SummaryIn mammals, the circadian system is hierarchical — a brain pacemaker located within the suprachiasmatic nucleus (SCN) is responsible for regulating locomotor activity rhythms and for synchronizing peripheral oscillators [1,2]. Recent genetic evidence in mice indicates that the bHLH transcription factors CLOCK and NPAS2 have partially redundant functions within the SCN [3,4]. To further examine the roles of CLOCK and NPAS2, we generated CLOCK-deficient (Clock−/−), NPAS2-deficient (Npas2−/−) and double-mutant (Clock−/−;Npas2−/−) mice carrying the mPer2Luciferase reporter gene [5]. We monitored the bioluminescence rhythms of tissue explants in culture and found that while CLOCK or NPAS2 is able to maintain SCN bioluminescence rhythmicity (Supplemental Data) [4], peripheral oscillators are arrhythmic without CLOCK. Thus, there are fundamental differences between the clock machinery of different tissues

DeBruyne, Jason P.

Reppert, Steven M.

Weaver, David R.

Elsevier - Publisher Connector 

Current Biology Vol 17 No 14
R538Correspondences
Peripheral 
circadian 
oscillators require 
CLOCK
Jason P. DeBruyne, David R. 
Weaver, and Steven M. Reppert*
In mammals, the circadian system 
is hierarchical — a brain pacemaker 
located within the suprachiasmatic 
nucleus (SCN) is responsible 
for regulating locomotor activity 
rhythms and for synchronizing 
peripheral oscillators [1,2]. Recent 
genetic evidence in mice indicates 
that the bHLH transcription factors 
CLOCK and NPAS2 have partially 
redundant functions within the SCN 
[3,4]. To further examine the roles of 
CLOCK and NPAS2, we generated 
CLOCK-deficient (Clock–/–), 
NPAS2-deficient (Npas2–/–) and 
double-mutant (Clock–/–;Npas2–/–) 
mice carrying the mPer2Luciferase 
reporter gene [5]. We monitored the 
bioluminescence rhythms of tissue 
explants in culture and found that 
while CLOCK or NPAS2 is able to 
maintain SCN bioluminescence 
rhythmicity (Supplemental Data) [4], 
peripheral oscillators are arrhythmic 
without CLOCK. Thus, there are 
fundamental differences between 
the clock machinery of different 
tissues.
We determined CLOCK’s role in 
peripheral oscillators by focusing 
on liver and lung tissue. The 
circadian oscillator in the liver has 
been useful for understanding 
the biochemical interactions that 
comprise the mouse circadian 
clockwork, e.g., [6,7]. Moreover, 
the livers of CLOCK-deficient 
mice rhythmically express mPer1 
and mPer2 mRNAs and proteins 
during the first day in constant 
darkness [3]. This suggested that 
the circadian clock in the liver might 
also be maintained in the absence 
of CLOCK due to the partially 
redundant function of NPAS2. 
Surprisingly, however, 
bioluminescence profiles of liver  
and lung explants from Clock–/–  
mice are arrhythmic (Figure 1A), N
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Npas2
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Current Biology
Figure 1. Analysis of clock function in cultured tissue explants.
(A) Representative bioluminescence profiles of tissue explants from liver (left column) 
or lung (right column). Data were normalized to the average luciferase activity produced 
over the duration of the recording and are plotted as the difference from the centered 
24 hour moving average (Supplemental Data). The two representative profiles shown 
for each genotype are from separate experiments. Genotypes are indicated in the lower 
right of each panel. Our animal studies were approved by the Institutional Animal 
Care and Use Committee of the UMass Medical School. (B) Normalized amplitudes 
of the bioluminescence profiles from liver (left panel) or lung (right panel) explants. 
See Supplemental Data for a detailed description of amplitude determination. Geno-
types are indicated below each panel. Each bar is the mean ± sem with the following 
n’s (n(explants)/n(animals)): Liver: Clock+/+;Npas2+/+ = 19/9, Clock+/+;Npas2–/– = 10/4, 
Clock–/–;Npas2+/+ = 23/10, Clock–/–;Npas2–/– = 8/3. Lung: Clock+/+;Npas2+/+ = 18/9, 
Clock+/+;Npas2–/– = 7/4, Clock–/–;Npas2+/+ = 21/10, Clock–/–;Npas2–/– = 6/3. Asterisks in-
