Light-Dependent Regulation of Circadian Clocks in Vertebrates

*Izawa Junko, Yoshimi Okamoto-Uchida, Akari Nishimura and Jun Hirayama*

#### **Abstract**

Circadian clocks are intrinsic time-tracking systems that endow organisms with a survival advantage. The core of the circadian clock mechanism is a cellautonomous 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 lightdependent regulation of the cellular clock. Here, selected light-dependent regulatory mechanisms of circadian clocks in mammals and zebrafish are described.

**Keywords:** circadian clock, cellular clock, zebrafish, light, photolyase

#### **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

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**References**

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 [14–18]. 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 [19–23].

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 [26–28].

#### **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,

**23**

**Figure 1.**

*Light-Dependent Regulation of Circadian Clocks in Vertebrates*

hypothalamic tract to the SCN central clock in mammals [33, 34].

**ipRGC**

**Light**

Strong

Gap junction

G ju

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 retino-

**SCN**

**RHT**

**Cone cell**

*DOI: http://dx.doi.org/10.5772/intechopen.86524*

Biopolar cells

**Rod cell** Weak

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

RGCs

#### *Light-Dependent Regulation of Circadian Clocks in Vertebrates DOI: http://dx.doi.org/10.5772/intechopen.86524*

*Chronobiology - The Science of Biological Time Structure*

**2. Cellular-clock regulation in mammals**

circadian machinery.

various pathologies [26–28].

products work remain to be elucidated at the cellular level.

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

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 [14–18]. 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 [19–23]. Here we describe selected light-dependent regulatory aspects of vertebrate

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

**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,

**22**

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].
