Glucocorticoids Play a Key Role in Circadian Cell Cycle Rhythms
Citation: Dickmeis T, Lahiri K, Nica G, Vallone D, Santoriello C, et al. (
Glucocorticoids Play a Key Role in Circadian Cell Cycle Rhythms
Thomas Dickmeis 0 1
Kajori Lahiri 0 1
Gabriela Nica 0 1
Daniela Vallone 0 1
Cristina Santoriello 0 1
Carl J. Neumann 0 1
Matthias Hammerschmidt 0 1
Nicholas S. Foulkes 0 1
0 Academic Editor: Ueli Schibler, University of Geneva , Switzerland
1 1 Max-Planck-Institut f u r Entwicklungsbiologie , T u bingen, Germany , 2 Max-Planck-Institut f u r Immunbiologie , Freiburg, Germany , 3 European Molecular Biology Laboratory Heidelberg , Heidelberg , Germany
Clock output pathways play a pivotal role by relaying timing information from the circadian clock to a diversity of physiological systems. Both cell-autonomous and systemic mechanisms have been implicated as clock outputs; however, the relative importance and interplay between these mechanisms are poorly understood. The cell cycle represents a highly conserved regulatory target of the circadian timing system. Previously, we have demonstrated that in zebrafish, the circadian clock has the capacity to generate daily rhythms of S phase by a cell-autonomous mechanism in vitro. Here, by studying a panel of zebrafish mutants, we reveal that the pituitary-adrenal axis also plays an essential role in establishing these rhythms in the whole animal. Mutants with a reduction or a complete absence of corticotrope pituitary cells show attenuated cell-proliferation rhythms, whereas expression of circadian clock genes is not affected. We show that the corticotrope deficiency is associated with reduced cortisol levels, implicating glucocorticoids as a component of a systemic signaling pathway required for circadian cell cycle rhythmicity. Strikingly, high-amplitude rhythms can be rescued by exposing mutant larvae to a tonic concentration of a glucocorticoid agonist. Our work suggests that cell-autonomous clock mechanisms are not sufficient to establish circadian cell cycle rhythms at the whole-animal level. Instead, they act in concert with a systemic signaling environment of which glucocorticoids are an essential part.
The physiology of most plants and animals changes
significantly between day and night. These daily rhythms
are generated by endogenous clocks or pacemakers, and
persist under constant conditions with a period length of
approximately 24 h (hence, they are termed circadian). In
vertebrates, cell-autonomous circadian clocks are present in
most cell types, and are termed peripheral clocks. In addition,
a limited number of specialized central pacemakers such as
the suprachiasmatic nucleus (SCN) of the hypothalamus [1,2]
appear to play a key role in coordinating the function of
peripheral clocks. Although it is known that ocular
photoreception synchronizes the SCN pacemaker with the
environment, the identity of the pathways that subsequently transmit
timing information to the peripheral clocks remains elusive.
Current models implicate multiple humoral signals that
result indirectly from the SCN circadian control of systemic
function, such as feeding behavior .
Both cell-autonomous and systemic regulatory mechanisms
have been implicated in clock output pathways that relay
timing information from the clock to physiological systems.
Circadian E box enhancers represent key regulatory elements
within the core transcriptiontranslation feedback loop of
the vertebrate clock. These promoter elements direct
circadian rhythms of transcription of clock genes by acting
as binding sites for the clock components CLOCK and
BMAL1. Circadian E boxes are also encountered in the
promoters of many non-clock genes (so-called
clock-controlled genes, e.g., see ). Via such target genes and their
downstream effectors, peripheral circadian clock
components directly regulate many aspects of cell physiology, such
as membrane trafficking, detoxification, nutrient metabolism,
and the cell cycle . The central SCN pacemaker, in contrast,
has been documented to influence systemic functions ranging
from locomotor activity rhythms and the sleepwake cycle to
endocrine activity. Thus, the circulating levels of many
hormones are under circadian control and so exert their
effects only during specific times of day. A major unexplored
issue is the relative contribution of cell-autonomous and
systemic factors in directing circadian clock outputs. Do
certain clock outputs rely solely upon direct peripheral clock
regulation, or do they require input from systemic factors,
acting either upstream or downstream of the peripheral
clocks? Are other outputs driven solely by circadian
oscillations of systemic signals?
A particularly interesting clock output is the timing of cell
proliferation. Daily rhythms of cell division are conserved
across huge evolutionary distances, from cyanobacteria to
humans [5,6]. This property has been proposed as a strategy
for minimizing the ultraviolet damaging effects of sunlight
To guarantee normal growth and to avoid tumor formation, the
timing of cell division must be under strict control. Remarkably, cells,
from bacteria to man, often divide only at certain times of day,
suggesting the influence of internal biological clocks. A central
pacemaker structure in the brain controls diurnal rhythms of
behavior and hormone release. However, biological clocks are also
encountered in almost every cell type (so-called peripheral
clocks), in which they regulate daily changes in cell biology,
including cell division. Very little is known to date about how the
two clock systems interact. Here, by examining zebrafish strains with
defects in hormone production, we find that peripheral clocks
require the steroid hormone cortisol to generate daily rhythms of
cell proliferation. Interestingly, the daily changes in cortisol levels
observed in normal zebrafish are not required to achieve this
control; treating the cortisol-deficient strains with constant levels of
a drug that mimics the effects of cortisol restores normal
celldivision rhythms. Thus, it appears that internal cell timers cooperate
with hormonal signals to regulate the timing of cell division.
during critical steps of the cell proliferation. In vertebrates,
circadian gating of certain cell cycle steps also occurs in cell
lines [7,8]. Furthermore, clock components have been
implicated in controlling the transcription of cell cycle
regulatory genes . These observations imply that the
circadian clock may regulate cell cycle progression via
cellautonomous mechanisms. However, given that systemic
factors such as hormones are well-known regulators of cell
proliferation [13,14], one important question is whether
cellautonomous regulatory mechanisms are sufficient to direct
circadian cell cycle rhythms at the whole-animal level.
