Light signaling to the zebrafish circadian clock
by Cryptochrome 1a
T. Katherine Tamai*, Lucy C. Young, and David Whitmore*
Department of Anatomy and Developmental Biology, Centre for Cellular and Molecular Dynamics, University College London,
21 University Street, London WC1E 6DE, United Kingdom
Edited by Jeffrey C. Hall, Brandeis University, Waltham, MA, and approved July 30, 2007 (received for review May 17, 2007)
Zebrafish tissues and cells have the unusual feature of not only
containing a circadian clock, but also being directly light-responsive.
Several zebrafish genes are induced by light, but little is known about
their role in clock resetting or the mechanism by which this might
occur. Here we show that Cryptochrome 1a (Cry1a) plays a key role in
light entrainment of the zebrafish clock. Intensity and phase response
curves reveal a strong correlation between light induction of Cry1a
and clock resetting. Overexpression studies show that Cry1a acts as
a potent repressor of clock function and mimics the effect of constant
light to ‘‘stop’’ the circadian oscillator. Yeast two-hybrid analysis
demonstrates that the Cry1a protein interacts directly with specific
regions of core clock components, CLOCK and BMAL, blocking their
ability to fully dimerize and transactivate downstream targets, providing a likely mechanism for clock resetting. A comparison of entrainment of zebrafish cells to complete versus skeleton photoperiods
reveals that clock phase is identical under these two conditions.
However, the amplitude of the core clock oscillation is much higher on
a complete photoperiod, as are the levels of light-induced Cry1a. We
believe that Cry1a acts on the core clock machinery in both a
continuous and discrete fashion, leading not only to entrainment, but
also to the establishment of a high-amplitude rhythm and even
stopping of the clock under long photoperiods.
entrainment 兩 oscillator 兩 phase shift 兩 photoperiod
A
n essential and defining feature of a circadian clock is that it
can be set or entrained to the local light–dark (LD) cycle.
Because light is the most typically used environmental cue, most
plants and animals have evolved circadian light detection mechanisms and signaling pathways that convey this light information to
the core clock machinery. Zebrafish represent an alternative, if as
yet relatively unexplored, vertebrate model system for the study of
the circadian clock. Their circadian system has some similarities to
that of Drosophila, particularly in regard to peripheral clock entrainment (1, 2). Zebrafish tissues contain endogenous circadian
oscillators that are directly light-responsive and entrainable to LD
cycles in vitro (3). This direct light sensitivity extends to the earliest
stages of development, as well as to embryonic cell lines, making
zebrafish cells distinct from their mammalian counterparts (3, 4).
This unusual feature means that the clock mechanism and entire
photoentrainment pathway are contained within a single cell.
Consequently, by transfecting zebrafish cell lines with luminescent
reporters driven by the promoters of rhythmic and light-responsive
genes, we have generated a model system where we can follow
circadian oscillations and their direct entrainment by light at the
cellular level (5, 6).
Recently, by employing single-cell luminescent imaging, we have
shown that an asynchronous population of zebrafish cells can be
strongly reset by a single light pulse to a common phase of the
circadian cycle (6). This is the consequence of a high-amplitude,
Type 0 phase response curve (PRC) that zebrafish clocks possess
(5). The cellular and molecular events involved in entrainment of
the zebrafish clock, however, have not been extensively studied. We
and others have identified several acutely light-responsive genes,
including Cryptochrome 1a (Cry1a) and Period 2 (Per2) (4, 7–9).
These molecules have been proposed to play key roles in entrain14712–14717 兩 PNAS 兩 September 11, 2007 兩 vol. 104 兩 no. 37
ment, although evidence for their mode of action is lacking. The aim
of this study, therefore, is to explore the molecular changes that
occur in a zebrafish clock-containing cell in response to light, with
particular emphasis on the role of Cry1a. Here we examine the
effect of light on Cry1a induction and phase shifting and establish
how the Cry1a protein interacts with the core clock machinery to
bring about light-dependent changes in clock function. Results from
intensity and PRCs demonstrate a strong correlation between light
induction of the Cry1a gene and clock resetting. Overexpression
analysis reveals that Cry1a is a potent transcriptional repressor and
appears to mimic the effect of sustained light to ‘‘stop’’ the circadian
oscillator. Biochemical studies show that the Cry1a protein binds to
multiple domains of central clock components, CLOCK and
BMAL. This binding then blocks transactivation, providing a likely
mechanism for clock resetting and establishment of high-amplitude
rhythms on a LD cycle. Thus, Cry1a appears to play a critical role
in the response of the zebrafish clock to light but, interestingly, as
part of a signaling pathway to the circadian pacemaker. Zebrafish
possess a number of cryptochromes (10), and it appears that a
‘‘division of labor’’ has occurred in this circadian system, with Cry1a
acting as a light-signaling molecule and other cryptochromes,
potentially taking on the role of circadian photopigment or core
clock protein (7, 10).
