Genetic and Molecular Analysis of Wild-Derived
Arrhythmic Mice
Tsuyoshi Watanabe1, Tohru Suzuki1,2, Akira Ishikawa3, Yuki Yokota1, Hiroki R. Ueda4,5, Rikuhiro G.
Yamada4, Hajime Tei6, Saki Imai1, Shigeru Tomida1, Junya Kobayashi1, Emiko Naito1, Shinobu Yasuo1,
Nobuhiro Nakao1, Takao Namikawa3, Takashi Yoshimura1, Shizufumi Ebihara1*
1 Division of Biomodeling, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan, 2 Department of Infectious Disease, Hamamatsu University
School of Medicine, Hamamatsu, Japan, 3 Division of Applied Genetics and Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan,
4 Laboratory for Systems Biology, Center for Developmental Biology, RIKEN, Hyogo, Japan, 5 Functional Genomics Subunit, Center for Developmental Biology, RIKEN,
Hyogo, Japan, 6 Research Group of Chronogenomics, Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan

Abstract
A new circadian variant was isolated by screening the intercross offspring of wild-caught mice (Mus musculus castaneus).
This variant was characterized by an initial maintenance of damped oscillations and subsequent loss of rhythmicity after
being transferred from light-dark (LD) cycles to constant darkness (DD). To map the genes responsible for the persistence ofrhythmicity (circadian ratio) and the length of free-running period (t), quantitative trait locus (QTL) analysis was performed
using F2 mice obtained from an F1 cross between the circadian variant and C57BL/6J mice. As a result, a significant QTL witha main effect for circadian ratio (Arrhythmicity; Arrh-1) was mapped on Chromosome (Chr) 8. For t, four significant QTLs,
Short free-running period (Sfp-1) (Chr 1), Sfp-2 (Chr 6), Sfp-3 (Chr 8), Sfp-4 (Chr 11) were determined. An epistatic interaction
was detected between Chr 3 (Arrh-2) and Chr 5 (Arrh-3). An in situ hybridization study of clock genes and mouse
Period1::luciferase (mPer1::luc) real-time monitoring analysis in the suprachiasmatic nucleus (SCN) suggested thatarrhythmicity in this variant might not be attributed to core circadian mechanisms in the SCN neurons. Our strategy
using wild-derived variant mice may provide a novel opportunity to evaluate circadian and its related disorders in humanthat arise from the interaction between multiple variant genes.
Citation: Watanabe T, Suzuki T, Ishikawa A, Yokota Y, Ueda HR, et al. (2009) Genetic and Molecular Analysis of Wild-Derived Arrhythmic Mice. PLoS ONE 4(1):e4301. doi:10.1371/journal.pone.0004301
Editor: Aimee K. Zaas, Duke University, United States of America
Received September 26, 2008; Accepted December 3, 2008; Published January 28, 2009
Copyright: ß 2009 Watanabe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported in part by Grant-in-Aid for Scientific Research to S.E.. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ebihara@agr.nagoya-u.ac.jp.

of these disorders. To study these issues, appropriate model micethat show aberrant circadian rhythms are substantially useful. So
far, several circadian mutant mice, most of which were created bymutagenesis or gene targeting, have been used for the model
system in human circadian and its related disorders [11].
However, it appears that most of human diseases result fromdeleterious combinations of polymorphic alleles with small effects
on the phenotypes and the diseases elicited by single gene defects
are fairly rare.
To establish model systems useful for circadian and its relateddisorders in human, we attempted to obtain variants from wildcaught mice (Mus musculus castaneus) because natural populations oforganism conserve genetic diversity (the subset of polymorphic loci)that give rise to phenotypic variation (i.e., many naturally occurring
variant genes, including harmful and beneficial genes, are conservedin wild populations). In Drosophila, for example, numerous variants
have been isolated from wild populations [12]. Because commonlyused laboratory mice are known to have originated from a limited
founder population in a few laboratories [13,14], new genetic variants
affecting circadian rhythms, which we cannot find in commonly usedlaboratory mice, might be obtained from wild mouse population.
In this attempt, we successfully isolated mice in which
behavioral circadian rhythms were abolished under conditions ofIntroduction
Circadian rhythms are fundamental properties of organisms
living on earth and numerous physiological and behavioral
functions are under the control of the circadian clock. In
mammals, the circadian pacemaker resides in the suprachiasmatic
nucleus (SCN), in which the self-sustained oscillation is generated
by the interaction of a set of activated clock genes [1]. Over the
past decade, multiple clock genes have been identified, and it is
now generally accepted that the generation of circadian rhythms isbased on transcription-translation feedback loops comprising theactivation of multiple clock genes. Meanwhile, several genomewide genetic analyses have demonstrated that many genes otherthan clock genes are involved in the circadian clock function [2–7].
In addition, recent studies analyzing genome-wide gene expression
patterns have revealed that many genes in the SCN and peripheralorgans oscillate with circadian rhythmicity [8–10]. Thus, it is likelythat a variety of genes are involved in refining and amplifying thecore circadian oscillation and maintaining the normal temporalphysiology of the complex circadian system.
It is now appreciated that disturbed circadian rhythms areassociated with many mental and physiological disorders, but little
is known about how circadian rhythms are implicated in the onset
PLoS ONE | www.plosone.org

