- Email: [email protected]
Analysis of Circadian Rhythms in Zebrafish
186
genetic approaches to circadian clocks
[5]
[5] Analysis of Circadian Rhythms in Zebrafish By Jun Hirayama, Maki Kaneko, Luca Cardone, Gregory Cahill, and Paolo Sassone-Corsi Abstract
The zebrafish probably constitutes the best animal system to study the complexity of the circadian clock machinery and the influence that light has on it. The possibilities of producing transgenic fishes, to establish lightresponsive cultured cells, and to directly explore light phototransduction on single clock cells are all remarkable features of this circadian system. This article describes some of the most useful methodologies to analyze the behavioral, cellular, and molecular aspects of the zebrafish circadian clock system.
Introduction
The zebrafish (Danio rerio) constitutes an attractive alternative to the mouse in the study of circadian rhythms in vertebrates. In addition, the zebrafish is useful for a comparative analysis of the molecular organization of the circadian clock in various systems, thereby providing essential information into the mechanisms governing rhythmicity (Cahill, 2002; Pando et al., 2002). In the zebrafish, the pineal gland and the retina have been identified as the primary pacemakers that regulate its physiology and behavior (Cahill, 1996), although to date no functional equivalent to the SCN (suprachiasmatic nucleus) has been described in the zebrafish system. Both pineal gland and retina are directly light entrainable and contain circadian oscillators that drive rhythmic melatonin synthesis. As in mammals, the zebrafish circadian system is composed of both central and peripheral clocks (Schibler and Sassone-Corsi, 2002). 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 (Cahill, 1996; Cermakian et al., 2000; Whitmore et al., 1998, 2000). In addition, cultured lines of embryonal zebrafish cells have been established, which display light responsiveness and an intrinsic autonomous clock mechanism (Pando et al., 2001; Whitmore et al., 2000). The Z3 cell line, which recapitulates most features of the zebrafish clock system (Pando et al., 2001), has been instrumental for the dissection of the intracellular
METHODS IN ENZYMOLOGY, VOL. 393
Copyright 2005, Elsevier Inc. All rights reserved. 0076-6879/05 $35.00
[5]
circadian rhythms in zebrafish
187
signaling pathways implicated in light transduction and clock function (Cermakian et al., 2002). Characterization of the molecular components of the zebrafish circadian oscillator has revealed duplications for most clock genes. There are three homologs of Clock genes (Ishikawa et al., 2002; Whitmore et al., 1998), three Bmal1 (Cermakian et al., 2000; Ishikawa et al., 2002), four Per (Pando et al., 2001; Vallone et al., 2004), and six Cry genes (Cermakian et al., 2002; Kobayashi et al., 2000). CLOCK:BMAL heterodimers provide the central transcriptional potential that drives the cell autonomous clocks. In zebrafish, both Clock and Bmal display rhythmic oscillations in gene expression, which, on average, peak early during the night phase. Both genes are expressed in most tissues of the animal but display differences in the peak, levels, and kinetics of expression. Expression variations are also observed for the same gene when comparing between different tissues (Cermakian et al., 2000). This suggests that the exact composition of CLOCK:BMAL heterodimers changes during time and between tissues. The two zfBmal genes are most divergent in their carboxy-terminal transcription activation domains. This is thought to allow the central transcription complex of the circadian oscillator to have precise control over its transcriptional potential, facilitating the proper response to general and tissue-specific entraining stimuli. Similarly, circadian expression profiles, light inducibility, and regulation of the numerous Per and Cry genes are also differential (Cermakian et al., 2002; Kobayashi et al., 2000; Pando et al., 2001), indicating the high molecular complexity of the zebrafish oscillator and suggesting a differential contribution of the various components in clock regulation for various peripheral tissues. General Methods
The following general guidelines are used in various laboratories, although slight variations are common. It is useful to refer to methods described (Westerfield, 1995) and to recommandations presented and updated in the ZFIN (The Zebrafish Information Network) Web site (http:// www.grs.nig.ac.jp:6070/). Fish Food. Fish are fed twice daily. The food consists either of ground dry trout pellets or of dry flake such as Tetra brand or daphnia, both of which are available at most pet stores. Water. Adult fish are maintained in distilled water, to which a small amount of salts and minerals is added. The water is heated at 26–29 ,
188
genetic approaches to circadian clocks
[5]
filtered, and recycled continuously. Embryos and young larvae are raised and maintained in egg water (60 mg/L Instant ocean in deionized water, pH 7.0, conductivity 100 S/cm, aerated at least 12 h). Light/Dark Cycle. Fish are classically maintained in a 14 light:10 dark cycle. Because zebrafish is photoperiodic in breeding, keeping a proper cycle is important for embryo preparation. Fish Dissection. Fish are killed by rapid immersion in chilled water followed by decapitation. After dissection, tissues are immediately frozen on dry ice for subsequent analysis. Ex Vivo Organ Study Zebrafish peripheral tissues (e.g., heart, kidney, and spleen) have been shown to be light responsive even when explanted from the animal and isolated in a culture dish. This remarkable feature allows direct entrainment of peripheral circadian clocks, independently from the central clock system (Cermakian et al., 2000; Whitmore et al., 1998, 2000). Therefore, ex vivo tissue systems represent a very useful tool to study light-dependent circadian gene induction. Materials. Prepare forceps, six-well plates, anatomical microscope, and culture medium (composition: L15 medium supplemented with 15% fetal calf serum, 2 mM glutamine, gentamycin, streptamycin, and penicillin). Procedure 1. Raise and sacrifice fish as described earlier. 2. Isolate tissues with sharp forceps from an individual fish between ZT9 and ZT12 (Zeitgeber time, ZT0 corresponding to light on and ZT14 to light off) under an anatomical microscope. 3. Place the dissected tissues in a six-well plate in L15 medium. 4. Keep the tissues at 25 and atmospheric CO2 concentration. Preparation of Embryo Breeding. Adult zebrafish (aged between 7 and 18 months) should be used for breeding. Zebrafish lay eggs every morning, shortly after sunrise. It is not advisable to collect embryos more than 2 days in a row from the same couple of fish. In our experience, zebrafish breed better if fed adult brine shrimp (Artemia sp.) once a day. Procedure 1. Keep males and females in separate tanks with up to 8 females or 16 males per 10-gal tank. Clean the tank once per day by replacing onethird water.
[5]
circadian rhythms in zebrafish
189
2. On the breeding day, feed the fish and clean the tank 1 or 2 h before the end of the light period. Transfer the males to the tank with the females at a rate of one male to two females. 3. Add marbles to cover the bottom of the tank. 4. After the beginning of the next light cycle, collect the embryos (see later). 5. Transfer the males back to their tank, and scoop out the marbles with a net and clean them by autoclaving. Collecting Embyros Material. Prepare a siphon made of a plastic or glass tube (1 cm i.d. and 30–50 cm long) covered at one end with a piece of tygon tubing. Procedure 1. Draw water with a siphon through a medium-mesh nylon net, sweeping the bottom of the tank from side to side. 2. Invert the net over a petri dish filled with egg water to let the embryos fall off into the dish. 3. Culture embryo at 26 under atmospheric CO2 concentration. Locomotor Activity Rhythms
Locomotor (swimming) activity is rhythmic in larval (5–20 day old) zebrafish maintained individually in 0.5-ml wells for a week or more in constant conditions. An automated infrared video image analysis system was developed for high-throughput recording of these rhythms. The system described here can monitor simultaneously the activity of up to sixty-three 5- to 12-day-old fish that have never been fed or up to 150 larger, 10- to 20-day-old fish that have been fed Paramecium ad libitum from day 5 to days 10–12. The maximum numbers are limited by the size of the image (related to the volume of water required to keep fish healthy in the absence of water changes) and the resolution of the digitized image. This system has been used to screen for clock mutations, to characterize mutant phenotypes, and to characterize the development of circadian rhythmicity in zebrafish (Cahill, 1998, 2002; DeBruyne et al., 2004; Hurd and Cahill, 2002). Strong behavioral rhythmicity can be recorded from larvae of AB and SJD strains; less robust rhythmicity was observed in C32, TU, and some pet store strains. During activity monitoring, larval zebrafish are maintained in a rectangular array of oval wells drilled in a translucent white polyethylene specimen plate. To avoid disturbance, the fish are not fed and water is
190
genetic approaches to circadian clocks
[5]
Fig. 1. Video-recording apparatus for monitoring behavioral rhythmicity of larval zebrafish. The diffuse axial illuminator ensures even illumination and eliminates glare from the water surface.
not changed during activity recording. The temperature is lowered to 24 to slow metabolism, and virtually all fish survive and can be recovered after a week. A CCD video camera is focused on the plate from above, and an infrared diffuse axial illuminator is used to produce even illumination without glare from the water surface (Fig. 1). The specimen plate is placed on a mirror to backlight the wells. The key imaging challenge is to ensure that all fish are darker than all background so that a single threshold can be set for automated object identification. Image acquisition and analysis are controlled by customized software running on a desktop computer. For each activity sample, a series of images is captured at a rate of 1/s, digitized, and stored in RAM (Fig. 2). The position of each fish in each image is determined, and the distance moved during the sample period is calculated from the series of coordinates. Data are stored to a text file, the images are erased from memory, and a new cycle of image capture and analysis is initiated. We typically sample 30–60 s of activity every 4 min and then average six samples to produce activity records with 24-min resolution. Materials Light-tight refrigerated incubator maintained at 24 ; monochrome video camera with a 50-mm macro lens, and a 1.7-cm CCD sensor with the IR-blocking filter removed, automatic gain control, shading correction
[5]
circadian rhythms in zebrafish
191
Fig. 2. Measurement of larval zebrafish behavioral rhythms by automated video image analysis. (A) Infrared video image of 6 wells of the 150-well specimen plate with 10-day-old zebrafish. (B) The image is divided into a grid of cells, each containing 1 well. (C) Thresholds are set to detect fish. (D) Simulation of paths swum by fish during a 1-min sampling period.
