Drosophila clock can generate ectopic circadian clocks.
ABSTRACT Circadian rhythms of behavior, physiology, and gene expression are present in diverse tissues and organisms. The function of the transcriptional activator, Clock, is necessary in both Drosophila and mammals for the expression of many core clock components. We demonstrate in Drosophila that Clock misexpression in nai;ve brain regions induces circadian gene expression. This includes major components of the pacemaker program, as Clock also activates the rhythmic expression of cryptochrome, a gene that CLOCK normally represses. Moreover, this ectopic clock expression has potent effects on behavior, radically altering locomotor activity patterns. We propose that Clock is uniquely able to induce and organize the core elements of interdependent feedback loops necessary for circadian rhythms.
- [show abstract] [hide abstract]
ABSTRACT: Pax-6 is a transcription factor containing both a homeodomain (HD) and a Paired domain (PD). It functions as an essential regulator of eye development in both Drosophila and vertebrates, suggesting an evolutionarily conserved origin for different types of metazoan eyes. Classical morphological and phylogenetic studies, however, have concluded that metazoan eyes have evolved many times independently. These apparently contradictory findings may be reconciled if the evolutionarily ancient role of Pax-6 was to regulate structural genes (e.g., rhodopsin) in primitive photoreceptors, and only later did it expand its function to regulate the morphogenesis of divergent and complex eye structures. In support of this, we present evidence that eyeless (ey), which encodes the Drosophila homolog of Pax-6, directly regulates rhodopsin 1 (rh1) expression in the photoreceptor cells. We detect ey expression in both larval and adult terminally differentiated photoreceptor cells. We show that the HD of Ey binds to a palindromic HD binding site P3/RCS1 in the rh1 promoter, which is essential for rh1 expression. We further demonstrate that, in vivo, P3/RCS1 can be replaced by binding sites specific for the PD of Ey. P3/RCS1 is conserved in the promoters of all Drosophila rhodopsin genes as well as in many opsin genes in vertebrates. Mutimerized P3 sites in front of a basal promoter are able to drive the expression of a reporter gene in all photoreceptors. These results suggest that Pax-6/Ey directly regulates rhodopsin 1 gene expression by binding to the conserved P3/RCS1 element in the promoter.Genes & Development 06/1997; 11(9):1122-31. · 12.44 Impact Factor
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ABSTRACT: The treatment of cultured rat-1 fibroblasts or H35 hepatoma cells with high concentrations of serum induces the circadian expression of various genes whose transcription also oscillates in living animals. Oscillating genes include rper1 and rper2 (rat homologs of the Drosophila clock gene period), and the genes encoding the transcription factors Rev-Erb alpha, DBP, and TEF. In rat-1 fibroblasts, up to three consecutive daily oscillations with an average period length of 22.5 hr could be recorded. The temporal sequence of the various mRNA accumulation cycles is the same in cultured cells and in vivo. The serum shock of rat-1 fibroblasts also results in a transient stimulation of c-fos and rper expression and thus mimics light-induced immediate-early gene expression in the suprachiasmatic nucleus.Cell 07/1998; 93(6):929-37. · 31.96 Impact Factor
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ABSTRACT: In Drosophila melanogaster four circadian clock proteins termed PERIOD (PER), TIMELESS (TIM), dCLOCK (dCLK), and CYCLE (CYC/dBMAL1) function in a transcriptional feedback loop that is a core element of the oscillator mechanism. dCLK and CYC are members of the basic helix-loop-helix (bHLH)/PAS (PER-ARNT-SIM) superfamily of transcription factors and are required for high-level expression of per and tim and repression of dClk, whereas PER and TIM inhibit dCLK-CYC-mediated transcription and lead to the activation of dClk. To understand further the dynamic regulation within the circadian oscillator mechanism, we biochemically characterized in vivo-produced CYC, determined the interactions of the four clock proteins, and calculated their absolute levels as a function of time. Our results indicate that throughout a daily cycle the majority of the dCLK present in adult heads stably interacts with CYC, indicating that CYC is the primary in vivo partner of dCLK. dCLK-CYC dimers are bound by PER and TIM during the late evening and early morning, suggesting the formation of a tetrameric complex with impaired transcriptional activity. Although dCLK is present in limiting amounts and CYC is by far the most abundant of the four clock proteins that have been examined, PER and TIM appear to interact preferentially with dCLK. Our results suggest that dCLK is the main component regulating the daily abundance of transcriptionally active dCLK-CYC complexes.Journal of Neuroscience 04/2000; 20(5):1746-53. · 6.91 Impact Factor
Cell, Vol. 113, 755–766, June 13, 2003, Copyright 2003 by Cell Press
Drosophila Clock Can Generate
Ectopic Circadian Clocks
to Clk (Emery et al., 1998). The mechanism of Clk inhibi-
tion of Clk and cry gene expression has recently been
et al., 2003; Glossop et al., 2003). Interestingly, the ex-
pression of vri and Pdp1, like per and tim, is dependent
on Clk (Blau and Young, 1999; McDonald and Rosbash,
2001). As CLK appears to be the limiting factor for CLK/
CYC (Bae et al., 2000), it may be the critical factor that
directs these interdependent loops and coordinates cir-
cadian gene expression.