1

January 2009 | Volume 4 | Issue 1 | e4301

Wild-Derived Arrhythmic Mice

The peaks of the relative power in the circadian (18–30 h; 1/18–
1/30 cycle/h) and non-circadian (1–18 h; 1/1–1/18 cycle/h)ranges were determined for each animal; the rhythm with a periodranging from 18 to 30 h was arbitrarily assigned as the circadianrhythm (Fig. 1J, K). The rhythmicity was evaluated using thefollowing method. The ratio (%) for the relative power of thelargest circadian peak to the sum of the largest circadian and noncircadian peak values was calculated at 15-d intervals (we term thisvalue the circadian ratio). The minimum ratio was defined as anindividual phenotype. If the ratio exceeded 50%, the activity
pattern was judged as circadian, and if not as non-circadian. In
DD conditions, t, which was determined by a chi-square (x2)periodogram analysis, was calculated at 15-d intervals, and thelowest value was used for QTL analysis because t was positively
correlated with the circadian ratio (Fig. S2). After beingtransferred from the LD 4:4 to DD conditions, most of thecircadian variants (n = 89) and some of the F2 mice (n = 10)
immediately became arrhythmic; therefore, these mice were notincluded in the QTL analysis for t. Daily activity counts under DD
conditions were obtained using the Chronobiology Kit, taking adaily average for the 15-d interval in which the minimum
circadian ratio was exhibited. The difference in phase angle
between the onset of activity and the offset of the light wascalculated based on the activity profile using the Chronobiology
Kit. The time elapsed at a quarter of the maximum counts was
defined as the onset of activity.

constant darkness (DD). We designated this variant the ‘‘circadianvariant.’’ In the present study, therefore, we initially performed aquantitative trait locus (QTL) analysis to map the QTLs
responsible for the persistence of circadian rhythmicity and thefree-running period (t) in this circadian variant. We thenundertook sequencing analysis to search the candidate genes for
QTLs. Finally we investigated how the expression of clock genes
was altered in the SCN of these mice.

Materials and Methods
Animals and housing
Sixteen adult wild mice (Mus musculus castaneus) were captured atseveral locations in the vicinity of University of the Philippines, Los
Banos, in June 1994 and shipped to our laboratory. A pair of themice captured in the market (a female mouse in Bayan market anda male mouse in New market) were bred to produce theexperimental progenies. Three female mice obtained from thepaired mice were backcrossed to a wild-caught male mouse,producing a total of 24 backcross progeny. These mice werescreened for locomotor activity rhythms and we identified two
individuals (one male and one female) that exhibited abnormal
patterns of activity rhythms: a large positive phase angle under
12 h light-12 h dark (LD 12:12) cycles and spontaneous rhythm
splitting under constant darkness (DD) conditions. These mice
were mated and their offspring were tested for activity rhythms. Of
these mice, some exhibited an abnormal activity rhythm patternsimilar to that of their parents. One abnormal male wasbackcrossed to the mother, and among the progeny of this mating
we discovered one mouse that became arrhythmic under DD
conditions. This variant mouse was crossed to an abnormal
littermate exhibiting a significant positive phase angle. In thisintercross, we obtained several circadian variants that were used
for subsequent breeding to maintain this phenotype. Because
reproductive efficiency was reduced during the intercross of thewild mice, one circadian variant was crossed to a C57BL/6J
mouse (CLEA Inc., Tokyo, Japan) and, subsequently, an F1 female
offspring was backcrossed to the parental circadian variant,producing one male and one female circadian variant. These
mice were mated and consecutive sib mating was continued for 7
generations. For genetic analysis, 18 F1 and 236 F2 progeny(males, n = 125; females, n = 111) obtained from the cross between
one male circadian variant from the colony of 7 generations and
C57BL/6J mice were analyzed.
During the experiment, the mice (50–80 days old at the
beginning of the experiment) were individually housed in a cageequipped with a running wheel (diameter, 10 cm; width, 5 cm) ina light-tight box. The light intensity inside the cages was 300–
500 lux. The room temperature was maintained at 2362uC
throughout the experiment. Food and water were available adlibitum. In all the experiments, the animals were treated inaccordance with the guidelines of Nagoya University.

Marker analysis
The microsatellite (Mit) markers used in this study werepurchased from Research Genetics Inc. (Huntsville, AL). Since
the size of marker alleles in wild-derived mice was unknown,hundreds of markers, which were selected on the basis of allelicinformation on the CAST/Ei and C57BL/6J strains from the
Mouse Genome Database on all chromosomes (Chrs), were tested
using the parents and F1 mice. Finally, a total of 271 informative
markers distributed throughout all the Chrs could be reliably
distinguished among the circadian variant, C57BL/6J, and the F1
mice (Table S1). In this study, alleles from the circadian variant
and C57BL/6J mice were referred to as C and B, respectively.
The genomic constitution of the parental variant male mouse wasshown (Fig. S1). Marker linkage maps were constructed using thecomputer software Map Manager QTXb20 [16].
Genomic DNA was extracted from the tail of each mouse by thestandard phenol:chloroform method. Polymerase chain reaction(PCR) amplifications of the Mit markers were performed according
to the PCR protocol described in our previous paper [17].

QTL analysis
Initially, interval mapping using a least-squares regression
method (Map Manager QTXb20) was performed on 236 F2
hybrids using 60 Mit markers (2 to 4 markers per chromosome).
When suggestive or significant QTLs were detected, further
analysis using additional markers (total 104) flanking potential
QTLs was conducted. The likelihood of odds (LOD) scores at the
genome-wide significant (5%) and suggestive (10%) levels were
determined by 1000 runs of a permutation test [18]. The LOD
scores for the circadian ratio at the genome-wide 5% and 10%
significant levels were 3.65 and 2.09, and 3.43 and 2.04 for t,
respectively.
The epistatic interaction analysis was performed as described by
Ishikawa et al. [19]. To declare the significant epistatic QTLs, twotests were conducted. First, the significance of the overall effect wasestablished by 1000 runs of a permutation test using Map
Manager QTXb20 and then, if the overall effect exceeded the