Results
A 1-h Light Pulse at Circadian Time (CT) 16 Leads to Large Phase Shifts
and Induction of Cry1a and Per2. To investigate how light phase shifts
the zebrafish clock, we monitored clock function by using a Per1luciferase zebrafish cell line (5) (Per1 is also called Per4). After 3 days
of entrainment, cells were given a ‘‘white’’ light pulse (400–700 nm
at 2,500 ␮W/cm2) for 1 h at CT16 and returned to constant darkness
(DD) for a further 3 days. Bioluminescent traces show that this 1-h
light pulse leads to a dramatic phase shift of ⬇12 h (Fig. 1A). To
identify potential molecules involved in phase shifting, we compared the expression of a number of Cryptochrome (Cry1a, Cry1b,
Cry2a, Cry2b, Cry3, and Cry4) and Period (Per1, Per2, and Per3)
genes in light-pulsed versus dark control cells by RNase protection
analysis. From these two gene families, only Cry1a and Per2 were
strongly induced by light (Fig. 1B and data not shown). Shorter light
pulses of 15 min also led to a significant increase in Cry1a and Per2
mRNA levels (data not shown) and were therefore used for all
subsequent experiments.
Author contributions: T.K.T. and D.W. designed research; T.K.T., L.C.Y., and D.W. performed research; T.K.T. and D.W. analyzed data; and T.K.T. and D.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: LD, light– dark; DD, constant darkness; CT, circadian time; bHLH, basic
helix–loop– helix; PRC, phase response curve; PAS, PER-ARNT-SIM.
*To whom correspondence may be addressed. E-mail: ktamai@yahoo.com and
d.whitmore@ucl.ac.uk.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0704588104/DC1.
© 2007 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0704588104
CELL BIOLOGY
Fig. 1. One-hour light pulse at CT16 leads to large phase shifts and induction
of Cry1a and Per2 mRNA. (A) Bioluminescent traces of Per1-luciferase cells
light pulsed for 1 h (light gray squares) or maintained in DD (dark gray
diamonds). The average (⫾ SEM) of quadruplicate wells is presented. (B) RNase
protection assay of Cry1a and Per2. RNA was prepared from light-pulsed and
dark control cells harvested at 0, 1, 2, 4, and 6 h after the light treatment. Tim2
served as an internal RNA control.
Light Induction of Cry1a Correlates with the Size of Phase Shift.
Zebrafish cells were pulsed with a range of light intensities to
examine whether there is a correlation between the magnitude of
Cry1a induction and the size of the resulting phase shift. To
facilitate this analysis, we generated a Cry1a-luciferase reporter cell
line by stably transfecting zebrafish PAC2 cells with 4.7 kb of the
zebrafish Cry1a promoter fused to luciferase. After entrainment,
Cry1a- and Per1-luciferase cells were exposed to light (400–700 nm)
at different intensities (0, 100, 1,000, 5,000, and 10,000 ␮W/cm2) for
15 min at CT16 and then returned to DD for 3 days. These
experiments show a clear correlation between light intensity, Cry1a
induction, and the size of the Per1 phase shift (Fig. 2A). Although
these light intensities may appear ‘‘unnaturally’’ high, they are
similar to the levels of sunlight we experience here in London
during winter (3,000–16,000 ␮W/cm2). Thus, for a tropical, diurnal
animal like zebrafish, these intensities are well within the average
levels experienced in nature.
Is Cry1a induction phase-dependent, and, if so, does it correlate
with the shape of the PRC? After entrainment, both reporter cell
lines were pulsed with white light (400–700 nm at 5,000 ␮W/cm2)
for 15 min at different CTs (CT0, CT4, CT8, CT12, CT16, and
CT20). These results demonstrate that, although Cry1a is induced
throughout the circadian cycle, the magnitude of its induction varies
significantly according to circadian phase, from 1.59 ⫾ 0.11-fold at
CT8 up to 4.38 ⫾ 0.20-fold at CT20 (Fig. 2B). Moreover, there is
a strong correlation between the magnitude of Cry1a induction and
the size of the resulting phase shift, from a 2.10 ⫾ 0.46-h phase delay
at CT8 to a 15.68 ⫾ 0.67-h phase delay at CT20 (Fig. 2C).
