Diabetes, Obesity and Metabolism 17 (Suppl. 1): 47–54, 2015.
© 2015 John Wiley & Sons Ltd

Seeing the forest and trees: whole-body and whole-brain imaging
for circadian biology
K. L. Ode1,2 & H. R. Ueda1,2
1 Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
2 Quantitative Biology Center, RIKEN, Osaka, Japan

Recent advances in methods for making mammalian organs translucent have made possible whole-body fluorescent imaging with single-cell resolution.
Because organ-clearing methods can be used to image the heterogeneous nature of cell populations, they are powerful tools to investigate the hierarchicalorganization of the cellular circadian clock, and how the clock synchronizes a variety of physiological activities. In particular, methods compatible withgenetically encoded fluorescent reporters have the potential to detect circadian activity in different brain regions and the circadian-phase distribution
across the whole body. In this review, we summarize the current methods and strategy for making organs translucent (removal of lipids, decolourizationof haemoglobin and adjusting the refractive index of the specimen). We then discuss possible applications to circadian biology. For example, the couplingof circadian rhythms among different brain regions, brain activity in sleep–wake cycles and the role of migrating cells such as immune cells and cancer
cells in chronopharmacology.
Keywords: light-sheet microscope, single-cell resolution, tissue clearing
Date submitted 10 April 2015; date of final acceptance 26 May 2015

The Circadian Clock as a Whole-body System
Many physiological processes, including the sleep–wake cycle,
hormone secretion and body temperature, change according toa day–night 24 h cycle [1]. Most of these processes are under
the control of a system called the circadian clock. The circadianclock works as an autonomous oscillator: the physiologicalactivities of organisms with a clock can cycle every 24 h without
the need for external stimuli for timing.
A series of seminal experiments established that the centerof the mammalian circadian clock lies in the suprachiasmatic
nucleus (SCN) located in the hypothalamus of the brain. The
initial demonstrations of the role of the SCN were done bya brain-lesion experiment where ablation of the brain area
including SCN disrupted the circadian cycle of behaviouralactivityand hormonal secretion in the absence of environmental light information (constant darkness condition) [2,3]. The
critical role of the SCN for autonomous circadian behaviour
was established by a SCN transplantation carried out by
Ralph et al. [4]. They used mutant hamsters with a shortcircadian period in behavioural rhythmicity. When SCN
from the mutant hamster was transplanted into the brain of
a wild-type hamster, the transplanted hamster had a shorter
period in behavioural rhythms. Similarly, a mutant hamstertransplanted with a wild-type SCN had normal circadianrhythms. These results clearly indicated that behaviouralrhythmicity is established by the circadian period encodedwithin the SCN.
Correspondence to: Hiroki R. Ueda, Department of Systems Pharmacology, Graduate School of
Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-0033, Japan.
E-mail: uedah-tky@umin.ac.jp

Because the SCN is a neuronal assembly, its activity can bemonitored using electrophysiological recording. Intriguingly,
explanted SCN cultured in vitro has rhythmic activity changes
over a 24-h period [5,6]. The rhythmicity is preserved even inneurons dissociated from the SCN [7]. Thus, each SCN neuron
works as an autonomous oscillator. Central components ofthis oscillator were revealed by genetic screening of mutant
organisms; the period (per) gene was originally identified in
fruit flies [8,9] and the clock gene was firstly identified in mice[10]. Currently ∼20 genes are ‘clock genes’, which comprise themammalian circadian oscillator [11]. Most clock genes encode
transcriptional regulators that activate or repress the expression of other genes. The resultant transcriptional network isinherently complex, but its core architecture can be understoodas a simple negative feedback loop (Figure 1). Clock and its
binding partner Bmal1 bind to a promoter sequence called an
E-box. The E-box regulates thousands of downstream genes
including the clock genes per and cry. The expressed PER and
CRY proteins form a heterodimer that binds to and inhibits the
BMAL1–CLOCK complex. The total action therefore forms anegative feedback, where elevated CRY and PER inhibit theirown transcription. The negative feedback loop can exhibit
oscillatory dynamics, and in the mammalian circadian clock,
this feedback loop of E-box regulation is thought to be the coreof the cellular circadian oscillator.
With this feedback loop, the expression of several clock genes
has robust 24 h rhythmicity. These genes are useful reporters
to analyze circadian rhythmicity even in non-neuronal cells,
because cell-autonomous circadian oscillation occurs in almost
all cell types in mammals including lung and liver [12], andin cultured cell lines such Rat-1 [13], NIH3T3 [14] and U2OS
cells [15]. The finding of circadian oscillation in non-SCN