Behavioral analysis
Wheel-running activity was continuously monitored andrecorded by a computer system (The Chronobiology Kit; Stanford
Software Systems, CA). The locomotor activity rhythms were
measured either under LD 12:12 or LD 4:4 conditions for 2 weeks
and subsequently under DD conditions for 7 weeks. To assess thepersistence of circadian rhythmicity, fast Fourier transform
analysis in the MatLab environment (ClockLab; Actimetrics,
Evanston, IL) was performed at 15-days intervals in DD. The
power spectral densities for frequencies ranging from 0 to 1 cycle/
h were determined and normalized to a total power of 1.0 [15].
PLoS ONE | www.plosone.org

2

January 2009 | Volume 4 | Issue 1 | e4301

Wild-Derived Arrhythmic Mice

Figure 1. Activity profiles of wild-derived circadian variant, C57BL/6J mice, and their F1, F2 offspring. Representative locomotor activity
rhythms (double-plotting) of the circadian variant (A and C) and C57BL/6J (B and D) mice maintained under LD 12:12 and LD 4:4, and then transferred
to DD. One F1 and three F2 mice with different activity patterns are shown in E–H. The arrhythmic mouse (I) received a 6-h light pulse (indicated by anarrow head) under DD (see circadian rhythms recovered). Light and dark cycles are represented by white and black bars above each record. The
results of fast Fourier transform analysis applied to the actograms of (F) and (G) for the final 15 days are shown in (J) and (K), respectively. Open andClosed arrows represent the maximum peaks of relative power in the circadian (18–30 h) and ultradian (1–18 h) range, respectively.
doi:10.1371/journal.pone.0004301.g001

coding regions of 69 genes located between D8Mit205 (30 cM,
52.3 Mb) and D8Mit249 (37 cM, 83.8 Mb) on Chr 8; these genes
are likely to be expressed in the SCN (H.R. Ueda et al.,unpublished observation).

genome-wide 5% significance level, the interaction was tested
using approximate genome-wide thresholds obtained as describedby Knott et al. [20].
The parental male mouse retains a heterozygous allele for loci(Fig. S1); therefore, we excluded the F2 mice produced by the B/
C6B/B backcross from the QTL analysis using Map Manager
QTXb20. Instead, we conducted QTL analysis for these loci withQTL Express. Using QTL Express, it is possible to analyze the
combined populations of backcrosses and F2 crosses [21]. At theloci where the genotype of the parental variant male were B/B(Fig. S1), these markesr were excluded from the analyses using
Map Manager QTXb20 and QTL Express.
The above QTL analysis is based on a one-QTL model. When
two LOD peaks were observed on a chromosome, a two-QTL
model was implemented using QTL Express. A statisticalcomparison between one- and two-QTL models was performed,
as described by Wei et al. [22].

In situ hybridization
Arrhythmic mice were selected prior to the sampling. The mice
maintained under LD 12:12 for 2 weeks or DD for 1–2 weeks
following transfer from LD were killed by decapitation and thebrains were immediately removed. In situ hybridization was carriedout according to Yoshimura et al. [24]. Antisense 45-meroligonucleotide probes (mClock: nucleotides 642–686 of GenBank
accession number AF000998; mBmal1: 1755–1799 of AB015203;
mPer1: 3239–3283 of AB002108; mPer2: 242–286 of AF035830;
and mPer3: 2301–2345 of AF050182) were labeled with [35S]dATP(New England Nuclear, Boston, MA) using terminal deoxyribonucleotidyl transferase (GIBCO-BRL). In the light-pulse experiment, mice were exposed to a 15-min light pulse (100 lux offluorescent lamps) at circadian time (CT) 16 on the first day after
transfer from LD 12:12 to DD (16 h after transfer to DD; CT0 is
defined as the time when lights would have come on under LD
12:12). Samples were collected 90 min after the light pulse.

Sequencing analysis of the candidate genes
Total RNA was extracted from the whole brains of circadianvariants, C57BL/6J and CASP mice — the last of which had been
established as an inbred strain by brother-sister mating from thesame pair as founder mice of the circadian variant [23]— using
TRIzol reagent (GIBCO BRL, Frederick, MD), then reverse
transcription PCR was performed using SuperScript III FirstStrand Synthesis System (Invitrogen, Carlsbad, CA). The CASP
mice exhibited no obvious abnormalities in the circadian rhythmsof locomotor activity under DD conditions. All nucleotide
sequencing was performed bidirectionally by direct sequencing
using a BigDye terminator v3.1 cycle sequencing ready-reaction
kit (Applied Biosystems, Foster City, CA). We sequenced the
PLoS ONE | www.plosone.org

mPer1::luc real-time monitoring
To know temporal changes of SCN physiology when thecircadian variant became behaviorally arrhythmic, the mouse
Period1::luciferase (mPer1::luc) real-time monitoring technique wasused. For this, the variant mice were mated with mPer1::luctransgenic mice, in which the expression of luciferase is driven by a
6.7-kb genomic fragment of the mouse promotor (C57BL/6J) [25].
The F1 mouse was backcrossed to the parental variant and their
3

January 2009 | Volume 4 | Issue 1 | e4301

Wild-Derived Arrhythmic Mice

offspring were screened for both behavioral rhythms and themPer1::luc transgene. The mice that became arrhythmic under DD
conditions and possessed mPer1::luc transgene were humanely
sacrificed by cervical translocation and decapitation between
either 0500 and 0600 or 1700 and 1800 (local time). Their brains
were rapidly removed and placed in cold Hank’s balanced salt
solution (Invitrogen). Coronal sections (200-mm thick) of the SCN
were prepared using a DTK-3000W vibratome. The SCN slices
were placed on culture membranes (Millicell-CM, PICM030-50;
Millipore, Bedford, MA) and cultured in 35-mm petri dishes with
1.2 ml of Dulbecco’s modified Eagle’s medium (Sigma, St. Louis,MO) supplemented with 3.5 g/l D-glucose, 10 mM HEPES,
25 U/ml penicillin, 25 mg/ml streptomycin, and 0.1 mM luciferin. We did not use any chemicals to stimulate the slice. Each dishwas sealed using a coverslip and vacuum grease. Theseexperiments were performed according to the methods previously
described in Watanabe et al. [26]. Slice culture was performed at
36uC in a light-tight black box, and bioluminescence from theSCN was visualized using an Olympus BX microscope equippedwith a Hamamatsu C4742-98-24NR ORCA camera. Images wereacquired and analyzed using AquaCosmos software (Hamamatsu
Photonics, Hamamatsu, Japan). The mPer1::luc bioluminescencerhythms from the whole SCN and individual SCN cells were
measured during 0–60 h in vitro, and the distribution of the peaktime for the individual SCN cells (30–40 cells per SCN slice) was
measured.