Although these intracellular feedback loops are pres-
ent in many tissues, they are not ubiquitous. In the adult
fly brain, clock gene expressing neurons are the excep-
tion rather than the rule. These rhythmic neurons can
be roughly divided into groups of dorsal neuron (DNs)
and lateral neuron (LNs). Indeed, the most prominent
circadian rhythm in fruit flies, the daily rhythm of rest
and activity, appears to be mediated by the LNs (Ka-
neko, 1998; Renn et al., 1999). The LNs can be subdi-
vided into a dorsal subgroup (LNd) and two ventral sub-
groups (LNv), consisting of small (LNvS) and large
neurons (LNvL). The LNv subgroup is distinguished by
its expression of the neuropeptide gene, pigment dis-
persing factor (pdf). Null mutants of pdf nearly abolish
circadian rhythms, indicating that it is a key circadian
component (Renn et al., 1999). Although there is no
apparent circadian cycling of pdf RNA, PDF is rhythmi-
cally expressed in the synaptic termini of the LNvS (Park
et al., 2000). Genetic disruption of Clk dramatically re-
duces pdf expression and alters the neuronal projec-
that it may be a decisive factor in determining circadian
To investigate this potential role of Clk, we used the
GAL4/UAS system to misexpress Clk. We show that Clk
cells that apparentlydo not express clockgenes in wild-
type flies. Furthermore, the appearance of these newly
rhythmic neurons is correlated with striking effects on
diurnal behavior, suggesting that some of these new
put programs. We propose that Clk acts as a critical
switch to generate circadian rhythmicity.
Jie Zhao,1Valerie L. Kilman,2Kevin P. Keegan,2
Ying Peng,1Patrick Emery,1,3Michael Rosbash,1
and Ravi Allada2,*
1Howard Hughes Medical Institute
Department of Biology
Waltham, Massachusetts 02454
2Department of Neurobiology and Physiology
Evanston, Illinois 60208
Circadian rhythms of behavior, physiology, and gene
expression are present in diverse tissues and organ-
isms. The function of the transcriptional activator,
Clock, is necessary in both Drosophila and mammals
for the expression of many core clock components.
We demonstrate in Drosophila that Clock misexpres-
sion in naı ¨ve brain regions induces circadian gene ex-
pression. This includes major components of the
mic expression of cryptochrome, a gene that CLOCK
normally represses. Moreover, this ectopic clock ex-
pression has potent effects on behavior, radically al-
tering locomotor activity patterns. We propose that
Clock is uniquely able to induce and organize the core
for circadian rhythms.
Circadian rhythms of gene expression and behavior are
widespread in biology. These rhythms are the result of
cell-autonomous intracellular clocks that are based in
large part on the expression of transcriptional feedback
loops (Allada et al., 2001; Panda et al., 2002b). In the
fruit fly, Drosophila melanogaster, circadian gene ex-
pression is driven by circadian clocks present in many
tissues and requires the function of the transcriptional
activator Clock (Clk) (Allada et al., 1998; McDonald and
Rosbash, 2001). CLK and its heterodimeric partner CY-
dian rhythm genes, period (per) and timeless (tim) (Dar-
lington et al., 1998; Rutila et al., 1998). PER and TIM
feed back directly on CLK and CYC to inhibit their own
synthesis (Darlington et al., 1998; Lee et al., 1998, 1999).
Clk itself is also rhythmically expressed, but its expres-
sion is inhibited by CLK/CYC rather than activated (Bae
et al., 1998; Darlington et al., 1998; Glossop et al., 1999).
cycle of sunlight involves the blue light photoreceptor,
CRYPTOCHROME (CRY; Helfrich-Forster et al., 2001).
Of note, cry expression is regulated in a similar manner
CLOCK Misexpression Results in Ectopic
Circadian timeless Expression
To characterize the consequences of Clk misexpres-
mon, 1993). Binding sites for the yeast transcription fac-
tor GAL4 (upstream activating sequence; UAS) were
fused upstream of Clk cDNA (UASClk). The pattern of
by the spatial and temporal expression pattern of the
GAL4 driver. In combination with numerous GAL4 driv-
ers, UASClk resulted in developmental lethality (see Ex-
perimental Procedures). However, we were able to gen-
chusetts Medical School, Worcester, Massachusetts 01655.
Figure 1. UASEGFP Expression Driven by Different Circadian Gene Promoters
Brains of female flies expressing UASEGFP under the indicated promoters were dissected, fixed, and imaged with confocal microscopy.
(A) pdfGAL4 promoter drove GFP expression in the lateral neurons only (LNvL; LNvS).
(B) With the crypiGAL4-13 promoter, GFP expression was only visible in the LNvL.
(C) crypGAL4-24females showed a broad distribution ofGFP expression, including regions of novelexpression (EB ?ellipsoid body, AN? antennal
neuropils) in addition to canonical circadian cells (DN, LNd, LNvL, LNvS) Circadian neuronal projections are traced with a solid white line.
(D) timGAL4 promoter drove GFP expression in a broad distribution, but not in all the same cells showing tim RNA expression in crypGAL4-
(E) crypGAL4-16 brains expressed GFP in a pattern similar to but distinct from crypGAL4-24 females. See text for details.
erate viable adult progeny with three clock-relevant
drivers: the pdf promoter (pdfGAL4), a previously de-
scribed cry promoter also containing the large cry first
intron (crypiGAL4; p ? promoter; i ? intron) and a cry
promoter without the first intron (crypGAL4; Emery et
al., 2000; Park et al., 2000).
crossed to a UAS-EGFP strain and the adult progeny
assayed for brain GFP expression (Figure 1). Consistent
with previous reports, pdfGAL4 and crypiGAL4 (line 13;
crypiGAL4-13) expression is limited to a small number
of adult neurons (Emery et al., 2000; Park et al., 2000).
pdfGAL4 expression is restricted to two cell groups
whose morphology and position is consistent with the
LNvS and LNvL (Figure 1A). The crypiGAL4 appears to
be expressed predominantly in the LNvL (Figure 1B). A
previous report indicated that this driver is also ex-
pressed in the LNd and the LNvS in addition to the
Drosophila Clock Induces Ectopic Clocks
LNvL (Emery et al., 2000). These three cell groups are
a substantial subset of neuronal clock gene expression
in the brain. The limited expression from these GAL4
lines contrasts markedly with the broader expression
of two independent inserts of crypGAL4, crypGAL4-24,
and crypGAL4-16 (Figures 1C and 1E). In addition to
the canonical circadian cells, expression is observed in
other areas, such as the ellipsoid body (EB). Based on
their characteristic morphology, many of these cells ap-
pear to be neuronal. Interestingly, we observed differ-
ences between the two inserts (Figures 1C and 1E).