dicate significant differences from wild type (ANOVA p < 0.001, Tukey’s HSD post hoc). 
Note, amplitude measurements were not significantly different between Clock–/–;Npas2+/+ 
and Clock–/–;Npas2–/– using the same test (p > 0.98).  
Magazine
R539despite a significant elevation in 
Npas2 expression in the livers 
of Clock–/– mice [3]. Comparison 
of rhythm amplitudes confirmed 
that bioluminescence profiles of 
Clock–/– liver and lung explants 
are indistinguishable from those of 
arrhythmic Clock–/–;Npas2–/– mice 
(Figure 1B). Arrhythmicity was 
not due to low luciferase activity; 
explants from either Clock–/– or 
Clock–/–;Npas2–/– mice produced 
levels of bioluminescence that were 
comparable to  those of explants 
from wild-type mice (Supplemental 
Data). 
The arrhythmic phenotype 
of CLOCK–deficient peripheral 
oscillators could result from 
rapid desynchronization of a 
population of cells each containing 
a functional intracellular oscillator. 
To examine this possibility, we 
exposed explants from Clock– /– 
mice to a media change, a stimulus 
known to resynchronize individual 
oscillators and thereby restore 
population rhythm amplitude [5,8]. 
A media change on the 9th day  
in vitro was sufficient to produce 
an acute increase in luciferase 
activity in all explant cultures, 
indicating that the explants were 
healthy and responsive (Figure 2).  
Media change reinstated 
rhythmicity in liver and lung 
explants from wild-type and 
Npas2–/– animals (Figure 2). In 
Clock–/– and Clock–/–;Npas2– /– 
explants, however, a media 
change produced only a single 
peak within about 24 hours and 
bioluminescence levels returned to 
approximately the mean level by 
the second day (Figure 2). Notably, 
similar bioluminescence patterns 
after initial placement in culture 
or after media change were also 
observed with SCN explants from 
arrhythmic Clock–/–;Npas2–/– mice 
(Supplemental Data and data 
not shown). Thus, the mPer1 
and mPer2 mRNA and protein 
rhythms previously detected in 
CLOCK– deficient livers in vivo [3] 
are most likely being driven by 
systemic cues secondary to the 
SCN-based rhythmicity in these 
animals, as recently described [7]. 
We conclude that —unlike the 
circadian pacemaker in the SCN— 
peripheral oscillators in liver and 
lung are dependent on CLOCK. 
NPAS2 maintains rhythmic SCN N
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Current Biology
Figure 2. A media change does not restore rhythmicity to CLOCK–deficient peripheral 
oscillators. 
Each panel contains bioluminescence profiles from lung explants, plotted as described 
for Figure 1. Genotypes are indicated in the lower right of each panel. The yellow ar-
rowhead indicates the time of the media change.function in the absence of CLOCK, 
but NPAS2 alone is unable to 
maintain rhythmicity in peripheral 
tissues. This could be due to 
fundamental differences in the 
clock mechanism between tissues 
or due to differences at the level 
of intercellular interactions. There 
may be a small subpopulation 
of cell-autonomous, NPAS2-
dependent oscillators in the SCN 
of Clock–/– mice, which drives 
rhythmicity in the SCN as a whole 
due to networking among SCN 
neurons [9,10]. Peripheral tissues 
probably lack robust within-tissue 
networking, and thus, may lack the 
ability to disseminate a rhythmic 
signal from a small subset of cells 
throughout the entire tissue.
Supplemental data
Supplemental data including  
experimental procedures are available 
at http://www.current-biology.com/cgi/
content/full/17/14/R538/DC1
References
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Coordination of circadian timing in 
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Dept. of Neurobiology, University 
of Massachusetts Medical School, 
364 Plantation St., Worcester, 
Massachusetts 01605 USA. 
*E-mail: steven.reppert@umassmed.edu


Peripheral circadian oscillators require CLOCK 

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Peripheral circadian oscillators require CLOCK

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Peripheral circadian oscillators require CLOCK

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