Cell Tissue Res (2002) 309:11–26
DOI 10.1007/s00441-002-0569-0
REVIEW
Ralf Stanewsky
Clock mechanisms in Drosophila
Received: 12 February 2002 / Accepted: 8 April 2002 / Published online: 29 May 2002
© Springer-Verlag 2002
Abstract Mechanisms underlying circadian clock function in Drosophila melanogaster have been revealed by
genetic and molecular approaches. Two interlocked transcriptional feedback loops involving at least the period,
timeless, Clock, and cycle genes generate molecular oscillations that are believed to control behavioral rhythmicity and other clock outputs. These oscillations are
further enhanced and fine-tuned to match the duration of
the solar day by post-transcriptional and post-translational mechanisms depending on the PERIOD and TIMELESS proteins and on the protein kinases DOUBLETIME and SHAGGY. Light is the principal zeitgeber for
synchronizing molecular and behavioral rhythmicity via
the blue-light photoreceptor CRYPTOCHROME and the
TIMELESS protein. In addition, light seems required for
maintaining robust molecular oscillations at least in peripheral clock-gene-expressing tissues like the eyes, antennae, or Malpighian tubules. Relaying temporal information to cells and tissues expressing overt biological
rhythms involves regulation of “output genes” at multiple levels. Although their regulation depends on the major clock genes, the majority of the clock-controlled
genes are not direct targets of clock factors.
Keywords Circadian rhythms · Clock genes ·
Cryptochrome · Drosophila · Feedback loops
Introduction
The study of clock mechanisms in Drosophila started in
1968 when R.J. Konopka systematically screened for
Research in my group is sponsored by the Deutsche Forschungsgemeinschaft (DFG)
R. Stanewsky (✉)
Universität Regensburg, Institut für Zoologie,
Lehrstuhl für Entwicklungsbiologie, Universitätsstrasse 31,
93040 Regensburg, Germany
e-mail: ralf.stanewsky@biologie.uni-regensburg.de
Tel.: +49-941-9433083, Fax: +49-941-9433325
mutants showing altered temporal pupal eclosion patterns in light:dark (LD) cycles. In the wild, Drosophila
melanogaster individuals eclose from their pupal case at
dawn; Konopka’s mutant strains were aperiodic, or
showed either advanced or delayed eclosion peaks
(Konopka and Benzer 1971). When tested in constant
conditions (constant darkness, DD), the phase-altering
mutants had short (19 h) and long (29 h) free-running periods, respectively. Subsequent genetic mapping experiments revealed that all three mutations map to the same
locus, which was then dubbed period (per); the mutant alleles per01 (aperiodic), perS (Short), and perL (Long). Not
only the eclosion rhythms were affected by these variants: the daily rest:activity cycle of mutant individuals
was altered in the same way, both in DD and LD cycles
(Konopka and Benzer 1971; Hamblen-Coyle et al. 1992).
This placed per function right in the heart of the circadian
clock and prompted cloning and molecular analysis of
this “clock gene,” as well as the search for novel rhythm
variants. Now, more than 30 years later (and mainly by
continuing Konopka’s approach) several new clock genes
have been identified and characterized, so that the wheels
and turns of Drosophila’s clock seem unraveled.
In this article I will summarize the current view of
core clock mechanisms in flies. Special care will be taken in the interpretation of results drawn from in vitro experiments and those involving clock-gene-expressing
cells other than those controlling the rhythmic behaviors
described above. This is because clock gene expression
is rather widespread throughout the fly and is even under
circadian control in most of these tissues (Hall 1995;
Plautz et al. 1997). But both the pupal eclosion and adult
activity rhythms mentioned above are controlled by only
a small number of per-expressing neurons (Dushay et al.
1989; Zerr et al. 1990; Ewer et al. 1992; Frisch et al.
1994; Helfrich-Förster 1998; Renn et al. 1999; Kaneko
et al. 2000a; Blanchardon et al. 2001). These “lateral
neurons” (LNs) are located bilaterally between the optic
lobes and the central brain (Fig. 1; cf. Kaneko and Hall
2000). In the adult they consist of two groups of more
ventrally located cells: typically five small LNvs (s-LNv)
12
Fig. 1A, B PERIOD proteinexpressing neurons and their
axonal projections in the brain
of adult Drosophila. A Fluorescence image of a whole-mount
brain-half immunostained with
an anti-serum against PER. The
fly was synchronized to a
12 h:12 h light-dark cycle and
sacrificed for staining at ZT 23.
Neuronal and glial staining is
indicated by arrows or arrowheads, respectively.
B Projections of PER-expressing neurons as revealed by
Helfrich-Förster (1995) and
Kaneko and Hall (2000). Note
that most LNvs also express the
neuropeptide PDF (filled circles). The projections from the
DN3 to the s-LNv were revealed by C. Helfrich-Förster
(personal communication). All
neuronal groups are present
symmetrically in both brain
hemispheres (see A), but
shown only on one side here
for clarification. Drawing
adapted from Kaneko and Hall
(2000) (OL optic lobe, Me medulla, La lamina, Es esophagus,
POT posterior optic tract).
Scale bar 400 µm (A)
and four large LNvs (l-LNv) projecting to the dorsal protocerebrum and to the second optic lobe neuropil (medulla) respectively (Fig. 1; cf. Stanewsky et al. 1997a;
Kaneko and Hall 2000). In addition, the l-LNv also project to the contralateral LNvs and the medulla. Both LNv
groups express the pigment dispersing factor (pdf) gene,
which was initially used to reveal their projection pattern
(Helfrich-Förster 1995). The neuropeptide encoded by
this gene is crucial for transmitting temporal information
from the LNs to downstream neurons in the dorsal brain
(Renn et al. 1999; Helfrich-Förster et al. 2000; Park et al.
2000a). A third, more dorsally located group of ca. six
LNds also projects to the dorsal brain but does not express pdf (Fig. 1; Kaneko and Hall 2000). The dorsal
brain region receiving input from the LNs is also connected to another group of clock-gene-expressing neurons, the “dorsal neurons” (DNs; see Fig. 1). They split
into three groups characterized by their position within
the dorsal brain: ca. 15 DN1, 2 DN2, and ca. 40 DN3, all
terminating in the same dorsal brain region as the LNs
(Fig. 1). In addition the DN1 and DN3 also project towards the s-LNv cell bodies so that most likely all clockgene-expressing neurons are interconnected (Fig. 1;
Kaneko and Hall 2000; Helfrich-Förster 2002 and personal communication). The role of the DNs is less well
understood, but from genetic mosaic studies it seems
clear that they are not crucial for maintaining behavioral
rhythms (Ewer et al. 1992; Frisch et al. 1994). Neverthe-
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less they seem to contribute to the behavioral pattern observed in wild-type flies since animals with a mutant
version of the disconnected (disco) gene (leading to the
loss of PER expression in all three LN groups, but not
affecting the DNs; Zerr et al. 1990; Blanchardon et al.
2001) still show entrained behavior under LD conditions
and stay rhythmic for the first 2 days upon transfer to
DD before becoming arrhythmic (Dushay et al. 1989;
Hardin et al. 1992; Helfrich-Förster 1998). Moreover,
per expression remains rhythmic in the DNs of disco mutants, even for 2 days in constant conditions (Blanchardon
et al. 2001), and it is therefore likely that the residual
rhythmicity is mediated by at least some of the DN
groups.
In larvae, only the precursors of the s-LNv and a subset of the DNs are present (e.g., Helfrich-Förster 2002).