Overexpression of Cry1a Represses Circadian Rhythms and Mimics the
Effect of Sustained Light. To further analyze the role of Cry1a, we
transfected Per1-luciferase cells with HA-tagged Cry1a by using
retroviruses. Western blot and immunocytochemical analyses demonstrate that this tagged protein is expressed and localized to the
nucleus [supporting information (SI) Fig. 7]. Bioluminescent traces
reveal that Cry1a overexpression abolishes rhythmic expression of
Tamai et al.
Fig. 2. Light induction of Cry1a correlates with size of Per1 phase shift. (A)
Cry1a- and Per1-luciferase cells were exposed to light (400 –700 nm) at CT16 at
the indicated intensities for 15 min. The average (⫾ SEM) from quadruplicate
wells is presented. The fold induction of Cry1a is plotted as a bar graph, and
the size of Per1 phase shift in hours is plotted as a line graph. Cry1a-luciferase
(B) and Per1-luciferase (C) cells were light-pulsed (400 –700 nm at 5,000
␮W/cm2) at the indicated CT. The histograms represent the average (⫾ SEM)
fold induction of Cry1a (B) and size of Per1 phase shift in hours (C) from three
independent experiments.
Per1 and significantly reduces basal levels in a dose-dependent
manner (Fig. 3A and data not shown). These results indicate that
Cry1a is a potent repressor of circadian clock function and, interestingly, appears to mimic the effect of constant light. Indeed,
sustained light treatment at 2,500 ␮W/cm2 appears to stop or
dramatically reduce the amplitude of the circadian oscillator (Fig.
3B). The oscillation then resumes from this point upon entry into
DD (Fig. 3B). This is most clearly demonstrated in experiments
with variable, long-duration light pulses starting at the same phase
(‘‘forward wedge’’) (SI Fig. 8A). Under such conditions, the zebrafish oscillator appears to stop during the light treatment and then
restarts at a predictable, fixed point immediately after the light-todark transition. By ending the variable-duration light pulses at a
fixed time (‘‘reverse wedge’’), the clock can be seen to restart from
a common phase, equivalent to CT12 (SI Fig. 8B). These observations fit well with the ‘‘clock stopping’’ action of light previously
described in classic studies, where circadian rhythms in eclosion of
PNAS 兩 September 11, 2007 兩 vol. 104 兩 no. 37 兩 14713
Fig. 3. Cry1a overexpression abolishes rhythmic expression of Per1 and mimics
constant light. (A) Bioluminescent traces of Per1-luciferase cells transfected with
HA-tagged Cry1a (black squares) or pCLNCX empty vector control (gray diamonds). After 3 days of entrainment, cells were transferred into DD for 3 days. (B)
Bioluminescent traces of Per1-luciferase cells entrained for 3 days, exposed to
constant light at 2,500 ␮W/cm2 for 60 h, and transferred into DD.
fly pupae and flight activity of mosquitoes have been examined (11,
12). We believe that this represents a molecular correlate of that
response and, in the case of zebrafish, is mediated through a
sustained increase in Cry1a levels. What is the mechanism of Cry1a
action?
Cry1a Interacts Directly with the PER-ARNT-SIM (PAS) B Domain of
CLOCK, As Well As Multiple Regions of BMAL. In vitro studies have
shown that the Cry1a protein binds directly to core clock components, CLOCK and BMAL (13). Using the yeast two-hybrid system,
we confirm most of these interactions and extend this analysis to
identify specific domains of CLOCK and BMAL involved in
protein binding. These experiments demonstrate that CLOCK1
and BMAL1 interact strongly at the basic helix–loop–helix (bHLH)
and PAS B domains, with little or no binding between the two PAS
A domains (Fig. 4C and SI Fig. 9A). Interaction assays with Cry1a
reveal that Cry1a binds strongly to the PAS B domain of CLOCK1
and to multiple regions of BMAL1, including the bHLH, PAS B,
and C-terminal transactivation domains (Fig. 4 and SI Fig. 9).
Additional analysis demonstrates that Cry1a interacts with
CLOCK3 and BMAL3, but not CLOCK2 or BMAL2 (SI Fig. 9B).
