© 1998 Nature America Inc. • http://neurosci.nature.com
articles
Zebrafish Clock rhythmic expression
reveals independent peripheral
circadian oscillators
David Whitmore, Nicholas S. Foulkes, Uwe Strähle and Paolo Sassone-Corsi
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, 1 rue Laurent Fries, 67404 Illkirch Cédex, C.U. de Strasbourg, France
The first two authors contributed equally to this work.
© 1998 Nature America Inc. • http://neurosci.nature.com
Correspondence should be addressed to P.S.-C. (paolosc@igbmc.u-strasbg.fr)
The only vertebrate clock gene identified by mutagenesis is mouse Clock, which encodes a bHLH-PAS
transcription factor. We have cloned Clock in zebrafish and show that, in contrast to its mouse homologue, it is expressed with a pronounced circadian rhythm in the brain and in two defined
pacemaker structures, the eye and the pineal gland. Clock oscillation was also found in other tissues,
including kidney and heart. In these tissues, expression of Clock continues to oscillate in vitro. This
demonstrates that self-sustaining circadian oscillators exist in several vertebrate organs, as was previously reported for invertebrates.
The genes involved in the generation of circadian rhythms are
being identified by genetic screens in a variety of organisms,
including Drosophila and Neurospora. The first vertebrate circadian clock gene, Clock, was cloned in the mouse via a large-scale
mutagenic screen1–3. This gene encodes a protein that is a member of the bHLH-PAS-domain transcription-factor family. A small
deletion within the putative transcription activation domain of
the CLOCK protein leads to period lengthening, as measured by
wheel-running activity, and eventual arrhythmicity in constant
darkness1,3. Expression of Clock does not oscillate in the mouse4,
a feature expected for a component of a circadian pacemaker5–7.
A homolog of Clock (dclock) identified in Drosophila8 does
show a circadian oscillation of expression9,10. This Drosophila
gene seems to be central for regulating expression of two key clock
genes in the fly, timeless (tim) and period (per)8,9. Expression of
both tim and per oscillates at the RNA and protein levels11–14. In
the same mutant screen that identified dclock, a separate mutation, cyc, was also discovered 15. The CYC (CYCLE) protein,
another bHLH-PAS factor, heterodimerizes with dCLOCK and
acts as a transcriptional activator. This heterodimer binds to
enhancer elements termed E-boxes, which are present in the promoters of the per and tim genes, and thereby activates their transcription8,9,15,16. BMAL1, the mouse homolog of CYC, has been
identified as a CLOCK partner by a yeast two-hybrid screen17.
Expression of the per gene oscillates in a number of tissues
even in vitro, as shown in per-luciferase transgenic Drosophila18.
These data demonstrate that many autonomous pacemaker structures exist in Drosophila. The situation may be similar in vertebrates. Three homologs of per have been cloned in the mouse
(mper1, 2 and 3), which show circadian rhythms of expression
not only in the two defined pacemaker structures, the retina and
the suprachiasmatic nucleus (SCN)19–21, but also in a number of
other tissues4,22–25. However, this per gene oscillation has not
been shown in vitro in organ or primary cultures. Therefore, in
the animal, it is still not clear whether these oscillations are simply driven from a central master circadian pacemaker, such as
nature neuroscience • volume 1 no 8 • december 1998
the SCN. Expression of the per gene has been described in vitro in
immortalized cell lines26. Serum treatment followed by starvation induces rhythmic gene expression with timing similar to
that reported in vivo in the organs from which the cell lines were
established26. Two central questions emerge from the current situation. Does the Clock gene oscillate in a vertebrate system and,
perhaps more importantly, do peripheral tissues in vertebrates
really contain self-sustained circadian pacemakers?
Results
THE ZEBRAFISH CLOCK GENE
We studied the expression of the Clock gene in the zebrafish, another vertebrate model system. Strong conservation of the Clock gene
sequence between vertebrate species was already suggested by
genomic Southern blot analysis using the mouse cDNA probe1.