and horizontal center resolution >750 TVL; a desktop computer with a 640 480 pixel, 8-bit gray scale frame capture card (Flashpoint 128), and a Windows operating system; Optimate 6.2 (MediaCybernetics, Silver Spring, MD) image processing software with the Swimming1.1 macro (Meyer Instruments, Houston, TX); a diffuse axial illuminator with infrared light source (custom made, but smaller versions are available commercially from Edmund Industrial Optics, Barrington, NJ; RVSI/NER, Weare, NH; or Advanced Illumination, Rochester, VT), a translucent white polyethylene specimen plate, 18.5 cm 13.5 cm 8 mm thick, with oval wells, 12 6 7 mm, arranged in 10 15 array with 1-mm-thick walls; an 18.5 13.5-cm flat mirror; egg water; and large-bore transfer pipettes. Procedure 1. Raise zebrafish to desired age (up to 100/1-liter beaker) at 28.5 in a 14:10 LD cycle. Change water daily and feed liberally with Paramecium from day 5 to the day before transferring them to the recording apparatus. 2. In the morning, wash larvae repeatedly with fresh egg water and starve the fish for at least 6 h before transferring them to recording wells.
192
genetic approaches to circadian clocks
[5]
3. Strain fish from the beaker with fine nylon mesh and wash into a 100-mm petri plate with fresh egg water. 4. Use a large-bore pipette to transfer fish to wells filled with fresh egg water. 5. Install the specimen plate on the reflecting surface of the mirror and under the diffuse axial illuminator (Fig. 1). The camera and illumination system are installed in the incubator, which is humidified by bubbling air through a large open reservoir of water. 6. Launch Optimate and the Swimming macro (Fig. 3), and click on Acquire to view the live-digitized video. Focus the camera so that the array of occupied wells fills the image field. 7. Set the camera controller to use automatic gain control. Use the four shading correction dials on the camera controller to even out any variation in background shading from center to periphery, side to side, or up and down. Freeze the image and open the Monochrome Threshold dialog window; pixels with values between the lower and the upper threshold will be highlighted. Set the lower threshold to 0 and scan the upper threshold down until all fish are below and all background is above the upper threshold. 8. With the image frozen, click and drag to set the Region of Interest, a rectangle that encompasses all occupied wells. Set the number of Rows and Columns of wells. Use the Test Layout button to superimpose a grid dividing the region of interest into the specified rows and columns onto the image. Repeat these steps if every cell in the grid does not encompass one and only one well. 9. Set sampling parameters, including Images/Cycle, Delay between image captures within a series, Cycle Length, which must be greater than the time required for acquisition and analysis of an image series, and the number of Cycles to Process before automatically shutting down. 10. Create an ASCII Data File with a .txt extension for the measurements. The first column of this tab-delimited file will contain time stamps, and each subsequent column will have data for one well. At the end of the recording period, transfer the data file to your favorite time series analysis program. We find Chrono 4.5.1 (Roenneberg and Taylor, 2000) to be a useful and versatile Macintosh program. Bioluminescence Rhythms in per3-luc Transgenic Zebrafish
In a variety of species, circadian rhythms of bioluminescence have been produced by expressing a luciferase transgene under the control of a clockdriven promoter (Brandes et al., 1996; Millar et al., 1992). This provides a
[5]
circadian rhythms in zebrafish
193
Fig. 3. Control panel for Swimming macro. Copyright Matthew Batchelor.
convenient method for high-throughput assays of molecular circadian rhythmicity. Zebrafish lines carrying a transgene with luciferase driven by the zebrafish period3 promoter(per3-luc) were produced. When incubated with luciferin, live larval fish and cultured adult organs, including retina, heart, spleen, and gall bladder from these transgenic fish, glow rhythmically (Kaneko and Cahill, 2004a,b). The transgene is a modified bacterial artificial chromosome (BAC) with a zebrafish genomic DNA insert that included a 50 coding sequence of the per3 gene as well as >8.3 kb of an upstream sequence (clone 8M06). The per3 sequence from the initiation codon to the end of the first coding exon was replaced with a gene cassette containing luciferase and kanamycin resistance genes (Muyrers et al., 1999). This construct was linearized by digestion with NotI and injected into one to two cell stage embryos
194
genetic approaches to circadian clocks
[5]
(Higashijima et al., 1997). Five germline transformant lines were recovered; luc expression levels were variable among the lines. Luc expression was highest in line 23, and we have focused further studies on this line. These fish will be made available through the Zebrafish International Resource Center (http://zfin.org/zirc).
Bioluminescence Rhythms in Live Larval Zebrafish
Materials TopCount multiplate scintillation counter (Perkin-Elmer) in a refrigerated incubator maintained at 22 , white 96-well Optiplates (Perkin-Elmer), TopsealA sheets (Perkin-Elmer), Holtfreter/luciferin solution (7.0 g NaCl, 0.4 g NaHCO3, 0.2 g CaCl2, 0.1 g KCl in 21 ml ddH2O, pH 7.0, with 0.5 mM d-luciferin potassium salt and 0.013% Amquel Instant Water Detoxifier) aerated overnight, and a large-bore pipette. Procedure 1. Raise zebrafish to 6 days of age at 22 under a 14:10 LD cycle. 2. Pipette fish into every other well of Optiplates. 3. Replace water in each occupied well with 200 L Holtfreter/ luciferin solution. 4. Cover each plate with a sheet of TopsealA and perforate over each occupied well twice with a 23-gauge needle. 5. Install loaded plates in the TopCount plate stacker and set the TopCount to monitor each well for 4.8 s every 30 min. 6. At the end of the experiment, transfer data to your favorite time series analysis program. Bioluminescence Rhythms in Cultured Organs
Materials TopCount multiplate scintillation counter (Perkin-Elmer) in a refrigerated incubator maintained at desired temperature (20–32 ), white 96-well Optiplates (Perkin-Elmer), TopsealA sheets, dissecting medium [Liebowitz L-15 culture medium (GIBCO) diluted 2:1 in ddH2O, 10% fetal bovine serum (FBS), 10% antibiotic/antimycotic solution (GIBCO)], and culture medium [Liebowitz L-15 culture medium (GIBCO) diluted 2:1 in ddH2O, 10% FBS, 1% penicillin/streptomycin (GIBCO), 0.5 mM luciferin potassium salt].
[5]
circadian rhythms in zebrafish
195
Procedure 1. Maintain adult zebrafish at 28.5 under a 14:10 LD cycle. 2. Dissect organs into dissecting medium. 3. Transfer organs to every other well of Optiplates with 260 l of culture medium. 4. Cover each plate with a sheet of TopsealA. 5. Install loaded plates in the TopCount plate stacker and set the TopCount to monitor each well for 15 s every hour. 6. At the end of the experiment, transfer data to your favorite time series analysis program. Recapitulating the Zebrafish Clock in Cultured Cells
The most remarkable and unique feature of the zebrafish system is the ability to respond to light. Several clock-related genes show circadian expression in ex vivo peripheral tissues like heart, kidney, and spleen (Whitmore et al., 1998, 2000) and in zebrafish-derived cell lines such as the Z3 line (Pando et al., 2001). Remarkably, circadian gene expression in Z3 cells can be synchronized (i.e., entrained) by light, proving that zebrafish cells contain not only a circadian oscillator, but also phototransduction mechanisms sufficient for light entrainment. In particular, we have established a zebrafish cell line, designated Z3, which derives from zebrafish embryos, and have demonstrated that several circadian clock components display distinct and differential light-dependent activation and expression profiles under various light conditions (Pando et al., 2001). Z3 cells, therefore, nicely recapitulate the zebrafish clock system and constitute an invaluable tool for investigating the link between light-dependent gene activation and the signaling pathways responsible for the generation of vertebrate circadian rhythms. Establishment of the Z3 Cell Line
Materials Prepare sterile forceps, hood, sterile beaker, 0.25% trypsin, 0.5% bleach, 1 phosphate-buffered saline (PBS), L15 medium [GIBCO/BRL, supplemented with 15% fetal calf serum (FCS), 2 mM glutamine, gentamycin, streptamycin, and penicillin], 25-cm2 flasks, and incubator (26 , atmospheric CO2 concentration). Procedure 1. Rinse the 24-h-old embryos in 0.5% bleach for 2 min and then rinse three times in sterile PBS.