Because all forms of CLOCK and BMAL are able to dimerize and
transcriptionally activate target genes (13, 14), these results indicate
that Cry1a can potentially block all but the CLOCK2:BMAL2
heterodimer (i.e., eight of the nine potential CLOCK:BMAL
combinations). Based on our Cry1a overexpression data above,
however, the CLOCK2:BMAL2 heterodimer alone does not appear sufficient to drive rhythmic expression of Per1. From these
results, we believe that Cry1a interferes with CLOCK:BMAL
function in two ways: first, by inhibiting transactivation directly by
binding to the C-terminal domain of BMAL, and, second, by
physically blocking the ability of new CLOCK and BMAL protein
to form active dimers by competing for the bHLH and PAS B
domains, regions where CLOCK and BMAL themselves directly
interact (Fig. 5).
Clock Entrainment to Skeleton Photoperiods Reveals the Continuous
Influence of Light on the Core Clock Mechanism. We propose that
light can influence the motion of the circadian oscillator by inter14714 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0704588104
Fig. 4. Yeast two-hybrid analysis of Cry1a binding to CLOCK1 and BMAL1. (A)
CLOCK1 deletion constructs were fused to the GAL4 DNA binding domain (BD)
and tested for interactions against full-length Cry1a or BMAL1 fused to the
GAL4 activation domain (AD). Colored rectangles represent functional domains of CLOCK1:bHLH (yellow), PAS A (green), and PAS B (blue). (B) Four yeast
transformants from each plasmid combination were patched onto agar plates
containing synthetic media without leucine or tryptophan (⫺L⫺W) to select
for plasmids. Interactions were assayed by monitoring growth on media
without adenine (⫺Ade). ⫹⫹ represents a strong interaction, and ⫺ indicates
no interaction. (C) BMAL1 deletion constructs were fused to the GAL4 activation domain (AD) and tested for interactions against full-length Cry1a or
CLOCK1 (amino acids 1–390) fused to the GAL4 DNA binding domain (BD).
Colored rectangles represent functional domains of BMAL1:bHLH (yellow),
PAS A (red), and PAS B (pink). Interactions were assayed as above. ⫹/⫺ and
⫺/⫹ represent weak interactions. NT, not tested.
fering with the ability of CLOCK and BMAL proteins to dimerize
and transactivate downstream genes, such as Per1, through an
increase in Cry1a levels. Short light pulses cause a transient increase
in Cry1a transcript levels, with a resultant strong repression and
phase shift of the Per1 rhythm. Moreover, constant light or constitutive overexpression of Cry1a appears to stop the circadian
oscillator, showing a sustained action of light on the clock. To
compare directly the continuous versus discrete action of light on
the clock, we examined entrainment to both complete and complementary skeleton photoperiods (‘‘two-pulse’’ entrainment).
Cells were taken from an unentrained state and placed on either a
complete 12:12 LD cycle or a skeleton light cycle with 15 min of
light, corresponding to dawn and dusk, of matching intensity (2,500
␮W/cm2). An examination of the Per1 oscillation on a complete
photoperiod shows the rapid establishment of a high-amplitude
rhythm from the starting DD condition (Fig. 6A). Similarly, cells on
the skeleton photoperiod show rapid entrainment and achieve a
stable phase relationship within 24 h, with one peak of Per1
expression showing a phase angle identical to that seen on a
complete photoperiod. However, there are clear differences in the
Per1 rhythm on the two lighting regimes. On the skeleton light cycle,
the ‘‘second’’ of the 15-min light pulses induces an additional
transient peak in Per1 expression at a phase equivalent to dusk,
before light-dependent repression (see Discussion). Entering DD
from this photoperiod reveals, however, that the first peak in Per1
Tamai et al.
is sustained, with the oscillator using this light pulse as the dawn
signal. The second ‘‘dusk’’ light pulse appears to generate a phase
delay in the rhythm, as predicted by the shape of the PRC. This then
leads to precise and stable timing of the Per1 peak at zeitgeber time
3, as would be expected from a nonparametric entrainment model
where each light pulse causes an equal and opposite phase shift (15).
Another, perhaps more dramatic, difference between the two
lighting regimes relates to the amplitude of the Per1 rhythm, which
is significantly reduced in cells on a skeleton photoperiod. In
particular, the trough in Per1 expression is much greater in the late
afternoon on a complete photoperiod, suggesting a much higher
level of repression by sustained light.