The zebrafish represents a valuable model for exploring the
vertebrate circadian clock and its regulation of cell cycle
timing. Robust daily S-phase rhythms are observed in larvae
raised under lightdark (LD) cycles . The persistence of
these rhythms following transfer of the larvae to constant
darkness (DD) conditions demonstrates that they are under
control of the circadian clock. Furthermore, consistent with
other clock outputs , exposure to a LD cycle is essential
for the establishment of these rhythms because they are
absent in larvae raised in DD. Circadian rhythms of S phase,
albeit with lower amplitude, are also observed in zebrafish
primary cell lines, implicating cell-autonomous regulation by
peripheral clock mechanisms . Interestingly, peripheral
clocks in this species can be entrained by direct exposure to
LD cycles . However, zebrafish also possess central
pacemakers: a structural counterpart of the SCN and a
photosensitive pineal complex where nighttime synthesis of
the hormone melatonin is directed by an endogenous clock
Extensive panels of zebrafish mutants that show specific
developmental defects in a range of organ systems have been
assembled, thanks to large-scale screening efforts . These
animals represent potentially powerful tools to dissect the
functional contribution of specific organs and tissues to the
generation of clock outputs at the whole-animal level. Here,
by studying a set of blind mutants, we have demonstrated that
ocular photoreception is not required to establish circadian
cell cycle rhythms during early larval development. In
contrast, a severe attenuation of cell cycle rhythms is
observed in mutants that exhibit a reduction or absence of
the corticotrope cell lineage in the pituitary gland.
Importantly, high-amplitude circadian cell cycle rhythms can be
rescued by exposing corticotrope-deficient larvae to tonic
concentrations of the glucocorticoid receptor (GR) agonist
dexamethasone. Our work reveals the contribution of
systemic factors to establishing circadian cell cycle rhythms
at the whole-animal level.
Ocular Photoreception Is Not Required for Zebrafish
Circadian Cell Cycle Rhythms
We have previously demonstrated that exposure to a LD
cycle is a prerequisite for circadian cell cycle rhythms to be
established during early larval development. Whereas
zebrafish peripheral clocks are directly light entrainable in vitro,
light input through the eyes also plays an important role in
entraining the circadian timing system in most vertebrates.
We therefore asked whether ocular photoreception might
contribute to establishing circadian cell cycle regulation in
the zebrafish. We examined cell cycle rhythms in a set of
functionally blind mutants using bromodeoxyuridine (BrdU)
incorporation as a marker for the S phase of the cell cycle.
lakritz/ath5 (lak) mutants, which carry a null mutation in a
basic Helix-Loop-Helix transcription factor gene, the atonal
homologue 5, lack the retinal ganglion cell layer . These cells
relay light information from the inner retina to the brain.
Furthermore, in mammals, the retinal ganglion cells
themselves function as a circadian photoreceptor . These
mutants are thus particularly well suited for examining the
role of ocular photoreception in clock outputs. Mutant and
wild-type siblings were raised under a LD regime, and BrdU
incorporation was tested at four time points on the sixth day
post-fertilization (dpf), before feeding starts. BrdU
incorporation rhythms in the lak mutant larvae are indistinguishable
from their wild-type siblings (Figure 1A) and thus not affected
by the absence of ocular light input. To confirm these results,
we examined chokh/rx3 mutant fish (carrying mutations in the
retinal homeobox gene 3), which show a severe impairment of eye
and retinal development . We analyzed two alleles
(weak, chkt25181 chkw, and strong, chkt25327 chks) with
differing severity of the morphological phenotype . As for
the lak mutants, in the weak rx3 larvae, BrdU incorporation
rhythms are indistinguishable from the wild-type siblings
(Figure 1B). Surprisingly, however, the strong rx3 mutant
larvae show a severe attenuation of the circadian S-phase
rhythm compared with their wild-type siblings (amplitude
reduced from 4.0-fold to 2.3-fold, Figure 1C and Figure S1).
Since both lak and weak rx3 mutants are blind, we conclude
that the attenuated S-phase rhythm of the strong rx3 mutant
is functionally unrelated to its blindness. This suggests the
presence of additional distinct defects in this mutant.
chokh/rx3 Mutant Fish Show Normal Clock Gene
What is the cause of the attenuated cell cycle rhythms in
the strong rx3 mutants? Given the proposed direct link
between the cell cycle and the circadian clock , we first
tested whether the cell cycle phenotype was due to a
deregulation of the circadian clock itself. We examined
mRNA expression of clock genes in larval RNA extracts from
wild-type and rx3 mutant siblings raised in the same
(A) Quantification of BrdU-labeled nuclei in lak mutant larvae and
wildtype siblings at four circadian time points. The mean number of positive
nuclei between swim bladder and anus (y axis) was plotted against ZT,
hours after lights on, lights off at ZT12) (x axis) for mutant (blue) and
sibling (red) larvae. Error bars indicate the 95% confidence interval for
the mean, and asterisks indicate the statistical significance of differences
between mutants and wild-type siblings as measured by the
MannWhitney-Test. ***, p , 0.001.
(B) Quantification of BrdU incorporation analogous to (A) for mutants
and siblings of the weak rx3 allele.
(C) Left: quantification analogous to (A) for strong rx3 mutants. Right:
representative examples of whole-mount stainings used for the
quantification. Mutant (mut) and sibling (sib) larvae at peak (ZT9) and
trough (ZT21) points of the cell cycle rhythm are shown. Mutant larvae
appear darker, because their melanophores are expanded (lack of visual
background adaptation in blind fish). A high-resolution image of this part
of the figure is provided as Figure S1.