The most salient features of crypGAL4-16 relative to
crypGAL4-24 were more prominent diffuse glial expres-
sion and much less (or absent) expression in the anten-
nal neuropils (AN) as well as in the DNs and in the LNv
GAL4-17) did not exhibit any detectable GAL4-driven
GFP expression, further indicating that the crypGAL4
expression pattern is dependent on insert location (data
not shown). The ectopic expressionpatterns do not cor-
respond with that of any known circadian gene and are
distinct from that observed for timeless promoter-GAL4
(timGAL4; Figure 1D).
We then assayed the spatial and temporal expression
of the direct Clk target gene, tim, in crypGAL4-24/?;
rescent in situ hybridization (Figure 2). tim expression
in wild-type flies showed expression restricted to the
canonical clock-geneexpressing cell groups(Figure 2A,
right images). These are the three groups of lateral neu-
rons, including the LNvL, LNvS, and LNd as well as the
dorsal neurons (DN1 and DN2; Figure 2A). In contrast,
many ectopic tim-expressing cells were observed in
cry24 females (Figure 2A, left images). In contrast to tim,
pdf is not expressed ectopically in cry24 flies (data not
shown). To quantify ectopic tim expression, we arbi-
trarily defined three ectopic locations as New1, New2,
and New3. The New1 cells in cry24 flies do not corre-
spond to cells in a similar area identified in timGAL4
(Figure 1D; Kaneko and Hall, 2000). As both cell groups
only appear in the context of GAL4-driven expression,
this precludes simple double-labeling experiments. The
results are consistent with UASClk activation of tim in
the broadexpression pattern of thecrypGAL4-24 driver.
We next determined whether ectopic tim mRNA is
rhythmically expressed. Under 12 hr light: 12 hr dark
(LD) conditions, y w flies show robust oscillations in tim
RNA with a peak at ZT14 and a trough at ZT2 (Figure
2A, right images). The cry24 flies also displayed robust
oscillations, not only in the normal circadian neurons
but also in all ectopic locations (Figure 2A, left images).
tude of cry24 tim cycling is similar to those in wild-type
flies in the LNs, DNs, as well as the three new locations
(Figure 2B). Indeed, cycling is evident even in many
scattered ectopic cells outside of these three groups.
The comparable phase and amplitude is remarkable
given that tim expression levels were substantially
higher in cry24 than in wild-type flies (Figure 2; at ZT8,
ZT14, and ZT20). tim mRNA oscillations also persisted
at least into the second day of constant darkness (DD;
Figure 3), indicating that the ectopic oscillations are not
purely light-driven. Interestingly, ectopic DD rhythms
also occur with comparable phase despite the higher
tim mRNA levels (Figure 3).
Clock Misexpression Induces Ectopic Rhythmic
We hypothesized that if CLK is inducing the entire pro-
gram of circadian gene expression then it should not
only induce genes that it directly activates (e.g., tim)
but also other rhythmically expressed genes that are
indirectly regulated or even repressed by CLK. cry is
rhythmically expressed with a peak and trough anti-
phase to those of per, tim, and vri (Emery et al., 1998).
As opposed to these CLK-activated genes, levels of cry
are high in a ClkJrkbackground (Emery et al., 1998). We
therefore compared cry expression in cry24 with wild-
type flies and made two important observations (Figure
4). First, the locations in which we observed ectopic
rhythmic tim expression do not express detectable cry
in wild-type flies (Figure 4A). These cells therefore do
not phenocopy a ClkJrkmutant (in which cry levels are
elevated) and do not otherwise appear primed for circa-
dian gene expression. Second, we clearly observe ec-
topic rhythmic cry expression (Figure 4B). Indeed, we
observe significantly higher levels of cry at ZT 2 than at
ZT 14, the opposite of that observed for tim. These data
are consistent with cry cycling antiphase to that of tim
in the ectopic cells. These observations suggest that
Clk expression in certain cells is sufficient to create
ectopic circadian clocks.
Induction of Ectopic Clocks Using an Independent
The GAL4 driver used to induce ectopic clocks was
derived from the cry gene. Although we do not observe
cry expression in the broad pattern of this driver in wild-
type flies, the use of a clock-relevant promoter may still
suggest that these ectopic cells already harbor some
clock gene expression or properties. In testing numer-
ous GAL4 lines, we found that one noncircadian line,
MJ162a, was adult viable in combination with UASClk.
This previously characterized line expresses GAL4 pre-
dominantly in the mushroom bodies and the antennal
ated with circadian gene expression (Joiner and Griffith,
1999). MJ162a, in combination with UASClk, gave rise
to ectopic cycling tim expression (Figures 5C, 5D, and
5F). As expected, the patterns of ectopic rhythmic gene
expression are distinct from crypGAL4-24 induced
clocks. This distinction was especially evident when we
optically sectioned brains from flies collected at ZT14
and compared the two patterns (Figures 5E and 5F). The
ectopic expression in cry24 is clustered more ventrally,
which is noticeable in the vicinity of the lateral neurons.