Table 1. Summary of circadian parameters of parents, F1 andF2 mice.

Characteristics

Arrhythmic

Rhythimic

p-value

Circadian variant (LD12:12RDD)
Number28

24

Free-running period (hours)

22.7060.16

22.6160.10

Circadian ratio (%)

n.s.a25.7662.73

72.9162.11,0.0001

Daily activity in DD (counts/day) 1459362977

1308662409

n.s.

Phase angle difference (hours)

2.1860.54

n.s.

1.7960.40

Circadian variant (LD 4:4RDD)
Number101

21

Free-running period (hours)

22.3660.12

22.5560.13

Circadian ratio (%)

20.2461.27

65.162.09,0.0001

1337961029

n.s.

Daily activity in DD (counts/day) 134616955

n.s.

C57BL/6J (LD 4:4RDD)
Number4

Free-running period (hours)

23.2860.09-

Circadian ratio (%)

78.6566.29-

Daily activity in DD (counts/day)

4276262682-

F1 (LD 4:4RDD)
Number4

14

Statistical analysis

Free-running period (hours)

23.4060.39

22.6260.17

n.s.

The differences between two groups were analyzed with a twotailed Student’s t test or the Mann-Whitney U test. Comparison
among the multiple groups was performed using a one-way
ANOVA. The correlation between the circadian ratio and t was
examined using Spearman’s rank correlation test.

Circadian ratio (%)

39.3862.81

73.0961.94,0.0001

Daily activity in DD (counts/day) 4641668411

3344565229

n.s.

F2 (LD 4:4RDD)

Results
Behavioral analysis of parental, F1, and F2 mice

43

193

n.s.

Free-running period (hours)

22.6460.11

22.8160.05

n.s.

Circadian ratio (%)

26.8362.20

77.8960.84,0.001

Daily activity in DD (counts/day) 3763063495

4405461709

n.s.

a
not significant.
Free-running period of arrhythmic group was obtained only during 15-days
intervals with the circadian ratio more than 50%.
doi:10.1371/journal.pone.0004301.t001

Of the 52 circadian variant mice that were entrained to LD
12:12 and transferred to DD, 28 mice (54%) lost and 24 mice(46%) maintained circadian rhythms of locomotor activity during
7 weeks (Table 1). There were no significant sex differences in thefrequency of the mice with abolished circadian rhythms (p.0.5;
Student’s t test). To increase the ratio of arrhythmic mice for QTL
analysis, we exposed the mice to an LD 4:4 schedule before
transferring them to DD; this is because SCN organization can be
disrupted by abnormal LD cycles [26]. Indeed, the percentage ofthe arrhythmic phenotype dramatically increased (83%) and thecircadian ratio decreased in both the arrhythmic and rhythmic
phenotypes (Table 1). The arrhythmic state was not irreversible
since a single light pulse could restore the rhythm (Fig. 1I). In
addition, a few arrhythmic mice exhibited the spontaneousrecovery of free-running rhythms without any specific treatments(data not shown). In the C57BL/6J mice, activity rhythms weredivided into three components by this abnormal LD cycle andthese animals exhibited short-term disturbed patterns after release
into DD (Fig. 1B, D). The mean values of t and daily activity
counts in the circadian variant mice transferred from LD 4:4 to
DD were significantly lower than those in C57BL/6J mice(p,0.01; Student’s t test).
In the F2 mice, various patterns of circadian rhythm wereobserved, which is reflected in the wide distribution of thecircadian ratio (Fig. 1F–H, Fig. 2A). t was also widely distributed,
encompassing the range of parents and F1 mice (Fig. 2B). It was
generally observed that the shorter t became, the less clear was the
PLoS ONE | www.plosone.org

Numbercircadian rhythm; this was reflected in the significant positive
correlation between the circadian ratio and t in the F2 mice(r = 0.197, p = 0.003; Spearman’s test) (Fig. S2).

QTL analysis
To map the genes responsible for disorganized circadianrhythms, QTL analysis for the circadian ratio and t was
performed. Because there were no significant differences in thefrequency of the mice with abolished circadian rhythm, both maleand female data were combined to analyze. With regard to thecircadian ratio, QTL analysis using Map Manager QTXb20
revealed a significant QTL (Arrhythmicity; Arrh-1, LOD 5.26) on
Chr 8 (Fig. 3A, Table 2). Moreover, a suggestive QTL for thecircadian ratio was mapped on Chr 3 (LOD 2.63). With regard tot, four significant QTLs were mapped on Chr 1 (Short free-running
period; Sfp-1, LOD 4.71), Chr 6 (Sfp-2, LOD 4.00), Chr8 (Sfp-3,
LOD 4.95) and Chr 11 (Sfp-4, LOD 4.84) (Fig. 3B, Table 2). The
one LOD confidence intervals of Arrh-1 and Sfp-3 on Chr 8 wereobserved to overlap (Fig. S3).
To detect epistatic interaction, we performed two-locus
interaction analysis for all possible markers using Map Manager
QTXb20. If the overall effect exceeded the genome-wide 5%
4