Pooled results from two (A), four (B), and nine (C) independent
experiments are shown.
conditions as those of the BrdU experiments. We assayed
expression of clock as a representative of the positive limb 
and of per4 for the negative limb  of the circadian
feedback loop . As shown in Figure 2A, rhythmic
expression of clock and per4 in both rx3 alleles is equivalent
to that of their wild-type siblings. Thus, the cell cycle defects
in the strong allele cannot be explained by a global
deregulation of the circadian clock.
Could the attenuated cell cycle rhythms of the strong rx3
mutants be attributed to the disruption of a systemic pathway
conveying circadian timing information to the cell cycle?
Nocturnal production of the hormone melatonin by the
pineal gland is a key central clock output . Recently,
melatonin has also been implicated in regulating cell
proliferation in larval zebrafish . Pineal mRNA expression
of the rate-limiting enzyme of the
pathway, arylalkylamine N-acetyltransferase (AANAT), is
under circadian regulation, with expression high during the
night and low during the day ( and Figure 2B, upper row).
This expression pattern has been used widely as a reliable
Circadian Clock Gene Expression or Disturbed Pineal Melatonin Output
(A) Circadian expression of clock genes in mutant larvae of both weak
and strong alleles of rx3. Representative RNase protection assay results
for per4, clock1, and b-actin expression using RNA harvested at the
indicated ZTs. t represents a tRNA negative control.
(B) Circadian expression of aanat2 in strong rx3 mutants. Whole-mount in
situ hybridization for aanat2 in strong rx3 mutants (strong) and
wildtype siblings (wildtype) at four circadian time points (6 dpf). Images
show dorsal views of the pineal gland (arrow), rostral up.
(C) Quantification of BrdU labeled nuclei in luzindole treated wild-type
larvae. Means of BrdU-positive nuclei determined as described in Figure
1 are shown at four circadian time points on 6 dpf for luzindole-treated
larvae (, blue) and control larvae ( , red). Error bars show 95%
confidence interval of the mean.
. The whole-mount in situ hybridization for aanat2 in
strong rx3 mutant larvae shown in Figure 2B (lower row)
reveals a circadian expression rhythm indistinguishable from
wild-type siblings. Thus, the attenuated cell cycle rhythms in
strong rx3 larvae seem unlikely to be explained by defects in
this endocrine pathway. As an additional test of the
contribution of melatonin, we treated wild-type larvae with
luzindole, a specific antagonist of the MT1 and MT2
highaffinity melatonin receptors [31,36]. Cell cycle rhythms were
not significantly affected by this treatment, confirming the
hypothesis that melatonin production is not required for
establishing cell cycle rhythms (Figure 2C). In lower
vertebrates, the pineal is a directly photosensitive structure, and
light directly affects the production of melatonin .
Thereindicator of the levels of melatonin synthesis in the zebrafish
fore, our results also indicate that direct pineal
photochokh/rx3 Mutant Fish Show Pituitary Defects
The hypothalamicpituitary axis is another endocrine
pathway with a crucial role in the control of cell proliferation
that shows circadian variations of activity . We examined
expression of a set of specific pituitary cell-lineage markers in
the rx3 mutants: The transcription factor pit1 , growth
hormone, gh , prolactin, prl , and glycoprotein
hormone alpha subunit, a-gsu . Expression of these
markers is equivalent in rx3 mutants of both alleles when
compared with their wild-type siblings (Figure 3A). Thus, the
somatotrope (gh), lactotrope (prl), and
gonadotrope/thyrotrope (a-gsu) lineages appear to be normally formed in the
strong rx3 mutants. However, for the
corticotrope/melanotrope lineage marker proopiomelanocortin (pomc, ), two
expression domains show a marked reduction in strong allele
rx3 mutants. The anterior pituitary domain is strongly
reduced (arrowhead), and the expression corresponding to
the b-endorphin/MSHa synthesizing cells of the arcuate
nucleus (, arrow) is essentially absent, whereas the
posterior pituitary expression domain (asterisk) appears
normal. All these domains have a wild-typelike appearance
in the weak allele mutant larvae (Figure 3A).
To test whether these differences reflect a general
disorganization of the diencephalon, we examined the
expression of a number of hypothalamic markers
(somatostatin3, isotocin, and corticotropin releasing factor; for details, see
Figure S2). The structures labeled by these markers are
present in both mutant alleles and show no major disruption,
despite the lack of normal eyecups. Thus, the strong rx3
mutation specifically seems to affect the pomc-expressing cells
in the anterior pituitary and the arcuate nucleus. Previous
studies have established that the anterior pituitary expression
domain of pomc consists mainly of cells of the corticotrope
lineage, whereas the posterior domain also contains
melanotrope cells . Furthermore, the arcuate nucleus has been
implicated in regulation of the corticotrope axis in mammals
. Thus, the reduced number of corticotrope cells in the
strong rx3 mutants might additionally lack normal
hypothalamic control. These findings implicate the corticotrope
lineage in circadian cell cycle regulation.
Corticotrope Deficiency Attenuates Cell Cycle Rhythms
Given the pituitary defect in the strong rx3 mutant, we
asked whether disruption of the hypothalamicpituitary axis
would cause similar circadian cell cycle defects to those seen
in the strong rx3 mutants. To address this issue, we examined
rhythms of BrdU incorporation in a series of zebrafish
mutants that lack either the entire pituitary or specific
subsets of pituitary lineages (Figure 3B3E) [38,40,4346]. The
fibroblast growth factor 3 mutant lia/fgf3 (two alleles, ) and the
proneural basic Helix-Loop-Helix transcription factor achaete
scute-complex like 1a mutant pia/ascl1a , which lack the entire
pituitary, show severely attenuated rhythms (Figure 3B and
unpublished data). Thus, genetic ablation of the pituitary
creates a circadian cell cycle phenotype highly similar to that
observed for the strong rx3 mutant. Since lia and pia mutants
show normal pomc expression in the arcuate nucleus, we can
also exclude a non-pituitarymediated contribution of
bendorphin/MSHaexpressing arcuate nucleus neurones to
cell cycle rhythm generation.