Light pulses given during the early larval stages are able
to synchronize pupal eclosion and adult activity rhythms
(Brett 1955; Sehgal et al. 1992), and PER expression in
the larval LNs has been correlated with this “time memory” (Kaneko et al. 1997, 2000b). Lack of the larval LNs
or disruption of rhythmic PER results in an arrhythmic
eclosion pattern, demonstrating that fluctuating PER
levels in these cells are required for this clock output
(Dushay et al. 1989; Blanchardon et al. 2001; cf.
Helfrich-Förster 2002 for a further discussion concerning
the role of individual larval, pupal, and adult clock-geneexpressing brain neurons and a detailed description of
their connections).
The biological function of per in the great majority of
the remaining, mainly non-neuronal tissues is completely
unknown. Nevertheless, most of the current clock models are based on findings obtained from experiments involving those “peripheral tissues” or from in vitro studies involving embryonic Drosophila or clockless yeast
cell lines. Undoubtedly these studies were important and
led to major breakthroughs unraveling molecular clock
function, but one should be careful extrapolating these
results to the few tissues with a known biological clock
function like the LNs.
That this is not “overcautious” becomes clear after
considering a different gene related to the circadian
clock: cryptochrome (cry) encodes a factor that is crucial
for the light response of the circadian clock and a mutation in this gene (cryb) results in the loss of overt clockgene oscillations in peripheral tissues (Stanewsky et al.
1998). Surprisingly cryb flies showed normal locomotor
rhythms in DD, and sure enough it turned out that clockgene cycling in the LNs was not abolished by this mutation, pointing to different functions of this gene in peripheral versus pacemaker tissues (Stanewsky et al. 1998
and see below). Moreover, even the different groups of
LNs were affected differentially by cryb (Helfrich-Förster
et al. 2001), so one should be even more cautious in generalizing results. A similar differential effect was also
observed for two other clock-gene mutations, which will
be discussed in more detail below: ClockJrk and cycle01
mutant flies inhibit expression of the pdf gene in the
s-LNv but not in the l-LNv (Park et al. 2000a). Consis-
tent with this, per expression levels are normal in the
l-LNv but reduced in all other cells of the two mutants,
including the s-LNv and LNd (Allada et al. 1998; Rutila
et al. 1998; Kaneko and Hall 2000). Another example involves the behaviorally arrhythmic disco flies, which
show normal molecular rhythms of clock-gene expression when peripheral tissues are examined but a reduction of LN cell number combined with abolished PER
expression within the remaining neurons (Zerr et al.
1990; Hardin et al. 1992; Blanchardon et al. 2001).
Components of the DD clock
The players
Which factors comprise the clock that drives lifelong
rhythmic behavior in the absence of external zeitgebers
(which can be observed for up to 8 weeks depending on
the experimental setup and the care of the researcher)?
The most straightforward way to identify such factors
would be to search for mutants that behave arrhythmically or show altered periods under these conditions using
forward genetic screens. Indeed, in addition to finding
new mutated versions of per (Dushay et al. 1992;
Konopka et al. 1994; Hamblen et al. 1998), this simple
strategy led to the discovery of several novel clock factors: timeless (tim), Clock (Clk), cycle (cyc), and doubletime (dbt). Whereas the original tim01 mutation was isolated as an aperiodic eclosion variant (Sehgal et al.
1994), the ClkJrk (Allada et al. 1998) and cyc01 (Rutila et
al. 1998) mutants emerged as arrhythmic strains from
screens for altered locomotor activity rhythms. Subsequent testing revealed that (as in the case of per) arrhythmic behavior was also observed for the respective other
clock output. Therefore it was concluded that the loci
identified by these mutations also encode crucial clock
factors. Subsequently, period-lengthening and -shortening alleles of tim were isolated as well as one novel lossof-function allele each for the tim and cyc loci (Rutila et
al. 1996; Rothenfluh et al. 2000a, 2000b; Stempfl et al.
2002; Park et al. 2000a).
In the case of dbt, originally two period-altering alleles were isolated in a screen where locomotor activity
rhythms were measured: one with short free-running
rhythms (18 h; dbtS), and a long variant (27 h; dbtL).
Again, eclosion rhythms were affected in the same way
by both mutations, suggesting that the locus encodes a
clock factor (Price et al. 1998). Subsequently, two additional period-lengthening alleles, dbth and dbtg, were isolated, exhibiting free-running periods of 29 h (dbth) or
28.5 h (dbtg/+) (Suri et al. 2000). When dbtg was placed
over a deletion of the locus the resulting flies were behaviorally arrhythmic (Suri et al. 2000): the same phenotype that was observed for homozygous dbtar flies
(Rothenfluh et al. 2000c). It turned out that completely
eliminating dbt gene function resulted in lethality, pointing to an additional role of the dbt gene product during
development (Price et al. 1998; Zilian et al. 1999). This
14
also indicates that even the two arrhythmia-inducing dbt
alleles are not loss-of-function alleles.
Different approaches uncovered rhythm-related functions for the shaggy (sgg) and vrille (vri) genes, which
had previously been shown to be developmental factors
(e.g., Siegfried et al. 1992; George and Terracol 1997).
Sgg was reidentified in a genetic screen where individual
genes covering a fair fraction of the whole Drosophila
genome were specifically overexpressed in all clockgene-expressing cells including the LNs (Martinek et al.
2001). In the case of sgg this resulted in shortenings of
the free-running period (20–21 h) and reducing sgg gene
function led to period lengthenings (26 h). Due to the vital function of this gene during development it was not
possible to study the behavioral consequences of a total
loss of function mutant (Martinek et al. 2001). However,
the specific effects of altered sgg expression on the expression of other clock genes within the LNs (discussed
below) suggest that this gene also encodes an important
clock factor. Finally, vri was identified in a molecular
screen aimed at isolating genes that are rhythmically expressed and controlled by the circadian clock (Blau and
Young 1999). Like sgg, vri has a vital function during
development, militating against behavioral analysis of
vri loss-of-function mutants. But reduction of vri gene
dosage resulted in mild period-shortenings (23 h), and
experimentally induced constant expression in the LNs
resulted in lengthened locomotor periods or even arrhythmic behavior (Blau and Young 1999). In addition,
vri is normally expressed in the LNs (LNv and LNd) and
overexpression in these cells blunts the expression of
other clock genes therein (Blau and Young 1999).
The behavioral and molecular phenotypes just described suggest that the per, tim, Clk, cyc, dbt, sgg, and
vri genes each comprise a crucial part of the Drosophila
clock. In the following I will summarize what is known
about their gene products and how they interact to generate self-sustained oscillations.
Interactions among clock factors
The transcription factors CLOCK and CYCLE
The genes Clk and cyc both encode transcription factors
containing a PAS protein-dimerization domain (for PERARNT-SIM; the founders of the PAS-protein family) and
a basic helix-loop-helix (bHLH) domain involved in
DNA binding (Crews and Fan 1999). Both genes show a
broad RNA expression pattern in heads including the
photoreceptor cells and the brain cortex, including the
region where the different LN groups are located (So et
al. 2000). Clk and cyc expression in the LNs is not surprising given the behavioral phenotypes of the respective
mutants described above. However, a subset of the LNs,
the l-LNv (Fig. 1), likely does not express those two
genes given that the ClkJrk and cyc01 mutations do not affect expression of per and tim within these cells (Kaneko
and Hall 2000). This suggests that other factors drive
Fig. 2 Model describing the daily patterns of activity, cellular distribution, and interactions of important clock genes under constant
conditions. Transcriptional activity is indicated by wavy lines, reduced RNA production by dotted lines. Protein concentrations are
coded by the intensity of the color chosen for a certain protein
(e.g., light blue indicates low TIM abundance, dark blue refers to
high TIM levels). Colored dots indicate progressive degradation of
a given protein. The phosphorylation status of PER and TIM is depicted by flags. CLK, CYC, PER and TIM proteins are abbreviated with single letters (C, B, P, and T, respectively) due to space
constraints. For other abbreviations, see text. Regulation of vri
RNA is identical to that of per and tim and was therefore not included. Levels and function of the VRI protein are entirely hypothetical, and the activator of Clk transcription is also not known
(for details, see text)
clock-gene expression within the l-LNv. The following
description of the temporal expression pattern of both
genes and their interactions is based on experiments that
were performed with crude RNA or protein extracts from
whole fly heads, and may not necessarily reflect the processes in the pacemaker neurons.