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Transcription repression

Synchronization cues

PER
CRY

SCN
BMAL1

CLOCK
Cry, Per
E-box

Peripheral clocks

Figure 1. Regulation of E-box promoter activity by BMAL1-CLOCK and PER-CRY. Almost all cells in mammals have a cell-autonomous circadian clock.
The core of the clock is thought to be a transcriptional/translational negative feedback loop, where the PER-CRY complex represses its own transcription.
The clocks in an entire body are orchestrated by synchronization cues from the suprachiasmatic nucleus (SCN).

cells, namely ‘peripheral clocks’, leads to a hierarchical viewof the circadian clock system; the SCN works as a masterclock that generates a signal to synchronize peripheral clocks.
This system is like adjusting a computer’s current time – atime server delivers time information to all computers on thesame network and each computer adjusts the time of its own
clock accordingly. One fundamental difference between theclock in our computers and the circadian clock is the phase
distribution; the phase of the circadian clock is not identicalin all peripheral clocks [12,16] – each organ may be delayed
relative to the central circadian clock. In other words, a bodyis an assembly of heterogeneous cells with different circadianphases.
Assuming the heterogeneous yet synchronized assembly ofcellular circadian clocks in the whole body, the next challenge
is to understand how the SCN synchronizes peripheral clocks
throughout the body. How does the signal from SCN propagate
and synchronize other peripheral clocks? What is the role ofheterogeneity in peripheral clocks? To answer these questions,the first step is to observe the circadian clock in the entire body.
The hierarchical organization of circadian system from cells toorgans requires a technology to observe centimetre-scale 3D
samples with cellular resolution. Recent advances in microscopic imaging and sample preparation techniques are opening
up the possibility of whole-body imaging at single-cell resolution. In this review, we will revisit a few different state-of-the-art
imaging methods and discuss what questions can be asked byusing such whole-body imaging techniques.

Technology for Imaging Circadian Activities
Throughout the Body
Method for Clearing Brain Tissue
The brain is the most complex organ in our body. The complexity comes from ‘heterogeneity’ in the brain – the nature of thebrain as an information processing organ lies in the network

48 Ode and Ueda

architecture of wired neurons and glia cells. A homogeneous
brain lysate loses this essential property.

“

The combination of tissue-clearing methods andlight-sheet microscopy allows for whole-organ
imaging with single-cell resolution.

”

Optical microscopes use light. As long as we use light toobserve the specimen, the transparency of the object greatly
limits the depth of observation. This is one reason why transparent biological samples such as sea urchin embryos havecontributed to our understanding of developmental processes.
Adult mammals in general, however, are not transparent. There
are at least two reasons for the opacity of biological samples.
One reason is the presence of scattering media in organs. Suchmaterials, typically lipid layers and red blood cells, scatter lightand hide the image behind opaque materials. The second reason
is different refractive indexes within the sample and between
the sample and its environment (e.g. air or immersion oil filing the space between the sample and the objective lens). To
overcome these limiting factors for the transmittance of light,
several techniques have been developed to make fixed organs
from mammals translucent.
Table 1 categorizes the tissue-clearing reagents/methods inthree classes, each of which has advantages and limitations. The
first category is methods that use organic solvent. The clearing
chemical cocktail composed by benzyl alcohol and benzyl
benzoate (BABB) [17], or the combination of tetrahydrofuran(THF) and dibenzyl ether (DBE) [18,19] are used in thesemethods. These chemicals effectively remove lipids and modify
the refractive index, therefore that the whole brain becomestranslucent within a few days. Because organic chemicals
denaturize protein, reporters based on fluorescent proteins are,in general, incompatible with BABB. THF/DBE protocols are
somewhat compatible with fluorescent proteins but fluorescent

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Table 1. Summary of major tissue-clearing reagents/protocols.
Category