To pinpoint the precise pituitary cell type responsible for
the establishment of normal circadian cell cycle rhythms, we
examined BrdU incorporation rhythms in two other pituitary
mutants that lack subsets of the pituitary lineages (Figure 3E):
The protein tyrosine phosphatase eyes absent 1 mutant aal/eya1
[44,45], which possesses only the lactotropes, and the
POUdomain transcription factor pit1 mutant , in which only
the corticotropes/melanotropes and the gonadotropes are
present. The aal mutants show a similar phenotype to the lia,
pia, and the strong rx3 mutants (Figure 3C), demonstrating
that the lactotrope lineage alone is not sufficient for
establishing circadian cell cycle rhythms. In contrast, the
pit1 mutants are indistinguishable from their wild-type
siblings (Figure 3D). Thus, the presence of only the
corticotrope/melanotrope and gonadotrope lineages is sufficient to
establish wild-type circadian cell cycle rhythmicity. Together
with the reduced number of corticotropes observed in the rx3
mutant embryos, this result strongly suggests that the
corticotropes are required for the establishment of the
circadian cell cycle rhythms.
Corticotrope Deficiency Leads to Lowered Larval Cortisol
The principal target organ of signaling by the corticotrope
axis is the medulla of the adrenal gland (interrenal gland in
fish ), where it regulates production of glucocorticoids
such as cortisol. To explore the mechanism of the cell cycle
defect in the strong rx3 mutants, we measured cortisol levels
in 6-d-old mutant and wild-type sibling larvae of both alleles
raised under a LD cycle (Figure 4A). All larvae tested show
higher cortisol levels at zeitgeber time (ZT)17 than at ZT1.
However, mutant larvae of the strong allele have significantly
lower levels (p , 0.0001) than all other larvae at both time
points. Thus, the reduction of corticotrope cells in the strong
allele mutant pituitary seems to strongly reduce cortisol
levels, pointing to cortisol as a candidate systemic signal
required for circadian cell cycle rhythmicity.
The GR Agonist Dexamethasone Can Rescue the Mutant
Cell Cycle Rhythm Phenotype
If cortisol is indeed the systemic signal, it should be
possible to rescue circadian cell cycle rhythms by artificially
stimulating glucocorticoid signaling in the strong rx3
mutants. Mutant larvae and wild-type siblings were raised in the
presence of the potent glucocorticoid agonist
dexamethasone. We then measured BrdU incorporation at four time
points on day 6 of development. Strikingly, dexamethasone
treatment fully restores high-amplitude circadian cell cycle
rhythms in the mutants (Figure 4B). Non-treated control
mutant larvae show the typical severely attenuated rhythms
(Figure 4C). Similarly, aal mutant larvae treated with
dexamethasone are indistinguishable from their wild-type
siblings (Figure 4D and 4E). In conclusion, tonically activating
glucocorticoid signaling during the early days of development
can rescue the circadian cell cycle rhythms in cortisol
Our previous work has shown that the diurnal cell cycle
rhythms of zebrafish larvae are under control of the circadian
clock, because these rhythms persist upon transfer into
constant darkness . We therefore asked whether the rescue
effect of dexamethasone treatment could also operate
without direct light input. Tonic dexamethasone application
could indeed rescue BrdU incorporation rhythms in mutant
larvae that were transferred to DD after 5 d of entrainment
under a LD cycle (Figure S3), clearly showing that the rescue
is due to interaction of glucocorticoids with the circadian
clock and not dependent on direct light input.
Tonic GR Gene Expression
We wished to explore in more detail how cortisol affects
cell cycle rhythmicity. We first tested the hypothesis that the
circadian clock might regulate expression levels of the
glucocorticoid receptor gene (GR) and thereby confer a
circadian rhythm of sensitivity to the receptor ligand. Such a
mechanism would enable even tonic levels of the ligand to
activate GR signaling pathways with a circadian rhythm.
Furthermore, recent reports have highlighted that many
members of the nuclear receptor superfamily show circadian
cycling of transcript levels . We thus prepared a time
course of total RNA and protein extracts from wild-type
sibling larvae during one LD cycle. Quantitative real-time
PCR analysis failed to detect any significant change in GR
mRNA levels during the course of the LD cycle (Figure 5A).
Consistently, levels of an 82-kDa immunoreactive protein
corresponding to the zebrafish GR also did not cycle as
determined by Western blotting analysis (Figure 5B). These
results indicate that the circadian clock does not simply affect
global expression levels of the GR.
Shorter Dexamethasone Treatments Also Rescue Cell
Many studies have documented the functional complexity
of the glucocorticoid signaling pathway in vivo . Lack of
cortisol during development and larval growth might
generally alter larval physiology and thereby also indirectly
affect cell cycle rhythms. Rescue of high-amplitude cell cycle
rhythms in rx3 mutant larvae by continuous exposure to
dexamethasone from early development onwards could act
via rescuing developmental defects as well as by affecting cells
more directly. In order to address this point, we
systematically tested the effect of reducing the duration of exposure to
dexamethasone on cell cycle rhythms. We supplemented the
medium with dexamethasone at progressively later stages
before harvesting on day 6 of larval development (Figure 5C
and unpublished data). Addition of dexamethasone as late as
the night before sampling (day 5) still resulted in a significant
increase in cell cycle amplitude. Dexamethasone delivered at
later time points failed to rescue the rhythm. Given that all
the major organ systems have developed and are functional at
this freely feeding larval stage , this would exclude a major
role for indirect developmental mechanisms.