A comparison of Cry1a expression in parallel shows that, under
the skeleton photoperiod, each of the entraining 15-min light pulses
induces a short peak in Cry1a levels (Fig. 6B). Therefore, a peak in
Cry1a occurs to mark both dawn and dusk, with each light pulse
phase shifting the oscillator to produce steady-state entrainment.
After the first entraining cycle, the level of Cry1a induction is very
similar, as would be predicted for light pulses producing advances
and delays of equal but opposite magnitude. On a complete
photoperiod, only a single peak in Cry1a is observed, but the
duration and amplitude of this induction are much greater than on
a skeleton photoperiod. The timing of this broader peak in Cry1a
corresponds perfectly to the enhanced level of repression we see in
the Per1 oscillation. Consequently, we believe that the enhanced
levels of Cry1a during the light phase of the complete photoperiod
act to increase Per1 repression and, subsequently, to increase the
amplitude of the circadian oscillation. So, although the phase of the
rhythm is set accurately by the skeleton photoperiod (demonstrating nonparametric entrainment), there is a clear consequence of
sustained light on the clock, which is to establish a high-amplitude
molecular oscillation.
We then examined entrainment to a single 15-min light pulse and
compared this to a complete photoperiod. Cells exposed to the
‘‘one-pulse’’ regime show rapid synchronization, with a peak in Per1
expression occurring at a phase identical to cells on a complete or
Tamai et al.
two-pulse skeleton photoperiod (Fig. 6C). However, both the
amplitude of the Per1 rhythm and the induction of Cry1a are higher
on a one-pulse than a two-pulse lighting regime but still fail to reach
the levels seen on a complete photoperiod (Fig. 6D). Moreover,
entrainment to the one-pulse skeleton is not complete, because the
oscillator appears to free-run between single light pulses, with the
rising phase of Per1 expression clearly advancing each cycle. Thus,
to fully entrain the Per1 oscillation, either the continuous presence
of light during the day or an acute light pulse in the evening to
generate a critical phase delay is required. These results are very
similar to the relative coordination or ‘‘bouncing’’ phenomenon
described by Pittendrigh and Daan (16) in wheel running behavior
of rodents exposed to short, one-pulse lighting regimes. In our
experiments, the single light pulse at dawn will strike the phase
advance region of the zebrafish PRC, which may generate ‘‘aftereffects’’ in the clock mechanism and lead to a shortening of the
free-running period (17). This ‘‘history dependence’’ or aftereffect
can be clearly seen when the cells are placed into DD, because cells
exposed to the one-pulse regime show a much shorter free-running
period than those exposed to the complete photoperiod (24.48 ⫾
0.39 versus 27.31 ⫾ 0 h, respectively) (Fig. 6C). This history
dependence, as well as partial entrainment of the Per1 waveform,
may suggest multioscillator complexity even in cultured cells, either
between cells in the population or possibly within a single cell.
Discussion
The circadian pacemakers in zebrafish cell lines show highamplitude phase shifts in response to short, 15-min pulses of white
light (400–700 nm). An examination of genes believed to be
involved in circadian clock function reveals that Cry1a, among
others, is strongly induced by such light pulses. Several lines of
evidence support the idea that Cry1a is critical for light-induced
phase shifts. We have shown a strong correlation between light
intensity, induction of Cry1a expression, and size of the resulting
phase shift. Furthermore, light induction of Cry1a shows a key
phase dependency, with modest increases during the day and strong
PNAS 兩 September 11, 2007 兩 vol. 104 兩 no. 37 兩 14715
CELL BIOLOGY
Fig. 5. Model for light-induced interactions of Cry1a with CLOCK1 and BMAL1. (A) Yeast two-hybrid assays indicate strong binding between the bHLH and
PAS B domains of CLOCK1 and BMAL1. (B) Cry1a interacts directly with the PAS B domain of CLOCK1 and the bHLH, PAS B, and C-terminal domains of BMAL1.
These data support the hypothesis that light-induced Cry1a inhibits CLOCK:BMAL function by binding directly to the transactivation domain of BMAL and to
critical regions where CLOCK and BMAL themselves directly interact to form an active dimer.
