A cell-based system that recapitulates the dynamic light-dependent regulation of the vertebrate clock Matthew P. Pando, Anna B. Pinchak, Nicolas Cermakian, and Paolo Sassone-Corsi* Institute de Ge´ne´ tique et de Biologie Mole´culaire et Cellulaire, Centre National de la Recherche Scientifique–Institut National de la Sante´et de la Recherche Me´dicale–Universite´Louis Pasteur, 1 Rue Laurent Fries, 67404 Illkirch, Strasbourg, France Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved July 6, 2001 (received for review May 9, 2001) The primary hallmark of circadian clocks is their ability to entrain at oscillating RNA and protein levels, protein localization, and to environmental stimuli. The dominant, and therefore most phys- posttranslational modifications that play a role in maintaining an iologically important, entraining stimulus comes from environmen- oscillation (8). However, this system is unable to offer any tal light cycles. Here we describe the establishment and character- assistance in the study of light-dependent signaling and mech- ization of a new cell line, designated Z3, which derives from anisms that regulate circadian rhythms. zebrafish embryos and contains an independent, light-entrainable We have previously demonstrated that the zebrafish is a very circadian oscillator. Using this system, we show distinct and dif- attractive system for studying light-dependent circadian rhyth- ferential light-dependent gene activation for several central clock micity in vitro (3, 4, 13). The ability to look at light-dependent components. In particular, activation of Per2 expression is shown signaling, oscillation, and entrainment has yet to be accom- to be strictly regulated and dependent on light. Furthermore, we plished by any in vitro mammalian system. These are all aspects demonstrate that Per1, Per2, and Per3 all have distinct responses to of the vertebrate circadian system that can be studied in ze- light–dark (LD) cycles and light-pulse treatments. We also show brafish-based in vitro systems. In the present study, we describe that Clock, Bmal1, and Bmal2 all oscillate under LD and dark–dark a newly developed, highly light-responsive, zebrafish cell line that conditions with similar kinetics, but only Clock is significantly displays robust and tightly regulated circadian oscillations. In this induced while initiating a light-induced circadian oscillation in Z3 cell line, designated Z3, we observe distinct and differential cells that have never been exposed to a LD cycle. Finally, our results light-dependent gene activation for several central clock com- suggest that Per2 is responsible for establishing the phase of a ponents. The gene expression profiles displayed under various circadian rhythm entraining to an alternate LD cycle. These findings light cycles and conditions further elucidate specific light- not only underscore the complexity by which central clock genes dependent regulation of clock genes and their functions. are regulated, but also establishes the Z3 cells as an invaluable system for investigating the links between light-dependent gene Materials and Methods activation and the signaling pathways responsible for vertebrate Fish and Embryo Harvest. Zebrafish were fed twice daily and kept circadian rhythms. at 29°C. They were maintained under a 14-h day, 10-h night cycle. Mating tanks were set up just before the beginning of the n recent years, a new and exciting dimension has been added night phase. Embryos were collected the following morning, just after the beginning of the day phase. The collected embryos were Ito our knowledge of the vertebrate circadian clock system. The Ϸ classical view of the circadian system describes it as diverse kept at 29°C and allowed to age for 24 h. physiological rhythms, which are regulated by a centralized clock structure (1, 2). Over the past few years, the idea that the clock Cell Culture. Embryo isolation, dispersion, and culture prepara- consists exclusively of a few centralized structures has been tion was done by using the following procedure. The 24-h challenged. Data coming from both vertebrate and invertebrate embryos were rinsed in 0.5% bleach for 2 min and then rinsed systems have demonstrated that the circadian timing system is three times in sterile PBS. After the final rinse in PBS, embryos dispersed throughout the animal (3–7), and that possibly every were manually dechorionated by using sterile forceps. Dechori- cell contains a functional circadian clock (8). In these studies, it onated embryos were then transferred to a tissue culture hood has been revealed that a variety of tissues and cells contain and placed in a sterile beaker containing sterile PBS and rinsed functional autonomous clocks. These clocks are able to maintain two times for 5 min. The PBS from the final wash was removed, and the embryos were dissociated by placing the embryos in an oscillation when placed in vitro and removed from any ͞ external cues or signals that originate from the classical clock 