The zebrafish represents an attractive model to explore
how circadian clock outputs are regulated at the
wholeanimal level. We have previously implicated a contribution of
directly light-entrainable peripheral clocks in the
cellautonomous control of circadian rhythms of S phase. Here,
by studying panels of zebrafish mutants affecting
development of the eye and hypothalamicpituitary axis, we have
been able to define the regulatory contribution of these
structures to establishing circadian cell cycle rhythms. This
study illustrates the power of using complementary sets of
zebrafish mutants in the genetic dissection of physiological
pathways in vivo.
We have shown that ocular photoreception is dispensable
for establishing this clock output function, potentially
reinforcing the notion that cell-autonomous light sensing
plays a key role in cell cycle entrainment in the zebrafish.
However, in common with most lower vertebrates, zebrafish
possess additional extraocular specialized photoreceptor
tissues: the pineal complex  and also the so-called deep
brain photoreceptors that line the third ventricle of the
diencephalon [51,52]. Since no zebrafish mutants are
available to date that specifically lack these photoreceptors, it is
problematic to assess their contribution. We used an
alternative pharmacological approach to interfere with the
melatonin signal, the major output of the pineal gland, and
thereby tested whether photoreception through the pineal
complex might affect cell cycle rhythms. Because treatment of
larvae with the melatonin receptor antagonist luzindole did
not change circadian cell cycle rhythms, pineal light
reception is not strictly required for the timing of circadian
cell cycle progression. However, it is still conceivable that
light input from the pineal conveyed via neuronal pathways
may contribute to the timing of the cell cycle . Also,
one type of dedicated photoreceptor might be able to
substitute for lack of input from the other types, leading to
functional redundancy of inputs from, e.g., the eye and the
pineal complex. Finally, the direct peripheral light reception
alone might also be sufficient to time this clock output in the
context of the whole animal. Ultimately, mutant zebrafish
that lack all specialized photoreceptor cells will be required
to assess the relative contribution of directly light-sensing
peripheral clocks to entraining the circadian cell cycle.
By studying pituitary mutants with overlapping cell-lineage
defects, and by our demonstration that pituitary
corticotropes are severely reduced in strong allele rx3 mutant larvae,
we have implicated this pituitary cell lineage in the
establishment of circadian cell cycle rhythms. Furthermore, levels of
cortisol in strong rx3 mutants are reduced and, importantly, a
GR agonist rescues the circadian cell cycle defects in both
strong rx3 and pituitary mutants. These findings point to
glucocorticoids as a requirement for high-amplitude cell
Previously, glucocorticoids have been implicated in the
entrainment of peripheral circadian clocks in mammals.
Injection of dexamethasone into mice can reset the phase of
peripheral oscillators . However, mice lacking the GR
show normal clock gene expression rhythms in the liver .
Furthermore, here we observe normal circadian clock gene
cycling in the strong rx3 mutant larvae (Figure 2A), despite
their low levels of cortisol. Consistently, also in the aal
mutants, per4 exhibits normal circadian rhythms of
expression (Figure S4). Finally, zebrafish cells grown in
cortisoldepleted medium still show normal clock expression under a
LD cycle (Figure S5B, see below). Thus, glucocorticoid
signaling is not an absolute requirement for the regulation
of the peripheral pacemakers themselves. Our data directly
implicate glucocorticoids in the regulation of clock output.
Glucocorticoids are well known to influence cell
proliferation, both in vitro and in vivo. For example, dexamethasone
can stimulate myoblast proliferation  and enhance the
mitogenic response of fibroblasts to epidermal growth factor
(EGF) , whereas it inhibits cell division in a
lymphosarcoma cell line . In addition, at the whole-animal level,
corticosteroids have been reported to affect the capacity of
the liver to regenerate after hepatectomy in rats . Our
results indicate that part of the effect of corticosteroids on
cell proliferation might be brought about by cooperation
with circadian clock output pathways.
At which level of organization might this interaction occur?
Given the wealth of physiological targets of glucocorticoids,
they might function through indirect, systemic pathways. For
example, in zebrafish larvae, they might be involved in the
maturation of organ systems and their physiological
functions during development. However, our finding that
dexamethasone treatment starting 10 h before sampling still
rescues cell cycle rhythms in rx3 mutants makes a long-term
effect on development unlikely. Nevertheless, other indirect
systemic pathways might act within this time frame. For
example, glucocorticoids could stimulate the release of
mitogens from neighboring tissues or influence the release
of other hormones (e.g., see ). Alternatively, they might
act directly at the level of the proliferating cells themselves.
We tested a potential cell-autonomous role for
glucocorticoids in circadian cell cycle rhythms by examining zebrafish
cell cultures grown in charcoal-treated (and thereby
steroiddepleted) medium (Figure S5). Whereas circadian clock gene
expression is normal in these cultures (Figure S5B), circadian
cell cycle rhythms are severely attenuated (Figure S5A), thus
mimicking the situation in cortisol-deficient larvae. However,
in this cell culture system, treatment with dexamethasone
does not rescue the attenuation (unpublished data). One
explanation for this result could be that glucocorticoids
might need to act synergistically with other substances that
are also depleted from the medium by charcoal treatment.
Taken together, although there are some hints for a direct
cell-autonomous action of glucocorticoids on circadian cell
cycle rhythms, more indirect systemic effects certainly cannot
be excluded. Indeed, given the multifaceted actions of
glucocorticoids, confinement of their effects on cell cycle
progression to a single level would be rather surprising. In the
animal, glucocorticoids might help to create a systemic
signaling environment, within which they could also exert
more direct effects.