In contrast, MJ162a-induced expression is primarily
suggest that the ability of Clock to ectopically induce
GAL4 driver but reflects a more general function of this
Clock Overexpression Dramatically Alters
Behavior in Light-Dark Cycles
In LD cycles, wild-type flies exhibit a bimodal activity
pattern, with a peak centered around lights-on (morning
peak) and a second peak around lights-off (evening
peak;Figure 6A).Thecry13 (crypiGAL4-13/UASClk)pat-
tern is also bimodal, and the evening activity peak is
Figure 2. Cycling of tim RNA in cry24 Flies During LD Cycles
Flies were entrained to LD cycles (12 hr light: 12 hr dark) for 3 days at 25?C and collected during the last day of LD at indicated Zeitgeber
time (ZT, where ZT0 is lights-on, ZT12 is lights-off). In situ mRNA hybridization was performed on adult brain whole mounts from cry24 female
flies and y w female flies to detect tim expression.
(A) SenSys camera images of adult brain whole mounts in situ hybridization. Left images, brains from crypGAL4-24/?;UASCLK/? (cry24)
female flies; right images, brains from wild-type (y w) female flies. Normal spatial tim expression seen in wild-type is characterized as tim-
expressing cell groups: large and small ventral lateral neurons (LNvL; LNvS), lateral neuron dorsal group (LNd), and two dorsal neuron groups:
DN1 and DN2. In cry24 flies, in addition to the normal tim-expressing neurons, widespread novel tim-expressing cell groups were detected,
as indicated in New1, New 2, and New3. Results show a representative of three experiments.
(B) Quantification of tim expression during the circadian cycle. The relative staining intensity of one experiment was quantified and plotted
as mean ? SEM for each time point of 4–6 brains. Peak tim RNA levels of each cell group were set to 100. Five groups of normal tim-
expressing neurons of cry24 flies are plotted in upper image (cry24, LD, tim); the three ectopic new groups of cells of cry24 flies are plotted
in middle image (cry24, LD, tim); and the five groups of normal tim-expressing neurons of y w flies are plotted in lower image (yw, LD, tim).
phase advanced, consistent with the shortened period
(Figure 6A; Table 1). Remarkably, cry24 females have
an LD activity pattern that is radically different from any
bimodal profile, the diurnal pattern has only a single
peak dominating the light phase with little or no evening
this pattern (see below).
Weconsidered thepossibility thatthe eveningactivity
peak in cry24 females was so advanced (by ?6 hr) that
it merged with the morning peak. To infer the phase of
the evening peak, we assayed the DD behavioral phase
Drosophila Clock Induces Ectopic Clocks
Figure 3. Cycling of tim RNA in cry24 Flies
Under DD Conditions
Flies were entrained to LD cycles for 3 days
and then transferred to DD. They were col-
lected at the indicated circadian time (CT)
during the second day of DD and fly brains
were subjected to in situ hybridization.
(A) SenSys camera images of brain whole
mounts in situ hybridization from cry24 fe-
male flies. Cyclic expression of tim in normal
and ectopic cell groups persist in DD.
(B) Quantification of tim expression during
the circadian cycle. The relative staining in-
tensity was quantified and plotted as mean ?
SEM for each time point of 4–6 brains. Peak
tim RNA levels of each cell group were set to
100. Five groups of normal tim-expressing
neurons of cry24 flies are plotted in upper
image and the three ectopic cell groups of
cry24 flies are plotted in lower image.
of cry24 flies. As period also affects phase in DD, we
compared the cry24 and cry13 lines that have almost
identical periods (Table 1). The first four days of DD
reveal little difference between the two genotypes, sug-
gesting that an advanced evening activity peak cannot
explain the altered diurnal behavior (Figure 6B). Given
the progressive reduction in rhythmicity observed in
these strains (Table 1), phase assessments beyond four
days of DD were not informative. Interestingly, these
data argue for a specific effect of Clk overexpression on
LD behavior. As an independent measure of pacemaker
phase under LD conditions, we performed anchored
phase-response curves (PRCs; Figure 6C). In principle,
PRCs define pacemaker phase by describing phase
change in response to brief light pulses administered
at different times of day. Both cry24 and cry13 were
very similar with respect to phase, marginally advanced
compared to the control UASClk strain (Figure 6C). We
observed, however, that the overall amplitude of the
cry24 PRC is suppressed. The data are consistent with
an inhibitory effect on circadian phototransduction or
on altered pacemaker amplitude but not on pacemaker
phase in LD. We also considered the possibility that
ectopic Clk expression results in an exaggerated light
response that swamps a mildly advanced evening activ-
ity peak. Indeed, it has been reported that CLK overex-
pression increases the locomotor activity response as
a result of light exposure (Kim et al., 2002). However,
cry13 and cry24 flies had similar levels of activity after
lights-on (data not shown).
The strong effects of Clk overexpression on the LD
behavior pattern are probably not due to alterations in
similarly shortened periods as cry13 flies, in which only
near normal LD profiles. Second, we did not observe
Figure 4. CyclingofcryRNAinEctopicLoca-
tions in cry24 Flies
Flies were entrained to LD cycles (12 hr light:
12 hr dark) for 3 days at 25?C and collected
during the last day of LD at indicated Zeit-
geber time (ZT, where ZT0 is lights-on, ZT12
is lights-off). Double-labeling in situ mRNA
hybridization was performed on adult brain
whole mounts from cry24 and y w female flies
to detect cry expression. SenSys camera im-
ages of adult brain whole mounts in situ hy-
bridization were taken from wild-type (y w)
(A) and crypGAL4-24/?;UASCLK/? (cry24)
(B) flies. cry expression seen in wild-type is
only observed in circadian cell groups: LNvL,
LNvS, lateral neuron dorsal group (LNd), and
two dorsal neuron groups: DN1 and DN2. In
cry24 flies, in addition to the normal cry-
expressing neurons, widespread novel cry-
expressing cell groups were detected, as
indicated in New1, New 2, and New3. cry cy-
cling is observed in canonical and ectopic
cells antiphase to that observed for tim.