0.25% trypsin at a concentration of 50 embryos ml and incu- structures and͞or the environment. bating them at room temperature. Trypsinization was accom- The discovery of a number of genes involved in the generation panied by manual dispersion by pipetting the embryos 5–10 times and maintenance of circadian oscillations (9, 10) and the recent through a P1000 (Gilson) every 3–5 min. Dissociation was realization that the circadian system consists of a complex continued until mostly single cells were obtained. The cell network of independent clocks, which are somehow synchro- suspension was rinsed two times in 10 ml of L15 medium nized to properly regulate all physiological rhythms (5, 11, 12), supplemented with 15% FCS, 2 mM glutamine, gentamycin, streptamycin, and penicillin (GIBCO͞BRL). After the final has necessitated the development of new tools and methodolo- ϫ gies for deciphering the circadian system. An ideal tool is an in rinse in L15, the cells were spun down at 300 g and resus- pended in complete L15 medium at a concentration of 20 vitro cell-based system that displays robust circadian rhythms. ͞ Cultured cells may be used to fully understand the complex embryos ml. Then 5 ml of the resuspended culture was placed molecular mechanisms, signal coupling, and regulatory feedback loops that are responsible for the proper timing of a circadian This paper was submitted directly (Track II) to the PNAS office. oscillation. A remarkable example of such a system is the serum Abbreviations: Bmal, brain and muscle ARNT-like; CT, circadian time; Cry, cryptochrome; shock-induced circadian oscillation that can be initiated in DD, dark–dark; LD, light–dark; Per, period; ZT, Zeitgeber time. immortalized cells (8). The physiological relevance of this system *To whom reprint requests should be addressed. E-mail: [email protected]. was bolstered by the demonstration that an in vitro rhythm in The publication costs of this article were defrayed in part by page charge payment. This cultured rat liver and lung could be reinitiated by a simple article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. medium change (5). The serum shock system is ideal for looking §1734 solely to indicate this fact. 10178–10183 ͉ PNAS ͉ August 28, 2001 ͉ vol. 98 ͉ no. 18 www.pnas.org͞cgi͞doi͞10.1073͞pnas.181228598 Downloaded by guest on September 24, 2021 in sealed 25-cm2 flasks and placed in an incubator maintained at 25°C. Cultures were replated at a 1:2 dilution once reaching confluence. Over the first several passages, a subpopulation able to survive under the given culture conditions grew out and established the Z3 line. RNA Analysis. Total RNA extraction was done by using RNA-Solv (Omega Bio-tek, Doraville, GA) as recommended by the man- ufacturer. A miniaturized RNase protection assay was per- formed as described (3). RNA was equilibrated on agarose gels by ethidium bromide staining. The zebrafish Clock, Bmal1, and Bmal2 probes for RNase protection assay were generated as described (3, 13). Zebrafish Per1, Per2, and Per3 probes corre- sponding to nucleotides 1–223, 1981–2369, and 1218–1572 of each respective ORF were used, and all riboprobes were made by using an in vitro transcription kit (Promega). Each point was prepared from one confluent 25-cm2 flask of Z3 cells. Lighting and Temperature Control. Cultured flasks were kept in a water-jacketed, thermostatically controlled, and light-sealed in- cubator. Illumination was achieved by using a halogen light source fed into the incubator through a fiber optic line. A programmable timer connected to the light source controlled the light cycles. All light–dark (LD) cycles consisted of 12 h of light and 12 h of dark, unless otherwise noted. Results Oscillating Gene Expression in Z3 Cells Exposed to a LD Cycle. Recent Fig. 1. Oscillation of clock components under a LD cycle. RNase protection CELL BIOLOGY analysis of Per1, Per2, Per3, Clock, Bmal1, and Bmal2 gene expression in Z3 cells findings (4) demonstrate that peripheral tissues in the zebrafish entrained for 5 days under a 12:12 LD cycle. RNA was harvested at the are directly light-responsive in vitro. With the aim of creating a indicated ZTs during day 6 of the LD cycle. The bar above indicates light (white) cell-based system to study the molecular mechanisms that reg- and dark (black) periods. tRNA serves as a negative control reaction (t). ulate light-entrainable circadian rhythms, we established a new Relative amounts of total RNA used for each sample are displayed (RNA). The zebrafish cell line designated Z3. The Z3 cell line was derived ZT0 and ZT24 represent two independent samples that were harvested at the from 24-h-old zebrafish embryos and maintained in constant represented dark to light transition points. darkness. To determine whether or not the Z3 cells were light-responsive, we placed them on a 12:12 LD cycle for 5 days before harvesting RNA to study possible oscillations in gene Oscillating Gene Expression in Z3 Cells Is Conserved in Constant expression.