Reagent/protocol nameKey chemical components or procedures

ReferenceI

BABB
THF-DBE
3DISCOiDISCO
Scale
SeeDB
ClearT
CUBIC
PACT-RIMS-PARS
TDE
CLARITY/Advanced CLARITY

Benzyl alcohol and benzyl benzoate
Tetrahydrofuran and dibenzyl ether
Tetrahydrofuran and dibenzyl ether
Tetrahydrofuran and dibenzyl ether
Urea
Fructose
Formamide, polyethylene glycol
Aminoalcohol
Hydrogel-embedded samples and perfusion-mediated reagent circulation
2,2′ -thiodiethanol
Hydrogel-embedded samples and electrophoretic lipid removal

[17]
[18]
[19]
[20]
[21]
[22]
[24]
[23,29][28][25][26,27]IIIII

BABB, benzyl alcohol and benzyl benzoate; CUBIC, clear, unobstructed brain imaging cocktails and computational analysis; DBE, dibenzyl ether; TDE,
2,2′ -thiodiethanol; THF, tetrahydrofuran.

photoquenching by organic solvent still occurs. Nonetheless,the immunostaining protocol of iDISCO allows the use of
non-protein reporters such as Alexa Fluor dyes and q-dot tolabel specific proteins [20].
By contrast, methods in the second category use
water-soluble chemicals, so that genetically encoded reporters
are more functional. A major component in the Scale
method – urea – is mostly responsible for lipid removal fromthe brain [21], whereas the fructose used in the SeeDB method
aims to adjust the refractive index within the brain [22]. The
clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) method adopts both strategies with aslightly different set of chemicals [23]: a mixture of urea andaminoalcohol for the removal of lipids and sucrose to adjust therefractive index. ClearT applies polyethylene glycol to stabilize
proteins [24] and 2,2′ -thiodiethanol (TDE) solutions for brain
clearing [25]. The use of water-soluble chemicals is also advantageous for easy handling and safety for the user. The third
category also uses water-soluble chemicals but the sample isembedded in an acrylamide gel [26,27]. The molecular mesh ofacrylamide is thought to maintain the detailed structure of theneuronal network in the brain and enables an electrophoresis
force to facilitate the removal of lipids.

“

The clearing method with water-soluble
chemicals is compatible with genetically encoded
reporters for neuronal activities, which depict
integrated neuronal activities over the time range
from minutes to hours.

”

Method for Clearing the Whole Organism
While the primarily application of these clearing methods isthe brain, it should be possible to apply these techniques toother organs. Indeed, methods in each category were extended
to other organs and even whole animals [28,29]. In the case of
several non-brain organs (e.g. liver), however, red blood cells
cannot be completely removed by the perfusion of buffers in
the fixation process. The leftover colouring limits transparencyof the organs. Amino-alcohols used in the CUBIC method canVolume 17 Suppl. 1 September 2015

overcome this problem because the chemical effectively elutes
heme from haemoglobin and clears the colour of blood [29].
The CUBIC method can clear the whole body of adult micewith a simple protocol; soaking the whole body in clearing
mixtures for several weeks. The cleared whole mice sample issufficiently translucent for fluorescent light-sheet microscopy(see next section) with resolution to distinguish nuclei ofindividual cells.

Light-sheet Microscopy for Efficient Image Acquisitionof Large Samples
In order to obtain 3D images of the cleared organs, opticalsectioning with high-resolution on the z-axis is also required.
Conventional confocal and two-photon microscopes can focus
excitation light at a specific voxel within the 3D sample. To
reconstruct the whole 3D image, the voxel is scanned along
every x-, y- and z-axis, limiting the throughput of image
acquisition. By contrast, light-sheet microscopy generates athin laser plane to excite an optical section within a specimen[30]. The image is then captured by a wide-field camera placedat the orthogonal direction of illumination. This optical setting
makes it possible to acquire a whole image section in a single
acquisition. Scanning is required only in the z-axis direction.
This optical configuration, however, requires the specimen tobe highly translucent, otherwise the sheet laser cannot illuminate the center of optical section and the fluorescence fromthe excited surface cannot reach the detector. Highly clearedorgans are one of the best applications for this state-of-the-art
microscopy; and several clearing methods outlined above
can be used with light-sheet microscopy. Figures 2 and 3 areexamples of whole-brain and whole-mice imaging obtained by
the combination of clearing treatment (CUBIC) and light-sheet
microscopy. The brain image has enough resolution to distinguish axon projections and the heart image reveals the detailed
mesh structure of blood vessels.