any significant LD behavioral effect of driving Clock only
in the LNv with pdfGAL4 (Figure 6D). Third, we assayed
the behavior of cry24 flies in a pdf01background. PDF
is thought to be the principal effector molecule of the
effect of pdf01on the LD behavior of cry24 flies (Figure
6D). Although we cannot exclude behavioral effects me-
diated by other circadian neurons, these data indicate
ioral effect of Clk overexpression.
altered diurnal behavior for crypGAL4-16/UASClk flies
(cry16; Figures 7C and 7D): In cry16 as well as cry24
flies, there is a single peak of activity around the time
6A, 7E, and 7F). However, there are behavioral differ-
ences between cry16 and cry24, consistent with the
anatomic differences in gene expression (Figures 1C
and 1E; Figures 7A and 7C). For example, the single
peak of activity present in cry16 flies is more advanced
than in cry24. In addition, ectopic gene expression and
behavioral phenotypes are present in both cry16 males
and females (Figures 7C–7F; Table 1). Although we find
subtle behavioral differences between cry16 males and
ences are much more subtle than the differences be-
tween cry24 males and females. Considering both cryp-
GAL4 lines as well as crypiGAL4, there is an excellent
correlation between ectopic tim expression and abnor-
that ectopic clocks are capable of making functional
connections with the locomotor output program.
In contrast, the presence of Clock-induced changes
in DD behavior does not require ectopic clock gene
expression. Clk overexpression is associated with re-
ductions in rhythmicity and shortened periods in UAS-
Clock flies in combination with crypiGAL4 as well as
with the two crypGAL4 lines that induce ectopic clocks
(Table 1). Given that GAL4 expression in the crypiGAL4
lines is restricted to canonical clock cells, it is likely that
these shortened periods and reduced rhythmicity are
the result of Clk overexpression in the lateral neurons.
Loss of rhythmicity may also be related to the toxicity
of Clk overexpression observed as developmental le-
thality. We propose that failure to observe period short-
ening effects of UASClk in combination with pdfGAL4
may reflect insufficient expression levels in the pace-
The Presence of Ectopic Clocks Is Correlated With
Altered Behavior in Light-Dark Cycles
To extend the correlation between ectopic Clk expres-
pared cry24 males and females. In contrast to cry24
females, cry24 males have a wild-type bimodal pattern
in LD (Figure 6A; Table 1). Importantly, ectopic tim ex-
pression was nearly absent from cry24 males (Figure
7B). We also did not find any ectopic tim expression in
crypiGAL4-13/UASClk flies, consistent with their bi-
modal activity patterns (Figure 6A; data not shown). Al-
thoughthe crypGAL4-24insertis onthe Xchromosome,
there are no strong differences in GAL4 levels between
males and females as monitored by GFP expression
(data not shown). However, we did observe only a low
lation of male transgenic GAL4 expression by dosage
compensation. Given the male-specific lethality, we hy-
pothesize that the GAL4-induced CLK toxicity selects
against cry24 males that express high levels of ectopic
Clk. As a result, the surviving adult males express low
levels of Clk and thus fail to exhibit ectopic clocks or
We also examined a second independent insert of
the crypGAL4 line, crypGAL4-16. Identical to cry24, we
observed ectopic rhythmic gene expression and strongly
Drosophila Clock Induces Ectopic Clocks
Figure 5. Induction of Ectopic tim Cycling with a Noncircadian GAL4 Line
Flies were entrained to LD cycles (12 hr light: 12 hr dark) for 3 days at 25?C and collected during the last day of LD at indicated Zeitgeber
time (ZT, where ZT0 is lights-on, ZT12 is lights-off). In situ mRNA hybridization was performed on adult brain whole mounts from crypGAL4-
24/?;UASCLK/? (cry24) female flies and MJ162a/UASClk female flies to detect tim expression. SenSys camera images of adult brain whole
mount in situ hybridization. Left images (A and B) show brains from cry24 female flies; right images (C and D) show brains from MJ162a/
UASClk female flies. tim expression is seen in normal tim-expressing cell groups: large and small ventral lateral neurons (LNvL; LNvS), lateral
neuron dorsal group (LNd), and two dorsal neuron groups: DN1 and DN2. In cry24 flies (A and B) and MJ162a/UASClk flies (C and D) additional
tim-expressing cell groups were also detected (see also Figure 2 and asterisks). Distinct ectopic cells in MJ162a/UASClock are more clearly
visible in confocal sections. (E and F) Ectopic cells in cry24 and MJ162a/UASClock are distinct. Double-labeling in situ mRNA hybridization
was performed on adult brain whole mounts from cry24 female flies and MJ162a/UASClk female flies to detect tim (red) and pdf (green)
expression.Confocal sectionscontaining thelargeventral LNs(LNvL) areusedto comparethe twogenotypes.In MJ162a/UASClock,prominent
dorsal expression of tim is observed, while more ectopic expression is observed ventrally including in the vicinity of the lateral neurons in
Ectopic period Expression Does Not Induce
Given that the expression of several key clock members
is dependent on Clk, we reasoned that its ability to
induce rhythmic gene expression would be unique
among clock genes. To verify this experimentally, we
induced ectopic per expression using the crypGAL4-16
driver (Figure 7G). In these per overexpressing flies, we
still observed evidence of central clock function (rhyth-
mic tim expression and anticipation of LD transitions)
but with reduced behavioral rhythmicity in DD (Figure
7G; data not shown). Consistent with our hypothesis,
we observed neither induction nor cycling of ectopic
tim expression in these flies. Thus, the ability to induce
Figure 6. Assessment of Diurnal and Circa-
dian Behavior in Clock Overexpressing Strains
(A) Altered diurnal behavior in Clock overex-
pressing strains. Activity profiles display av-
erage relative activity throughfour days of LD
(12 hr light: 12 hr dark) for each indicated
group of flies. Light bars display times of
lights-on; dark bars times of lights-off. Geno-
types are all heterozygous for the indicated
transgenes except male crypGAL4-24 that
are hemizygous for this X-linked transgene.