What is the relative importance of systemic and peripheral
circadian clock mechanisms in glucocorticoid-mediated
circadian cell cycle regulation? Circadian rhythms of
circulating glucocorticoids have long been recognized as a clock
output in various vertebrates . However, here we show
that the role played by glucocorticoids in circadian cell cycle
rhythms does not necessarily involve conveying timing
information via changes in circulating levels. Rather, a
constant glucocorticoid signal can rescue rhythms of cell
cycle in cortisol-deficient mutant larvae. In the animal, the
timing information might stem from another cycling systemic
signal, or it might be provided by peripheral circadian clocks.
Circadian changes in glucocorticoid levels might then
reinforce the peripheral timing information in cell cycle
control, or they might be required for other physiological
How can a constant glucocorticoid signal lead to a
rhythmic output? We propose a working model in which a
certain level of glucocorticoid signaling may be permissive
(Figure 6A, green arrow) for peripheral circadian clock
regulation of genes involved in cell proliferation (Figure 6A,
red arrow). Alternatively, the clock could gate responsiveness
to the glucocorticoid signal, which would then act to regulate
cell proliferation (Figure 6A, blue arrow). In either scenario,
in corticotrope-deficient mutants, GR signaling is attenuated,
and then peripheral clock input alone would be insufficient
to generate full cell cycle rhythms (Figure 6B). Furthermore,
in the rescue experiments, tonic dexamethasone treatment
activates GR signaling and would restore cell cycle rhythms in
the mutants with timing information being provided by the
(A) In wild-type larvae, corticotrope function is associated with normal
cortisol levels (that exhibit circadian cycling). In target cells, cortisol
activates GR signaling (green arrow) that may be permissive for the
peripheral clock regulation of cell cycle. In addition, cycling cortisol levels
may confer circadian rhythms of GR signaling that potentially reinforce
cell cycle rhythmicity. The red arrow indicates the peripheral circadian
clock control of cell cycle, and the blue arrow symbolizes the potential
gating of GR signaling by the peripheral pacemaker. Cooperation
between the peripheral clock mechanism and the GR signaling pathway
generates a circadian rhythm of cell cycle.
(B) In corticotrope-deficient mutants, circulating cortisol levels are
significantly reduced. In the absence of normal GR signaling, peripheral
clock input is not sufficient to generate cell cycle rhythms. Dashed
arrows indicate attenuated pathways.
(C) Cell cycle rhythms are rescued in corticotrope-deficient mutants by
tonic dexamethasone (DEX) treatment. Thus timing information is
provided by peripheral circadian clocks and is not reliant on cycling
peripheral circadian clock (Figure 6C). In this model, cycling
An attractive mechanism for gating responsiveness to
glucocorticoids would involve circadian clock regulation of
expression of the GR itself. Expression of many nuclear
receptor transcripts is under circadian clock control in
different peripheral tissues . However, here we show that
the levels of the GR transcript as well as the protein are not
subject to significant daynight variation. Thus, a simple
scenario in which transcription of the GR gene is a regulatory
target of peripheral clock components, e.g., via E box
elements, appears unlikely. One can speculate that if indeed
the receptor is under clock control, then this may occur at the
post-translational level. Alternatively, circadian clock control
might operate at various levels on other elements of the
glucocorticoid signaling pathway, perhaps downstream of the
receptor itself [64,65].
Glucocorticoids can increase the amplitude of cell cycle
rhythms in strong rx3 mutants even when they are delivered
the night before assaying BrdU incorporation. Specifically,
they have to be present at least before ZT17, or 16 h before
the peak of BrdU incorporation at ZT9 is reached. Adding
dexamethasone at ZT21 (12 h before the expected peak) is not
sufficient. This might reflect either a minimum time needed
to exert downstream effects on the cell cycle, or a
requirement for the presence of glucocorticoids at a certain time of
day. Interestingly, the last time point with rescue capability
coincides with the natural peak in cortisol levels in wild-type
larvae (Figure 4A). Experiments involving a precisely
controlled temporal activation and inactivation of the
glucocorticoid signaling pathway will be needed to decide between
In summary, our results call for a re-evaluation of existing
models that account for control of circadian cell cycle timing
purely via the direct regulation of gene expression by
peripheral clock components. We reveal a requirement for
endocrine regulation involving glucocorticoids that operates
downstream of the clock mechanism itself. It is tempting to
speculate that many other clock outputs may involve similar
contributions from cell-autonomous and systemic control
Materials and Methods
Fish care, RNase protection assay, BrdU labeling, and
dexamethasone treatment. Fish were raised and bred according to standard
procedures . RNase protection analysis was carried out as
described previously , and the per4, clock1, and b-actin probes
have been described [27,28,66]. The raising of larvae under controlled
lighting and temperature conditions, and the BrdU labeling
procedure have been described in . Briefly, ten mutant and ten
wild-type sibling larvae each were sorted into cell culture flasks on
day 2 post-fertilization and raised at 25 8C under a 12-h light:12-h
dark cycle until day 6 of development. Three hours after lights on
(ZT 3), the larvae of one flask were incubated for 20 min with BrdU
before fixation in 4% paraformaldehyde/PBS, and this procedure was
repeated at three additional time points at 6-h intervals. The cell
culture BrdU incorporation experiments (AB.9 cells, ATCC) were
carried out as described in . Cells were raised in L15 medium
supplemented with gentamycin, streptomycin, penicillin , and
either 15% fetal bovine serum (FBS) or 15% charcoal-treated FBS
(both Biochrom, Berlin, Germany).