Fig. 6. Zebrafish cells entrain to complete and skeleton photoperiods. Shown are bioluminescent traces of Per1-luciferase (A and C) and Cry1a-luciferase (B
and D) cells exposed to a complete versus two-pulse skeletal photoperiod (A and B) or a complete versus one-pulse skeletal photoperiod (C and D). The average
(⫾ SEM) of quadruplicate wells is presented. The rectangular bars above (complete photoperiod) and below (skeleton photoperiod) represent light (white) and
dark (black) periods.
inductions in the late night. This is of particular interest, because we
have shown that a single light pulse can shift an asynchronous
population of clock cells to a common phase of the circadian cycle,
equivalent to the early day or zeitgeber time 4 (6). One would
therefore predict that, if cells were close to zeitgeber time 4, only a
small phase shift and modest increase in Cry1a would be necessary
for entrainment. In the late night, however, a much larger phase
shift and higher level of Cry1a induction would be required, which
is precisely what we observe experimentally.
Because such correlations are not proof of a functional role for
Cry1a in phase shifting, we explored this issue further by overexpressing Cry1a in our Per1 luminescent cell line. The consequences
of this are quite dramatic, in that the clock oscillation is completely
abolished. Thus, Cry1a can clearly act as a strong transcriptional
repressor but, more importantly, as a light-induced repressor of
clock function. If Cry1a is a key element in the input pathway, then
increasing its levels independent of light (i.e., in the dark) should
mimic the natural action of light. This is true in the case of Cry1a
overexpression, which mimics remarkably well the consequences of
constant light. Both bright light and tonically overexpressed Cry1a
appear to stop the circadian clock. As described in Drosophila (12),
the core molecular oscillator of zebrafish seems to be held relatively
motionless for light exposures ⬎12 h and begins to oscillate from
that phase onwards when released back into darkness. This has
interesting implications for the zebrafish clock on photoperiods
⬎12 h of light, where it appears likely that this system may act more
like an ‘‘hourglass’’ than an oscillator.
Our yeast two-hybrid assays show that Cry1a can clearly interact
with the core clock transcriptional activators, CLOCK and BMAL.
The consequence of this binding is to strongly disrupt transactivation by the CLOCK:BMAL heterodimer, and, as such, Cry1a
strongly represses the positive limb of the circadian clock mechanism. Upon light exposure, Cry1a is strongly induced. Per1 expression levels fall rapidly and are tonically suppressed under constant
14716 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0704588104
light conditions. Phase shifts and clock entrainment appear, at least
in part, to be a result of this light-dependent transcriptional
repression by Cry1a. As shown above, the circadian oscillator
appears to be held ‘‘motionless’’ when the day length begins to
exceed 12 h at about CT12. What is special about this circadian
phase? It in fact corresponds to the time when expression of both
CLOCK and BMAL is becoming strongly activated (ref. 3 and data
not shown). We believe that newly synthesized CLOCK and BMAL
proteins may be targets for Cry1a, which binds to these key
transcriptional activators, prevents their downstream action, and,
consequently, stops the clock at this phase.
It is apparent from both the literature and a closer examination
of our Per1-luciferase rhythms under various lighting conditions that
Cry1a is not the only ‘‘player’’ in the light input pathway to the
zebrafish clock. Light pulses also induce Per2 expression, and
injection of a Per2 antisense morpholino into early-stage zebrafish
embryos has been shown to block subsequent synchronized rhythms
in zfaanat2 expression in the pineal gland (8). However, the
mechanism by which Per2 may be involved in clock resetting is not
yet known. Curiously, light pulses also lead to the acute induction
of Per1, which occurs before the increase in Cry1a levels and
subsequent repression in Per1 expression. It takes ⬇3 h for Cry1a
to reach peak transcript levels after light exposure, during which
time a transient increase in Per1 is observed. The mechanism and
potential role of this transient Per1 increase are not yet understood.
We have demonstrated that zebrafish cells entrain to both
complete and skeleton (one- and two-pulse) photoperiods. Although the phase angle of the Per1 peak is identical under these
conditions, the amplitude of the rhythm is very different. Moreover,
using single-pulse treatments, entrainment does not appear to be
complete across the entire cycle, and there are clear aftereffects on
the resulting free-running period in DD. The duration and amplitude of Cry1a induction are also considerably greater under complete versus skeleton photoperiods. Nevertheless, we believe that
Tamai et al.
Materials and Methods
Zebrafish Cell Lines. The generation of a Per1-luciferase zebrafish cell
line has been described (5). To establish a Cry1a-luciferase cell line,
a 4.7-kb fragment of the Cry1a promoter was amplified by PCR
from bacterial artificial chromosome clone HUKGB735F08222Q
(German Resource Center for Genome Research, Berlin, Germany) and subcloned into pGL3-Basic (Promega, Madison, WI).