Thus, we initially screened an embryo cDNA library with the mouse
Clock probe at low stringency. Following the isolation of a single
partial-length clone, we completed the fish Clock cDNA sequence by
3’ RACE PCR. The 894 amino-acid zebrafish CLOCK protein shares
80% identity with mouse CLOCK and 53% identity with dCLOCK
(Fig. 1). The strongest conservation among the zebrafish, Drosophila and mouse proteins is at the amino (N) terminus, within the
bHLH-PAS DNA binding and protein–protein interaction domain
(Fig. 1). In particular, a long region of sequence identity is centered
on the PAS B domain. The stretch of 51 amino acids that is deleted
from the mouse CLOCK protein in the Clock mutant is also a region
of strong homology with zebrafish CLOCK, but is not conserved
in the dCLOCK sequence (Fig. 1). Another feature of fish CLOCK
is a stretch of 51 residues composed of glutamines interspersed with
only 9 leucine or histidine residues (amino acids 825 to 876). This
extended glutamine stretch is reminiscent of sequences composing
the carboxyl (C)-terminal region of dCLOCK8.
RHYTHMIC CLOCK EXPRESSION IN THE EYE AND PINEAL GLAND
To explore the pacemaker role of CLOCK in the zebrafish, we initially studied its temporal expression pattern in the adult eye and
701
© 1998 Nature America Inc. • http://neurosci.nature.com
© 1998 Nature America Inc. • http://neurosci.nature.com
articles
Fishclock
mclock
dclock
1
60
.........M TSSIDRDDSS IFDGLMEEDE KDKAK....R VSRNRSEKKR RDQFNVLIKE
MVFTVSCSKM SSIVDRDDSS IFDGLVEEDD KDKAK....R VSRNKSEKKR RDQFNVLIKE
.......... .......... .MDD..ESDD KDDTKSFLCR KSRNLSEKKR RDQFNSLVDD
Fishclock
mclock
dclock
61
120
LGTMLPGNTR KMDKSTILQK SIDFLRKHKE IAAQSESSEI RQDWKPPFLS NEEFTQLMLE
LGSMLPGNAR KMDKSTVLQK SIDFLRKHKE TTAQSDASEI RQDWKPTFLS NEEFTQLMLE
LSALISTSSR KMDKSTVLKS TIAFLKNHNE ATDRSKVFEI QQDWKPAFLS NDEYTHLMLE
Fishclock
mclock
dclock
121
180
ALDGFFLAIM TDGNIIYVSE SVTSLLEHLP SDLVDQNLLN FLPLGEHSEV YKAL..STHM
ALDGFFLAIM TDGSIIYVSE SVTSLLEHLP SDLVDQSIFN FIPEGEHSEV YKIL..STHL
SLDGFMMVFS SMGSIFYASE SITSQLGYLP QDLYNMTIYD LAYEMDHEAL LNIFMNPTPV
Fishclock
mclock
dclock
181
240
LEGETLTPDY LKTKNQLEFC CHMLRGTIDP KEPPVYEYVK FIGNFKS... ........LN
LESDSLTPEY LKSKNQLEFC CHMLRGTIDP KEPSTYEYVR FIGNFKS... ........LT
IEPR...QTD ISSSNQITFY THLRRGGMEK VDANAYELVK FVGYFRNDTN TSTGSSSEVS
Fishclock
mclock
dclock
241
300