Image Processing and its Computational Analysis
Comparison of different samples is an issue for data processing,
especially when the images are complex three-dimensional
ones. For this purpose, brain imaging using magnetic resonancedoi:10.1111/dom.12511 49

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A

DIABETES, OBESITY AND METABOLISM

A

B

500 μm

C

2 mm

B

5 mm

500 μmFigure 3. Whole-body imaging with the CUBIC method. (A)
3D-reconstituted whole-body image of P1 mouse acquired with alight-sheet fluorescent microscope. The mouse expressed EGFP (shown ingreen) under a CAG promoter and was stained with propidium iodide (PI)(shown in purple). (B and C) Coronal plane images of the mouse lung (B)and intestine (C). See reference [29] for the detailed process.

Whole Brain Imaging of Neuronal Network
and its Activity
Tracing Neuronal Connections

500 μmFigure 2. Whole-brain imaging with the CUBIC method. (A)
3D-reconstituted whole-brain image of an adult mouse acquiredwith a light-sheet fluorescent microscope. The mouse expressed YFP(shown in yellow) under a Thy1 promoter and was stained with propidium
iodide (PI) (shown in green). (B) Magnified view of the white-box regionin A. See reference [23] for the detailed process.

imaging (MRI) or magnetoencephalography (MEG) use thestandard brain atlas. By fitting obtained images to the standard
brain, we can compare and annotate each image based on
the same format. A similar approach was used in the CUBIC
protocol [23]. Each brain image is transformed to fit the sharedstandard brain by a method based on affine transformation.
This fitting works well in the case of the brain, an organ withrelatively simple structure, but it will be highly difficult to
fit the other organs, especially whole body images because
the position of the arms and legs are variable for individual
specimens and digestive organs (e.g. a stomach or intestines)change shape upon food intake. An algorithm to standardize
such complex 3D images will be required for future image
processing.

50 Ode and Ueda

In the following section, we will discuss the potential application of whole-brain and whole-organism imaging to circadian
biology. A typical application of whole-brain imaging may beto illustrate the network architecture of neuronal connections
between SCN and other brain areas. Cleared brains withwater-soluble chemicals keep the detailed structure of axon
projections and synapses, and are compatible with geneticallycoded fluorescent reporters. Thus, it is possible to track neuronal connections. For example, tracing of axon projections
from the SCN will be possible by inducing fluorescent proteins
under the control of SCN-specific promoters (see below) or
by introducing viral neuronal tracers to identify functional
synaptic connections.
Although, it is well-known that the SCN regulates various
physiological activities, which are also regulated by different
areas in the brain, the molecular and cellular mechanisms ofthe co-ordination among the SCN and other brain regions
is not completely understood. A pioneering experiment of
SCN transplantation demonstrates indirect (not throughsynaptic connection) regulation of behavioural rhythmicity by
the SCN; transplanted SCN encapsulated in semi-permeable
material sets the behavioural rhythm in host hamsters [31].
It is also known that SCN neurons directly project to several
other regions, primarily the hypothalamus [32]. Retrograde
tracing revealed direct or multi-synaptic projections fromseveral brain areas to the SCN. Through this network, theSCN delivers circadian signals and SCN afferent pathways may
contribute to reset the circadian clock in the SCN. Because
the current afferent/efferent projection models of the SCN

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neuronal network are largely based on the ensemble of manystudies with different species and techniques, and there areseveral inconsistent observations between studies, it should be
worthwhile to carry out a systematic investigation of projection
patterns to and from the SCN on a whole-brain scale.