PAC2 cells were electroporated with 5 ␮g each of linearized
Cry1a-luciferase DNA and pcDNA3.1/myc-His A (Invitrogen,
Carlsbad, CA). After neomycin selection, bioluminescence was
monitored on a Packard TopCount scintillation counter (28°C), and
cells from one strongly luminescent well were selected for these
studies. RNase protection assays showed that 4.7 kb of the Cry1a
promoter regulated expression of luciferase, which matched that of
endogenous Cry1a (data not shown). For all experiments, cells were
plated at 2.5–5.0 ⫻ 105 cells per milliliter.
RNA Analysis. Cells were maintained on a 12:12 LD cycle for 3 days
and then transferred into DD. At CT16 of the following day, cells
were exposed to white light (250 ␮W/cm2) for 1 h or kept in DD as
controls. Samples were harvested at the times indicated in the figure
legends. Total RNA was extracted in TRIzol (Invitrogen) following
the manufacturer’s instructions. The full-length coding regions of
the Cryptochrome (Cry1a, Cry1b, Cry2a, Cry2b, Cry3, and Cry4) and
Period (Per1, Per2, and Per3) genes were amplified by PCR from
existing plasmids or from PAC2 cells by RT-PCR and subcloned
into pGEM-Teasy (Promega). These plasmids were linearized and
used as templates for riboprobe synthesis (Promega). RNase protection assays were carried out as previously described (3).
Bioluminescence Assays. Per1- and Cry1a-luciferase cells were plated
in quadruplicate wells of a 96-well plate in media containing 0.5 mM
beetle luciferin (Promega). Unless otherwise indicated, cells were
placed on a 12:12 LD cycle for 3 days and transferred into DD. On
the following day, samples were light-pulsed at the time, intensity,
and duration indicated in Results. Bioluminescence was monitored
on a Packard TopCount NXT scintillation counter (28°C).
collected 2 days later, and Per1-luciferase cells were infected twice
a day for 2 days. Transfection efficiencies of 60–90% were typically
obtained. Single cells from a transfected population were sorted by
FACS into individual wells of a 96-well plate. Several clones were
expanded and examined by Western blot and immunocytochemistry (Fig. 3 and data not shown).
Western Blots. Cells were harvested in 300 ␮l of cracking buffer (8
M urea/5% SDS/40 mM Tris, pH 6.8/0.1 mM EDTA/0.4 mg/ml
bromophenol blue/147 mM 2-mercaptoethanol) plus protease and
phosphatase (type I and type II) inhibitors (Sigma, St. Louis, MO)
and boiled for 10 min. Proteins were separated by SDS/PAGE and
transferred to nitrocellulose (Schleicher and Schuell, Dassel, Germany). Blots were probed with rat anti-HA antibody high-affinity
3F10 (Roche, Basel, Switzerland) diluted 1:500 and developed by
using the ECL Plus Western Blotting Detection System (GE
Healthcare, Chalfont, St. Giles, U.K.).
Immunocytochemistry. Cells were fixed on day 3 at zeitgeber time 3
in 4% paraformaldehyde in PBS for 20 min at room temperature,
washed with PBS, and permeabilized in 0.2% Triton X-100 in PBS
for 5 min at room temperature. Samples were washed with PBS,
blocked in 5% albumin, and then incubated at 4°C overnight in rat
anti-HA antibody high-affinity 3F10 (Roche) diluted 1:500 in 1%
albumin in PBS. Cells were washed with PBS and incubated at room
temperature for 1 h in Alexa Fluor 568 goat anti-rat (Molecular
Probes, Eugene, OR) diluted 1:1,000 in 1% albumin in PBS.
Samples were washed with PBS, incubated in DAPI (1:50,000) for
5 min at room temperature, washed again, and mounted.
Yeast Two-Hybrid Assays. The full-length coding regions of zebrafish
Cry1a, CLOCK (1, 2, and 3) and BMAL (1, 2, and 3) were fused
in-frame to the GAL4 DNA-binding domain of pGBKT7 or the
GAL4 activation domain of pGADT7 (Clontech). Deletion constructs encoding the amino acids indicated in the figures were also
generated. pGBKT7 and GAL4 DNA binding domain fusions were
transformed into yeast strain AH109 (MATa), and pGADT7 and
GAL4 activation domain fusions were transformed into strain Y187
(MAT␣). The two strains were mated on rich media YPDA, and
diploids were selected on synthetic dropout media minus leucine
and tryptophan (Clontech). Protein interactions were routinely
assayed by monitoring growth on synthetic media minus adenine.