TVPNSTRNGF EGVIQRSLRH AFEDRVCFIA TVRLAKPQFI KEMCTVEEPN EEFTSRHSLE
SVSTSTHNGF EGTIQRTHRP SYEDRVCFVA TVRLATPQFI KEMCTVEEPN EEFTSRHSLE
NGSNGQPAVL PRIFQQNPNA EVDKKLVFVG TGRVQNPQLI REMSIIDPTS NEFTSKHSME
Fishclock
mclock
dclock
301
360
WKFLFLDHRA PPIIGYLPFE VLGTSGYDYY HVDDLETLAK CHEHLMQYGK GKSCYYRFLT
WKFLFLDHRA PPIIGYLPFE VLGTSGYDYY HVDDLENLAK CHEHLMQYGK GKSCYYRFLT
WKFLFLDHRA PPIIGYMPFE VLGTSGYDYY HFDDLDSIVA CHEELRQTGE GKSCYYRFLT
Fishclock
mclock
dclock
361
420
KGQQWIWLQT HYYITYHQWN SRPEFIVCTH TVVSYAEVRA EQRRE...LG IEESPPEISA
KGQQWIWLQT HYYITYHQWN SRPEFIVCTH TVVSYAEVRA ERRRE...LG IEESLPETAA
KGQQWIWLQT DYYVSYHQFN SKPDYVVCTH KVVSYAEVLK DSRKEGQKSG NSNSITNNGS
Fishclock
mclock
dclock
421
480
DK...SQDSG SESQLNTSSL KE........ .......ALE RFDHSRTPSA SSRSSRKSSS
DK...SQDSG SDNRINTVSL KE........ .......ALE RFDHSPTPSA SSRSSRK.SS
SKVIASTGTS SKSASATTTL RDFELSSQNL DSTLLGNSLA SLGTETAATS PAVDSSPMWS
Fishclock
mclock
dclock
481
540
HTAVSDPTST QTK.LQTDRS TPPRQSVSAI EMTSQRR... .........S SISSQSMSSQ
HTAVSDPSST PTK.IPTDTS TPPRQHLPAH EKMTQRR... .........S SFSSQSINSQ
ASAVQPSGSC QINPLKTSRP ASSYGNISST GISPKAKRKC YFYNNRGNDS DSTSMSTDSV
Fishclock
mclock
dclock
541
600
TTGQTMGTSL VSQPQQPQTL QATVQPVLQF STQMDAMQHL KEQLEQRTRM IEANIQRQQE
SVGPSLTQPA MSQAANLPIP QGMSQ..FQF SAQLGAMQHL KDQLEQRTRM IEANIHRQQE
TSRQSMMTHV SSQSQRQRSH HREHHRENHH NQSHHHMQQQ QQHQNQ.... .....QQQHQ
Fishclock
mclock
dclock
601
660
ELRQIQDELQ RVQGQGLQMF LQP....... .......SGG GLNLSSVQL. TQS..SSVQT
ELRKIQEQLQ MVHGQGLQMF LQQ....... .......SNP GLNFGSVQLS SGN..SNIQQ
QHQQLQQQLQ HTVGTPKMVP LLPIASTQIM AGNACQFPQP AYPLASPQLV APTFLEPPQY
Fishclock
mclock
dclock
661
720
AGTLSMQGAV VPTATLQSSL QSTHSSTQHT VTQHPQQTAV QQQNLLRDQT TNLNQQSQRS
LTPVNMQGQV VPANQVQSGH IST...GQHM IQQQTLQSTS TQQS....QQ SVMSGHSQQT
LTAIPMQ.PV IAPFPVAPVL SPLPVQSQTD MLPDTVVMTP T.QSQLQDQL QRKHDELQKL
Fishclock
mclock
dclock
721
780
THTLQSPQGA LPASLYNTMM ISQPTQANVV QISTSLAQNS STSGAAVDLL TKDPTDYRFP
SLPSQTP.ST LTAPLYNTMV ISQPAAGSMV QIPSSMPQN. STQSATVTTF TQDR.QIRFS
ILQQQNELRI VSEQLLLSRY TYLQPMMSM. ....GFAPGN MTAAAVGNLG ASGQRGLNFT
Fishclock
mclock
dclock
781
840
ATQQLLTKLV TGPMACGAVM VPTTMFMGQ. ...VVTA... FAPQ.....Q GQPQTISIAQ
QGQQLVTKLV TAPVACGAVM VPSTMLMGQ. ...VVTAYPT FATQ.....Q QQAQTLSVTQ
GSNAVQPQF. ...NQYGFAL NSEQMLNQQD QQMMMQQQQN LHTQHQHNLQ QQHQSHSQLQ
Fishclock
mclock
dclock
841
900
QPSAQTADQQ THTQAQTQAA ATAQQ...QG QNQAQLTQQQ TQFLQAPRLL HSNQSTQ...
