**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):BMALdependent 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, 53–55]. 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 (nonrepressor 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 z*Per2* 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*<sup>−</sup>/<sup>−</sup> *zPer2*<sup>−</sup>/<sup>−</sup> *zCry2a*<sup>−</sup>/<sup>−</sup> 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

**27**

*Light-Dependent Regulation of Circadian Clocks in Vertebrates*

light-dependent-induced PER1 and PER2 contribute to the synchronization of

**9. Light signaling pathway regulating cellular clocks in zebrafish cells**

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

decreased expression of the *zCry1a* and *zPer2* genes.

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

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

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 lightinduced expression of *zCry1a* and *zPer2* [62, 63]. The reason for these contradictory

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

cellular clocks in the SCN of mammals.

results is unknown.

**zebrafish**

*Chronobiology - The Science of Biological Time Structure*

**7. Cellular-clock regulation in zebrafish**

able feature of being light responsive [10, 13].

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 remark-

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, 53–55]. 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 z*Per2* 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** 

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

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*<sup>−</sup>/<sup>−</sup> *zPer2*<sup>−</sup>/<sup>−</sup> *zCry2a*<sup>−</sup>/<sup>−</sup> 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

**ontology of circadian clocks during development**

for studies investigating the ontology of vertebrate clocks.

**26**

light-dependent-induced PER1 and PER2 contribute to the synchronization of cellular clocks in the SCN of mammals.
