structures in amphibians and exclusively in retinal RGCs in primates and rodents. Photic information received by RGCs is conveyed through the retinohypothalamic tract to the SCN central clock in mammals [33, 34].

Figure 1.

Retinal cells responsible for vision and photoreception for circadian clock regulation.

4. Light-dependent transcription in the mammalian retina

Dopamine is the major catecholamine in the vertebrate retina and plays a central role in neural adaptation to light. Indeed, light stimulates the synthesis, turnover, and release of retinal dopamine, which makes dopamine an important mediator of light signaling to retinal cellular clocks [35, 36, 37]. Among the members of the dopamine-receptor family, the dopamine D2 receptor (D2R) has been shown to control light-induced reset of the circadian clock in the mouse retina [38, 39, 40]. At the molecular level, it has been reported that signaling mediated by the D2R enhances the transcriptional capacity of the CLOCK:BMAL complex. This effect involves the extracellular signal-regulated kinase (ERK)/MAPK transduction cascade and is associated with a D2R-induced increase in phosphorylation of the transcriptional coactivator, cAMP-responsive element-binding protein (CREB) and its recruitment to the CLOCK:BMAL complex [40]. Importantly, this activation of CLOCK:BMAL1-dependent transcription is responsible for the induction of the Per1 gene by light in the retina, which is in turn responsible for the reset of the retinal cellular clock. These findings provide evidence for the physiological links among the ERK/MAPK signaling pathway, dopamine, and the light input pathway of circadian clocks.

5. Light-dependent transcription in mammalian SCN

Light resets the circadian clock by its phase-shifting properties. In particular, the phase-shifting effects of light only occur during the nighttime period of the circadian cycle. In nocturnal mammals kept in darkness, a light pulse during the subjective night (that is, the time of day corresponding to the dark period in a normal light-dark cycle) can reset the clock by evoking changes in the SCN-controlled rhythms [41, 42, 43]. If the light pulse is given at an early point in time during the subjective night, it induces a shift in SCN-controlled rhythms to a later time (phase delay). Conversely, if the light pulse is provided at the end of the subjective night, the SCN-controlled rhythms will be shifted to an earlier position in the circadian cycle (phase advance). Photic signals perceived by the retina are conveyed to the SCN through the RHT [32]. Glutamate has been identified as the major neurotransmitter responsible for transducing the photic information to the SCN along the RHT [44] (Figure 2). Once glutamate is released by the SCN, it binds to N-methyl-d-aspartate (NMDA) receptors, which in turn leads to the Ca²⁺ influx, that is, finally responsible for the activation of calcium-/calmodulin-dependent protein kinase (CaMK).

Figure 2.

Signaling cascade transducing photic signal perceived by the retina to the transcription of clock genes in SCN.

The involvement of the ERK/MAPK pathway in the light-input system of the circadian clock in the SCN has been well established. Mice exposed to light pulses during their subjective night display rapid ERK upregulation (phosphorylation) in the SCN [45]. Furthermore, disruption of the MAPK pathway has been shown to block light-induced phase shifting of the circadian clock at the behavioral level [46]. This finding suggests that the ERK cascade is integrally involved in photic entrainment of mammalian circadian rhythms. Events downstream of the light-induced signaling pathway in the SCN lead to the phosphorylation of cAMP-response element-binding protein (CREB), which then stimulates expression of Per1 and Per2 genes, which contain a calcium-/cAMP-response element (CRE) in their promoters [6, 47, 48]. Although the exact mechanism by which light induces early gene expression remains to be elucidated, it has been shown that a single light pulse engenders chromatin remodeling via the phosphorylation of histone H3 at Ser10 [49].

6. Light-dependent synchronization of cellular clocks in the mammalian SCN

The appropriate synchronization of cellular clocks in tissues and organs is required for the generation of circadian rhythms in a variety of physiological processes, such as sleep and metabolism [50]. In addition, the light-dependent induction of Per1 and Per2 is thought to contribute to the synchronization of cellular clocks in the SCN [6, 8]. However, this idea has not been fully elucidated using adequate genetically modified mice. Mouse Per1 and Per2 genes are induced by the CLOCK (NPAS2):BMAL complex and by light. In particular, the CLOCK (NPAS2):BMAL-dependent regulation of Per1 and Per2 is essential for establishment of the circadian clock’s rhythmicity. Thus, genetic inhibition of both mouse Per1 and Per2 genes disrupts the cellular clock, preventing the analysis of synchronization [51, 52]. This problem has been solved by using zebrafish models, as described below.

7. Cellular-clock regulation in zebrafish

The zebrafish constitutes an attractive alternative to the mammalian system with which to study the complexity of circadian clock machinery and light’s influence on it [9]. Characterization of the molecular components of the zebrafish circadian oscillator has revealed that the negative feedback loop in zebrafish consists of components similar to those of mammals [11]. Organ- and tissue-culture explant experiments have demonstrated that peripheral circadian oscillators are present throughout the tissues and organs of the zebrafish and that they display the remarkable feature of being light responsive [10, 13].