While Clk expression is itself circadianly regulated –
resulting in cycling Clk mRNA and CLK protein levels –
cyc is constitutively transcribed, leading to high, steadystate cyc RNA and CYC protein levels (Bae et al. 1998,
2000; Lee et al. 1998; Rutila et al. 1998). Both Clk RNA
and protein show a similar temporal profile, reaching
peak levels around subjective dawn (the time when in
nature or in an LD cycle the lights would go on) and
their lowest levels in the early subjective night (Fig. 2).
15
In addition, CLK (like the per and tim gene products, see
below) is post-translationally modified by phosphorylation (Lee et al. 1998). Yet, it is not clear whether the
phosphorylation status is circadianly regulated as in the
case of PER and TIM.
CLK and CYC proteins heterodimerize and bind to
so-called E-Box sequences – six consensus nucleotides
that are a target for bHLH transcription factors (reviewed
by Kyriacou and Rosato 2000) – in the promoters of the
per and tim genes, thereby activating their transcription
(Darlington et al. 1998; Lee et al. 1999; Fig. 2). The interaction of CLK and CYC has been shown to occur in
fly heads (Bae et al. 2000), whereas the binding of the
heterodimer to the per and tim promoter sequences has
been inferred from cell culture experiments (Darlington
et al. 1998) or from the low levels of PER and TIM proteins in ClkJrk or cyc01 mutant backgrounds (Allada et al.
1998; Rutila et al. 1998). Direct binding has so far only
been shown with in vitro translated CLK and CYC proteins (Lee et al. 1999). Since CLK levels vary throughout the day and CYC is constitutively present in high
amounts, it is likely that CLK dictates the number of
CLK-CYC heterodimers in the cell (Bae et al. 2000).
This in turn would explain why activation of the per and
tim promoters occurs rhythmically. A caveat of this model results from the fact that levels of the CLK-CYC heterodimer are decreasing rapidly during the time per and
tim transcription is activated in the middle of the day
(Bae et al. 2000; So and Rosbash 1997). These two studies followed the temporal profile of both events only in
LD, but the temporal CLK expression is similar in LD
and DD (Lee et al. 1998), suggesting that this is the case
for the dimer too. Thus, some mechanism has to exist
that prevents the CLK-CYC dimer from activating per
and tim at earlier times (Fig. 2 and see below).
The multiple roles of PERIOD and TIMELESS
PER, a founding member of the PAS protein family, is
an odd one, since it lacks the bHLH DNA-binding domain. The PAS domain functions as a binding site for
PER’s partner TIM (Gekakis et al. 1995), although the
latter protein itself has no such protein-protein interaction domain. Instead TIM contains three ARMADILLOlike dimerization domains, two of which overlap with
the region that can bind to PER in cultured Drosophila
cells (Kyriacou and Hastings 2001; Saez and Young
1996). The spatial expression pattern of per and tim is
very similar (see above and Fig. 1), although in general
tim seems to be active in more cells compared to per
(Kaneko and Hall 2000). The temporal profiles of gene
activity as well as the protein-protein interactions between PER, TIM and other proteins discussed below
were mainly extracted from “whole head” preparations.
PERIOD and TIMELESS as repressors
CLK- and CYC-mediated transcription of per and tim
starts in the middle of the day and peak levels of each
mRNA are reached in the early evening (Hardin et al.
1990; Sehgal et al. 1995; So and Rosbash 1997). The
PER and TIM proteins accumulate with a ca. 4- to 6-h delay – reaching peak levels late in the night – compared to
their mRNAs. This delay is a potentially crucial feature
of the clock mechanism (Zerr et al. 1990; Zeng et al.
1994, 1996; Myers et al. 1996; and see below). Interestingly, already in the early stage of clock-molecule studies, a difference in the cyclical PER expression profile
was found between a peripheral tissue (the photoreceptors) and the LNs, where peak and trough levels of PER
were reached later compared to the photoreceptors (Zerr
et al. 1990). PER and TIM accumulate in the cytoplasm
of photoreceptor cells, Malpighian tubules, and LNs and
eventually enter the nucleus probably as a heterodimer
(Vosshall et al. 1994; Curtin et al. 1995; Hege et al. 1997;
Fig. 2). In fact, heterodimerization with TIM is likely to
be a prerequisite for PER accumulation (cf. Price et al.
1995) since newly synthesized monomeric PER is destabilized by the dbt-encoded kinase (Price et al. 1998;
Kloss et al. 1998; Fig. 2). DBT function therefore delays
accumulation of the PER-TIM dimer until sufficient
amounts of TIM are present in the cytoplasm, allowing
per and tim transcription to continue. This delay is further
increased by temporal gating of PER and TIM nuclear
entry, a process likely to involve dbt and sgg gene functions (see below). The temporal gap between transcriptional activity and nuclear entry of the repressors is probably important to maintain robust molecular oscillations
(e.g., Price et al. 1998), although the relevance of this delay is still under debate (e.g., Suri et al. 2000; see below).
Once in the nucleus PER and TIM are thought to repress transcription of their own genes by interfering with
their activators CLK and CYC (Lee et al. 1998, 1999;
Bae et al. 2000; Fig. 2). Most likely this occurs via a direct binding of the PER-TIM dimer to the CLK-CYC dimer in the late night and early morning (Bae et al. 2000).
But this would not explain why transcription of per and
tim is turned off ca. 4 h before the PER-TIM dimer enters the nucleus (see above). It is more likely that the initial deactivation of per and tim expression is caused by
the trough levels of CLK reached around the same time
(at subjective dusk; Fig. 2). Later, the repressor activity
of PER and TIM prevents the CLK-CYC dimer from reinitiating per and tim transcription during the rising
phase of CLK in the late night and its initial decline in
the morning. Only after PER and TIM have disappeared
from the nucleus around midday can a new round of
transcription be initiated by the CLK-CYC dimer.
PERIOD and TIMELESS as activators
In addition to negatively regulating their own expression, PER and TIM are part of a second, interlocked
16
feedback-loop, which further regulates Clk gene expression. The initial finding was that in per01 and tim01 mutant animals levels of Clk RNA and protein were low,
suggesting that PER and TIM positively influence Clk
activity (Bae et al. 1998; Lee et al. 1998). Surprisingly,
in per01 ClkJrk and per01 cyc01 double-mutant, levels of
Clk RNA were at peak levels throughout the circadian
day, suggesting that the CLK-CYC dimer normally represses Clk expression and that this repression is released by the PER-TIM dimer (Glossop et al. 1999).