Tracing the Circadian-phase Variation in the Brain
The direct/indirect signals from SCN will also be importantto reset and synchronize the peripheral clocks in non-SCN
neurons. Direct projection may be important for regulating
specific brain areas, while indirect spreading of neurotransmitters will affect a broad area next to the SCN. Heterogeneous
distribution of circadian phase is also true in the brain. Interestingly, the phase of circadian clock may vary depending on
the brain area [33], and food restriction may selectively affectthe clock phase of the cerebral cortex and hippocampus [34].
The role of clocks in the brain outside the SCN may be more
actively important for the development of neuronal network,
not just for keeping the robust circadian rhythms. A recent
study revealed that selective deletion of Clock or Bmal1 in
the parvalbumin-expressing inhibitory neuron, which is not
involved in the SCN, delays the critical period of visual cortex
plasticity upon monocular deprivation [35].
A detailed illustration of circadian phase in a cleared brain
would be done by using fluorescent-protein-based circadianreporters. Although phase determination by real-time monitoring of circadian reporters (e.g. Per2-luciferase monitoring)is impossible for a fixed brain, relative values of two or threecircadian-expressing reporters at a few time points can determine the phase of oscillation [36–38]. With such a strategy,
the heterogeneous phase distribution of circadian phase in thebrain (or ultimately in the entire body) may be quantified.
In order to trace circadian synchronization originating fromthe SCN, it is important to perturb the circadian phase specifically at the SCN, and trace how the altered phase at the SCN
affects the phase of other brain regions. The phase adjustmentof the SCN may be accelerated by knockdown of the arginine
vasopressin (AVP) receptor or elevated by vasoactive intestinal peptide (VIP) action [39,40]. These conditions disrupt theneuronal coupling within the SCN and make the circadianphase more flexible to the environmental light phase (i.e. reducing the ‘jet lag’ effect). In these cases, non-SCN brain regions
will experience unusually drastic phase shifts. Do other brain
regions faithfully trace the altered time? Alternatively, possible circadian coupling between brain regions may resist drastic phase shifts. Neuromedin S promoter is another promising
tool to observe the response of other brain regions to the selective inhibition of circadian output from the SCN. Recently, Leeet al. succeeded in disrupting circadian rhythmicity by inducing abnormal expression of clock genes at the subpopulation of
SCN neurons by using promoter of neuromedin S [41], a neuropeptide that selectively expresses in the SCN [42]. Those studies have mainly focused on the circadian time-keeping mechanism within the SCN, but with whole-brain imaging, the observation can be extended to the response to and coupling of circadian signals within the whole brain, and ultimately within thewhole body.

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Illustration of the Neuronal Activity in the Whole Brain
The firing activity of a neuron is the physical nature of information processing in the brain. Current brain-clearing protocols require fixation of samples, which make it impossible toobtain the millisecond dynamics of electrophysiological neuronal actions. Fortunately, by using makers for the expressionof immediate-early genes (IEGs), such as c-fos and Arc/Arg3.1
the imaging of a fixed whole brain can be an excellent method
to investigate the integrated change in neuronal activity over aminutes-to-hours scale [43]. This time scale also matches withthe time resolution necessary for tracking circadian dynamics.
Genetically encoded fluorescent protein under the control ofthe promoter of IEGs has been a reliable tool to monitor neuronal activity, and such tools work well with the water-soluble
brain clearing methods. Thus whole-brain imaging of IEG
activity provides complementary information that one may not
expect from in vivo electrophysiological recording or real-time
imaging of voltage/ion sensitive reporters, which are generally
limited to a local area of the brain.

Tracking Sleep–Wake Activity Throughout the Brain
Sleep–wake cycle is one of the most significant phenomena
regulated by the circadian system. Nevertheless, the brain center responsible for controlling sleep is still under discussion.
Sleep–wake cycle is believed to contain homeostatic regulation, namely sleep homeostasis independent of the circadian regulation. With this homeostasis, an animal’s sleep-time
to wake-time ratio is kept constant so that an animal that
could not sleep enough would take extended sleep time in thenext sleep phase (recovery sleep). Given this hypothesis, brain
regions activated under the condition of sleep loss would beregions responsible for sleep homeostasis, and thereby regulate the normal sleep-to-wake activity ratio. The ventrolateralpreoptic nucleus (VLPO) is one of the best understood brain
regions matching these criteria. Recent findings suggest differences between natural sleep cycles and sleepiness elicited by
the forced loss of sleep time. Inhibition of gliotransmission by
the induction of a dominant-negative form of SNARE protein(dnSNARE) in glia cells caused a defect in sleep homeostasis.
Mice expressing dnSNARE had normal sleep time/quality evenafter significant loss of sleep [44]. The mice were also insensitive
to the induction of increased sleep pressure by lipopolysaccharide (LPS) injection [45]. Strikingly, the mice exhibit almosta normal time-course of sleep–wake cycle in the absence ofsleep deprivation or LPS injection. These results suggest decoupling between the mechanisms responsible to generate a normal sleep–wake pattern and those responsible for homeostatic
recovery sleep. A study focusing on the effect of dexmedetomidine, an 𝛼2 adrenergic agonist clinically used for sedatives,
may be consistent with this decoupling [46]. Dexmedetomidine
induced elevated activity of VLPO and several other regions
judged by c-fos promoter activation. A similar activation pattern was obtained by forced sleep deprivation, but surprisingly,
natural sleep did not induce the activation of VLPO and theother regions, at least not to the same extent of dexmedetomidine application or sleep deprivation. Of course, the uncoupling
may be due to a quantitative difference: natural sleep may only