The characterization of components of the zebrafish cellular clock has revealed duplication of most clock genes. There are two, three, four, and eight homologues of the Clock, Bmal, Per, and Cry genes, respectively. Their circadian expression profiles and light inducibility are different, indicating the differential contribution of various clock components in the regulation of cellular clocks [28, 53, 54, 55]. For example, an investigation into the in vitro functions of the protein products of zebrafish Cry genes revealed that they fall into one of two groups: one group inhibits CLOCK:BMAL-mediated transcription (repressor-type CRYs: zCRY1a, zCRY1b, zCRY2a, and zCRY2b), while the other group does not inhibit transcription (non-repressor type CRYs: zCRY3, zCRY4, zCRY Dash, and plant-type zCRY).

The CLOCK (NPAS2):BMAL complex and/or light regulates the expression of zebrafish repressor types of Crys and Pers [50, 56]. The zCry2a and zCry2b genes are induced both by the CLOCK (NPAS2):BMAL complex and by light; zCry1b, zPer1a, zPer1b, and zPer3 are induced by the CLOCK (NPAS2):BMAL complex but not by light; and zCry1a and zPer2 are induced by light but not by the CLOCK (NPAS2):BMAL complex. These distinct dependencies of zPer and zCry gene expressions recently enabled us to uncover the role of light-induced zPER2, zCRY1a, and zCRY2a in the light-dependent synchronization of cellular clocks.

8. zPER2, zCRY1a, and zCRY2a are required for the light-dependent ontology of circadian clocks during development

In vertebrates, cellular clocks in zygotes and early embryos are not functional and become gradually set in motion during development [57, 58]. In mammals, it is quite difficult to analyze the processes of cellular-clock formation during development because embryogenesis proceeds inside the maternal uterus. Thus, the molecular mechanisms underlying the establishment of cellular clocks during vertebrate development are not well understood. Zebrafish eggs are externally fertilized and are transparent [11, 54]. In addition, zebrafish embryos develop rapidly from fertilized eggs to larvae that swim, making them an excellent model for studies investigating the ontology of vertebrate clocks.

During zebrafish development, organogenesis is completed within 2 days postfertilization (dpf) [59]. Zebrafish larvae hatch within four dpf and start to display locomotor behavior. Zebrafish cellular clocks are autonomously set in motion during development within 1–4 dpf but are out of phase with each other in tissues and organs. Light synchronizes the phases of the cellular clocks to establish behavioral rhythms [50, 60]. Our recent study generated zCry1a^−/−zPer2^−/−zCry2a^−/− triple knockout (TKO) zebrafish and used these TKO animals to show that light-induced zPER2, zCRY1a, and zCRY2a help to synchronize cellular clocks in early embryos and larvae in a light-dependent manner, thus contributing to behavioral rhythm formation in zebrafish larva [50]. Notably, these findings provide evidence that light-dependent-induced PER1 and PER2 contribute to the synchronization of cellular clocks in the SCN of mammals.

9. Light signaling pathway regulating cellular clocks in zebrafish cells

Studies using cultured zebrafish cells have identified cellular signaling cascades involved in the light-dependent regulation of cellular clocks. In several organisms, external stimuli are connected to a cell’s nucleus via MAPK signaling pathways [61]. There are three major MAPKs: c-JUN N-terminal kinase (JNK), p38, and ERK. Light has been reported to activate these signaling cascades in zebrafish cells. Using a pharmacological approach, it was established that light-induced zPer2 transactivation requires the ERK signaling pathway [15]. It has also been proposed that light-induced ERK activation triggers zCry1a transcription, whereas light-induced p38 activation suppresses it, highlighting a MAPK-mediated cross-regulatory mechanism for the expression of circadian-clock genes [21]. As mentioned above, evidence strongly suggests the involvement of the ERK pathway in the light-input system of the mammalian circadian clock. Thus, these findings are consistent with the idea that several aspects of the complex mammalian photo-signal transduction pathway involved in the regulation of circadian clocks are more easily investigated, both pharmacologically and molecularly, using cultured zebrafish cells. In addition, it was reported that the light-activated JNK signaling pathway induces expression of zCry1a and zPer2 [62]. Notably, in contrast to these studies, it has recently been reported that the light-activated p38 pathway facilitates the expression of zCry1a and zPer2 and that the ERK/MAPK signaling pathway is not involved in the light-induced expression of zCry1a and zPer2 [62, 63]. The reason for these contradictory results is unknown.