This scenario raised the hypothesis that Clk expression
would be activated after the nuclear entry of PER and
TIM in the middle of the night, consistent with what is
observed experimentally (Bae et al. 1998; Fig. 2). Since
CLK and CYC are positively acting transcription factors,
it is hard to imagine that they can act as repressors as
well. Instead of directly repressing Clk, it seems more
likely that they upregulate a yet unknown repressor in a
mode similar to per and tim. This factor would then also
be downregulated by nuclear PER and TIM, an assumption that jibes with the observation that Clk RNA levels
are low in per01 and tim01 (because the repressor is up)
and high in per01 ClkJrk and per01 cyc01 double mutants
(because the repressor is down due to missing transcriptional activation). A potential candidate molecule for this
function is discussed in the next paragraph, but yet another question remains open: what activates Clk expression in the absence of functional CLK, CYC, PER, and
TIM proteins?
The role of vrille
Vri encodes a basic-Zipper transcription factor and is
expressed with the same temporal profile as per and tim
(Blau and Young 1999). Its spatial expression pattern in
the fly head is similar to that of per and tim (including
the LNs) although vri seems to be present in more CNS
cells compared to the other two clock genes (Blau and
Young 1999). Since vri function is required for normal
clock-gene oscillations, its function was placed within
the core of the circadian pacemaker mechanism. Moreover vri is involved in post-transcriptional regulation of
PDF, indicating that it also plays a role in clock output
(Blau and Young 1999). There is also circumstantial
evidence that VRI indeed could be the Clk repressor and
in the following I will present some arguments in favor
of this hypothesis. First, vri is activated by CLK and
CYC, since vri RNA levels are low in ClkJrk or cyc01
mutants. This activation is likely to be a consequence of
direct CLK-CYC binding to an E-Box in the vri promoter, since both the dimer and the promoter element are
crucial for activating vri transcription in vitro (Blau and
Young 1999). The low repressor-encoding RNA levels
in ClkJrk or cyc01 would be consistent with the high
levels of Clk RNA in the same genetic backgrounds
(Glossop et al. 1999; see above). Second, in per01 or
tim01 mutant flies vri RNA levels are intermediate, similar to the per and tim steady-state RNA amounts in the
same mutant backgrounds. This indicates that vri is indeed regulated by the same mechanism as per and tim
(Blau and Young 1999). The predicted constant and intermediate VRI amounts would explain why Clk RNA is
low in per01 and tim01. Third, vri overexpression reduces
per and tim RNA levels (Blau and Young 1999), probably as a result of reduced Clk activity. If VRI is indeed
the repressor, one would expect its phase of expression
to be just the opposite to that of Clk, whose gene products reach their trough levels around dusk, just about
when vri RNA is at its peak. This implies that – unlike
for per and tim – there would be no delay expected to
exist between vri RNA and protein accumulation and
also no gated nuclear entry of VRI. As a result (and as
in the case of Clk) the RNA and protein profiles of vri
would be very similar. VRI would be made and enter the
nucleus closely following the temporal vri RNA pattern,
repressing Clk from afternoon until midnight when PER
and TIM enter the nucleus and block VRI’s function
(Fig. 2).
What gene product activates Clk transcription? Arguments favoring certain candidates are even more handwaving than those for VRI being a repressor. Nevertheless, microarray analysis aimed at identifying direct target genes of CLK revealed two transcription factors that
are normally rhythmically expressed in the fly head, with
a similar temporal profile to per and tim (McDonald and
Rosbash 2001). The two genes in question, pdp1 and
sticky ch1, encode basic-Zipper and bHLH transcription
factors, respectively, and could therefore be involved in
CLK regulation via positive feedback regulation. Alternatively, especially in case VRI turns out to be a rhythmically active Clk repressor, production of its activator
could be temporally flat (Fig. 2).
The kinases encoded by double-time and shaggy
DOUBLE-TIME regulates PERIOD at several points
throughout the day
Dbt encodes the Drosophila ortholog of the mammalian
casein kinase Iε (Kloss et al. 1998). Considering the behavioral phenotypes of dbt mutations discussed above,
the kinase function of the DBT enzyme could be responsible for the circadian fluctuation of the phosphorylation
status observed for both the PER and TIM proteins
(Edery et al. 1994; Zeng et al. 1996) or even CLK (Bae
et al. 1998). It turned out that dbt function influences
PER post-translationally, although at present it cannot be
ruled out that TIM and CLK are also substrates of this
enzyme (Kloss et al. 1998; Price et al. 1998; Blau 2001).
Dbt RNA and protein are expressed constitutively in
the photoreceptor cells and the brain cortex including the
LNs (Kloss et al. 1998, 2001). Yet, the spatial dbt expression, at least during development, is probably much
broader compared to per and tim because strong hypomorphic alleles (dbtP) result in pupal lethality and altered
brain architecture (Price et al. 1998; Zilian et al. 1999).
17
Although constitutively expressed, DBT undergoes daily
changes in subcellular localization in photoreceptor cells
and the LNs at least under LD conditions (Kloss et al.
2001).
One role of DBT is to destabilize cytoplasmic PER in
the absence of sufficient amounts of TIM that normally
stabilize PER by forming a heterodimer. This can be inferred from the accumulation of high amounts of PER in
the strong dbtP allele, although only very little TIM is
expressed in this mutant. Moreover, PER isolated from
dbtP larvae is hypophosphorylated, indicating that DBT
destabilizes PER by phosphorylation (Price et al. 1998;
Fig. 2). Evidence for this also comes from a different set
of experiments, involving flies expressing per-lacZ fusion genes. One of these (SG-lacZ), encoding the N-terminal half of PER, is constitutively expressed although
its encoding RNA exhibits per-like oscillations (Zwiebel
et al. 1991; Dembinska et al. 1997). Adding the next 231
PER amino acids in the C-terminal direction – encoded
by BG-lacZ – not only results in robust protein oscillations, but also in daily changes of the phosphorylation
pattern (Dembinska et al. 1997; Stanewsky et al. 1997a).
This implies that PER phosphorylation is required for
rhythmic turnover and defines a likely DBT target within
the PER protein.
This view is further supported by the direct physical
interaction between DBT and PER, which can be observed both in vitro and in vivo (Kloss et al. 1998,
2001). The interaction occurs throughout the day, resulting in the observed daily rhythms in subcellular localization of DBT (Fig. 2). It is likely that DBT just
sticks to PER when the latter protein is newly synthesized in the cytoplasm, leading to substantial amounts
of cytoplasmic DBT in the early night. After the shift
of the PER-TIM complex into the nucleus around midnight, DBT is also observed predominantly in this compartment, indicating that the three proteins enter the nucleus as a complex (Kloss et al. 2001). Strikingly, in
per01 flies DBT is mainly nuclear, suggesting that this
is its default localization, and that cytoplasmic PER is
needed to accumulate DBT in the cytoplasm. However,
the same nuclear localization is observed in tim01 flies,
but since TIM accumulates to rather high amounts in
the cytoplasm of per01 flies (Myers et al. 1996) and
PER levels are very low in tim01 flies (Price et al. 1995,
1998), it is much more likely that PER is responsible
for cytoplasmic accumulation of DBT (Kloss et al.
2001).
PER phosphorylation is not restricted to the cytoplasm since the highest molecular weight forms are observed at times when PER is in the nucleus (Edery et al.
1994; Curtin et al. 1995). Since PER persists in nuclei of
dbtP larvae in the absence of TIM, it was suggested that
DBT affects the stability of PER also in this compartment (Price et al. 1998). That this is likely to be the case
was shown by applying yet another clock gene mutation,
the timUltraLong (timUL) allele. Flies carrying this variant
exhibit free-running periods of 33 h (!) caused by a dramatically increased stability of the nuclear TIMUL-PER
complex (Rothenfluh et al. 2000b). In agreement with
this finding PER is hypophosphorylated (and therefore
probably stable) for an extended period of time in timUL
flies (Rothenfluh et al. 2000b; Kloss et al. 2001). When
TIMUL is removed from this complex by light (see next
section), PER becomes rapidly phosphorylated, indicating that TIM normally blocks excessive PER phosphorylation as long as the proteins are part of the same complex (Kloss et al. 2001; Fig. 2).