QQQQQQQQPP QQQQQQQQSS QEQQLPSVQQ PAQAQLGQPP QQFLQTSRLL HGNPSTQ...
QHTQQQHQQQ QQQQQQQQQQ QQQQQQQQQQ QQQQQQQQQQ LQLQQQNDIL LREDIDDIDA
Fishclock
mclock
dclock
901
960
.LILQAAFPL QQQGTFTT.. .......... .......... .......... ..........
.LILSAAFPL .QQSTFPP.. .......... .......... .......... ..........
FLNLSPLHSL GSQSTINPFN SSSNNNNQSY NGGSNLNNGN QNNNNRSSNP PQNNNEDSLL
Fishclock
mclock
dclock
961
1020
.......... .......... .......... .........A TQQQQQLHQQ QQQLQQQQQL
.......... .......... .......... .........S HHQQHQ.... ..........
SCMQMATESS PSINFHMGIS DDGSETQSED NKMMHTSGSN LVQQQQQQQQ QQQILQQHQQ
Fishclock
mclock
dclock
1021
1073
QQQQQQQQQQ LQQQHQQQQQ QLQQQHQQQQ QQLAAHRSDS MTERSNPPPQ *..
.......... .......... ......PQQQ QQLPRHRTDS LTDPSKVQPQ *..
QSNSFFSSNP FLNSQNQNQN QLPNDLEILP YQMSQEQSQN LFNSPHTAPG SSQ
pineal gland, structures that rhythmically synthesize melatonin in
vitro and under constant darkness27. Using a miniaturized RNAse
protection assay (RPA) and a riboprobe generated from the 5’ end
of the Clock transcript, we analyzed Clock expression in eyes and
702
bHLH
PAS A
PAS B
Fig. 1. Conservation of zebrafish,
mouse and Drosophila CLOCK.
The amino-acid sequences of
mouse1, Drosophila8 and zebrafish
CLOCK are aligned by the program Pileup (GCG). Amino acids
in magenta are conserved among
all three proteins, and amino
acids in cyan are conserved
between two proteins, whereas
amino acids in black are not conserved. The bHLH, PAS A and
PAS B regions are boxed and
labeled. The deletion in the
mouse CLOCK protein associated with the Clock mutant
mouse (∆mClock), and the region
truncated from dCLOCK in the
Drosophila mutant Jrk (∆dClock),
are delineated by dashed arrows.
Dots indicate spaces inserted
into the sequences to provide
optimal alignment.
∆ mClock
∆ dClock
pineal glands throughout a light–dark cycle (LD 14:10). In both
tissues, we found a robust daily induction of the Clock transcript,
with a major peak of expression at the onset of the dark period
(Fig. 2a and b). A minor increase was also visible at zeitgeber time
nature neuroscience • volume 1 no 8 • december 1998
© 1998 Nature America Inc. • http://neurosci.nature.com
articles
a
pineal gland
b
eye
clock
clock
IRBP
c
DD
© 1998 Nature America Inc. • http://neurosci.nature.com
IRBP
eye
clock
pineal
clock
CREB
Fig. 2. Expression of the zebrafish Clock gene oscillates in pacemaker structures. (a) RNAse protection analysis of Clock, IRBP and CREB
expression in zebrafish eye RNA samples. Fish were maintained under a 14:10 light:dark cycle and killed at the indicated zeitgeber times (ZT).
The bar above demonstrates light (white) and dark (black) periods. tRNA serves as a negative control reaction (t). Clock expression shows a
major peak at the beginning of the night and also a minor increase at ZT 4. IRBP oscillates out of phase with the Clock transcript. Expression of
CREB is stable throughout the cycle. (b) RNAse protection analysis of Clock and IRBP expression in the pineal gland. The pattern of expression
of both genes is equivalent to that in the eye. Expression of CREB did not oscillate (data not shown). (c) Clock expression in the eye and pineal
gland continues to oscillate during two days under constant darkness. The gray and black bar above represents constant darkness (DD), with
gray the subjective day and black the subjective night. CREB expression did not oscillate through the period of the experiment (data not
shown). Each experiment was repeated four times, and representative autoradiographs are shown. (For quantitative data, see Fig. 4b.)