196
genetic approaches to circadian clocks
[5]
2. After the final rinse in PBS, dechorionate manually the embryos using sterile forceps. 3. Transfer dechorionated embryos to a tissue culture hood and place them in a sterile beaker containing sterile PBS and rinse twice for 5 min. 4. Place the embryos in 0.25% trypsin at a concentration of 50 embryos/ml and incubate them at room temperature to dissociate them. During trypsinization, pipette the embryos 5–10 times through a P1000 (Gilson) every 3–5 min, until mostly single cells are obtained. 5. Rinse the cell suspension twice in 10 ml of culture medium. 6. Spin down the cells at 300g and resuspend in complete L15 medium at a concentration of 20 embryos/ml. 7. Place 5 ml of the resuspended cells in sealed 25-cm2 flasks and place in an incubator maintained at 26 and atmospheric CO2 concentration. 8. Split cells at a 1:2 dilution once reaching confluence. After several passages, the Z3 cellular subpopulation is able to survive under the given culture conditions by growing steadily (Fig. 4). Culture Conditions for Z3 Cells
Temperature and CO2 concentration: 26 and atmospheric CO2 concentration. Medium: L-15 medium (GIBCO-BRL), containing 10% FCS and gentamycin. Incubator: should be water jacketed, thermostatically controlled, and light sealed (Fig. 5A). Control of light/dark conditions:
Fig. 4. Confluent Z3 cells in culture. Magnification 100.
[5]
circadian rhythms in zebrafish
197
Fig. 5. Incubator for Z3 cell culture. (A) Z3 cells exposed to light. Illumination is achieved by a halogen light (short arrow) fed into the incubator through a fiber-optic line (long arrow). The incubator is water jacketed, thermostatically controlled, and light sealed. (B) A programmable timer, which controls light–dark cycles (short arrow) and the halogen light source (long arrow).
Illumination is achieved by using a halogen light source fed into the incubator through a fiber-optic line (Fig. 5A). A programmable timer connected to the light source controls the light cycles (Fig. 5B). Passage: Tripsinize and split cells at a 1:3 dilution once reaching confluence. Under the culture conditions described, Z3 cells should attain confluence at 3–4 days after passage. Passages should be done under the dark, using a red lamp. Other Zebrafish-Derived Cells for Circadian Studies
In addition to Z3 cells, two other zebrafish cell lines have been employed for the study of the circadian clock: PAC2 cells, established by
198
genetic approaches to circadian clocks
[5]
Nancy Hopkins at the Massachussets Institute of Technology (Cambridge, USA), and BRF41 cells (Ishikawa et al., 2002). These lines are derived from the embryo and caudal fin, respectively, and are cultured under conditions highly similar to Z3 cells. Molecular Methods
RNA Isolation RNASolv reagent (Omega Bio-tek) can be used for isolating RNA from Z3 cells as well as from zebrafish tissues. RNA Analysis The RNase protection assay (RPA) has been essential for studying circadian clock gene expression at the RNA level in zebrafish because of its robustness, reproducibility, sensitivity, and reliability (Cermakian et al., 2000; Ishikawa et al., 2002; Kobayashi et al., 2000; Pando et al., 2001; Whitmore et al., 1998, 2000). To visualize circadian clock gene expression within fish tissues, in situ hybridization has also been applied successfully (Cermakian et al., 2000; Whitmore et al., 2000). These methodologies have been described in detail elsewhere (Macho and Sassone-Corsi 2003; Sassoon and Rosenthal 1993; Tessarollo and Parada 1995). DNA sequences selected for protection or in situ analyses are cloned into a suitable vector to obtain the antisense probe by RNA polymerase. A suitable restriction site is required for linearization of the plasmid that would give rise to probes in the range of 100–400 nucleotides. To obtain a reliable riboprobe, several fragments need to be tested. Successfully utilized riboprobes for zebrafish clock genes are listed in Table I. Signaling Inhibitors Treatment To study light-induced signaling pathways leading to the regulation of clock gene expression, a panel of inhibitors can be used. We used this approach on Z3 cells. For example, U0126 and Ro-31-8220, MEK, and PKC inhibitors, respectively, have been shown to block Per2 light induction (Cermakian et al., 2002). To study Per2 induction, inhibitors are added directly on confluent Z3 cells. After a 1-h treatment, cells are exposed to light and then harvested at each time point of interest for RNA analysis. Inhibitors are dissolved in dimethyl sulfoxide (DMSO). For control experiments, cells are exposed to DMSO without the inhibitor. It is important to note that the treatment should be done under a dim red light. Similar approaches can be used to analyze the signaling pathways utilized by the clock machinery at different times of the circadian cycle, as well as to explore the transduction routes implicated in the temperature compensation process.
[5]
199
circadian rhythms in zebrafish TABLE I Riboprobes for Expression Study of Zebrafish Circadian Clock Genes
Gene
From–to
Protected fragment
Ref.
zfClock1 zfClock2 zfClock3 zfBma11
639–731 1404–1527 1301–1462 1328–1759
92 123 161 431
Ishikawa et al. (2002) Ishikawa et al. (2002) Ishikawa et al. (2002) Cermakian et al. (2000) Cermakian et al. (2000) Ishikawa et al. (2002) Pando et al. (2001) Pando et al. (2001) Pando et al. (2001) Vallone et al. (2004) Cermakian et al. (2002) Cermakian et al. (2002) Cermakian et al. (2002)
zfBma12
1199–1599
400
zfBma13 zPer1 zPer2 zPer3 zPer4 zCry1a
1646–1971 1–223 1981–2369 1218–1572 265–675 1535–1671
231 222 88 354 410 136
zCry2a
1679–1854
175
zCry1b
1661–1821
200
zCry2b
Plus 40 nt after stop codon 1491–1804
313
zCry3
1767–1797
353
zCry4
Plus 323 nt after stop codon 1621–1677
113
Cermakian et al. (2002)
zfBeta-actin
Plus 56 nt after stop codon 957–1070
113
Kobayashi et al. (2000)
Cermakian et al. (2002) Cermakian et al. (2002)
Action Spectrum Analysis of Z3 Cells To elucidate phototransduction pathways and the respective photoreceptors, the action spectrum analysis is an essential step. For details on how to perform an action spectrum, please look at specialized literature (Payne and Sancar, 1990). This section provides simple information on the preparation of Z3 cells for this analysis. Indeed, we used the direct light-responsiveness of Z3 cells by scoring for Per2 induction to perform the action spectrum of Z3 cells (Cermakian et al., 2002).
200
genetic approaches to circadian clocks
[5]
Materials. Integrated monochromator-actinometer and cuvette (Quantacount Photon Technology International). Procedure 1. Trypsinize Z3 cells and resuspend them in 8 ml of the culture medium. 2. Maintain the cells in suspension by rocking for 4 h. 3. Transfer cells to a cuvette and irradiate light with specific wavelengths through an integrated monochromator–actinometer. 4. Maintain cells in darkness for 2.5 h and then isolate RNA for analysis. Retroviral Infection
Transient transfection efficiency of cultured zebrafish-derived cells is relatively low (Hirayama et al., 2003). To circumvent this problem, we have found that retroviral infection of Z3 cells is a very attractive option. Solutions and Materials 2HBS: Dissolve HEPES (5 g) and NaCl (8 g) in 400 ml sterile MilliQ H2O. Adjust pH to 7.1 with 10 N NaOH and bring to 500 ml. Autoclave and store at room temperature. 100 phosphate solution: Dissolve 4.97 g Na2HPO4, 4.2 g NaH2PO4, and 400 ml sterile MilliQ H2O and bring to 500 ml. Autoclave and store at room temperature. 2 M CaCl2: Dissolve 58.8 g CaCl2 2H2O in sterile MilliQ H2O and bring to 200 ml. Filter sterilize with a 0.2-m filter and store at 4 . 0.1% gelatin (porcine skin, Sigma): Add gelatin (1 g) to 1 liter of sterile MilliQ H2O and autoclave (gelatin will not dissolve until autoclaved). Store at room temperature. 293gagpol packaging cell line : Gagpol selection can be done by adding 200 l of 1 mg/ml blastocidin per 10 ml medium. Medium: DMEM þ 4.5 g/L glucose þ 10% FCS þ gentamycin. Retroviral vectors: The RetroMax expression system (IMGENEX) can be used because it provides several kinds of retroviral vectors, such as pCLXSN, pCLNCX, pCLNRX, and pCLNDX, in which the cloned genes are under the control of SV40, CMV, RSV, and DHFR promoters, respectively. In our experience, pCLNCX seems to be one of the most useful vectors because of the high infection efficiency and the significant gene expression levels revealed in infected Z3 cells (unpublished data). As an
[5]
circadian rhythms in zebrafish
201
enveloping vector, pMD.G/vsv-g is recommended because it is available commercially and has given good results in Z3 cells. Preparation of Virus Solution Gelatin Coat 1. Add 3 ml 0.1% gelatin to a 10-cm plate. 2. Incubate at room temperature for 10 min. 3. Remove gelatin from plate and rinse the plate once with 5 ml PBS. Transfection 1. Split 293 gagpol packaging cells onto gelatin-coated 10-cm plate at 40% confluence and let cells recover overnight at 37 and 5% CO2. 2. Prepare solution A by mixing 7.5 l of 100 phosphate solution and 375 l of 2 HBS. 3. Prepare solution B by mixing 45 l of 2 M CaCl2 and 368 l of DNA containing 20 g of virusvector, 5 g of envelope vector, and 5 g of carrier vector (pBluescript). 4. Add solution B to solution A dropwise while tapping the tube. 5. Mix well and incubate at room temperature for 15 min. 6. Add mixture to cells dropwise and mix gently. 7. Incubate cells at 37 under 5% CO2 for 24 h. Refresh medium to remove growth-inhibiting factors 24 h after transfection and further incubate cells under the same conditions for an additional 24 h. Recovery of Viral Particles 1. Recover all the medium (around 9 ml) from dish and add 1 ml FCS and 10 l 4 mg/ml polybrene. 2. Filter the medium through a 0.22-m (low protein binding) syringe filter. 3. Store at 4 for short-term storage (2 days maximum) and freeze on dry ice and store at 20 for long-term storage. Z3 Cell Infection 1. Split Z3 cells onto a 6-cm plate to 80% confluence and let cells recover overnight at 25 under atmospheric CO2 concentration. 2. Dilute recovered viral particle solution fivefold or more. 3. Remove culture medium from the plate of Z3 cells and add 2.5 ml of diluted virus solution.