Blocking DBT function might indeed be a crucial
function of TIM in the nucleus: Using the same timUL
flies and the same strategy of clearing PER from the
mutant TIM protein, it was shown that monomeric PER
can act as a potent repressor of CLK-CYC-induced per
and tim expression (Rothenfluh et al. 2000b). Consistent
with this idea, a mutant version of PER that constitutively localizes to the nucleus disrupts behavioral
rhythms even in the presence of a wild-type per gene
copy. Moreover, in vitro expression of this nuclear form
of PER in the complete absence of TIM resulted in potent inhibition of CLK-CYC-induced per expression
(Rothenfluh et al. 2000b). Taken together this suggests
that nuclear TIM retards phosphorylation of PER, thereby prolonging the cycle. An important time cue for setting the period length of the molecular cycle would then
be the dissociation of TIM from PER and DBT in the
late night.
Regulation of nuclear PER stability by DBT (at least
under LD conditions) was also suggested after analysis
of period-lengthening and arrhythmia-inducing dbt alleles: dbtL, dbtg, and dbth/+ all show a prolonged presence of nuclear PER in the early morning. Although in
these alleles PER also accumulates a bit later in the day
compared to wild type, peak levels of PER are reached
around the same time (Price et al. 1998; Suri et al. 2000).
Therefore this delayed onset of PER accumulation in the
mutants cannot explain the significantly slower decay of
nuclear PER in the morning (Price et al. 1998; Suri et al.
2000). In LD conditions the extended repressing activity
of monomeric PER results in a delayed onset of per and
tim expression, thereby eliminating the delay usually observed between transcripts and proteins (Suri et al.
2000). Although the importance of “the delay” for feedback regulation has been questioned after those results,
the same study shows that it is not eliminated in constant
conditions, implying its importance in maintaining freerunning molecular oscillations.
Strikingly, the stable monomeric forms of PER observed in the period-lengthening alleles were hyperphosphorylated (Price et al. 1998; Suri et al. 2000), and even
in the arrhythmia-inducing dbtar mutants temporally flat
PER molecules represented a mixture between hypo- and
hyperphosphorylated molecules (Rothenfluh et al.
2000c). This indicates that other kinases are also involved in PER regulation. On the other hand, the dbtar
mutant underscored the specificity of DBT for PER: A
double mutant between perS and dbtar restored behavioral and molecular rhythms, indicating that the increased
turnover of nuclear PERS proteins (Curtin et al. 1995)
18
compensates for the increased stability of nuclear PER in
dbtar flies (Rothenfluh et al. 2000c).
Interestingly, DBT nuclear and cytoplasmic function
seems to have opposing effects on cycle progression. In
the cytoplasm PER accumulation is inhibited by DBT
and can only occur after enough TIM is present, resulting in a delay of PER increase compared to its RNA. In
contrast, nuclear DBT function seems to set the endpoint
for each molecular cycle by phosphorylating monomeric
PER and turning it into a potent repressor before it is degraded (Fig. 2).
DBT seems to affect yet another step in the circadian
cycle, i.e., the nuclear translocation of PER or the PERTIM complex. Evidence for this stems from a detailed
analysis of the period-shortening dbtS allele (Price et al.
1998; Bao et al. 2001). Although turnover of nuclear
PER and its cytoplasmic accumulation in photoreceptor
cells of this mutant is accelerated, the nuclear entry of
PER is delayed by several hours compared to wild type
(Bao et al. 2001). Since this effect was analyzed only in
LD conditions, it is not clear if it describes an additional
clock function of DBT, or its involvement in entrainment
to such environmental cycles (see below).
SHAGGY affects TIMELESS phosphorylation
and nuclear translocation of the PER-TIM dimer
Shaggy encodes the ortholog of the mammalian glycogen synthase kinase-3 (GSK-3) and is important for the
daily changes in phosphorylation pattern of TIM, which
is likely to be a substrate of this kinase (Martinek et al.
2001). Although the spatial and temporal expression pattern of sgg in adult flies is not known, its overexpression
in all tim-expressing cells results in an advanced nuclear
entry of PER and TIM proteins in the LNs correlated
with shorter free-running periods of locomotor rhythms
(Martinek et al. 2001). Reducing sgg expression results
in hypophosphorylated TIM throughout the circadian
day, whereas overexpression has the opposite effect. Together with the observation that mammalian GSK-3 in
vitro phosphorylates fly TIM, this shows that SGG is
crucial for TIM phosphorylation. Although it was proposed that SGG mainly targets cytoplasmic TIM during
early subjective night, SGG also affects the onset of TIM
phosphorylation in the middle of subjective night (Martinek et al. 2001). This event could normally trigger the
dissociation of TIM from the TIM-PER-DBT complex,
leading to DBT-dependent phosphorylation of PER,
thereby turning it into a potent monomeric repressor of
CLK-CYC-induced per and tim expression until the 24-h
cycle is completed (Fig. 2).
Since completely eliminating sgg gene function results in lethality, it is not certain if SGG is the only kinase involved in TIM phosphorylation. There is evidence that a tyrosine kinase is linked to degradation of
TIM by the proteasome (Naidoo et al. 1999) and TIM
has also been discussed to be a substrate of DBT (Blau
2001).
In summary it seems clear that both DBT and SGG
are involved in post-translational modifications of PER
and TIM, respectively. Their functions regulate the period of the circadian clock and seem to be a potential target for other regulatory events determining day length,
as indicated by the opposing effects both kinases have on
nuclear entry of the PER-TIM complex.
The LD-clock: light as a potent amplifier
Poor molecular rhythms in the absence of light as
zeitgeber
Although it is important to study the functions that generate sustained molecular and behavioral oscillations
under constant conditions, it is at least equally important
to study clock mechanisms under more natural conditions, like LD cycles. It should be noted that the LD cycles applied in most laboratories aimed at simulating the
natural change of night and day are far from appropriate. In nature, organisms synchronize their clock mainly
to twilight conditions occurring at dawn and dusk,
which are characteristic of dramatic changes in both
light quantity and quality (Roenneberg and Foster
1997). Moreover, light:dark changes in nature usually
go in hand with temperature changes that also can act as
potent zeitgebers (e.g., Wheeler et al. 1993). We are only starting to understand how light and temperature interact to mediate stable entrainment to environmental
cycles in flies (e.g., Tomioka et al. 1998; Majercak et al.
1999).
For a detailed description of light inputs into the circadian clock the reader is referred to more specific articles dealing with this subject (e.g., Foster and HelfrichFörster et al. 2001; Shafer 2001; Zordan et al. 2001).