Pairings that were negative were tested on media minus histidine
plus different concentrations of 3-aminotriazole.
N-terminally tagged HA-Cry1a was amplified by PCR and subcloned into the retroviral vector pCLNCX (Imgenex, San Diego,
CA). Expression of cDNAs subcloned into pCLNCX is driven by
the CMV promoter. The packaging cell line GP2–293 (Clontech,
Palo Alto, CA) was transfected as described (6). Retrovirus was
We thank Nick Foulkes (Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Karlsruhe, Germany), Daniela Vallone (Institute of
Toxicology and Genetics, Forschungszentrum Karlsruhe), Matt Pando
(Exon Hit Therapeutics, Gaithersburg, MD), Amanda Carr (Institute of
Ophthalmology, University College London), and Veronica Ferrer (Department of Anatomy and Developmental Biology, University College
London) for plasmids and reagents; Kirsty Allen and Derek Davies at
Cancer Research UK for expert cell sorting; and members of the D.W.
laboratory for valuable comments. We are especially grateful to Carl
Johnson and Terry Page (who bear no responsibility for any circadian
‘‘errors’’ in the text) for many helpful comments and suggestions. This work
was supported through funds from The Wellcome Trust and the Biotechnology and Biological Sciences Research Council.
1. Plautz JD, Kaneko M, Hall JC, Kay SA (1997) Science 278:1632–1635.
2. Tamai TK, Vardhanabhuti V, Arthur S, Foulkes NS, Whitmore D (2003) J Neuroendocrinol 15:344–349.
3. Whitmore D, Foulkes NS, Sassone-Corsi P (2000) Nature 404:87–91.
4. Tamai TK, Vardhanabhuti V, Foulkes NS, Whitmore D (2004) Curr Biol 14:R104–
R105.
5. Vallone D, Gondi SB, Whitmore D, Foulkes NS (2004) Proc Natl Acad Sci USA
101:4106–4111.
6. Carr AJF, Whitmore D (2005) Nat Cell Biol 7:319–321.
7. Cermakian N, Pando MP, Thompson CL, Pinchak AB, Selby CP, Gutierrez L, Wells DE,
Cahill GM, Sancar A, Sassone-Corsi P (2002) Curr Biol 12:844–848.
8. Ziv L, Levkovitz S, Toyama R, Falcon J, Gothilf Y (2005) J Neuroendocrinol 17:314–320.
9. Hirayama J, Cardone L, Doi M, Sassone-Corsi P (2005) Proc Natl Acad Sci USA
102:10194–10199.
10. Kobayashi Y, Ishikawa T, Hirayama J, Daiyasu H, Kanai S, Toh H, Fukuda I, Tsujimura
T, Terada N, Kamei Y, et al. (2000) Genes Cells 5:725–738.
11. Peterson EL (1980) J Theor Biol 84:281–310.
12. Pittendrigh CS (1966) Z Pflanzenphysiol 54:275–307.
13. Ishikawa T, Hirayama J, Kobayashi Y, Todo T (2002) Genes Cells 7:1073–1086.
14. Lahiri K, Vallone D, Gondi SB, Santoriello C, Dickmeis T, Foulkes NS (2005) PLoS Biol
3:e351.
15. Pittendrigh CS, Minis DH (1964) Am Nat 98:261–294.
16. Pittendrigh CS, Daan S (1976) J Comp Physiol 106:223–252.
17. Pittendrigh CS (1960) Cold Spring Harbor Symp Quant Biol 25:159–184.
Retroviral Constructs and Transfections. A DNA fragment encoding
Tamai et al.
PNAS 兩 September 11, 2007 兩 vol. 104 兩 no. 37 兩 14717
CELL BIOLOGY
Cry1a is playing a key role under both conditions. Cry1a can act as
a nonparametric or discrete entraining cue under skeletal photoperiods, causing an acute repression and phase shift of the Per1
rhythm. In addition, it can act in a parametric or continuous manner
under full photoperiods, where the sustained induction of Cry1a
acts to dramatically increase the amplitude of the circadian oscillator. These observations, in some ways, blur the distinction of
parametric and nonparametric entrainment as representing two
separate mechanisms. Taking this one step further, these results
suggest that, through its light induction and subsequent inhibitory
action on CLOCK:BMAL, Cry1a may actually stop the clock
completely.