doi:10.1111/dom.12511 51

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require weak gliotransmission or activation of VLPO. Still it
will be important to re-evaluate the role of VLPO and otherbrain regions on the normal sleep–wake cycle. A whole-brain
activity atlas during the sleep–wake cycle will be a good reference for that evaluation. For example, when we carried out
time-course c-fos imaging of the whole brain, there are several
brain regions that show elevated activity at specific times ofthe day. Although more detailed analysis is required to investigate neuronal control of sleep, imaging allows us to quantitatively compare the cumulative activity of neurons across different brain areas. We also would like to point out the potential
importance for having the brain atlas of sleep state in the view ofpharmacological treatment of mental disorders. Because sleep
disorders are associated with many mental disorders and viceversa, medicines targeting the central nerve system often affectsleep–wake dynamics. Thus, a detailed sleep–wake brain atlas
will contribute to understanding potential effects/side-effects ofmedicines by comparing activity maps between brains under
different medicines.

Tracking Non-circadian Autonomous Oscillators
Time-course comparison of brain activity also has the potential to identify brain centers for non-circadian oscillators.
A methamphetamine-induced oscillator (MPO) was proposed
based on the observation that chronic intake of methamphetamine alters the period of behavioural rhythms [47]. The
elicited rhythmicity is SCN independent – methamphetamine
induces behavioural oscillations even in circadian-deficientanimals [48]. Similarly, the restricted-feeding induced oscillator (FEO) was also shown to operate independently from theSCN [49,50]. If there is a brain center responsible for these
non-circadian oscillators, the center should show cyclic activity
changes with a period matched to the non-circadian oscillation.

Uncover the Role of Peripheral Clocks in the
Whole Body
Heterogeneous Phase Distribution in Body Clocks
As discussed in the previous sections, whole-body imaging withsingle-cell resolution would allow us to illustrate the heterogeneous distribution of circadian phases. Synchrony of the circadian clock in the entire body is often schematically described,
but the genuine distribution of circadian phase is unclear. The
phase distribution and its transition upon various environmental signals will elucidate how the signal from SCN propagates toperipheral clocks and vice versa.

“

Whole-organ imaging will be an ideal
technology to investigate the heterogeneous natureof cell assembly, such as circadian-phase variance of
peripheral clocks and their synchronization
process.

”

Circadian phase and period of peripheral clocks are determined by the combination of cell-intrinsic peripheral clocks

52 Ode and Ueda

DIABETES, OBESITY AND METABOLISM

and synchronization cues from the SCN. Pando et al. succeededin showing this relationship by an elegant clock-defective
mouse embryonic fibroblast (MEF) transplantation experiment [51]. When Per1−/− MEF with a short circadian period
is implanted into wild-type mice, the period of mutant MEF isreturns to a wild-type period. This result indicates the SCN’s
dominance over peripheral clocks. At the same time, theperipheral clock is not just a slave to the central clock – theintrinsic clock mechanism plays a significant role. The synchronization signal from the SCN is not sufficient to induce denovo circadian oscillation in non-oscillatory Clockc/c mutant
MEFs. The role of peripheral clocks were also demonstrated
by brain-specific rescue of clock function that can rescue thebehavioural rhythmicity but cannot rescue organ-specific gene
expression [52].