The pituitary and lak mutants are not morphologically
distinguishable from wild-type siblings early in development. Thus, two flasks
with 25 larvae each from a cross of heterozygous carriers were sorted
and processed as described above to ensure the presence of a
sufficient number of mutants per time point. lak mutant larvae can be
recognized by their expanded melanophore phenotype later in
development. To identify the pituitary mutants, larvae were stained
for growth hormone expression by whole-mount in situ hybridization
(see below) and then stained for BrdU incorporation.
Dexamethasone treatment was carried out essentially as described
by . Dexamethasone (Sigma, St. Louis, Missouri, United States) was
dissolved in distilled water at 1 mM as a stock solution, then diluted
further in E3 medium to a final concentration of 25 lM. Ten strong
allele rx3 mutants and wild-type siblings were sorted into each cell
culture flask on day 2 of development and raised in the presence or
absence of dexamethasone until BrdU labeling on day 6 as described
above. Luzindole treatments were performed similarly, with a stock
solution of 0.01 M luzindole (Sigma) in ethanol diluted further in E3
to a final concentration of 0.00001 M . Twenty larvae were raised
in 25 ml of E3 at this luzindole concentration from day 2 of
development, and on day 4, an additional 30 ll of luzindole stock
solution were added to compensate for potential degradation of the
compound. Control larvae were treated with equivalent solvent
(ethanol) concentrations only.
In situ hybridization. In situ hybridization was carried out
essentially as described , with the following modifications: The
4% paraformaldehyde fixation step after rehydration was omitted.
Larvae were washed twice in PBS0.1% Tween-20 (PBST), then rinsed
for 2 min in distilled water, incubated for 7 min in pre-cooled
acetone at 20 8C, passed through distilled water for 2 min at room
temperature, and washed 335 min in PBST. Then, larvae were
digested with 1 mg/ml of collagenase P (Roche, Basel, Switzerland) in
PBS with 1% BSA and 1% DMSO at room temperature for 45 min,
before fixation for 20 min at 4 8C in 4% paraformaldehyde. After five
washes with PBST, larvae were prehybridized with HYB buffer for 46
h, then incubated with the probes overnight. After antibody
incubation, larvae were washed 6 3 15 min in PBST, then staining
was developed for several hours at room temperature and up to
overnight at 4 8C. To remove pigmentation, larvae were incubated in
3 ml of 10% H2O2/methanol overnight, followed by addition of 12 ml
of PBST, incubation overnight, and several washes with PBST. Probes
used were aanat2 , pit1 , gh , prl , a-gsu , pomc ,
isotocin , ppss3 , and per4 . corticotrophin relasing factor (crf )
was cloned in our laboratory.
Quantitative RT-PCR. Total RNA was extracted from triplicate
samples using Trizol RNA isolation reagent (GIBCO-BRL, San Diego,
California, United States) according to the manufacturers
instructions. The RNA (3 lg) was reverse-transcribed using Oligo(dT) primer
(Amersham Biosciences, Little Chalfont, United Kingdom) and
SuperScript III reverse transcriptase (Invitrogen, Carlsbad,
California, United States). per4 mRNA levels were determined by real-time
PCR using the DNA Engine Opticon thermocycler (Bio-Rad, Hercules,
California, United States) following the manufacturers instructions.
First-strand cDNA aliquots from each sample were diluted 203 and
served as templates in a PCR consisting of master mix, SYBR Green I
fluorescent dye (Bio-Rad), and 400 nM gene-specific primers. Copy
numbers were normalized using b-actin controls. Primer sequences
were per4: 59-CCGTCAGTTTCGCTTTTCTC-39 and
59-ATGTGCAGGCTGTAGATCCC-39; glucocorticoid receptor:
59-CGGACAGAGCTTCCTCTTTG-39 and 59-CTGCTGCATTCCACTGACAT-39;
and b-actin: 59-TCCTGCTTGCTAATCCAC-39 and
Western blotting. Protein extracts were prepared by homogenizing
20 larvae per time point in 100 ll of Laemmli buffer. A total of 10 ll
of the homogenate was loaded on a 6% SDS polyacrylamide gel, then
Western blotting (BioRad) was carried out using an anti-human
glucocorticoid receptor a (sc-1002; Santa Cruz Biotechnology, Santa
Cruz, California, United States) and anti-mouse CREB antibodies
(Upstate Biotechnology, Billerica, Massachusetts, United States), and
visualized with the ECL detection system (Amersham Biosciences).
Cortisol luminescence immunoassay. Twenty-five larvae each were
raised in cell culture flasks with 20 ml of E3 medium under a LD cycle
and a constant temperature of 25.3 8C. At each time point, larvae
were rapidly transferred to 2-ml Eppendorf tubes, the medium was
removed, and the larvae were snap frozen in liquid nitrogen and
stored at 80 8C until further processing. For each time point and
condition, three identical flasks were used per experiment. Larvae
were homogenized on ice with a microgrinder (Eppendorf, Hamburg,
Germany), then extracted with 500 ll of cold ethanol. After
centrifugation for 10 min at 3,000 rpm at 4 8C, the supernatant was
recovered and evaporated in a SpeedVac. The resulting pellet was
resuspended in 20 ll of standard A buffer of the IBL cortisol LIA kit
(IBL, Hamburg, Germany) and measured following the instructions
provided by the supplier. The LIA plate was read by a VICTOR light
1420 luminometer (Wallac/Perkin Elmer, Wellesley, Massachusetts,
United States), and the raw data were analyzed using the
MikroWin2000 program, version 4.23 (Mikrotek Laborsysteme, Overath,
Statistical analysis. Statistical analysis was performed using the
GraphPad Prism version 4.00 for Windows (Graph Pad Software,
Figure S1. High-Resolution View of Representative Whole-Mount
BrdU Stainings Shown in Figure 1
Mutant (mut) and sibling (sib) larvae at peak (ZT9) and trough (ZT21)
points of the cell cycle rhythm are shown.