Error bars represent standard error for that
nation with UASClk (males/females): ? (16/
47), crypiGAL4-13 (47/79), crypGAL4-24 (37/
130). Detailed methods used to generate
these profiles are outlined in Experimental
(B) Phase of Clock overexpressing females is
not significantly altered in constant darkness
(DD). Each DD plot exhibits average activity
through the first four period-length (22.5 hr)
intervals of the DD record. Error bars repre-
sent the standard error across the four days.
Number of animals tested is crypGAL4-24/
UASClk ? 37 and crypiGAL4-13/UASClk? 22.
See Experimental Procedures for details.
(C) Phase-response curve suggests that in-
duced Clock expression does not dramati-
cally alter pacemaker phase. The phase-
of a population, with respect to a nonpulsed
indicated above; all flies tested were female.
pulse time point. Phase delays are seen in
are observed in the late night (ZT21-24) for
all strains indicating no large change in pace-
(D) Altered diurnal behavior in Clock overex-
pressing strains is not due to expression in
the ventral lateral neurons. Activity profiles
display average relative activity through four
days of LD (12 hr light: 12 hr dark) for each
indicated group of flies. Light bars display
times of lights-on; dark bars times of lights-
off. Genotypes are all heterozygous for the
indicated transgenes except UASClk-pdf01,
which is homozygous. Error bars represent
standard error for that bin. Number of female
animals analyzed for each line pdfGAL4/
UASClk (32), crypGAL4-24/UASClk-pdf01(13),
Several lines of evidence now place Clk at the top
of a genetic hierarchy controlling circadian clock gene
expression. Intact Clk is necessary for multiple aspects
of the fly and mouse circadian phenotype. In both sys-
tems, there is strong genetic and biochemical evidence
that CLK and its partner CYC (BMAL1 in mammals) form
a heterodimericcomplex anddirectly activatetranscrip-
tion of several important clock genes (reviewed in (Al-
circadian feedback loops essential for rhythmic gene
expression. Moreover, microarray analyses in both flies
and mice indicate that all detectable rhythmic gene ex-
pression is dependent on Clk (McDonald and Rosbash,
We have demonstrated that Clk misexpression induces
ectopic clocks. These ectopic clocks are evident by
measurements of clock gene expression under light-
dark andconstant darkness conditions. Thebasic result
is not dependent on a cry-derived GAL4 driver, as the
independent noncircadian GAL4 line MJ162a induces
ectopic clocks in distinct brain regions. Furthermore, it
is likely that Clk is inducing major components of the
clock gene program as it also induces rhythmic expres-
sion of cry, a gene that the CLK-CYC complex normally
represses. The ectopic clocks appear to have potent
effects on the LD behavioral program.
Drosophila Clock Induces Ectopic Clocks
that light and these new clocks may collaborate to an-
tagonize positive factors (such as PDF), which are nor-
mally released by canonical clock cells in a temporally
CLK to induce PDF in the ectopic locations is consistent
new neural connections are involved in the behavioral
changes. Alternatively, the ectopic clocks may alter the
coupling between the central pacemaker and outputs
under LD conditions.
To examine the mechanism of ectopic clock forma-
tion, we first considered that Clk might induce new
clocks only in cells that are highly predisposed to ex-
pressing rhythmicity. In this case, Clk expression would
induce one or only a few missing clock genes necessary
for molecular oscillator properties. An analogous case
from mammals may be that of cultured rat-1 fibroblasts,
which mimic the behavior of peripheral clocks such as
the liver. Exposure of the rat-1 cells to high concentra-
tions of serum (serum shock) can induce rhythmicity
in cells that otherwise exhibit no apparent rhythmicity
(Balsalobre et al., 1998). The predisposition of these
cells is reflected in their substantial level of clock gene
expression. In contrast, we found that tim and cry ex-
pression is undetectable in the ectopic cells without Clk
expression. This expression analysis is consistent with
prior reports indicating that there is no detectable per
and tim protein in adult brain neurons outside of the
LNs and DNs (Stanewsky et al., 1998; Kaneko and Hall,
2000 and references within). We also considered the
possibility that tim is expressed in these ectopic loca-
tions in wild-type flies but that tim mRNA levels are
simply below the level of detection. Consistent with this
possibility, tim promoter gal4-driven GFP can be visual-
ized in neurons without detectable tim expression (Fig-
ure 1D; Kaneko and Hall, 2000). Similarly, broader ex-
pression of the per gene has been observed with
grounds, suggesting some low level per expression in
other brain regions (Kaneko et al., 1997; Price et al.,
1998). The functional relevance of these transgene ex-
pression patterns without detectable per or tim expres-
sion remains unclear. Moreover, it is not even certain
that the expression of these reporters is Clk-dependent.
Nonetheless,a comparisonoftheectopic rhythmiccells
with the timGAL4:UASEGFP pattern indicates that they
are two distinct cell populations (Figures 1D and 2A).
is consistent with the notion that these cells are not fully
preprogrammed for rhythmicity. It will be of interest also
to compare expression of per-lacZ fusion proteins that
reveal potential cryptic per expression with the ectopic
clock locations shown here.