Potential Application to Chronopharmacology
and Chronotherapeutics
Given that peripheral circadian clocks are driven by the combination of an intrinsic molecular oscillator and environmental synchronization signals, what if a cell migrates to different
organs with different environmental conditions? Because thesemobile cells are a minority in the destination organ, a conventional bioluminescent assay that monitors average circadian
activity among cell populations is not suitable to analyze therhythms in these minority populations.
Whole-organ imaging is particularly important for focusing on smaller numbers of certain cells – one may easily imagine its potential application to cancer biology, where a small
number of transformed cells are critical for tumour invasion.
Numerous experiments have shown that circadian timing is a
critical factor for the effectiveness of anti-cancer drugs [53].
Most anti-cancer drugs (e.g. chemicals that induce DNA damage or inhibit cell division) target transformed cells using the
fast-replicating feature of the cell cycle in transformed cells.
Because the circadian clock regulates cell cycle progression andthe efficacy of DNA repair mechanisms, the clock affects the tolerance of transformed cells to anti-cancer drugs. The circadianclock also largely affects the pharmacokinetics of drugs, as clock
timing affects the activity of metabolic pathways to uptake,deliver and metabolize drugs. Therefore, a precise chronotherapeutic model requires understanding of the circadian phase in
the whole body at single-cell resolution.
The circadian clock also controls immune responses, where
the migration of cells is essential to defend the organism
from the threat of infection. It is well known that the level ofhormones, cytokines and the number of circulating immune
cells shows global circadian variation in the body [54]. Notably,
several studies have shown that the peripheral circadianclock in local cells is also critical for gating the induction of
inflammatory responses. Keller et al. showed that circadianclock acting in macrophage fulfils the circadian cytokine
response [55], and Gibbs et al. showed that the intrinsic clockin epithelial bronchiolar cells is responsible for rhythmic
secretion of chemokine, thereby controlling the circadiananti-inflammatory response in the lung [56]. Given the emerging role of circadian clocks in immune responses, the interplay
between clocks in migrating cells and clocks in local tissue

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cells will be of great interest. Such circadian interplay might
be involved in local, tissue-specific crosstalk among the local
environment and tissue-resident macrophage and memory T
cells [57,58]. Whole-organ imaging has the potential to survey
these heterogeneous minor populations.

review article
8. Reddy P, Zehring WA, Wheeler DA et al. Molecular analysis of the period locus in
Drosophila melanogaster and identification of a transcript involved in biologicalrhythms. Cell 1984; 38: 701–710.
9. Konopka RJ, Benzer S. Clock mutants of Drosophila melanogaster. Proc Natl
Acad Sci U S A 1971; 68: 2112–2116.
10. Vitaterna MH, King DP, Chang AM et al. Mutagenesis and mapping of a mouse
gene, Clock, essential for circadian behavior. Science 1994; 264: 719–725.

Conflict of interest
The authors declare that there is no conflict of interest regarding
this manuscript.

Acknowledgements
We thank all members in the department of systems pharmacology at UTokyo and cell design core in the Quantitative Biology Center, RIKEN for their continual discussion, especially Dr
Arthur Millius for critical reading and proof of this manuscript,
and Drs Kazuki Tainaka and Etsuo A. Susaki, and Mr. Shimpei
I. Kubota for helping us to make figures. Some of the resultspresented in this manuscript were obtained with the support of
the following grants: Program for Innovative Cell Biology by
Innovative Technology and the Brain Mapping by Integrated
Neurotechnologies for Disease Studies (Brain/MINDS) from
the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, a Grant-in-Aid for Scientific Research(S) (grant no. 25221004), a Grant-in-Aid for Scientific Researchon Innovative Areas (grant no. 23115006), and a Grant-in-Aid
for JSPS Fellows (grant no. 26-1180) from MEXT/Japan Society for the Promotion of Science (JSPS), the Strategic Programs for R&D (President’s discretionary fund) of RIKEN, an
intramural Grant-in-Aid from the RIKEN Center for Developmental Biology and RIKEN Quantitative Biology Center, agrant from Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), a
Grant-in-Aid from Japan Foundation for Applied Enzymology,
a Grant-in-Aid from the Shimabara Science Promotion Foundation, and by the RIKEN Foreign Postdoctoral Researcher
Program.

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