(ZT) 4 in the eye (where ZT 0 corresponds to lights on, local clock
time, and ZT 14 is lights off) (Fig. 2a). The Clock rhythm also persisted for at least two cycles under constant darkness (Fig. 2c),
demonstrating that it was not directly light driven.
We also tested expression of interphotoreceptor retinoid-binding protein (IRBP) and CRE binding protein (CREB) (Fig. 2a
and b). IRBP is involved in the regeneration of photopigment,
and its expression is higher at midday than at midnight in the
zebrafish retina28. CREB is a constitutively expressed transcriptional activator of the cAMP signal-transduction pathway29,30.
Consistently, in both the eye and pineal gland samples, IRBP had
a clear pattern of diurnal expression, completely out of phase
with that found for Clock, whereas CREB expression did not significantly oscillate throughout the 24-hour cycle (Fig. 2a and b).
These results confirm that the rhythm of Clock expression does
not reflect a global oscillation of gene expression.
CLOCK OSCILLATION IN THE BRAIN
To determine whether Clock expression oscillates in other tissues,
we analyzed the expression of Clock during the day-night cycle
in the zebrafish brain (Fig. 3). In the whole brain, we observed
a significant oscillation in Clock expression, similar in timing and
amplitude to that in the pineal gland and the eye. This rhythmic
expression persisted under constant darkness (Fig. 3a). In the
brain, as for the eye, the second, low-amplitude increase in Clock
expression at ZT 4 was visible (compare Figs 2a and 3a). Thus
the daily profile of Clock expression in zebrafish is similar to that
reported in Drosophila9, in that there are two peaks of expression, although the peaks are reported to be of equal amplitude
nature neuroscience • volume 1 no 8 • december 1998
in the fly8,9. Using in situ hybridization analysis on serial brain
cross sections with an antisense Clock probe, in the periventricular gray layer of the optic tectum, we observed a Clock-positive
signal at ZT 15, which was significantly reduced at ZT 3 (Fig. 3b).
This Clock expression was confirmed by RNAse protection assays
from dissected optic tecta (Fig. 3c). Additional analysis in the
telencephalon and hindbrain also revealed a day–night oscillation (Fig. 3c), demonstrating that Clock is rhythmically expressed
throughout the brain. Differential expression of the Clock and
mper genes in various regions of the brain has already been
shown in the mouse4,23,24. Furthermore, day–night oscillations of
expression have been reported for mper1, 2 and 3 in the
SCN4,22–25,31, and for mper1 in the pars tuberalis and Purkinje
neurons of the cerebellum4,23.
CLOCK OSCILLATION IN PERIPHERAL TISSUES
The oscillation in Clock expression observed in the brain prompted us to explore expression of Clock in different zebrafish organs
(Fig. 4). In the kidney, spleen and heart, Clock expression oscillated with a timing similar to that in the eyes, pineal gland and
brain (Fig. 4a). The magnitude of this oscillation varied between
tissues, with the greatest in the eye and no significant day-night
difference in the testis (Fig. 4b). The oscillatory Clock expression
was consistent with the existence of endogenous, self-sustaining
oscillators in the different fish organs. To test this hypothesis, we
dissected heart and kidney, tissues showing significant Clock oscillation, and placed them in organ culture. They were incubated
for two to three days under constant darkness, during which the
hearts continued to beat. The oscillation of Clock expression
703
© 1998 Nature America Inc. • http://neurosci.nature.com
articles
a
brain
b
ZT5
ZT3
ZT
clock
CREB
c
© 1998 Nature America Inc. • http://neurosci.nature.com
DD
clock
clock
telencephalon
optic
tectum
hindbrain
Fig. 3. Clock expression oscillates in the zebrafish brain. (a) RNAse protection analysis of Clock expression in whole brain RNA. Upper panels, fish were maintained under a 14:10 light:dark cycle and killed at the indicated times. Clock expression peaks at the beginning of the night
and shows a minor increase at ZT 4. Expression of CREB is stable throughout the cycle. Lower panel, Clock expression continues to oscillate
on the second day under constant darkness. (b) In situ hybridization analysis of Clock expression in cross sections through the diencephalon,
which includes the optic tectum. Strong hybridization with the Clock antisense probe is visible over the periventricular layer of the optic tectum at ZT 15 but not at ZT 3. Hybridization with a sense control probe produced no significant signals (data not shown). (c) RNAse protection analysis of Clock expression in dissected telencephalon, optic tectum and hindbrain. Each experiment was repeated five times, and
representative autoradiographs are shown. (For quantitation, see Fig. 4b.)