202
genetic approaches to circadian clocks
[5]
4. Incubate for 2–3 h at 25 . Remove virus solution and repeat infection four times. 5. Remove virus solution from the plate and replace with usual complete medium for Z3 cells and grow as usual. Score for expression 24–48 h postinfection. Preparation of Nuclear Extracts
Z3 cells represent a valuable tool for the study of general light-dependent gene induction. Using the electrophoretic mobility shift assay (EMSA) with nuclear extract from Z3 cells cultured in either light or dark conditions, light-responsive promoter sequences could be identified. The general method of EMSA (Rimbach et al., 2001) can be applied for the Z3 cell line. We have performed a wide-search analysis for transcription factors whose DNA binding to their respective specific recognition sequence would be induced by light in Z3 cells (J. Hirayama and L. Cardone, unpublished results). This approach proved very successful, and this section describes the methodology used. Solutions Hypotonic buffer (prepare just before use): 10 mM HEPES–KOH (pH 7.8), 10 mM KCl, 0.1 mM EDTA, 1 mM dithiotheitol (DTT), 0.15% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 protease inhibitor cocktail, 50 mM NaF, and 100 M Na3 VO4. Hypertonic buffer (prepare just before use): 20 mM HEPES–NaOH (pH 7.8), 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 protease inhibitor cocktail, 50 mM NaF, and 100 M Na3 VO4. Procedure 1. Rinse Z3 cells cultured in 75-cm2 flask two times in sterile PBS. 2. Add 500 l of hypotonic buffer directly to the cells and scrape and transfer them to a 1.5-ml tube. 3. Leave the tube on ice for 10 min. 4. Centrifuge for 5 min at 700g and 4 and remove the supernatant. The supernatant can be used as the cytosolic fraction. 5. Add 500 l of hypotonic buffer to the precipitate and resuspend. 6. Centrifuge at 700g for 5 min at 4 and remove the supernatant. 7. Add 50 l of hypertonic buffer to the precipitate and shake at 4 for 30 min. 8. Centrifuge at 10,000g for 30 min at 4 .
[5]
circadian rhythms in zebrafish
203
9. Transfer the supernatant to a 1.5-ml tube. The supernatant is the nuclear fraction. 10. Freeze the nuclear extract on dry ice and store at 80 for longterm storage. Normally, the expected yield from a 75-cm2 confluent flask is 50–60 g of total nuclear protein as determined by the Bio-Rad protein assay kit. This procedure could be extended to applications that include immunoprecipitation and coimmunoprecipitation of specific proteins and consequent analysis by Western blotting and chromatin-Immuno precipitation, valuable to unravel the transcriptional complex recruitment to specific gene regulatory elements. References Brandes, C., Plautz, J. D., Stanewsky, R., Jamison, C. F., Straume, M., Wood, K. V., Kay, S. A., and Hall, J. C. (1996). Novel features of Drosophila period transcription revealed by real-time luciferase reporting. Neuron 16, 687–692. Cahill, G. M. (1996). Circadian regulation of melatonin production in cultured zebrafish pineal and retina. Brain Res. 708, 177–181. Cahill, G. M. (1998). Circadian rhythmicity in the locomotor activity of larval zebrafish. Neuroreport 9, 3445–3449. Cahill, G. M. (2002). Clock mechanisms in zebrafish. Cell Tissue Res. 309, 27–34. Cermakian, N., Pando, M. P., Thompson, C. L., Pinchak, A. B., Selby, C. P., Gutierrez, L., Wells, D. E., Cahill, G. M., Sancar, A., and Sassone-Corsi, P. (2002). Light induction of a vertebrate clock gene involves signaling through blue-light receptors and MAP kinases. Curr. Biol. 12, 844–848. Cermakian, N., Whitmore, D., Foulkes, N. S., and Sassone-Corsi, P. (2000). Asynchronous oscillations of two zebrafish CLOCK partners reveal differential clock control and function. Proc. Natl. Acad. Sci. USA 97, 4339–4344. DeBruyne, J., Hurd, M. W., Gutie´ rrez, L., Kaneko, M., Tan, Y., Wells, D. E., and Cahill, G. M. (2004). Characterization and mapping of a zebrafish circadian clock mutant. J. Neurogenet. In press. Higashijima, S.-I., Okamoto, H., Ueno, N., Hotta, Y., and Eguchi, G. (1997). High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev. Biol. 192, 289–299. Hirayama, J., Fukuda, I., Ishikawa, T., Kobayashi, Y., and Todo, T. (2003). New role of zCRY and zPER2 as regulators of sub-cellular distributions of zCLOCK and zBMAL proteins. Nucleic Acids Res. 31, 935–943. Hurd, M. W., and Cahill, G. M. (2002). Entraining signals initiate behavioral circadian rhythmicity in larval zebrafish. J. Biol. Rhythms 17, 307–314. Ishikawa, T., Hirayama, J., Kobayashi, Y., and Todo, T. (2002). Zebrafish CRY represses transcription mediated by CLOCK-BMAL heterodimer without inhibiting its binding to DNA. Genes Cells 7, 1073–1086. Kaneko, M., and Cahill, G. M. (2004a). Development of circadian molecular oscillations revealed by bioluminescence in transgenic zebrafish. Manuscript in preparation.
204
genetic approaches to circadian clocks
[5]
Kaneko, M., and Cahill, G. M. (2004b). Real-time measurement of circadian gene expression in peripheral tissues from transgenic zebrafish. Soc. Res. Biol. Rhythms Abstr. 9, 90. Kobayashi, Y., Ishikawa, T., Hirayama, J., Daiyasu, H., Kanai, S., Toh, H., Fukuda, I., Tsujimura, T., Terada, N., Kamei, Y., Yuba, S., Iwai, S., and Todo, T. (2000). Molecular analysis of zebrafish photolyase/cryptochrome family: Two types of cryptochromes present in zebrafish. Genes Cells 5, 725–738. Macho, B., and Sassone-Corsi, P. (2003). Functional analysis of transcription factors CREB and CREM. Methods Enzymol. 370, 396–415. Millar, A. J., Short, S. R., Chua, N. H., and Kay, S. A. (1992). A novel circadian phenotype based on firefly luciferase expression in transgenic plants. Plant Cell. 4, 1075–1087. Muyrers, J. P. P., Zhang, Y., Testa, G., and Stewart, A. F. (1999). Rapid modification of bacterial chromosomes by ET-recombination. Nucleic Acids Res. 27, 1555–1557. Pando, M. P., Pinchak, A. B., Cermakian, N., and Sassone-Corsi, P. (2001). A cell-based system that recapitulates the dynamic light-dependent regulation of the vertebrate clock. Proc. Natl. Acad. Sci. USA 98, 10178–10183. Pando, M. P., and Sassone-Corsi, P. (2002). Unraveling the mechanisms of the vertebrate circadian clock: Zebrafish may light the way. Bioessays 24, 419–426. Payne, G., and Sancar, A. (1990). Absolute action spectrum of E-FADH2 and E-FADH2MTHF forms of Escherichia coli DNA photolyase. Biochemistry 29, 7715–7727. Rimbach, G., Saliou, C., Canali, R., and Virgili, F. (2001). Interaction between cultured endothelial cells and macrophages: In vitro model for studying flavonoids in redoxdependent gene expression. Methods Enzymol. 335, 387–397. Roenneberg, T., and Taylor, W. (2000). Automated recordings of bioluminescence with special reference to the analysis of circadian rhythms. Methods Enzymol. 305, 104–119. Sassoon, D., and Rosenthal, N. (1993). Detection of messenger RNA by in situ hybridization. Methods Enzymol. 225, 384–404. Schibler, U., and Sassone-Corsi, P. (2002). A web of circadian pacemakers. Cell 111, 919–922. Tessarollo, L., and Parada, L. F. (1995). In situ hybridization. Methods Enzymol. 254, 419–430. Vallone, D., Gondi, S. B., Whitmore, D., and Foulkes, N. (2004). E-box function in a period gene repressed by light. Proc. Natl. Acad. Sci. USA 101, 4106–4111. Westerfield, M. (1995). ‘‘The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio).’’ University of Oregon Press. Whitmore, D., Foulkes, N. S., Strahle, U., and Sassone-Corsi, P. (1998). Zebrafish Clock rhythmic expression reveals independent peripheral circadian oscillators. Nature Neurosci. 1, 701–707. Whitmore, D., Foulkes, N. S., and Sassone-Corsi, P. (2000). Light acts directly on organs and cells in culture to set the vertebrate circadian clock. Nature 404, 87–91.
genetic approaches to circadian clocks
[5]
[5] Analysis of Circadian Rhythms in Zebrafish By Jun Hirayama, Maki Kaneko, Luca Cardone, Gregory Cahill, and Paolo Sassone-Corsi Abstract
The zebrafish probably constitutes the best animal system to study the complexity of the circadian clock machinery and the influence that light has on it. The possibilities of producing transgenic fishes, to establish lightresponsive cultured cells, and to directly explore light phototransduction on single clock cells are all remarkable features of this circadian system. This article describes some of the most useful methodologies to analyze the behavioral, cellular, and molecular aspects of the zebrafish circadian clock system.