Here, I will only summarize how light resets the molecular clock-gene oscillations in flies. Indeed, light does
probably more than that, because molecular oscillations
dampen rapidly in DD. Robust gene-product rhythms in
flies can be observed for a maximum of 4 days in DD,
which is in stark contrast to the lifelong behavioral
rhythms. Therefore, the mechanisms described in the
previous chapter are likely not sufficient to explain sustained rhythmic behavior. Several arguments have been
presented trying to “rescue” the dogma that rhythmic
gene expression drives behavioral rhythms. Initially
DD-dampening was attributed to interindividual drift of
clock-gene oscillations. In this context it is noteworthy
that usually at least ca. 30 fly heads are required to determine the RNA or protein content at one given timepoint, which could result in flattening of a molecular
rhythm if the individuals had drifted out of phase from
each other. Introducing the luciferase reporter allowed
real-time measurement of clock-gene expression in individual flies (Brandes et al. 1996). But even here rapid
dampening of per-driven luciferase expression occurred
in DD, ruling out the desynchronization hypothesis
(Stanewsky et al. 1997b). In turn, this pointed to the
19
presence of multiple oscillators within an individual,
which could rapidly run out of phase with each other in
the absence of synchronizing time cues. The existence
of brain-independent oscillators and moreover their ability to be synchronized by light was demonstrated in several studies (e.g., Plautz et al. 1997; Giebultowicz et al.
2000), further nourishing the intraindividual desynchronization hypothesis. Alternatively, however, it is possible that these “peripheral oscillators” (which we generally use as a source to generate our models: see above)
are not able to maintain sustained oscillations in DD.
The observed dampening oscillations of per- and timdriven luciferase expression in isolated body tissues
favor the latter hypothesis (Plautz et al. 1997;
Giebultowicz et al. 2000). Moreover, the amplitude of
the only known biological rhythm controlled by a peripheral oscillator – circadian olfactory responses in the
fly antennae – is rather low (both in terms of sensitivity
changes and of clock-gene expression) and it is at least
questionable whether it persists for more than a week in
constant conditions (Krishnan et al. 1999, 2001).
This brings us back to the fly’s activity rhythm, which
we know persists in DD. It is controlled by the LNs, and
therefore at least in these cells the molecular cyclings
should continue without zeitgebers. This is difficult to
prove and would require the behavior of a fly to be monitored, before sacrificing it to determine the amount of a
certain clock-gene product. If molecular rhythms indeed
underlie the behavioral ones, a certain behavioral activity phase should always be correlated with a certain expression level of a clock gene. One hopes that this kind
of experiment will indeed show “lifelong” clock-gene
oscillations. Robust fluctuations of TIM protein and vri
RNA have been observed in the s-LNv after flies were
kept for 2 days in DD (Yang and Sehgal 2001). Strikingly, in the same animals oscillations had dampened to arrhythmicity in the l-LNv, supporting the crucial role for
the smaller neurons in controlling locomotor rhythms,
whereas the large cells may at best behave like peripheral oscillators.
How light interacts with clock-gene products
As discussed, the natural light:dark changes keep the different clock-gene-expressing tissues synchronized and
they are probably responsible for maintaining rhythmicity as well. How is light interacting with the clock works?
TIM is a key player, since it is rapidly degraded in response to light, both in light:dark cycles and after light
pulses (Hunter-Ensor et al. 1996; Lee et al. 1996; Myers
et al. 1996; Zeng et al. 1996). PER is stabilized by TIM
and this observation led to a model of how light resets
molecular oscillations. Late in the night, light causes advanced degradation of nuclear TIM (and subsequently
PER), resulting in the release of CLK-CYC repression
by the PER-TIM dimer (or monomeric PER), so that per
and tim transcription can be activated again (Fig. 3). In
the early evening, light also causes a reduction in TIM
Fig. 3 Light effects on clock molecules. The example shown here
illustrates how light pulses in the late night result in a phase advance of clock-gene cyclings. The natural equivalent would be the
day by day earlier occurring sunrise in winter and spring. The biological significance of the CRY-PER interaction is not known. In
any event, the earlier than usual disappearance of TIM probably
results in an earlier decline of PER. As a consequence per and tim
transcription start earlier each day. A similar CRY-dependent
mechanism is responsible for phase delays induced by light pulses
in the early night (or consecutive longer days in winter and
spring), and might also contribute to the maintenance of clockgene oscillations under the light-dark condition (for details, see
text) (NF postulated “nuclear factor,” TK postulated tyrosine kinase, U ubiquitin). Other symbols and abbreviations are described
in the legend to Fig. 2
levels, but here tim RNA levels are high so that the depleted proteins can be replaced by newly synthesized
TIM, resulting in phase delays. Since light pulses given
at these times have the same qualitative effect on the
phase of behavioral rhythms (e.g., Saunders et al. 1994),
it was tempting to speculate that the TIM response is
connected to the behavioral one. That this is indeed the
case was demonstrated by Suri et al. (1998) and Yang et
al. (1998), who independently showed that both TIM
degradation and behavioral resetting require a similar
light intensity and quality. In addition, the latter study
showed that this is true for the LNs too, and that the eyes
and the canonical phototransduction cascade are not required for TIM degradation within these neurons. This
suggested the existence of an extraocular, non-opsinphotoreceptor mediating circadian photoreception, which
turned out to be CRYPTOCHROME (Emery et al. 1998,
2000; Stanewsky et al. 1998). Although the eyes – and
probably other extraocular photoreceptors – also contribute to entrainment of molecular and behavioral rhythms
(Stanewsky et al. 1998; Helfrich-Förster et al. 2001),
CRY is the main mediator for light-induced TIM degradation in peripheral tissues and in most of the LN cell
types. Strikingly, however, the behaviorally important sLNvs are the exception. In cryb mutant flies, which are
devoid of functional CRY, TIM oscillations in these cells
and behavioral rhythms can be nicely entrained by LD
cycles (Stanewsky et al. 1998; Helfrich-Förster et al.
2001). This suggests that light-dependent, but CRY-inde-
20
pendent mechanisms exist that are able to synchronize
biologically meaningful TIM oscillations (cf. Yang et al.
1998).
Nevertheless, in peripheral oscillators CRY is clearly
crucial for light-induced TIM disappearance: In LD cycles TIM is stable in photoreceptors of cryb flies
(Stanewsky et al. 1998) and this phenotype can be reversed by expressing functional CRY in these cells
(Emery et al. 2000). In cryb mutant Malpighian tubules
and whole-head protein extracts, TIM does not respond
to light pulses, again indicating the importance of
functional CRY for light-dependent TIM degradation
(Ivanchenko et al. 2001; Lin et al. 2001). In fact, lightdependent interactions between TIM and CRY (Ceriani
et al. 1999) and also between PER and CRY (Rosato et
al. 2001) probably occur in the nuclei of yeast cells. Furthermore, interactions between these proteins are observed in the cytoplasm of Drosophila cells cultured in
darkness (Ceriani et al. 1999; Rosato et al. 2001), leading to the model that a nuclear factor binds to CRY, preventing it from binding the two clock proteins in the
dark. Light activation of CRY probably results in the dissociation or inactivation of the nuclear factor, allowing
the CRY-TIM and CRY-PER interactions to occur
(Rosato et al. 2001; Fig. 3). However, mutations in the
yeast genome could be induced (probably eliminating
the proposed “nuclear factor”), which rendered the nuclear PER-CRY interaction light independent (Rosato et
al. 2001). Therefore, it is more likely that PER (and
maybe TIM too) and CRY can always bind to each other,
unless a light-sensitive nuclear factor abolishes their interaction.
A likely result of the CRY-TIM interaction is the
phosphorylation of TIM by a tyrosine kinase and subsequent ubiquitination of this clock protein. As a consequence TIM is degraded via the proteasome pathway
(Naidoo et al. 1999; Fig. 3). This was further investigated by Lin et al. (2001), who showed that CRY is also
degraded by the same pathway in response to light (cf.
Emery et al. 1998). CRY degradation requires electron
transport, mediated by one of the CRY cofactors, flavin
adenine dinucleotide (FAD). Specifically blocking this
transfer probably results in the accumulation of activated
CRY protein since TIM ubiquitination is increased (Lin
et al. 2001). This suggests that light-activated CRY indeed promotes TIM turnover.
