The clock components Period2, Cryptochrome1a, and Cryptochrome2a function in establishing light-dependent behavioral rhythms and/or total activity levels in zebrafish

Jun Hirayama,Yusuke Maruyama,Yikelamu Alifu,Sachiko Nishina,Ken Takamatsu,Yoichi Asaoka,Ken-Ichi Nakahama,Sho Yamaguchi,Jun Tomita,Nobuhiko Takamatsu,Kazuhiko Kume,Atsuo Kawahara,Hiroshi Nishina,Noriyuki Azuma,Rin Hamabe,Teruya Tamaru,Atsuhiko Hattori

doi:10.1038/s41598-018-37879-8

Abstract

The circadian clock generates behavioral rhythms to maximize an organism’s physiological efficiency. Light induces the formation of these rhythms by synchronizing cellular clocks. In zebrafish, the circadian clock components Period2 (zPER2) and Cryptochrome1a (zCRY1a) are light-inducible, however their physiological functions are unclear. Here, we investigated the roles of zPER2 and zCRY1a in regulating locomotor activity and behavioral rhythms. zPer2/zCry1a double knockout (DKO) zebrafish displayed defects in total locomotor activity and in forming behavioral rhythms when briefly exposed to light for 3-h. Exposing DKO zebrafish to 12-h light improved behavioral rhythm formation, but not total activity. Our data suggest that the light-inducible circadian clock regulator zCRY2a supports rhythmicity in DKO animals exposed to 12-h light. Single cell imaging analysis revealed that zPER2, zCRY1a, and zCRY2a function in synchronizing cellular clocks. Furthermore, microarray analysis of DKO zebrafish showed aberrant expression of genes involved regulating cellular metabolism, including ATP production. Overall, our results suggest that zPER2, zCRY1a and zCRY2a help to synchronize cellular clocks in a light-dependent manner, thus contributing to behavioral rhythm formation in zebrafish. Further, zPER2 and zCRY1a regulate total physical activity, likely via regulating cellular energy metabolism. Therefore, these circadian clock components regulate the rhythmicity and amount of locomotor behavior.

Highlights

Diurnal animals display increased locomotor activity during the day and reduced locomotor activity at night[1]
The two clock genes zCry2a and zCry2b, as well as zCry1a and zPer[2], were among the 40 genes most strongly induced in double KO (DKO) animal exposed to 12-h light (Supplementary Table S1). zCry2a and zCry2b are evolutionarily related to zCry1a (Fig. 2B), and we previously reported that zCRY2a and zCRY2b transcriptionally repress the CLOCK(NPAS2):BMAL complex[33]
We identified target genes of interest whose expression levels change in the same direction between zebrafish that form behavioral rhythms (i.e. wild type (WT) and DKO animals exposed to 12-h light) and those that do not (i.e. DKO animals exposed to 3-h light, and triple KO (TKO) animals exposed to 3-h or 12-h light)

Summary

Introduction

Diurnal animals display increased locomotor activity during the day and reduced locomotor activity at night[1]. The circadian clock regulates sleep and resting behavior in organisms ranging from bacteria to humans, generating daily physiological rhythms that have been proposed to control the timing of sleep and locomotor activity, but not their total amount[4,5,6,7]. Light directly resets asynchronous cellular clocks to a common phase by suppressing their transcriptional activities[20,21,22] This in vitro system facilitates studies on the photic responses of clock genes encoding cellular clock regulators and has revealed several cellular signaling pathways involved in the light response of cellular clocks[23,24,25,26,27]

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Results

Conclusion