Introduction
The zebrafish (Danio rerio) constitutes an attractive alternative to the mouse in the study of circadian rhythms in vertebrates. In addition, the zebrafish is useful for a comparative analysis of the molecular organization of the circadian clock in various systems, thereby providing essential information into the mechanisms governing rhythmicity (Cahill, 2002; Pando et al., 2002). In the zebrafish, the pineal gland and the retina have been identified as the primary pacemakers that regulate its physiology and behavior (Cahill, 1996), although to date no functional equivalent to the SCN (suprachiasmatic nucleus) has been described in the zebrafish system. Both pineal gland and retina are directly light entrainable and contain circadian oscillators that drive rhythmic melatonin synthesis. As in mammals, the zebrafish circadian system is composed of both central and peripheral clocks (Schibler and Sassone-Corsi, 2002). 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 (Cahill, 1996; Cermakian et al., 2000; Whitmore et al., 1998, 2000). In addition, cultured lines of embryonal zebrafish cells have been established, which display light responsiveness and an intrinsic autonomous clock mechanism (Pando et al., 2001; Whitmore et al., 2000). The Z3 cell line, which recapitulates most features of the zebrafish clock system (Pando et al., 2001), has been instrumental for the dissection of the intracellular
METHODS IN ENZYMOLOGY, VOL. 393
Copyright 2005, Elsevier Inc. All rights reserved. 0076-6879/05 $35.00
[5]
circadian rhythms in zebrafish
187
signaling pathways implicated in light transduction and clock function (Cermakian et al., 2002). Characterization of the molecular components of the zebrafish circadian oscillator has revealed duplications for most clock genes. There are three homologs of Clock genes (Ishikawa et al., 2002; Whitmore et al., 1998), three Bmal1 (Cermakian et al., 2000; Ishikawa et al., 2002), four Per (Pando et al., 2001; Vallone et al., 2004), and six Cry genes (Cermakian et al., 2002; Kobayashi et al., 2000). CLOCK:BMAL heterodimers provide the central transcriptional potential that drives the cell autonomous clocks. In zebrafish, both Clock and Bmal display rhythmic oscillations in gene expression, which, on average, peak early during the night phase. Both genes are expressed in most tissues of the animal but display differences in the peak, levels, and kinetics of expression. Expression variations are also observed for the same gene when comparing between different tissues (Cermakian et al., 2000). This suggests that the exact composition of CLOCK:BMAL heterodimers changes during time and between tissues. The two zfBmal genes are most divergent in their carboxy-terminal transcription activation domains. This is thought to allow the central transcription complex of the circadian oscillator to have precise control over its transcriptional potential, facilitating the proper response to general and tissue-specific entraining stimuli. Similarly, circadian expression profiles, light inducibility, and regulation of the numerous Per and Cry genes are also differential (Cermakian et al., 2002; Kobayashi et al., 2000; Pando et al., 2001), indicating the high molecular complexity of the zebrafish oscillator and suggesting a differential contribution of the various components in clock regulation for various peripheral tissues. General Methods
The following general guidelines are used in various laboratories, although slight variations are common. It is useful to refer to methods described (Westerfield, 1995) and to recommandations presented and updated in the ZFIN (The Zebrafish Information Network) Web site (http:// www.grs.nig.ac.jp:6070/). Fish Food. Fish are fed twice daily. The food consists either of ground dry trout pellets or of dry flake such as Tetra brand or daphnia, both of which are available at most pet stores. Water. Adult fish are maintained in distilled water, to which a small amount of salts and minerals is added. The water is heated at 26–29 ,
188
genetic approaches to circadian clocks
[5]
filtered, and recycled continuously. Embryos and young larvae are raised and maintained in egg water (60 mg/L Instant ocean in deionized water, pH 7.0, conductivity 100 S/cm, aerated at least 12 h). Light/Dark Cycle. Fish are classically maintained in a 14 light:10 dark cycle. Because zebrafish is photoperiodic in breeding, keeping a proper cycle is important for embryo preparation. Fish Dissection. Fish are killed by rapid immersion in chilled water followed by decapitation. After dissection, tissues are immediately frozen on dry ice for subsequent analysis. Ex Vivo Organ Study Zebrafish peripheral tissues (e.g., heart, kidney, and spleen) have been shown to be light responsive even when explanted from the animal and isolated in a culture dish. This remarkable feature allows direct entrainment of peripheral circadian clocks, independently from the central clock system (Cermakian et al., 2000; Whitmore et al., 1998, 2000). Therefore, ex vivo tissue systems represent a very useful tool to study light-dependent circadian gene induction. Materials. Prepare forceps, six-well plates, anatomical microscope, and culture medium (composition: L15 medium supplemented with 15% fetal calf serum, 2 mM glutamine, gentamycin, streptamycin, and penicillin). Procedure 1. Raise and sacrifice fish as described earlier. 2. Isolate tissues with sharp forceps from an individual fish between ZT9 and ZT12 (Zeitgeber time, ZT0 corresponding to light on and ZT14 to light off) under an anatomical microscope. 3. Place the dissected tissues in a six-well plate in L15 medium. 4. Keep the tissues at 25 and atmospheric CO2 concentration. Preparation of Embryo Breeding. Adult zebrafish (aged between 7 and 18 months) should be used for breeding. Zebrafish lay eggs every morning, shortly after sunrise. It is not advisable to collect embryos more than 2 days in a row from the same couple of fish. In our experience, zebrafish breed better if fed adult brine shrimp (Artemia sp.) once a day. Procedure 1. Keep males and females in separate tanks with up to 8 females or 16 males per 10-gal tank. Clean the tank once per day by replacing onethird water.
[5]
circadian rhythms in zebrafish
189
2. On the breeding day, feed the fish and clean the tank 1 or 2 h before the end of the light period. Transfer the males to the tank with the females at a rate of one male to two females. 3. Add marbles to cover the bottom of the tank. 4. After the beginning of the next light cycle, collect the embryos (see later). 5. Transfer the males back to their tank, and scoop out the marbles with a net and clean them by autoclaving. Collecting Embyros Material. Prepare a siphon made of a plastic or glass tube (1 cm i.d. and 30–50 cm long) covered at one end with a piece of tygon tubing. Procedure 1. Draw water with a siphon through a medium-mesh nylon net, sweeping the bottom of the tank from side to side. 2. Invert the net over a petri dish filled with egg water to let the embryos fall off into the dish. 3. Culture embryo at 26 under atmospheric CO2 concentration. Locomotor Activity Rhythms
Locomotor (swimming) activity is rhythmic in larval (5–20 day old) zebrafish maintained individually in 0.5-ml wells for a week or more in constant conditions. An automated infrared video image analysis system was developed for high-throughput recording of these rhythms. The system described here can monitor simultaneously the activity of up to sixty-three 5- to 12-day-old fish that have never been fed or up to 150 larger, 10- to 20-day-old fish that have been fed Paramecium ad libitum from day 5 to days 10–12. The maximum numbers are limited by the size of the image (related to the volume of water required to keep fish healthy in the absence of water changes) and the resolution of the digitized image. This system has been used to screen for clock mutations, to characterize mutant phenotypes, and to characterize the development of circadian rhythmicity in zebrafish (Cahill, 1998, 2002; DeBruyne et al., 2004; Hurd and Cahill, 2002). Strong behavioral rhythmicity can be recorded from larvae of AB and SJD strains; less robust rhythmicity was observed in C32, TU, and some pet store strains. During activity monitoring, larval zebrafish are maintained in a rectangular array of oval wells drilled in a translucent white polyethylene specimen plate. To avoid disturbance, the fish are not fed and water is
190
genetic approaches to circadian clocks
[5]
Fig. 1. Video-recording apparatus for monitoring behavioral rhythmicity of larval zebrafish. The diffuse axial illuminator ensures even illumination and eliminates glare from the water surface.
not changed during activity recording. The temperature is lowered to 24 to slow metabolism, and virtually all fish survive and can be recovered after a week. A CCD video camera is focused on the plate from above, and an infrared diffuse axial illuminator is used to produce even illumination without glare from the water surface (Fig. 1). The specimen plate is placed on a mirror to backlight the wells. The key imaging challenge is to ensure that all fish are darker than all background so that a single threshold can be set for automated object identification. Image acquisition and analysis are controlled by customized software running on a desktop computer. For each activity sample, a series of images is captured at a rate of 1/s, digitized, and stored in RAM (Fig. 2). The position of each fish in each image is determined, and the distance moved during the sample period is calculated from the series of coordinates. Data are stored to a text file, the images are erased from memory, and a new cycle of image capture and analysis is initiated. We typically sample 30–60 s of activity every 4 min and then average six samples to produce activity records with 24-min resolution. Materials Light-tight refrigerated incubator maintained at 24 ; monochrome video camera with a 50-mm macro lens, and a 1.7-cm CCD sensor with the IR-blocking filter removed, automatic gain control, shading correction
[5]
circadian rhythms in zebrafish
191
Fig. 2. Measurement of larval zebrafish behavioral rhythms by automated video image analysis. (A) Infrared video image of 6 wells of the 150-well specimen plate with 10-day-old zebrafish. (B) The image is divided into a grid of cells, each containing 1 well. (C) Thresholds are set to detect fish. (D) Simulation of paths swum by fish during a 1-min sampling period.