Whether the CRY-dependent mechanism proposed
above is also involved in TIM turnover under constant
conditions is unknown. It should be kept in mind that, in
addition to the light-dependent tyrosine kinase function,
SGG is involved in TIM phosphorylation under constant
conditions (Martinek et al. 2001). Nevertheless, in several peripheral tissues a function for CRY under free-running conditions was demonstrated. The circadianly regulated olfactory responses and the clock gene cyclings
normally observed in fly antennae become arrhythmic in
cryb mutant animals (Krishnan et al. 2001). To rule out
that the observed arrhythmicity was due to the lack of
light-induced synchronization (caused by cryb) amongst
the antennal cells comprising this peripheral oscillator,
the animals were entrained by temperature cycles prior
to their release into constant conditions. As shown previously, PER and TIM oscillations in the eyes of cryb mutant flies can be entrained by this zeitgeber (Stanewsky
et al. 1998). Again, antennae isolated from cryb mutant
animals exhibited neither molecular nor sensitivity
rhythms in constant conditions, indicating that CRY is a
part of the clock in this tissue (Krishnan et al. 2001). A
similar result was obtained for another peripheral oscillator, ticking in the Malpighian tubules. Here the cryb mutation also led to constitutive clock gene expression under constant conditions (Ivanchenko et al. 2001). This
study also beautifully demonstrated the difference between the clock mechanisms in LN and peripheral tissues: In DD, both PER and TIM cycled with normal amplitude and phase in the LN of cryb mutant larvae in
agreement with the normal free-running locomotor period of adult cryb flies (Ivanchenko et al. 2001; cf.
Stanewsky et al. 1998).
In summary, the CRY-mediated turnover of TIM is
likely to be the driving force of the high-amplitude
rhythms of clock-gene cyclings observed in peripheral
oscillators under LD conditions. No other known clock
genes or any of their products are influenced by light directly, but since the different feedback loops are interconnected (see above) the TIM light-response probably
helps to amplify molecular oscillations in general. For
example, the amplitude of both Clk RNA and protein cycling is enhanced by light (Lee et al. 1999; Bae et al.
1998).
Other mechanisms contributing to overall molecular
oscillations of clock gene products
Transcriptional mechanisms
The cAMP response element-binding protein (CREB)
has been implicated in circadian rhythms both in mammals and flies (Ginty et al. 1993; Ding et al. 1997; Belvin et al. 1999). The Drosophila CREB2 gene encodes a
protein, whose activity is circadianly regulated (Belvin
et al. 1999). A functional per gene is required for
CREB2 rhythmicity, but at the same time CREB2 function is needed for normal rhythmic transcriptional activity of per and normal behavioral rhythmicity (Belvin et
al. 1999). That CREB2 indeed affects transcription was
indicated by the differential effects a CREB2 mutant had
on the real-time expression pattern of transgenic flies
transformed with two different per-luciferase reportergene fusions. Cycling of a reporter gene reflecting per’s
transcriptional activity was severely blunted, whereas
that of a construct which in addition mimics post-transcriptional regulation of per mRNA (see below) exhibited quite robust oscillations in the face of the mutant
(Belvin et al. 1999; cf. Stanewsky et al. 1997b). This
suggests that CREB2 is part of the regulatory feedback
loops, and in addition to CLK and CYC influences per
21
transcription. In fact, three potential CREB-binding sites
are present in the per promoter region (Belvin et al.
1999) and one in the tim promoter (Okada et al. 2001). It
is not known whether this transcription factor also regulates any of the other clock genes discussed above, but
there is evidence that transcriptional regulation by
CREB2 contributes to rhythmic gene expression downstream of the central clock (see below).
Post-transcriptional regulation of period RNA
The first hint that temporal regulation of per RNA involves more than transcriptional regulation mediated by
promoter sequences came from a study where the daily
profile of endogenous per mRNA was compared with
that of a reporter gene transcript driven by per-promoter
sequences (Brandes et al. 1996). Surprisingly, the daily
upswing of the reporter-RNA was advanced by several
hours as compared to that of per RNA, suggesting that
the transcriptional and mRNA profiles of per differ. That
this is indeed the case was shown by So and Rosbash
(1997): the comparison of the temporal profiles of per
transcription with those of per mRNA accumulation revealed the same advanced rising phase for the transcript
relative to the mature mRNA. This demonstrated that per
RNA is post-transcriptionally regulated during its rising
phase, most likely by temporal stabilization of the
mRNA (So and Rosbash 1997). Further reporter-gene
analyses of per-luciferase fusion genes containing various amounts of the transcribed region of per in addition
to the promoter revealed that this additional form of regulation is mediated by sequences in per’s 5’-transcribed
(but untranslated) region, probably within the first intron
(Stanewsky et al. 1997b, 2002). In addition, it was
shown that this event depends on functional PER and
TIM proteins. In tim01 mutant flies, a pulse of wild-type
TIM resulted in a temporally fairly normal accumulation
and decline of PER (Suri et al. 1999). This was mediated
by a purely post-transcriptionally regulated rise of per
RNA, which was also dependent on a functional per
gene. Thus, the PER-TIM dimer – in addition to its multiple roles in transcriptional regulation – may also mediate RNA regulation at the post-transcriptional level (Suri
et al. 1999).
The biological significance of this additional level of
regulation is further implied by altered free-running periods of flies containing a per gene lacking intron-1 (Liu
et al. 1991; Cooper et al. 1994). Moreover, a per-encoding transgene, completely lacking the promoter region –
but retaining parts of intron-1 – is able to restore rhythmic behavior in per01 mutant flies (Frisch et al. 1994),
and rhythmic RNA expression within these flies is regulated exclusively post-transcriptionally (So and Rosbash
1997).
The mechanism of this regulation is not known, but a
different – PER and TIM independent – mechanism of
post-transcriptional per mRNA turnover involves an alternative splicing event in per’s 3′-untranslated region
(UTR) (Majercak et al. 1999). At low temperatures it results in a phase advance of per mRNA compared to
warmer temperatures and is likely to be responsible for
adjusting the behavioral pattern to seasonal changes in
the daily temperature cycles (Majercak et al. 1999). In
contrast, post-transcriptional regulation mediated by the
5′-UTR of per is not influenced by temperature
(Stanewsky et al. 2002). Although this suggests a different mechanism, regulation of splicing events involving
intron-1 may participate in this form of PER- and TIMdependent RNA regulation (as discussed in Stanewsky
et al. 2002).
Reaching the end of our discussion concerning
“clock mechanisms” in the fly, one is left stunned by the
complexity of known processes contributing to clockgene oscillations. It is difficult, and probably unwise, to
judge which of the mechanisms is more important than
the other. That is because it has repeatedly been demonstrated that a particular mechanism on its own (e.g.,
post-translational regulation) can mediate behavioral
rhythmicity, but in no case neither the molecular nor the
behavioral rhythms obtained were even close to normal
(Ewer et al. 1988; Frisch et al. 1994; Vosshall and
Young 1995; Cheng and Hardin 1998; Yang and Sehgal
2001). A common theme of these studies involves pegging per or tim RNA (or both: Yang and Sehgal 2001) at
constant levels by driving their expression with constitutively active promoters in per01 or tim01 mutant animals. Although transcription and overall mRNA levels
are probably flat in these flies, rhythmic behavior was
observed, likely driven by rhythmic turnover of PER
and TIM proteins in the LNs (Ewer et al. 1988; Frisch et
al. 1994; Vosshall and Young 1995; Yang and Sehgal
2001). Nevertheless, the fraction of rhythmic flies was
far from being 100% and free-running periods were abnormal, usually substantially longer than the typical 24h rhythm.