January 2009 | Volume 4 | Issue 1 | e4301

Wild-Derived Arrhythmic Mice

Figure 2. Frequency histograms of phenotypic values for parental, F1, and F2 mice. The distribution of phenotypic values of circadian ratio(A) and free-running period (B) are shown. The circadian ratio (%) is the relative power of the largest circadian peak to the sum of the largest circadianpeak and non-circadian peak values. The free-running period (t) was determined by a chi-square (x2) periodogram analysis. Mean values of eachphenotype for these mice are summarized in Table 1.
doi:10.1371/journal.pone.0004301.g002

genes (Spcs3, Wdr17, Npy1r, Psd3, Gdf1, Fcho1, and Eps15l1) in thecircadian variant were found to have base substitutions causing
amino acid changes (Table 3). The sequences of the 7 genes in the
CASP mice were the same as those in the C57BL/6J mice.

significance level, the interaction effect was tested using approximate genome-wide thresholds. As a result, significant epistatic
QTLs (p = 0.0011) between D3Mit31 (Arrh-2) and D5Mit81 (Arrh-3)
were detected for the circadian ratio (Fig. 4). As shown in Fig. 4, if
the genotypes for D3Mit31 and D5Mit81 were both C/Chomozygous, the mean circadian ratio was 45.74 (67.65,
S.E.M), which was lower than the other combinations of genotype.
The QTLs determined in this study, and the clock and clockrelated genes are summarized in Fig. 5.

Clock gene expression in the SCN of the circadian variant
under LD conditions
The SCN is known to be a circadian pacemaker controlling
behavioral rhythms. Therefore, we studied the expression of
known clock genes (mPer1, mPer2, mPer3, Clock, and mBmal1) andthe photic induction of mPer1 and mPer2 genes in the SCN of thecircadian variant mice (Fig. 6A, B, C). The levels of all the mPer
genes were high during the light and low during the dark with apeak at zeitgeber time (ZT) 6 for mPer1 and ZT10 for mPer2 andmPer3. In contrast, the level of mBmal1 increased during the dark
and decreased during the light. Clock gene expression did notexhibit clear daily rhythmicity. Under DD conditions when
behavioral rhythms persisted, the same patterns as those of LD

Sequence of the candidate genes
To search the candidate genes for Arrh-1 and Sfp-3 on Chr 8, wesequenced the coding regions of the genes mapped between
D8Mit205 (30.0 cM, 52.3 Mb) and D8Mit249 (37.0 cM, 83.8 Mb)
that covered one LOD support interval of Arrh-1 and Sfp-3.
Among the genes located in this region, we selected 69 genes thatare highly or moderately expressed in the SCN (H.R. Ueda,
unpublished data, Table S2). When compared with C57BL/6J, 7
PLoS ONE | www.plosone.org

5

January 2009 | Volume 4 | Issue 1 | e4301

Wild-Derived Arrhythmic Mice

Figure 3. LOD score plots of QTLs with main effects. Results of interval mapping for the circadian ratio (A) and free-running period (B) using
Map Manager QTXb17 software. The horizontal lines show the genome-wide 10%, 5% significance levels estimated by a permutation test. No
statistical evidence for the presence of two QTLs was observed for these two traits.
doi:10.1371/journal.pone.0004301.g003(Fig. 7A, B, C) in all the arrhythmic mice examined. If the SCN
maintains the circadian rhythm in spite of the fact that the miceare behaviorally arrhythmic, it might be expected that the phasesof the bioluminescence rhythm in the SCN sampled at different
times would not coincide. However, the peak time for slices
sampled 12 h apart (1700–1800, 0500–0600) appeared approximately 23 h (230060055 h for 1700–1800, 233060029 h for
0500–0600) after the preparation (Fig. 7D, E).

12:12 were observed in all genes (data not shown). A 15-min lightpulse induced mPer1 and mPer2 expression in these mice in amanner similar to that in the C57BL/6J mice (Fig. 6C).

Real-time monitoring analysis of mPer1::luc
bioluminescence rhythms in the SCN
Since the in situ hybridization study described above was unable
to reveal the temporal changes of clock gene expression in thearrhythmic mice in which phase references were not determined
under DD, we performed real-time monitoring analysis ofmPer1::luc bioluminescence rhythms of the SCN in which activityof an individual SCN can be traced. Contrary to our expectation,
mPer1::luc bioluminescence in the SCN, regardless of a single
neuron or its population, exhibited clear circadian fluctuation
PLoS ONE | www.plosone.org

Discussion
In the present study, genetic screens were carried out on theprogeny of wild-caught castaneus mice. One reason for selecting the
castaneus mice is their high percentage of polymorphic genetic
markers, such as microsatellites, which is considerably useful for
6

January 2009 | Volume 4 | Issue 1 | e4301

Wild-Derived Arrhythmic Mice

Table 2. Summary of QTLs affecting the circadian ratio and free-running period.