and horizontal center resolution >750 TVL; a desktop computer with a 640 480 pixel, 8-bit gray scale frame capture card (Flashpoint 128), and a Windows operating system; Optimate 6.2 (MediaCybernetics, Silver Spring, MD) image processing software with the Swimming1.1 macro (Meyer Instruments, Houston, TX); a diffuse axial illuminator with infrared light source (custom made, but smaller versions are available commercially from Edmund Industrial Optics, Barrington, NJ; RVSI/NER, Weare, NH; or Advanced Illumination, Rochester, VT), a translucent white polyethylene specimen plate, 18.5 cm 13.5 cm 8 mm thick, with oval wells, 12 6 7 mm, arranged in 10 15 array with 1-mm-thick walls; an 18.5 13.5-cm flat mirror; egg water; and large-bore transfer pipettes. Procedure 1. Raise zebrafish to desired age (up to 100/1-liter beaker) at 28.5 in a 14:10 LD cycle. Change water daily and feed liberally with Paramecium from day 5 to the day before transferring them to the recording apparatus. 2. In the morning, wash larvae repeatedly with fresh egg water and starve the fish for at least 6 h before transferring them to recording wells.
192
genetic approaches to circadian clocks
[5]
3. Strain fish from the beaker with fine nylon mesh and wash into a 100-mm petri plate with fresh egg water. 4. Use a large-bore pipette to transfer fish to wells filled with fresh egg water. 5. Install the specimen plate on the reflecting surface of the mirror and under the diffuse axial illuminator (Fig. 1). The camera and illumination system are installed in the incubator, which is humidified by bubbling air through a large open reservoir of water. 6. Launch Optimate and the Swimming macro (Fig. 3), and click on Acquire to view the live-digitized video. Focus the camera so that the array of occupied wells fills the image field. 7. Set the camera controller to use automatic gain control. Use the four shading correction dials on the camera controller to even out any variation in background shading from center to periphery, side to side, or up and down. Freeze the image and open the Monochrome Threshold dialog window; pixels with values between the lower and the upper threshold will be highlighted. Set the lower threshold to 0 and scan the upper threshold down until all fish are below and all background is above the upper threshold. 8. With the image frozen, click and drag to set the Region of Interest, a rectangle that encompasses all occupied wells. Set the number of Rows and Columns of wells. Use the Test Layout button to superimpose a grid dividing the region of interest into the specified rows and columns onto the image. Repeat these steps if every cell in the grid does not encompass one and only one well. 9. Set sampling parameters, including Images/Cycle, Delay between image captures within a series, Cycle Length, which must be greater than the time required for acquisition and analysis of an image series, and the number of Cycles to Process before automatically shutting down. 10. Create an ASCII Data File with a .txt extension for the measurements. The first column of this tab-delimited file will contain time stamps, and each subsequent column will have data for one well. At the end of the recording period, transfer the data file to your favorite time series analysis program. We find Chrono 4.5.1 (Roenneberg and Taylor, 2000) to be a useful and versatile Macintosh program. Bioluminescence Rhythms in per3-luc Transgenic Zebrafish
In a variety of species, circadian rhythms of bioluminescence have been produced by expressing a luciferase transgene under the control of a clockdriven promoter (Brandes et al., 1996; Millar et al., 1992). This provides a
[5]
circadian rhythms in zebrafish
193
Fig. 3. Control panel for Swimming macro. Copyright Matthew Batchelor.
convenient method for high-throughput assays of molecular circadian rhythmicity. Zebrafish lines carrying a transgene with luciferase driven by the zebrafish period3 promoter(per3-luc) were produced. When incubated with luciferin, live larval fish and cultured adult organs, including retina, heart, spleen, and gall bladder from these transgenic fish, glow rhythmically (Kaneko and Cahill, 2004a,b). The transgene is a modified bacterial artificial chromosome (BAC) with a zebrafish genomic DNA insert that included a 50 coding sequence of the per3 gene as well as >8.3 kb of an upstream sequence (clone 8M06). The per3 sequence from the initiation codon to the end of the first coding exon was replaced with a gene cassette containing luciferase and kanamycin resistance genes (Muyrers et al., 1999). This construct was linearized by digestion with NotI and injected into one to two cell stage embryos
194
genetic approaches to circadian clocks
[5]
(Higashijima et al., 1997). Five germline transformant lines were recovered; luc expression levels were variable among the lines. Luc expression was highest in line 23, and we have focused further studies on this line. These fish will be made available through the Zebrafish International Resource Center (http://zfin.org/zirc).
Bioluminescence Rhythms in Live Larval Zebrafish
Materials TopCount multiplate scintillation counter (Perkin-Elmer) in a refrigerated incubator maintained at 22 , white 96-well Optiplates (Perkin-Elmer), TopsealA sheets (Perkin-Elmer), Holtfreter/luciferin solution (7.0 g NaCl, 0.4 g NaHCO3, 0.2 g CaCl2, 0.1 g KCl in 21 ml ddH2O, pH 7.0, with 0.5 mM d-luciferin potassium salt and 0.013% Amquel Instant Water Detoxifier) aerated overnight, and a large-bore pipette. Procedure 1. Raise zebrafish to 6 days of age at 22 under a 14:10 LD cycle. 2. Pipette fish into every other well of Optiplates. 3. Replace water in each occupied well with 200 L Holtfreter/ luciferin solution. 4. Cover each plate with a sheet of TopsealA and perforate over each occupied well twice with a 23-gauge needle. 5. Install loaded plates in the TopCount plate stacker and set the TopCount to monitor each well for 4.8 s every 30 min. 6. At the end of the experiment, transfer data to your favorite time series analysis program. Bioluminescence Rhythms in Cultured Organs
Materials TopCount multiplate scintillation counter (Perkin-Elmer) in a refrigerated incubator maintained at desired temperature (20–32 ), white 96-well Optiplates (Perkin-Elmer), TopsealA sheets, dissecting medium [Liebowitz L-15 culture medium (GIBCO) diluted 2:1 in ddH2O, 10% fetal bovine serum (FBS), 10% antibiotic/antimycotic solution (GIBCO)], and culture medium [Liebowitz L-15 culture medium (GIBCO) diluted 2:1 in ddH2O, 10% FBS, 1% penicillin/streptomycin (GIBCO), 0.5 mM luciferin potassium salt].
[5]
circadian rhythms in zebrafish
195
Procedure 1. Maintain adult zebrafish at 28.5 under a 14:10 LD cycle. 2. Dissect organs into dissecting medium. 3. Transfer organs to every other well of Optiplates with 260 l of culture medium. 4. Cover each plate with a sheet of TopsealA. 5. Install loaded plates in the TopCount plate stacker and set the TopCount to monitor each well for 15 s every hour. 6. At the end of the experiment, transfer data to your favorite time series analysis program. Recapitulating the Zebrafish Clock in Cultured Cells
The most remarkable and unique feature of the zebrafish system is the ability to respond to light. Several clock-related genes show circadian expression in ex vivo peripheral tissues like heart, kidney, and spleen (Whitmore et al., 1998, 2000) and in zebrafish-derived cell lines such as the Z3 line (Pando et al., 2001). Remarkably, circadian gene expression in Z3 cells can be synchronized (i.e., entrained) by light, proving that zebrafish cells contain not only a circadian oscillator, but also phototransduction mechanisms sufficient for light entrainment. In particular, we have established a zebrafish cell line, designated Z3, which derives from zebrafish embryos, and have demonstrated that several circadian clock components display distinct and differential light-dependent activation and expression profiles under various light conditions (Pando et al., 2001). Z3 cells, therefore, nicely recapitulate the zebrafish clock system and constitute an invaluable tool for investigating the link between light-dependent gene activation and the signaling pathways responsible for the generation of vertebrate circadian rhythms. Establishment of the Z3 Cell Line
Materials Prepare sterile forceps, hood, sterile beaker, 0.25% trypsin, 0.5% bleach, 1 phosphate-buffered saline (PBS), L15 medium [GIBCO/BRL, supplemented with 15% fetal calf serum (FCS), 2 mM glutamine, gentamycin, streptamycin, and penicillin], 25-cm2 flasks, and incubator (26 , atmospheric CO2 concentration). Procedure 1. Rinse the 24-h-old embryos in 0.5% bleach for 2 min and then rinse three times in sterile PBS.