10. Role of redox signaling in cellular-clock regulation by light in zebrafish

It has been proposed that the light-dependent transcription of zCry1a and zPer2 is controlled through the production and removal of cellular reactive oxygen species (ROS) [16]. ROS were originally thought to act solely as toxic metabolites, because they react with components of DNA, proteins, and lipids and exert oxidative stress [64]. However, ROS are also ideally suited to be signaling molecules because they are small and can easily diffuse over short distances within a cell. In addition, mechanisms for ROS production and their rapid removal (for example, via catalase) are present in almost all cell types [64, 65]. In various organisms, light induces ROS production, which leads to an altered redox status in cells [28]. In zebrafish cells, this light-induced redox change transduces photic signals and leads to the transactivation of zCry1a and zPer2 [16, 62, 66]. Importantly, light increases intracellular catalase activity by increasing the expression of catalase, an event that occurs after the maximum expression of the zCry1a and zPer2 genes has been reached [16]. This increased catalase activity diminishes light-induced cellular ROS levels, resulting in decreased expression of the zCry1a and zPer2 genes.

The toxic effects of oxidative stress have been linked to cellular ROS production induced by light-activated flavin-containing oxidases [67]. The absorbance of light in the near violet-blue region by these enzymes activates them and induces photoreduction of the flavin adenine dinucleotide (FAD) moiety, leading to ROS production. Accordingly, signaling by flavoproteins frequently induces a change in the redox state of cells [67]. Recent studies have provided evidence that flavin-containing oxidases are responsible for the light-dependent production of ROS that are second messengers coupling photoreception to photoreactivation and the circadian clock in zebrafish [62, 66] (Figure 3).

Figure 3.

Light signaling pathway regulating clock gene induction in zebrafish.

11. Link between circadian clocks and light-dependent DNA repair in zebrafish

Solar radiation has both beneficial and harmful effects for most species. Beneficial aspects include its role in photosynthesis and the entrainment of circadian clocks [28]. However, the UV component of solar radiation can produce cytotoxic, mutagenic, and carcinogenic lesions in DNA, which can transform or kill cells. In particular, the UV component of solar radiation produces cytotoxic and mutagenic lesions in DNA called cyclobutane pyrimidine dimers (CPDs) and pyrimidine [6-4] pyrimidone photoproducts. Photoreactivation is a light-dependent DNA repair mechanism mediated by DNA photolyases (PHRs), which bind to and repair UV-induced DNA damage using visible light as an energy source [43, 68]. Two classes of PHRs have been identified, one specific for CPDs (CPD PHRs) and the other specific for [6-4] photoproducts (64PHRs). Importantly, both the induction of PHRs in response to light and the subsequent light-dependent repair of DNA by PHRs are essential for successful photoreactivation in zebrafish cells [21]. Notably, the expression level of the z64Phr gene is regulated by the same light-induced MAPK cascades as those controlling the expression of the clock gene zCry1a, which is associated with the light-dependent regulation of the circadian clock [21, 66]. Light-induced ERK activation triggers the expression of z64Phr, whereas light-induced p38 activation inhibits it. Thus, both light-dependent DNA repair and regulation of the circadian clock are governed by shared regulatory pathways. Both CRYs and PHRs belong to the DNA photolyase/cryptochrome protein family and have highly similar amino acid sequences [43, 68]. Evolutionary studies have shown that the animal CRY protein functionally diverged first from the CPD photolyase and then further to generate 64PHR [69]. These facts, together with the observation that zCry1a and z64Phr share regulatory pathways, strongly indicate an evolutionary link between the circadian clock and DNA repair.

12. Conclusion

In mammals, light signals are received by the retina and then integrated with the SCN cellular clocks [7]. The SCN cellular clocks then transmit light information to peripheral cellular clocks via humoral signals and synchronize them. Recent studies have reported that factors other than cellular clocks in the SCN can synchronize peripheral cellular clocks in a light-dependent manner [42]. In contrast, in zebrafish, light directly synchronizes peripheral cellular clocks in addition to central cellular clocks [9]. Despite the differences between the light-dependent regulation of peripheral cellular clocks in mammals and zebrafish, both require similar MAPK signaling pathways and light induction of clock genes to regulate cellular clocks in a light-dependent manner.

The development of circadian clocks would be one way to segregate daytime from nighttime processes, with light-dark cycles acting as selective pressures [28]. In this scenario, increasing levels of oxygen free radicals during the daytime may have been a decisive factor in relegating the anabolic processes of mitosis, growth, and consolidation to the dark hours. Thus, it is reasonable to propose that redox signaling and stress responding pathways such as MAPKs are utilized in the light-dependent regulation of the circadian clock.

Acknowledgments

This work was supported in part by a Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research [19K12900 (J.I.) and 18KT0068 (J.H.)]. This work was also supported by grants from the Watanabe Foundation and the Smoking Research Foundation (J.H.).