Nearestmarker (cM)aPeak LOD
scorebPeakposition (cM)% variancec

Phenotype at the nearest locus(%)6S.E.M
C/C

B/C

B/B

-

D3Mit38 (70.3)

2.63*

70.3

5

59.1363.62

69.6862.16

73.4762.61,0.01

Arrh-1

D8Mit9 (33.5)

5.26**

33.5

10

54.5363.70

68.7562.05

76.0962.70,0.0001% variancePhenotype at the nearest locus(hours)6S.E.M

QTL
Circadian ratio

Nearestmarker (cM)

QTL

Peak LOD
score

Peak
position (cM)

Free-running period

C/C

B/C

p-valuedp-value

B/B

Sfp-1

D1Mit71 (19.5)

4.71**

22.3

9

22.5360.07

22.7960.06

23.0560.09,0.0001

Sfp-2

D6Mit188 (32.5)

4.00**

32.5

8

22.4860.08

22.8060.06

22.9960.09,0.001

Sfp-3

D8Mit7 (32.0)

4.95**

32.0

10

22.5060.08

22.7260.06

23.0760.08,0.0001

Sfp-4

D11Mit179 (52.0)

4.84**

52.0

10

22.5360.08

22.7360.06

23.0960.09,0.0001a

Map position is based on the Mouse Genome Database.
Genome-wide 5% (**), and 10% (*) level estimated by 1000 runs of a permutation test for each phenotype.
Percentage of the phenotypic variance accounted for by each QTL.
d
Estimated by one-way ANOVA analysis.
doi:10.1371/journal.pone.0004301.t002
bcgenetic mapping. A second reason is that the F1 progeny (bothsexes) of castaneus mice and laboratory mice (Mus musculus
domesticus), such as C57BL, are fertile, which enables us to use an
F2 intercross for genetic mapping [27]. Finally, we reasoned thatnatural populations of organisms conserve genetic diversity andmany naturally occurring variant genes are maintained within awild mouse population. In the absence of gene interactions, thevariant genes are assumed to have subtle effects on the phenotype.
Table 3. Mutations with amino acids substitution in thecircadian variant.

Gene name Position (Mb)

Figure 4. Epistatic interaction between D3Mit31(Arrh-2) andD5Mit81 (Arrh-3) for the circadian ratio. Open circles and open
squares represent the homozygous and heterozygous circadian variant(C) alleles for D5Mit81 (Arrh-3), respectively. Open triangles represent
the homozygous C57BL/6 (B) allele. Three genotypes for the D3Mit31(Arrh-2) locus are shown on the x-axis. Each value represents themean6S.E.M.
doi:10.1371/journal.pone.0004301.g004

Mutation

Amino acids substitution

Spcs3

56.0

495TRA

His165Gln

Wdr17

56.1

1732ART

Thr578Ser

Npy1r

69.6

361GRA

Ile121Val

Psd3

70.6

80CRT

Ala7Val

Gdf1

73.2

332CRT

Ser111Leu

520TRA

Val177Glu

Fcho1

74.6

1520TRC

Leu507Pro

Eps15l1

75.3

2456GRA

Ser819Asn

doi:10.1371/journal.pone.0004301.t003

Figure 5. Chromosomal locations of QTLs, and clock and clock-related genes. Each vertical bar represents a mouse chromosome. Blackcircles represent the centromeres. Symbols on the left side of each chromosome are the QTLs detected in the present study. Symbols on the rightside of each chromosome are the known clock and clock-related genes including previously mapped QTLs [5].
doi:10.1371/journal.pone.0004301.g005

PLoS ONE | www.plosone.org

7

January 2009 | Volume 4 | Issue 1 | e4301

Wild-Derived Arrhythmic Mice

However, these variants are important in considering the functionof a highly complex system since many common human diseases,
such as diabetes, hypertension and alcoholism, are caused bynaturally occurring variant genes and their interactions. Certainly,
induced mutations such as Clock are important for understanding
the mechanisms of the circadian clock [28]; however, ourapproach to isolate naturally occurring mutations that cannot be
achieved by mutagenesis or gene knockout may be useful forunderstanding the mechanisms underlying circadian and its
related disorders in human [29].
In this study, we detected QTLs with main effects affecting thecircadian ratio (Arrh-1) and t (Sfp-1, 2, 3, 4). Of these QTLs, one
LOD confidence interval for Arrh-1 and that for Sfp-3 overlapped,suggesting that the same gene might contribute to these
phenotypes. With regard to the circadian ratio, the effects ofepistatic interactions, which were not detected as main effects,

Figure 6. Expression of clock genes in the SCN of the circadian variant mice. (A) Representative autoradiographs of mPer1, mPer2, mPer3,
Clock, and Bmal1 signal at ZT 6 and 18 in arrhythmic circadian variant mice. (B) Temporal changes of expression of clock genes in arrhythmic circadianvariant mice (mean values6S.E.M., n = 4–5). The white and black bars represent the light and dark, respectively. (C) Light-induced expression of mPer1
and mPer2 in the circadian variants and C57BL/6J mice (mean values6S.E.M., n = 3–4, * p,0.05, Mann-Whitney U test).
doi:10.1371/journal.pone.0004301.g006

PLoS ONE | www.plosone.org

8

January 2009 | Volume 4 | Issue 1 | e4301

Wild-Derived Arrhythmic Mice

Figure 7. Real-time analysis of mPer1::luc bioluminescence in the SCN of arrhythmic circadian variant mice. (A) Representative activity
profile and the result of x2 periodgram analysis for the final 10 days. (B) Images of mPer1::luc bioluminescence in the SCN of the arrhythmic mouse
shown in (A). The number below each image represents time after SCN slice preparation. (C) Temporal profiles of mPer1::luc bioluminescenceobtained from the whole SCN (sum of the right and left SCN) (above) and 20 SCN neurons (below) of the arrhythmic mouse shown in (A). The x-axis
represents time (hours) after culture initiation and the y-axis is the intensity of mPer1::luc bioluminescence. Peak time distributions of individual SCN
neurons of arrhythmic mice prepared at 1700–1800 (D) and 0500–0600 (E). The temporal location of the first peak (from 12 h to 36 h in vitro) was
examined in each SCN neuron. Peak time distributions of each mouse (#1–4) and the piled data (the top graph) are shown.
doi:10.1371/journal.pone.0004301.g007