196
genetic approaches to circadian clocks
[5]
2. After the final rinse in PBS, dechorionate manually the embryos using sterile forceps. 3. Transfer dechorionated embryos to a tissue culture hood and place them in a sterile beaker containing sterile PBS and rinse twice for 5 min. 4. Place the embryos in 0.25% trypsin at a concentration of 50 embryos/ml and incubate them at room temperature to dissociate them. During trypsinization, pipette the embryos 5–10 times through a P1000 (Gilson) every 3–5 min, until mostly single cells are obtained. 5. Rinse the cell suspension twice in 10 ml of culture medium. 6. Spin down the cells at 300g and resuspend in complete L15 medium at a concentration of 20 embryos/ml. 7. Place 5 ml of the resuspended cells in sealed 25-cm2 flasks and place in an incubator maintained at 26 and atmospheric CO2 concentration. 8. Split cells at a 1:2 dilution once reaching confluence. After several passages, the Z3 cellular subpopulation is able to survive under the given culture conditions by growing steadily (Fig. 4). Culture Conditions for Z3 Cells
Temperature and CO2 concentration: 26 and atmospheric CO2 concentration. Medium: L-15 medium (GIBCO-BRL), containing 10% FCS and gentamycin. Incubator: should be water jacketed, thermostatically controlled, and light sealed (Fig. 5A). Control of light/dark conditions:
Fig. 4. Confluent Z3 cells in culture. Magnification 100.
[5]
circadian rhythms in zebrafish
197
Fig. 5. Incubator for Z3 cell culture. (A) Z3 cells exposed to light. Illumination is achieved by a halogen light (short arrow) fed into the incubator through a fiber-optic line (long arrow). The incubator is water jacketed, thermostatically controlled, and light sealed. (B) A programmable timer, which controls light–dark cycles (short arrow) and the halogen light source (long arrow).
Illumination is achieved by using a halogen light source fed into the incubator through a fiber-optic line (Fig. 5A). A programmable timer connected to the light source controls the light cycles (Fig. 5B). Passage: Tripsinize and split cells at a 1:3 dilution once reaching confluence. Under the culture conditions described, Z3 cells should attain confluence at 3–4 days after passage. Passages should be done under the dark, using a red lamp. Other Zebrafish-Derived Cells for Circadian Studies
In addition to Z3 cells, two other zebrafish cell lines have been employed for the study of the circadian clock: PAC2 cells, established by
198
genetic approaches to circadian clocks
[5]
Nancy Hopkins at the Massachussets Institute of Technology (Cambridge, USA), and BRF41 cells (Ishikawa et al., 2002). These lines are derived from the embryo and caudal fin, respectively, and are cultured under conditions highly similar to Z3 cells. Molecular Methods
RNA Isolation RNASolv reagent (Omega Bio-tek) can be used for isolating RNA from Z3 cells as well as from zebrafish tissues. RNA Analysis The RNase protection assay (RPA) has been essential for studying circadian clock gene expression at the RNA level in zebrafish because of its robustness, reproducibility, sensitivity, and reliability (Cermakian et al., 2000; Ishikawa et al., 2002; Kobayashi et al., 2000; Pando et al., 2001; Whitmore et al., 1998, 2000). To visualize circadian clock gene expression within fish tissues, in situ hybridization has also been applied successfully (Cermakian et al., 2000; Whitmore et al., 2000). These methodologies have been described in detail elsewhere (Macho and Sassone-Corsi 2003; Sassoon and Rosenthal 1993; Tessarollo and Parada 1995). DNA sequences selected for protection or in situ analyses are cloned into a suitable vector to obtain the antisense probe by RNA polymerase. A suitable restriction site is required for linearization of the plasmid that would give rise to probes in the range of 100–400 nucleotides. To obtain a reliable riboprobe, several fragments need to be tested. Successfully utilized riboprobes for zebrafish clock genes are listed in Table I. Signaling Inhibitors Treatment To study light-induced signaling pathways leading to the regulation of clock gene expression, a panel of inhibitors can be used. We used this approach on Z3 cells. For example, U0126 and Ro-31-8220, MEK, and PKC inhibitors, respectively, have been shown to block Per2 light induction (Cermakian et al., 2002). To study Per2 induction, inhibitors are added directly on confluent Z3 cells. After a 1-h treatment, cells are exposed to light and then harvested at each time point of interest for RNA analysis. Inhibitors are dissolved in dimethyl sulfoxide (DMSO). For control experiments, cells are exposed to DMSO without the inhibitor. It is important to note that the treatment should be done under a dim red light. Similar approaches can be used to analyze the signaling pathways utilized by the clock machinery at different times of the circadian cycle, as well as to explore the transduction routes implicated in the temperature compensation process.
[5]
199
circadian rhythms in zebrafish TABLE I Riboprobes for Expression Study of Zebrafish Circadian Clock Genes
Gene
From–to
Protected fragment
Ref.
zfClock1 zfClock2 zfClock3 zfBma11
639–731 1404–1527 1301–1462 1328–1759
92 123 161 431
Ishikawa et al. (2002) Ishikawa et al. (2002) Ishikawa et al. (2002) Cermakian et al. (2000) Cermakian et al. (2000) Ishikawa et al. (2002) Pando et al. (2001) Pando et al. (2001) Pando et al. (2001) Vallone et al. (2004) Cermakian et al. (2002) Cermakian et al. (2002) Cermakian et al. (2002)
zfBma12
1199–1599
400
zfBma13 zPer1 zPer2 zPer3 zPer4 zCry1a
1646–1971 1–223 1981–2369 1218–1572 265–675 1535–1671
231 222 88 354 410 136
zCry2a
1679–1854
175
zCry1b
1661–1821
200
zCry2b
Plus 40 nt after stop codon 1491–1804
313
zCry3
1767–1797
353
zCry4
Plus 323 nt after stop codon 1621–1677
113
Cermakian et al. (2002)
zfBeta-actin
Plus 56 nt after stop codon 957–1070
113
Kobayashi et al. (2000)
Cermakian et al. (2002) Cermakian et al. (2002)
Action Spectrum Analysis of Z3 Cells To elucidate phototransduction pathways and the respective photoreceptors, the action spectrum analysis is an essential step. For details on how to perform an action spectrum, please look at specialized literature (Payne and Sancar, 1990). This section provides simple information on the preparation of Z3 cells for this analysis. Indeed, we used the direct light-responsiveness of Z3 cells by scoring for Per2 induction to perform the action spectrum of Z3 cells (Cermakian et al., 2002).
200
genetic approaches to circadian clocks
[5]
Materials. Integrated monochromator-actinometer and cuvette (Quantacount Photon Technology International). Procedure 1. Trypsinize Z3 cells and resuspend them in 8 ml of the culture medium. 2. Maintain the cells in suspension by rocking for 4 h. 3. Transfer cells to a cuvette and irradiate light with specific wavelengths through an integrated monochromator–actinometer. 4. Maintain cells in darkness for 2.5 h and then isolate RNA for analysis. Retroviral Infection
Transient transfection efficiency of cultured zebrafish-derived cells is relatively low (Hirayama et al., 2003). To circumvent this problem, we have found that retroviral infection of Z3 cells is a very attractive option. Solutions and Materials 2HBS: Dissolve HEPES (5 g) and NaCl (8 g) in 400 ml sterile MilliQ H2O. Adjust pH to 7.1 with 10 N NaOH and bring to 500 ml. Autoclave and store at room temperature. 100 phosphate solution: Dissolve 4.97 g Na2HPO4, 4.2 g NaH2PO4, and 400 ml sterile MilliQ H2O and bring to 500 ml. Autoclave and store at room temperature. 2 M CaCl2: Dissolve 58.8 g CaCl2 2H2O in sterile MilliQ H2O and bring to 200 ml. Filter sterilize with a 0.2-m filter and store at 4 . 0.1% gelatin (porcine skin, Sigma): Add gelatin (1 g) to 1 liter of sterile MilliQ H2O and autoclave (gelatin will not dissolve until autoclaved). Store at room temperature. 293gagpol packaging cell line : Gagpol selection can be done by adding 200 l of 1 mg/ml blastocidin per 10 ml medium. Medium: DMEM þ 4.5 g/L glucose þ 10% FCS þ gentamycin. Retroviral vectors: The RetroMax expression system (IMGENEX) can be used because it provides several kinds of retroviral vectors, such as pCLXSN, pCLNCX, pCLNRX, and pCLNDX, in which the cloned genes are under the control of SV40, CMV, RSV, and DHFR promoters, respectively. In our experience, pCLNCX seems to be one of the most useful vectors because of the high infection efficiency and the significant gene expression levels revealed in infected Z3 cells (unpublished data). As an
[5]
circadian rhythms in zebrafish
201
enveloping vector, pMD.G/vsv-g is recommended because it is available commercially and has given good results in Z3 cells. Preparation of Virus Solution Gelatin Coat 1. Add 3 ml 0.1% gelatin to a 10-cm plate. 2. Incubate at room temperature for 10 min. 3. Remove gelatin from plate and rinse the plate once with 5 ml PBS. Transfection 1. Split 293 gagpol packaging cells onto gelatin-coated 10-cm plate at 40% confluence and let cells recover overnight at 37 and 5% CO2. 2. Prepare solution A by mixing 7.5 l of 100 phosphate solution and 375 l of 2 HBS. 3. Prepare solution B by mixing 45 l of 2 M CaCl2 and 368 l of DNA containing 20 g of virusvector, 5 g of envelope vector, and 5 g of carrier vector (pBluescript). 4. Add solution B to solution A dropwise while tapping the tube. 5. Mix well and incubate at room temperature for 15 min. 6. Add mixture to cells dropwise and mix gently. 7. Incubate cells at 37 under 5% CO2 for 24 h. Refresh medium to remove growth-inhibiting factors 24 h after transfection and further incubate cells under the same conditions for an additional 24 h. Recovery of Viral Particles 1. Recover all the medium (around 9 ml) from dish and add 1 ml FCS and 10 l 4 mg/ml polybrene. 2. Filter the medium through a 0.22-m (low protein binding) syringe filter. 3. Store at 4 for short-term storage (2 days maximum) and freeze on dry ice and store at 20 for long-term storage. Z3 Cell Infection 1. Split Z3 cells onto a 6-cm plate to 80% confluence and let cells recover overnight at 25 under atmospheric CO2 concentration. 2. Dilute recovered viral particle solution fivefold or more. 3. Remove culture medium from the plate of Z3 cells and add 2.5 ml of diluted virus solution.