indeed persisted in vitro, for two complete cycles in the case of the
heart and three cycles for the kidney (Fig. 4c–e). These data directly support the existence of independent endogenous circadian
oscillators. The peak of Clock expression seemed to be shifted relative to the in vivo profile in the kidney, whereas for the heart, in
vitro and in vivo expression patterns were equivalent. The phase
difference in the kidney was already present within the first cycle
in culture and may reflect a tissue-specific phase shift of the oscillator produced by the dissection or culture conditions.
Discussion
Here we show that the expression of the Clock gene in the
zebrafish oscillates with a robust day–night rhythm, which persists in constant darkness. Two modes of action were initially proposed for CLOCK in the mouse1,32. In one model, Clock would
oscillate functionally and be involved in a transcription–translation feedback loop, in which it would regulate its own level of
expression across the circadian cycle. In the second model, Clock
was proposed to act as a master regulator of pacemaker function,
controlling the expression of other pacemaker genes, but did not
need to oscillate itself1,32. Data from the mouse tend to support
the second model, in which CLOCK functions as a constitutively expressed activator 4. However, the data presented here in
zebrafish, as well as those from Drosophila, tend to support the
first hypothesis10. The Clock gene in zebrafish seems to oscillate
similarly to canonical clock genes in Drosophila and Neurospora6. CLOCK heterodimerizes with its protein partner BMAL1 in
Drosophila and mouse and activates the promoters of other clock
genes (mper 1 in mouse and per and tim in Drosophila)8,9,15–17.
In Drosophila, the Per and Tim proteins feed back to inhibit their
own expression11,33. Furthermore, this Per–Tim feedback is
704
thought to be mediated through a direct interaction with CLOCK
and BMAL19, possibly disrupting this heterodimer and consequently causing loss of transcriptional activation. The demonstration of Clock mRNA oscillations in zebrafish raises the
possibility that the Clock promoter is also a target of Per–Tim
regulatory feedback. Data from Drosophila, where Clock also oscillates, support this hypothesis10. However, Drosophila Per and Tim
have been proposed to activate the Clock promoter10. It is not yet
known whether this is also true for zebrafish.
Whereas our initial studies focused on tissues known to
contain circadian pacemakers, namely the pineal gland and
the eye, analysis of other tissues, such as the heart and kidney,
also revealed an oscillation in Clock. Similar oscillations
occur for mper 1,2 and 3 in the mouse testis, skeletal muscle
and liver25. The existence of molecular oscillations in vertebrate tissues in vivo is not surprising, as many tissues undergo dramatic daily changes in their physiology, and a number
of genes are rhythmically expressed, for example, the PARleucine-zipper transcription factors DBP, TEF34 and the gluconeogenic liver enzyme tyrosine aminotransferase 35 .
Circadian gene expression has even been described in
immortalized fibroblast and hepatocyte cell lines26. However, this expression pattern is only found after transient treatment of the cells with high serum concentrations and
subsequent incubation in serum-free medium26. Because the
oscillations are not detected under steady-state culture conditions, it has been suggested that serum treatment either
synchronizes single-cell independent oscillators or activates
a ‘dormant’ circadian oscillator26. To resolve this point, single-cell measurements of gene expression will be required 26.