Found at doi:10.1371/journal.pbio.0050078.sg001 (8.0 MB TIF).
Whole-mount in situ hybridization with probes against somatostatin3
(ppss3)  (A), isotocin (itnp)  (B), and corticotropin releasing factor (crf)
(C) of strong and weak rx3 mutants and wild-type siblings on 6 dpf.
For each panel: wild-type (wildtype), strong mutant (strong), and
weak mutant (weak); top row shows a lateral view with the anterior
end left (in wild-type: left eye removed to allow a better view of the
brain stainings), and the bottom row shows a dorsal view with the
anterior end left. Expression patterns of these representative probes
appear normal, despite the lack of normal eyecups in the mutants.
Found at doi:10.1371/journal.pbio.0050078.sg002 (6.4 MB TIF).
Figure S3. Dexamethasone Interacts with the Circadian Clock to
Rescue Circadian Cell Cycle Defects
Effects of dexamethasone treatment on BrdU incorporation in strong
rx3 mutants and wild-type siblings after transfer into constant
darkness. Larvae were raised from 2 dpf to 6 dpf in the presence
(A) or absence (B) of 25 lM dexamethasone (DEX). Lights were turned
off on day 5 at ZT12, and BrdU pulses and harvesting carried out at
circadian time (CT) 3, 9, 15, and 21 on the following day. Means of
BrdU-positive nuclei at four circadian time points on 6 dpf are shown
for DEX-treated mutants (mut, blue) and wild-type siblings (sib,
red) (A), and for untreated (CON) mutants (blue) and wild-type
siblings (red) (B). Untreated mutants do not show significant
circadian cycling under these conditions (Kruskal-Wallis test, p
0.3022). Error bars show the 95% confidence interval of the mean.
Asterisks indicate statistical significance of the difference between
mutant and wild-type as determined by the Mann-Whitney test: **, p
, 0.01; ***, p , 0.001. Pooled results of two independent
experiments are shown.
Found at doi:10.1371/journal.pbio.0050078.sg003 (3.1 MB TIF).
Figure S4. Normal Expression of per4 in aal Mutants
Whole-mount in situ hybridization with probes against per4 of aal
mutants and wild-type siblings. Mutants and wild-type siblings were
discriminated by co-hybridizing with a probe against growth hormone
(AD) Overview of staining results for mutants (C and D) and
wildtype siblings (A and B) at ZT 12 (A and C) and 23 (B and D).
(E and F) Close-ups of mutant and wild-type sibling larvae at ZT12 (E)
and ZT23 (F). Strong per4 staining is visible in both mutant and
wildtype siblings at ZT23, whereas barely any staining can be detected in
larvae of both genotypes at ZT12.
Found at doi:10.1371/journal.pbio.0050078.sg004 (5.5 MB TIF).
Figure S5. Low Cortisol Levels Attenuate Circadian Cell Cycle
Rhythms in Cultured Cells Downstream of the Circadian Clock
(A) BrdU incorporation in AB9 zebrafish cell culture cells raised in
charcoal-treated medium (a standard procedure to selectively reduce
steroid levels in culture medium , GCs) and normal medium
(GCs). For each time point, the mean OD450 (optical density)
measurement of the BrdU enzyme-linked immunosorbent assay
(ELISA) from eight independent wells plus the 95% confidence
interval of the mean are shown for cells raised in charcoal-treated
(blue) or normal serum (red). One representative experiment is
shown. Removal of glucocorticoids results in severe attenuation of
circadian cell cycle rhythms.
(B) Clock gene expression in AB9 cells raised in charcoal-treated and
normal medium. Quantitative RT-PCR results for per4 expression are
shown. Cells were grown in 25-cm2 culture flasks for 5 d under a LD
cycle before RNA isolation. Experiments were carried out in
triplicate. Error bars indicate the 95% confidence interval of the
mean. Clock gene expression is indistinguishable between cells raised
in glucocorticoid-depleted medium and those raised in normal
medium, indicating that glucocorticoids exert their effects on the cell
cycle downstream of the circadian clock.
Found at doi:10.1371/journal.pbio.0050078.sg005 (973 KB TIF).
The GenBank accession numbers (http://www.ncbi.nlm.nih.gov/
Genbank) for the genes discussed in this paper are a-gsu
(NM_205687), aal/eya1 (NM_131193), aanat2 (NM_131411), atonal
homologue 5 (AB049457), clock (NM_130957), corticotropin releasing factor
(DQ250674), glucocorticoid receptor (NM_001020711), lia/fgf3
(NM_131291), isotocin (AY069956), per4 (NM_212439), pia/ascl1a
(NM_131219), pit1 (AY421970), pomc (NM_181438), prl
(NM_181437), retinal homeobox gene 3 (NM_131227), and somatostatin3
(BI472739 and BI473045).
We wish to thank Agustin Rojas-Mun oz and Ralf Dahm for kindly
sharing with us rx3 mutants; Charles Plessy, David Whitmore, Ferenc
M uller, Darren Gilmour, and Patrick Blader for critically reading the
manuscript; and Andreas Heyd, Gillian Brunt, and Philipp Mracek for
excellent technical assistance. We especially thank Siegfried Ferner
and Victor Herbst (IBL, Hamburg) for help with data analysis of the
Author contributions. TD and NSF conceived and designed the
experiments. TD, KL, DV, CS, and NSF performed the experiments.
TD and NSF analyzed the data. TD, GN, CJN, and MH contributed
reagents/materials/analysis tools. TD, DV, and NSF wrote the paper.
Funding. We acknowledge funding by the Max-Planck-Gesellschaft,
Centre National de la Recherche Scientifique (NSF), Deutsche
Forschungsgemeinschaft (TD), and European Molecular Biology
Competing interests. The authors have declared that no competing
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