Most compelling perhaps is the absence of cry ex-
pression in these ectopic locations. If these cells were
simply missing Clk, they should behave as Clk mutants
against the hypothesis that these cells are largely pro-
grammed for circadian rhythmicity. In addition, we were
able to induce ectopic rhythms in distinct locations us-
ing a noncircadian GAL4 line, MJ162a.
Our analysis raises some intriguing parallels between
Clk and eyeless, a gene involved in the induction of eye
Table 1. Circadian Behavior of Clock Overexpressing Strains
GenotypePeriod ? SEM % Rhythmic (n)
23.3 ? 0.1
23.3 ? 0.1
22.4 ? 0.1
23.6 ? 0.1
22.2 ? 0.4
23.9 ? 0.1
23.5 ? 0.1
24.2 ? 0.1
24.7 ? 0.4
GenotypePeriod ? SEM % Rhythmic (n)
23.3 ? 0.1
24.1 ? 0.2
22.7 ? 0.1
22.5 ? 0.1
23.9 ? 0.1
23.4 ? 0.2
24.3 ? 0.1
24.0 ? 0.5
SEM is standard error of the mean; n ? number of flies analyzed.
2001; Panda et al., 2002a). The abnormal pacemaker
neuronal morphology in the fly mutant is consistent with
anadditional rolein regulatingcircadian neuronaldevel-
opment (Park et al., 2000). All of these data suggest that
the Clk gene may be necessary for many if not most
aspects of clock cell specification as well as function.
The ectopic clocks appear to strongly influence diur-
nal behavior, implying that these new clocks make func-
tional connections with locomotor output pathways.
There is an excellent correlation between the altered
diurnal behavior and ectopic tim expression, for exam-
ple in male versus female cry24 flies. In contrast, en-
hanced expression in the pacemaker lateral neurons
with pdfGAL4 and crypiGAL4-13 has little or no effect
on behavior under LD conditions. Consistent with this
notion, the potent effects of Clk on LD behavior are not
blocked in a pdf01background.
Although we cannot completely exclude a role for
increased expression in the lateral neurons or other
known circadian cells, we favor the notion that new
clock cells are responsible for the altered LD behavior.
One of the prominent regions of crypGAL4 driven gene
expression is the ellipsoid body, a brain region pre-
viously implicated in the higher order control of locomo-
tor activity (Martin et al., 1999). As such, Clk-driven ex-
pression here might be expected to influence locomotor
activity. Interestingly, the MJ162a line does not drive
detectable expression in the ellipsoid body (Joiner and
Griffith, 1999), nor does it have prominent behavioral
effects in combination with UASClk (data not shown).
Differences between cry16 and cry24 flies further sug-
gest that other neurons or even glia may mediate some
of the ectopic Clk behavioral effects. One possibility
is that the transgenic strains manifest a dramatically
suppressed evening activity peak, which is normally
tightly regulated by the circadian clock. This suggests
Figure 7. Differences in Ectopic Clocks Between Different Lines
All images are SenSys camera images of adult brain whole mounts in situ hybridization. Both crypGAL4-24/?;UASClk/? (cry24) and crypGAL4-
16;UASClk (cry16) female and male flies were entrained to LD cycles for 3 days at 25?C and collected during the last day of LD at ZT2 and
ZT14. Ectopic tim overexpression were observed cry24 female flies and in both and female and male cry16 flies. Ectopic cell groups are
indicated by New1, 2, and 3, based on morphology and location. We cannot definitively determine if these three clusters are the same between
cry16 and cry24 flies. No ectopic tim overexpression was found in male cry24 flies. Figure shows a representative of two experiments.
(E and F) Diurnal behavior in crypGAL4-16/UASClock strains. Activity profiles display average relative activity through four days of LD (12 hr
light: 12 hr dark) for each indicated group of flies. Light bars display times of lights-on; dark bars times of lights-off. Error bars represent
standard error for that bin. Number of animals analyzed is 36 for cry16 males and 23 for cry16 females.
(G) tim expression in UASper/?; crypGAL4-16/? flies. tim expression analyzed as in Figures 7A–7D. No ectopic tim induction or cycling is
morphogenesis. Like Clk for circadian rhythms, ectopic
expression of eyeless can induce the formation of ec-
topic eyes (Halder et al., 1995). Both eyeless and Clk
function in terminally differentiated neurons to control
highly specialized gene expression: opsins in the case
of eyeless and rhythm genes in the case of Clk (Sheng
et al., 1997). It has been proposed that activation of a
photoreceptor gene was the original function of eyeless
in evolution (Sheng et al., 1997). Similarly, we propose
that the original role of Clk was to activate expression
of a clock gene ancestor, and its ability to direct the
formation of temporally regulated feedback loops was
a more recent acquisition.
Despite its reported function as a “master control
gene,” not all cells are substrates for eyeless-induced
ectopic eye formation (Bonini et al., 1997). Similarly, we
believe the presence of other rhythm factors, such as
CYC, are likely required for Clk expression to induce
functional clocks. Furthermore, we were unable to in-
duce rhythmic gene expression with transfected Clk in
CYC-expressing S2 cells, suggesting that still other fac-
ments assessing ectopic clock formation in different
circadian mutant backgrounds and tissues should ad-
eyeless and Clk, we have reason to suspect that Clk
may have more far reaching functions. For example,
be functional, whereas we present substantial evidence
that ectopic clocks can alter behavior. Taken together
with the Clk mutant effects on LNv anatomy (Park et al.,
2000), normal Clk expression may even contribute to
pacemaker cell wiring properties. Given the similarities
between the fly and mammalian clock systems, we sug-
gest that the mammalian orthologs of Clk and cyc may
play similar roles.