Compared with the previous QTL analysis for t, the mapped
positions of Sfp-1, 2, 3 and 4 were completely different [4]. We also
found that Tgfa (35.8 cM) and Prok2 (41.0 cM) on Chr 6, and reverbA (57.0 cM) on Chr 11 were close to the map positions of Sfp-2
and Sfp-4, respectively [10,35–37]. However, there were nodifferences in the amino acid sequences of these genes between thecircadian variant and the C57BL/6J mice. Thus, we now
determined the multiple QTLs that were not identified in previous
studies using the established laboratory mouse strains.
In many mice exhibiting abnormal circadian behavioral
rhythms, the expression of clock genes is severely affected in the
SCN [15,38–44]. However, compared with previous studies, in situ
analysis of our mice revealed no abnormal patterns of clock gene
expression in the SCN when behavioral rhythms persisted under
LD 12:12 and DD conditions [39,40,45]. In addition, no obvious
change in photic induction of mPer1 and mPer2, or in the grossanatomy (Nissl stain) of the SCN was found. To overcome thelimitations of in situ methods for monitoring circadian rhythms ofeach individual SCN, we performed real-time monitoring analysis
using the mPer1::luc reporter system [46,47]. Unexpectedly,
mPer1::luc bioluminescence of an individual SCN neuron or its
population exhibited circadian fluctuations despite behavioral
arrhythmicity. For this result, there are two possible explanations;either the SCN oscillation continued, which was not reflected inbehavioral rhythms by impaired output pathway, or desynchronized SCN neurons were reset by the stimulation of the culture
preparation (e.g. the stimulation of decapitation, slicing the brain,or placing the slice on culture membranes etc.). To assess thesewere observed between Chr 3 (Arrh-2) and Chr 5 (Arrh-3),
indicating that circadian rhythms are sustained by the complex
interaction among multiple genes.
When we compared Arrh-1 with the mapped loci of known
clock-related genes, we found that Arrh-1 was closely mapped tothe position of the Rab3a gene, which has been reported to affect t;a point mutation in the conserved amino acid (Asp77Gly) within
the GTP-binding domain of the Rab3a protein shortened thecircadian period of locomotor activity [30]. Although we analyzed
the sequence of the open reading frame and the expression level of
Rab3a mRNA in the SCN, no difference was detected between thecircadian variant and the C57BL/6J mice. Instead, we found that
7 genes (Spcs3, Wdr17, Npy1r, Psd3, Gdf1, Fcho1, and Eps15l1) hadbase substitutions causing amino acid changes in the circadianvariant. Among these genes, Npy1r (neuropeptide Y receptor Y1)
might be an interesting candidate, since the mice lacking Npy1r
have been reported to exhibit reduced locomotor activity [31].
Other reports also indicated that NPY Y1 receptors are involved
to some degree in light-induced phase shifts [32,33]. In addition,
the region in which we detected amino acid substitution in thecircadian variant is well conserved among mice, rats, dogs, andhumans. Another potential candidate is the Gdf1 gene. Gdf1 is
known to regulate left-right patterning during development inmice. Although the function in the adult animal has not been
determined [34], Gdf1 mRNA is highly expressed in the SCN(online information in the Allen Brain Atlas; www.brain-map.org/
welcome.do), raising the possibility that this gene is related to clockfunctions in the SCN.
PLoS ONE | www.plosone.org

9

January 2009 | Volume 4 | Issue 1 | e4301

Wild-Derived Arrhythmic Micepossibilities, we compared the bioluminescence rhythm sampled
12 h out of phase. The results indicated that the peak time ofmPer1::luc rhythms was dependent on the preparation time andappeared almost 23 h after the preparation in both cases. These
results suggested that circadian oscillation of individual SCN
neurons might be reset and synchronized by culture stimulation.
This is consistent with the behavioral data demonstrating therestoration of circadian rhythms by a light pulse given toarrhythmic mice (Fig. 1I) [48]. Our results appear to becomparable with Yoshikawa et al. [49] who demonstrated thatthe peak time of SCN bioluminescence rhythms was completely
determined by the time of the culture preparation in constantlight-induced arrhythmic rats. Although it is possible thatbehavioral arrhythmicity in our mice results from desynchronization among individual SCN neurons, this does not eliminate
another possibility that the preparation stimulation itself regenerates the circadian oscillation of the SCN. In any case, our findingthat arrhythmic behavior may not be ascribed to core circadianmechanisms will provide an important insight into the downstream mechanisms that control circadian expression rhythms.
In summary, we isolated a novel variant from a wild mouse
population, which abolished behavioral circadian rhythms underDD, and mapped the positions of the multiple QTLs affecting thepersistence of circadian rhythmicity and t. The analysis of clock
gene expression in the SCN revealed that the dysfunction in thisvariant was possibly due to desynchronizing mechanisms among
the SCN neurons. Although further studies are required to identify
the genes responsible for this dysfunction and define themechanisms underlying the desynchronization, our variantsarising from the interaction between multiple genetic variants
may prove useful in understanding the complex circadian systemand the mechanisms underlying circadian and its related disorders
in human.

Found at: doi:10.1371/journal.pone.0004301.s001 (0.17 MB
DOC)
Table S2 The list of genes examined the coding sequence.
Found at: doi:10.1371/journal.pone.0004301.s002 (0.06 MB
DOC)
Figure S1 The genomic constitution of the parental variant
male mouse. Grey arrow heads with numbers show Mit markers
used for typing of F2 mice (total 104 markers), and black arrowheads for that of the parental variant male mouse. C/C:
homozygous allele for circadian variant, C/B: heterozygous allele,
B/B: homozygous allele for C57BL/6J.
Found at: doi:10.1371/journal.pone.0004301.s003 (1.01 MB TIF)

Correlation of phenotypic values of the circadianratio (x-axis) and free-running period (y-axis) in F2 mice.
Significant correlation was observed (y = 22.471+0.0056,
ñ = 0.198, p = 0.003 by Spearman’s test).
Found at: doi:10.1371/journal.pone.0004301.s004 (0.57 MB TIF)
Figure S2

Figure S3 Overlapping of Arrh-1 and Sfp-3 on Chr 8. Overlayof LOD score plots for the circadian ratio (black line) and freerunning period (gray line). 1-LOD support intervals are shown foreach phenotype.
Found at: doi:10.1371/journal.pone.0004301.s005 (4.19 MB TIF)

Acknowledgments
We thank the Nagoya University Radioisotope Center for making available
the use of its facilities and Dr. T. Shiroishi (National Institute of Genetics)
for providing us HMI/MS mice in the study of sequencing the candidate
genes.