Using zebrafish Clock as a marker, we provide evidence for
nature neuroscience • volume 1 no 8 • december 1998
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articles
ZT
a
b
n=4
OD
clock
n=5
n=4
kidney
Pineal
clock
© 1998 Nature America Inc. • http://neurosci.nature.com
n=5
Spleen Kidney Testis Heart Brain
heart in vitro
d
heart
n=5
testis
Eye
n=5
n=3
Day 1
Day 2
spleen
kidney in vitro
c
Day 1
Day 2
clock
CREB
clock
e
n=6
CREB
Day 1
Day 2
Day 3
OD
n=7
clock
OD
value
Kidney
Heart
Fig. 4. Clock expression in different zebrafish organs demonstrates the existence of independent circadian oscillators. (a) RNAse protection
analysis of Clock expression in the kidney, testis, heart and spleen from fish maintained on a 14:10 light:dark cycle. In all organs, Clock mRNA
oscillates with a peak at ZT 15. (b) Mean Clock expression (optical density, OD) in different organs, with standard error. Equal loading was
confirmed using CREB as an internal standard. By unpaired Student’s t-test, differences between ZT3 and ZT15 expression were significant
for kidney, heart and brain, (p < 0.001); eye and pineal, (p < 0.005) and spleen (p < 0.05), whereas for testis, the difference was not significant
(p = 0.68). Clock expression cannot be compared between organs because the amount of RNA assayed was different in each case. (c) Two
independent experiments showing oscillating Clock expression in the kidney in organ culture in constant darkness. CREB expression did not
oscillate during the culture period. The OD values are plotted for each band in the three-day experiment. (d) Oscillating Clock expression in
the heart in organ culture over two days in constant darkness. (e) Quantitation of Clock expression in vitro. Mean trough and peak Clock levels (OD) in the kidney and heart with standard error. CREB is used as an internal standard. By unpaired Student’s t-test, differences between
trough and peak expression were significant for kidney (p < 0.0001) and heart (p < 0.005).
self-sustained independent circadian oscillators in vertebrate
organs in primary culture.
The relative contribution of central versus peripheral pacemakers to the rhythmic physiology of these organs in the fish is
not yet clear. It is possible that each organ pacemaker controls all
rhythmic outputs within that structure, and central pacemakers
nature neuroscience • volume 1 no 8 • december 1998
exist to coordinate each of these separate peripheral tissue pacemakers. The nature of these synchronizing or internal entraining
signals is not known, but a good candidate might be pineal melatonin, released into the blood stream under the regulation of a
master clock36. The widespread description of cryptochrome photopigments in many mouse tissues, however, raises the interest705
© 1998 Nature America Inc. • http://neurosci.nature.com
articles
© 1998 Nature America Inc. • http://neurosci.nature.com
ing possibility that a master clock is not even necessary for entrainment, with each tissue being directly responsive to light37.
The classical view of vertebrates has been that a small number of master oscillators, pineal gland, retina and SCN, control
all aspects of rhythmic physiology 38. Our data in zebrafish add
to the growing evidence that individual tissues also generate their
own rhythms.
Methods
CLONING AND SEQUENCING. An oligo-dT-primed zebrafish embryo cDNA
library (18–40 hours) prepared in lambda ZapII was plated and replica
lifted on nitrocellulose, according to standard protocols. As a probe, the
PAS B region of the mouse Clock cDNA was amplified by RT-PCR from
mouse eye RNA1. The PCR fragment was labeled with [α32P]dCTP by
random hexamer priming (Multiprime labelling kit, Amersham) and
then hybridized with the nitrocellulose filters in a 6x SSC, 5x Denhardt’s
solution with 1% SDS and 0.1 µg/µl denatured salmon sperm DNA solution at 50°C. A single positive clone was purified, the insert excized in
vivo (Stratagene) and then sequenced using Taq DNA polymerase and a
cycle sequencing system (Big Dye Terminator, Applied Biosystems).
Sequencing reactions were analyzed on a 373 DNA sequencing machine
(Applied Biosystems). 3’ rapid amplification of cDNA ends ( 3’ RACE)
PCR was done using a Marathon cDNA synthesis kit and klentaq DNA
polymerase according to manufacturers instructions (Clontech).
Sequence analysis was done with the University of Wisconsin GCG computer package.
