A cell-based system that recapitulates the dynamic light-dependent regulation of the vertebrate clock

Abstract

The primary hallmark of circadian clocks is their ability to entrain to environmental stimuli. The dominant, and therefore most physiologically important, entraining stimulus comes from environmental light cycles. Here we describe the establishment and characterization of a new cell line, designated Z3, which derives from zebrafish embryos and contains an independent, light-entrainable circadian oscillator. Using this system, we show distinct and differential light-dependent gene activation for several central clock components. In particular, activation of Per2 expression is shown to be strictly regulated and dependent on light. Furthermore, we demonstrate that Per1, Per2, and Per3 all have distinct responses to light-dark (LD) cycles and light-pulse treatments. We also show that Clock, Bmal1, and Bmal2 all oscillate under LD and dark-dark conditions with similar kinetics, but only Clock is significantly induced while initiating a light-induced circadian oscillation in Z3 cells that have never been exposed to a LD cycle. Finally, our results suggest that Per2 is responsible for establishing the phase of a circadian rhythm entraining to an alternate LD cycle. These findings not only underscore the complexity by which central clock genes are regulated, but also establishes the Z3 cells as an invaluable system for investigating the links between light-dependent gene activation and the signaling pathways responsible for vertebrate circadian rhythms.I n recent years, a new and exciting dimension has been added to our knowledge of the vertebrate circadian clock system. The classical view of the circadian system describes it as diverse physiological rhythms, which are regulated by a centralized clock structure (1, 2). Over the past few years, the idea that the clock consists exclusively of a few centralized structures has been challenged. Data coming from both vertebrate and invertebrate systems have demonstrated that the circadian timing system is dispersed throughout the animal (3-7), and that possibly every cell contains a functional circadian clock (8). In these studies, it has been revealed that a variety of tissues and cells contain functional autonomous clocks. These clocks are able to maintain an oscillation when placed in vitro and removed from any external cues or signals that originate from the classical clock structures and͞or the environment.The discovery of a number of genes involved in the generation and maintenance of circadian oscillations (9, 10) and the recent realization that the circadian system consists of a complex network of independent clocks, which are somehow synchronized to properly regulate all physiological rhythms (5,11,12), has necessitated the development of new tools and methodologies for deciphering the circadian system. An ideal tool is an in vitro cell-based system that displays robust circadian rhythms. Cultured cells may be used to fully understand the complex molecular mechanisms, signal coupling, and regulatory feedback loops that are responsible for the proper timing of a circa...

Circadian rhythms in Mexican blind cavefish Astyanax mexicanus in the lab and in the field

“…2a,c). The expression of cry1a and per2 is strongly induced in surface fish, similar to previous reports in zebrafish 22,[34][35][36] . This acute light response is greatly reduced, though still statistically significant, in Pachón cavefish and does not occur in Chica cavefish.…”

“…In this study, we have explored, under both laboratory and field conditions, how the circadian clock has evolved in this species. In the laboratory, surface fish show a robust circadian oscillation in per1 gene expression, as has been previously reported for zebrafish and other teleost species 20,31,32,34,42 . A high-amplitude per1 rhythm is also apparent in surface fish under natural conditions.…”

“…Cry1a and per2 have been shown in teleosts to be important for light resetting of the circadian clock 22,33 . These genes are primarily regulated by light and are thus good markers of light sensitivity 22,[34][35][36] . Sequence analysis reveals a high degree of conservation between the three populations of Astyanax in all clock genes isolated.…”

A Common Origin: Signaling Similarities in the Regulation of the Circadian Clock and DNA Damage Responses

“…In addition, cultured lines of embryonal zebrafish cells, which recapitulate most features of the zebrafish clock system, have been established as an attractive vertebrate cell-based model suitable for the examination of the light signaling pathway and its impact on the circadian clock. 56) Studies using these cell lines have revealed critical roles for redox control and MAPK signaling pathways in light-dependent circadian entrainment 57) (Fig. 2).…”

Functional and Structural Analyses of Cryptochrome

“…The immunoprecipitated materials and whole cell extracts were analyzed by immunoblotting with anti-VP16 (zCRY1a and chimera 17), anti-zCRY3 (chimeras 11, 18, and zCRY3), anti-V5 (zB-MAL3), or anti-FLAG antibodies (zCLOCK1). The abbreviations c 5,11,12,13,14,16,17, and 18 respectively indicate chimeras 5, 11, 12, 13, 14, 16, 17, and 18. localization (Fig. 4C, lanes 7-18).…”