202
genetic approaches to circadian clocks
[5]
4. Incubate for 2–3 h at 25 . Remove virus solution and repeat infection four times. 5. Remove virus solution from the plate and replace with usual complete medium for Z3 cells and grow as usual. Score for expression 24–48 h postinfection. Preparation of Nuclear Extracts
Z3 cells represent a valuable tool for the study of general light-dependent gene induction. Using the electrophoretic mobility shift assay (EMSA) with nuclear extract from Z3 cells cultured in either light or dark conditions, light-responsive promoter sequences could be identified. The general method of EMSA (Rimbach et al., 2001) can be applied for the Z3 cell line. We have performed a wide-search analysis for transcription factors whose DNA binding to their respective specific recognition sequence would be induced by light in Z3 cells (J. Hirayama and L. Cardone, unpublished results). This approach proved very successful, and this section describes the methodology used. Solutions Hypotonic buffer (prepare just before use): 10 mM HEPES–KOH (pH 7.8), 10 mM KCl, 0.1 mM EDTA, 1 mM dithiotheitol (DTT), 0.15% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 protease inhibitor cocktail, 50 mM NaF, and 100 M Na3 VO4. Hypertonic buffer (prepare just before use): 20 mM HEPES–NaOH (pH 7.8), 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 protease inhibitor cocktail, 50 mM NaF, and 100 M Na3 VO4. Procedure 1. Rinse Z3 cells cultured in 75-cm2 flask two times in sterile PBS. 2. Add 500 l of hypotonic buffer directly to the cells and scrape and transfer them to a 1.5-ml tube. 3. Leave the tube on ice for 10 min. 4. Centrifuge for 5 min at 700g and 4 and remove the supernatant. The supernatant can be used as the cytosolic fraction. 5. Add 500 l of hypotonic buffer to the precipitate and resuspend. 6. Centrifuge at 700g for 5 min at 4 and remove the supernatant. 7. Add 50 l of hypertonic buffer to the precipitate and shake at 4 for 30 min. 8. Centrifuge at 10,000g for 30 min at 4 .
[5]
circadian rhythms in zebrafish
203
9. Transfer the supernatant to a 1.5-ml tube. The supernatant is the nuclear fraction. 10. Freeze the nuclear extract on dry ice and store at 80 for longterm storage. Normally, the expected yield from a 75-cm2 confluent flask is 50–60 g of total nuclear protein as determined by the Bio-Rad protein assay kit. This procedure could be extended to applications that include immunoprecipitation and coimmunoprecipitation of specific proteins and consequent analysis by Western blotting and chromatin-Immuno precipitation, valuable to unravel the transcriptional complex recruitment to specific gene regulatory elements. References Brandes, C., Plautz, J. D., Stanewsky, R., Jamison, C. F., Straume, M., Wood, K. V., Kay, S. A., and Hall, J. C. (1996). Novel features of Drosophila period transcription revealed by real-time luciferase reporting. Neuron 16, 687–692. Cahill, G. M. (1996). Circadian regulation of melatonin production in cultured zebrafish pineal and retina. Brain Res. 708, 177–181. Cahill, G. M. (1998). Circadian rhythmicity in the locomotor activity of larval zebrafish. Neuroreport 9, 3445–3449. Cahill, G. M. (2002). Clock mechanisms in zebrafish. Cell Tissue Res. 309, 27–34. Cermakian, N., Pando, M. P., Thompson, C. L., Pinchak, A. B., Selby, C. P., Gutierrez, L., Wells, D. E., Cahill, G. M., Sancar, A., and Sassone-Corsi, P. (2002). Light induction of a vertebrate clock gene involves signaling through blue-light receptors and MAP kinases. Curr. Biol. 12, 844–848. Cermakian, N., Whitmore, D., Foulkes, N. S., and Sassone-Corsi, P. (2000). Asynchronous oscillations of two zebrafish CLOCK partners reveal differential clock control and function. Proc. Natl. Acad. Sci. USA 97, 4339–4344. DeBruyne, J., Hurd, M. W., Gutie´ rrez, L., Kaneko, M., Tan, Y., Wells, D. E., and Cahill, G. M. (2004). Characterization and mapping of a zebrafish circadian clock mutant. J. Neurogenet. In press. Higashijima, S.-I., Okamoto, H., Ueno, N., Hotta, Y., and Eguchi, G. (1997). High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev. Biol. 192, 289–299. Hirayama, J., Fukuda, I., Ishikawa, T., Kobayashi, Y., and Todo, T. (2003). New role of zCRY and zPER2 as regulators of sub-cellular distributions of zCLOCK and zBMAL proteins. Nucleic Acids Res. 31, 935–943. Hurd, M. W., and Cahill, G. M. (2002). Entraining signals initiate behavioral circadian rhythmicity in larval zebrafish. J. Biol. Rhythms 17, 307–314. Ishikawa, T., Hirayama, J., Kobayashi, Y., and Todo, T. (2002). Zebrafish CRY represses transcription mediated by CLOCK-BMAL heterodimer without inhibiting its binding to DNA. Genes Cells 7, 1073–1086. Kaneko, M., and Cahill, G. M. (2004a). Development of circadian molecular oscillations revealed by bioluminescence in transgenic zebrafish. Manuscript in preparation.
204
genetic approaches to circadian clocks
[5]
Kaneko, M., and Cahill, G. M. (2004b). Real-time measurement of circadian gene expression in peripheral tissues from transgenic zebrafish. Soc. Res. Biol. Rhythms Abstr. 9, 90. Kobayashi, Y., Ishikawa, T., Hirayama, J., Daiyasu, H., Kanai, S., Toh, H., Fukuda, I., Tsujimura, T., Terada, N., Kamei, Y., Yuba, S., Iwai, S., and Todo, T. (2000). Molecular analysis of zebrafish photolyase/cryptochrome family: Two types of cryptochromes present in zebrafish. Genes Cells 5, 725–738. Macho, B., and Sassone-Corsi, P. (2003). Functional analysis of transcription factors CREB and CREM. Methods Enzymol. 370, 396–415. Millar, A. J., Short, S. R., Chua, N. H., and Kay, S. A. (1992). A novel circadian phenotype based on firefly luciferase expression in transgenic plants. Plant Cell. 4, 1075–1087. Muyrers, J. P. P., Zhang, Y., Testa, G., and Stewart, A. F. (1999). Rapid modification of bacterial chromosomes by ET-recombination. Nucleic Acids Res. 27, 1555–1557. Pando, M. P., Pinchak, A. B., Cermakian, N., and Sassone-Corsi, P. (2001). A cell-based system that recapitulates the dynamic light-dependent regulation of the vertebrate clock. Proc. Natl. Acad. Sci. USA 98, 10178–10183. Pando, M. P., and Sassone-Corsi, P. (2002). Unraveling the mechanisms of the vertebrate circadian clock: Zebrafish may light the way. Bioessays 24, 419–426. Payne, G., and Sancar, A. (1990). Absolute action spectrum of E-FADH2 and E-FADH2MTHF forms of Escherichia coli DNA photolyase. Biochemistry 29, 7715–7727. Rimbach, G., Saliou, C., Canali, R., and Virgili, F. (2001). Interaction between cultured endothelial cells and macrophages: In vitro model for studying flavonoids in redoxdependent gene expression. Methods Enzymol. 335, 387–397. Roenneberg, T., and Taylor, W. (2000). Automated recordings of bioluminescence with special reference to the analysis of circadian rhythms. Methods Enzymol. 305, 104–119. Sassoon, D., and Rosenthal, N. (1993). Detection of messenger RNA by in situ hybridization. Methods Enzymol. 225, 384–404. Schibler, U., and Sassone-Corsi, P. (2002). A web of circadian pacemakers. Cell 111, 919–922. Tessarollo, L., and Parada, L. F. (1995). In situ hybridization. Methods Enzymol. 254, 419–430. Vallone, D., Gondi, S. B., Whitmore, D., and Foulkes, N. (2004). E-box function in a period gene repressed by light. Proc. Natl. Acad. Sci. USA 101, 4106–4111. Westerfield, M. (1995). ‘‘The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio).’’ University of Oregon Press. Whitmore, D., Foulkes, N. S., Strahle, U., and Sassone-Corsi, P. (1998). Zebrafish Clock rhythmic expression reveals independent peripheral circadian oscillators. Nature Neurosci. 1, 701–707. Whitmore, D., Foulkes, N. S., and Sassone-Corsi, P. (2000). Light acts directly on organs and cells in culture to set the vertebrate circadian clock. Nature 404, 87–91.