FISH. Zebrafish were raised from our own stocks and kept at 29°C. They
were fed twice daily and maintained under a 14-hour day, 10-hour night
cycle, or under constant darkness. Adult fish (4 months old) were killed
by rapid emersion in chilled water and then decapitation. Dissections
were done under PBS using microdissection tools and a dissection microscope. Care of fish and all procedures were in full compliance with institutional guidelines for animal experimentation.
RNA ANALYSIS. Mini-scale RNA extractions from zebrafish tissues were
done with an acid-phenol method39. A miniaturized RPA was developed
for assaying Clock transcript expression in less than 1 µg total RNA. The
assay was based upon standard protocols 40 but adapted for 10-µl
hybridization volumes in micro-eppendorf tubes, with all incubations
done in a PCR thermal cycler. Protected products were resolved on denaturing 8 M urea and 6% polyacrylamide minigels. The Clock probe was
generated from the original 2.1 kb pBluescript cDNA clone by deleting an
internal HincII restriction fragment, linearizing with XbaI and then transcribing a [α32P]UTP-labeled 400-nucleotide riboprobe with T7 RNA
polymerase (Promega). The probe extends over the PAS A and bHLH
domains of CLOCK. Zebrafish CREB was isolated from the oligo-dTprimed embryo cDNA library by screening at low stringency with a
mouse cDNA probe29. The predicted amino-acid sequence is 95% identical to that of mouse CREB. A 260-nucleotide [α32P]UTP-labeled riboprobe was transcribed from a XhoI-linearized plasmid template by T3
RNA polymerase (Promega); this probe straddles the P-box region of
CREB. Zebrafish IRBP cDNA was amplified by RT-PCR from daytime
eye RNA using amplimers designed from the published sequence data28.
After subcloning in pAdvantage (Clontech), the plasmid was linearized in
the polylinker, and T7 RNA polymerase was used to generate a 260nucleotide antisense riboprobe extending over the 5’ end of the IRBP
transcript. In RNAse protection assays, 100 ng (pineal glands) or 500 ng
(eye and brain) of total RNA was assayed. A pool of RNA for each point
was prepared from 6 fish (50 fish for the pineal glands). RNAse protection
assay autoradiographs were scanned on an Imaging Densitometer (Biorad) and quantified using Molecular Analyst software (Biorad).
IN VITRO ORGAN CULTURE. For in vitro organ cultures, freshly dissected tissue was placed in modified 199 medium supplemented with 10% fetal
calf serum, 2 mM glutamine, and with gentamycin, streptamycin and
penicillin. Cultures were maintained for two days in constant darkness
in a 5% CO2 incubator at 24°C with a single change of medium. Organs
were dissected from fish between ZT 9 and ZT 12 and placed directly in
culture in constant darkness. Time point were taken first at ZT 21 on the
706
same day and subsequently every six hours during the following days.
Tissue was removed from the cultures, and RNA was extracted directly.
IN SITU HYBRIDIZATION. In situ hybridization with a 2.1-kb, [35S]ATPαSlabeled antisense probe for the zebrafish Clock cDNA was done as
described41. Sections of brain were prepared by embedding the fresh tissue in OCT, freezing on dry ice and then cutting 14-µm serial cross sections on a cryostat. Hybridized sections were exposed overnight on
X-OMAT X-ray films (Kodak) or dipped in photo-emulsion and developed after three to four days.
Acknowledgements
We thank Joseph S. Takahashi, Nicolas Cermakian, Dario De Cesare, Lucia
Monaco, Jean-Marie Garnier, Pilar Garcia-Villalba and Patrick Blader for
discussions, advice and gifts of materials. We also acknowledge the technical
assistance of Estelle Heitz, as well as Dominique Biellman, Odile Nkundwa,
Nadine Fisher, Serge Vicaire and Frank Ruffenach. D.W. was supported by an
EEC TMR fellowship. Our studies are funded by grants from CNRS, INSERM,
CHUR, Rhône-Poulenc Rorer (Bioavenir), Fondation pour la Recherche
Médicale and Association pour la Recherche sur le Cancer (P. S.-C.).
RECEIVED 21 AUGUST: ACCEPTED 24 OCTOBER 1998
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