In summary this suggests that all the different clock
mechanisms discussed above contribute and are necessary to generate normal behavioral rhythmicity. In the
following final part I will summarize our current knowledge about how the temporal information generated by
all these processes might be transferred from clock cells
to downstream target cells expressing overt circadian
rhythms. For detailed discussions regarding clock-output the reader is referred to special reviews dealing with
this subject (Williams and Sehgal 2001; Jackson et al.
2001).
Mechanisms controlling clock-output
Two of the three biological rhythms mentioned in this review, pupal eclosion and adult locomotor activity, depend on clock-gene expression in certain brain neurons
(LNs). In contrast, the clock gene-dependent sensitivity
rhythm in the fly antennae is probably independent of
these neurons, and the rhythm-generating cells are unknown (Krishnan et al. 1999, 2001). Therefore in the lat-
22
ter case it is possible that the rhythm-generating clock
cells are identical with those expressing the biological
rhythm so that it seems unnecessary to transmit temporal
information spatially.
But how is the time-of-day information relayed from
the LNs to the cells that directly regulate eclosion and
activity of the fly? Rhythmicity of both events is regulated by the neuropeptide pigment-dispersing factor (PDF),
which is expressed within LNs of larvae and LNv of
adults. Ectopic PDF expression during development disrupts sustained eclosion rhythms (Helfrich-Förster et al.
2000). In adults, a loss-of-function mutation in the pdf
gene results in short-period activity rhythms that gradually become arrhythmic (Renn et al. 1999). Expression
of pdf is both transcriptionally and post-transcriptionally
regulated by clock genes: In the larval LNs and the adult
s-LNv pdf RNA levels are low or undetectable in the
face of the cyc01 and ClkJrk mutations, respectively (Park
et al. 2000a; Blau and Young 1999), suggesting that
CLK and CYC activate pdf transcription. In fact, the pdf
promoter contains an E-box consensus-binding site for
CLK and CYC, but surprisingly neither do pdf mRNA
levels exhibit daily fluctuations (Park and Hall 1998),
nor is the E-box required for normal amounts and spatial
expression of pdf (Park et al. 2000a). This shows that the
transcriptional regulation by CLK and CYC is probably
indirect.
RNA levels of pdf are unaffected by the per01 and
tim01 mutations (Park and Hall 1998; Park et al. 2000a)
as well as by overexpression of vri within the larval LNs
(Blau and Young 1999). Nevertheless all three gene
products affect PDF expression post-transcriptionally. A
circadian rhythm of PDF accumulation in the nerve terminals of the s-LNv in the dorsal brain is disrupted in
per01 and tim01 mutant flies (Park et al. 2000a), and overall PDF levels in larval LNs are severely reduced in vrioverexpressing animals (Blau and Young 1999).
The role of PDF in timing pupal eclosion is not
known, but it seems likely that timed release of the neuropeptide serves as a signal for downstream cells expressing other factors involved in eclosion (cf. Jackson
et al. 2001). One of those is encoded by the lark gene,
which is expressed in a group of crustacean cardioactive
peptide (CCAP)-positive cells that are involved in eclosion (for other CCAP cells: see Zhang et al. 2000). Mutations in lark were shown to specifically alter the eclosion
rhythms without affecting adult activity rhythms (reviewed by Jackson et al. 1998). Although lark RNA is
expressed constitutively, LARK protein expression is
circadianly regulated in CCAP cells of late pupae, consistent with its role in regulating rhythmic eclosion
(Jackson et al. 1998; Zhang et al. 2000).
Surprisingly, in the adult brain PDF is required for accumulation of normal levels of MAP kinase, suggesting
that the Ras/MAPK-signaling pathway is the next downstream target of the clock and activated by PDF
(Williams et al. 2001). In fact, mutations in the Neurofibromatosis-1 (Nf1) gene, which encodes a Ras-GTPaseactivating protein, result in arrhythmic behavioral activi-
ty. MAP kinase levels are upregulated in Nf1 mutants
and the behavioral phenotype can be rescued by mutations that downregulate MAP kinase signaling, further
establishing a role for this signaling pathway in the output of the circadian clock (Williams et al. 2001). In addition, signaling by PKA is involved in the output controlling adult activity rhythms since mutations in the catalytic subunit of PKA (encoded by the DCO gene), or its
regulatory subunit RII, result in arrhythmic behavior
(Levine et al. 1994; Majercak et al. 1997; Park et al.
2000b). The cells in which these signaling pathways act
(and probably interact: Williams et al. 2001) to control
activity rhythms are unknown, but future studies will
certainly address these questions.
In summary, similar to the feedback loops comprising
the central clockworks, the known output mechanisms
also involve regulatory processes at different levels. Microarray analyses as well as molecular and genetic approaches aimed at determining the extent of circadianly
regulated gene expression suggested that about 1–6% of
all Drosophila genes are rhythmically expressed (Van
Gelder et al. 1995; Claridge-Chang et al. 2001; McDonald
and Rosbash 2001; Stempfl et al. 2002; Ueda et al.
2002). One way to generate these rhythms would be via
direct transcriptional regulation through clock genes, but
the few examples discussed above indicate that this is
rather unlikely. In fact McDonald and Rosbash (2001)
found only seven genes (in addition to tim and vri) that
seem to be direct targets of CLK, indicating that the vast
majority of transcript oscillations are controlled indirectly. Moreover, Stempfl et al. (2002) showed that the
rhythmic activity of certain circadianly regulated enhancers and expression of oscillating transcripts can be
affected differentially by the two clock mutants per01 and
tim01, pointing to novel mechanisms of gene regulation
in the clock output. Similar conclusions were drawn
from a study of per01, tim01, and ClkJrk effects on RNA
levels of usually rhythmically expressed genes. Considering the transcriptional feedback loops discussed above,
it was expected that levels of a given transcript were either low in per01 and tim01 mutant backgrounds and high
in ClkJrk, or vice versa. Those cases were found, but in
addition a similar number of genes were equally influenced by all three clock mutations (either up- or downregulated) (Claridge-Chang et al. 2001). Nevertheless,
the enrichment of binding sites for transcription factors
with a known function in the circadian clock (e.g., Eboxes and CREB-binding sites) in the promoters of
clock-regulated genes argues in favor of widespread
transcriptional control (Claridge-Chang et al. 2001;
Stempfl et al. 2002). This does not imply that the same
molecules and mechanisms as in the central clock are
used in the output pathway. In fact, the transcriptional
regulation of pdf and the rare direct CLK targets argue
against this view (see above).
Importantly, the “mass identification” of novel circadianly regulated genes revealed insight into the diversity
of processes likely to be regulated by the clock. These
involve functions like vision, olfaction, mechanorecep-
23
tion, detoxification, learning and memory, ion-channel
activity, and certain metabolic events (Claridge-Chang et
al. 2001; McDonald and Rosbash 2001; Stempfl et al.
2002; Ueda et al. 2002). Moreover, reproduction has
been shown to be regulated by the clock, in terms of both
mating behavior and physiological processes underlying
overall reproductive fitness (Sakai and Ishida 2001; Beaver
et al. 2002). Studying the temporal aspects of these processes will certainly help to understand how the circadian clock regulates these rhythms in order to optimize the
overall fitness of organisms.
Acknowledgements I thank Charlotte Helfrich-Förster and the
members of my laboratory for critical comments on the manuscript, and Patrick Emery for discussions.
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