Clock was first tagged with hemagglutinin (HA) epitope by PCR
cloning. Briefly, a C-terminal fragment of Clock was PCR-amplifed
using pSK(?) Clock cDNA and an oligonucleotide with HA epitope
was digested with ClaI/XhoI and ligated to ClaI/XhoI digested
pSKClock vector to generated pSK(?) ClockHA. ClockHA was sub-
sequently ligated into pUAST (EagI/XhoI) to generate pUAS-
ClockHA. y w; Ki pp(ry?delta 2-3)/? embryos were injected with
pUAST-ClockHA. A single line (UASClk) was obtained as a third
chromosome insert. HA epitope is not immunologically detectable
(data not shown).
Drosophila Clock Induces Ectopic Clocks
pdfGAL4, crypiGAL4, and timGAL4 have been previously de-
scribed (Emery et al., 2000; Kanekoand Hall, 2000; Park et al., 2000).
crypGAL4 was constructed similarly to the previously described
crypiGAL4 construct except that a NotI/NcoI fragment was cloned
in front of the GAL4 coding region of pPTGAL4. As a result, only
the promoter and a fraction of the first exon of the cry gene are
present. The NcoI site is a natural site in the coding region of the
cry gene. The 5? end of the GAL4 coding region was thus modified
by PCR to contain a 5? NcoI site and to be in phase with the few
cry codons present in the construct. The modified GAL4 region
was sequenced to ensure that no PCR errors had occurred. Three
independent lines were analyzed (16, 17, and 24). Line 17 did not
show any expression or behavioral phenotypes.
Adult viability was not observed with UASClk in combination with
the following brain GAL4 drivers: hsGAL4 (even in the absence of
heat shock), 7B, MJ94, MJ63, MJ126a, MJ250 (Joiner and Griffith,
1999), 201Y (Yang et al., 1995), and drlPGAL8(Moreau-Fauvarque et
al., 1998). Most lines were pupal lethal even when raised at 18?C.
For adult viable lines, flies were crossed at 25?C for three days then
transferred to 18?C to increase the number of healthy adult flies
obtained. All molecular and behavioral analyses were conducted on
flies entrained at 25?C.
for a single half-hour bin. Error bars indicate the standard error
across the four single day intervals for each half-hour bin.
To determine phase in constant darkness (DD), analyses were
performed as for LD analyses. Four days of data as defined by the
period of the genotypes in DD were used (22.5 hr for cry24 and
cry13). Data from the four days were averaged together and plotted
into a single day corresponding to this period. For determination of
phase response curves, a 10 min long light pulse was administered
during the dark period of the last full day of LD. After the pulse, flies
are monitored in constant darkness for five days for assessment of
phase. Data from populations of a given genotype or pulse time
were pooled together. Phase was defined as the time at which the
average fly activity was at 50% evening activity offset. We deter-
mined the time of the peak evening activity and the subsequent
trough of evening activity for each day after the light pulse. The
50% evening activity offset was calculated as the (evening peak
activity ? post-evening trough)/2. The time at which activity is near-
est this 50% evening activity offset defines the phase each day.
Differences in the time of activity offset between pulsed and non-
pulsed population were used to calculate phase changes on days
2–4 after the light pulse. The calculated phase changes for these
three days were then averaged together to produce the average
phase change for a given experiment. The final plot exhibits the
averaged results for three experiments. We obtained results similar
to published results for our wild-type control validating this method-
GFP Expression Analysis
dian promoters weredissected and the brains fixedin 3.7% formalin
in PEM. After rinses in PBS ? 0.3% Triton and PBS, brains were
mounted in Fluoromount (company) and imaged on a Leica laser
scanning confocal microscope. Optical sections were taken at 2–5
micron intervals and used to construct a maximum projection image
for each brain.
flies, Dan Eberl pPTGAL4, Mei-Ling Joiner and the Bloomington
Stock Center for GAL4 lines, and Amita Sehgal for UASper flies. We
also thank Myai Emery-Le and Orie Shafer for assistance in the
initial phases of this project and Rich Carthew for comments. R.A.
In Situ mRNA Hybridization on Adult Brain Whole Mounts
Adult fly brains were dissected in phosphate-buffered saline (PBS)
and fixed in 4% paraformaldehyde for 30 min at room temperature.
After prehybridization for minimum of two hours in Hybrix (50%
formamide, 5 ? SSD, 100 ?g/ml tRNA, 100 ?g/ml ssDNA, and 0.1%
Tween 20) at 55?C, the brains were incubated with probes overnight
at 55?C. Three tim probes were used in this study which correspond
to nucleotides 405–1253, 1584–2580, and 2851–4193. These probes
were used simultaneously. The pdf probe used corresponds to nu-
cleotides 282–570. The cry probe used is generated from the full-
length of the cry gene (nucleotide 1–1764). Antisense RNA probes
were synthesized and labeled using digoxigenin (tim, cry) or biotin
(pdf) RNA labeling kit from Boehringer Mannheim. The probes were
hydrolyzed in sodium bicarbonate buffer and stored in Hybrix at
?20?C until use. The hybridized RNA signals were detected using
fluorescent tyramides (NEN LifeScience). Brains were mounted in
glycerol with 4% n-propyl gallate and examined with a Zeiss Axio-
phot microscopy equipped with Photometrics SenSys CCD camera
(Photometrics Ltd., Tucson, AZ). SenSys camera images were as-
sembled in Adobe Photoshop and were used for quantification of
fluorescence signals. For a given time course experiments, 4–6 indi-
field at 20? magnification. The intensities of the fluorescence signal
were measured using Openlab software (Improvision).
Received: September 25, 2002
Revised: May 7, 2003
Accepted: May 7, 2003
Published: June 12, 2003
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