Author Contributions
Conceived and designed the experiments: SE. Performed the experiments:
TW TS YY SI ST JK EN SY NN. Analyzed the data: TW TS AI.
Contributed reagents/materials/analysis tools: HRU RGY HT TN TY.
Wrote the paper: TW SE.

Supporting Information
Table S1 Microsatellite markers genotyped and their map

position (cM).

References
12. Lindsley D, Zimm G (1992) The genome of Drosophila melanogaster. San
Diego, Tokyo: Academic Press.
13. Wade CM, Kulbokas EJ 3rd, Kirby AW, Zody MC, Mullikin JC, et al. (2002)
The mosaic structure of variation in the laboratory mouse genome. Nature 420:
574–578.
14. Wade CM, Daly MJ (2005) Genetic variation in laboratory mice. Nat Genet 37:
1175–1180.
15. Vitaterna MH, Selby CP, Todo T, Niwa H, Thompson C, et al. (1999)
Differential regulation of mammalian period genes and circadian rhythmicity by
cryptochromes 1 and 2. Proc Natl Acad Sci U S A 96: 12114–12119.
16. Manly KF, Cudmore RH Jr, Meer JM (2001) Map Manager QTX, crossplatform software for genetic mapping. Mamm Genome 12: 930–932.
17. Yoshimura T, Yokota Y, Ishikawa A, Yasuo S, Hayashi N, et al. (2002) Mapping
quantitative trait loci affecting circadian photosensitivity in retinally degenerate
mice. J Biol Rhythms 17: 512–519.
18. Churchill GA, Doerge RW (1994) Empirical threshold values for quantitative
trait mapping. Genetics 138: 963–971.
19. Ishikawa A, Hatada S, Nagamine Y, Namikawa T (2005) Further mapping ofquantitative trait loci for postnatal growth in an inter-sub-specific backcross ofwild Mus musculus castaneus and C57BL/6J mice. Genet Res 85: 127–137.
20. Knott SA, Marklund L, Haley CS, Andersson K, Davies W, et al. (1998)
Multiple marker mapping of quantitative trait loci in a cross between outbred
wild boar and large white pigs. Genetics 149: 1069–1080.
21. Seaton G, Haley CS, Knott SA, Kearsey M, Visscher PM (2002) QTL Express:
mapping quantitative trait loci in simple and complex pedigrees. Bioinformatics
18: 339–340.
22. Wei WH, de Koning DJ, Penman JC, Finlayson HA, Archibald AL, et al. (2007)
QTL modulating ear size and erectness in pigs. Anim Genet 38: 222–226.
23. Zhao WD, Ishikawa A, Yamagata T, Bolor H, Wakasugi N (2002) Female miceof DDK strain are fully fertile in the intersubspecific crosses with Mus musculus
molossinus and M. m. castaneus. Mamm Genome 13: 345–351.

1. Reppert SM, Weaver DR (2001) Molecular analysis of mammalian circadianrhythms. Annu Rev Physiol 63: 647–676.
2. Hofstetter JR, Mayeda AR, Possidente B, Nurnberger JI Jr (1995) Quantitative
trait loci (QTL) for circadian rhythms of locomotor activity in mice. Behav
Genet 25: 545–556.
3. Hofstetter JR, Possidente B, Mayeda AR (1999) Provisional QTL for circadianperiod of wheel running in laboratory mice: quantitative genetics of period in RI
mice. Chronobiol Int 16: 269–279.
4. Hofstetter JR, Trofatter JA, Kernek KL, Nurnberger JI, Mayeda AR (2003)
New quantitative trait loci for the genetic variance in circadian period of
locomotor activity between inbred strains of mice. J Biol Rhythms 18: 450–462.
5. Shimomura K, Low-Zeddies SS, King DP, Steeves TD, Whiteley A, et al. (2001)
Genome-wide epistatic interaction analysis reveals complex genetic determinantsof circadian behavior in mice. Genome Res 11: 959–980.
6. Suzuki T, Ishikawa A, Nishimura M, Yoshimura T, Namikawa T, et al. (2000)
Mapping quantitative trait loci for circadian behavioral rhythms in SMXA
recombinant inbred strains. Behav Genet 30: 447–453.
7. Suzuki T, Ishikawa A, Yoshimura T, Namikawa T, Abe H, et al. (2001)
Quantitative trait locus analysis of abnormal circadian period in CS mice.
Mamm Genome 12: 272–277.
8. Panda S, Antoch MP, Miller BH, Su AI, Schook AB, et al. (2002) Coordinatedtranscription of key pathways in the mouse by the circadian clock. Cell 109:
307–320.
9. Storch KF, Lipan O, Leykin I, Viswanathan N, Davis FC, et al. (2002) Extensive
and divergent circadian gene expression in liver and heart. Nature 417: 78–83.
10. Ueda HR, Chen W, Adachi A, Wakamatsu H, Hayashi S, et al. (2002) A
transcription factor response element for gene expression during circadian night.
Nature 418: 534–539.
11. Roybal K, Theobold D, Graham A, DiNieri JA, Russo SJ, et al. (2007) Manialike behavior induced by disruption of CLOCK. Proc Natl Acad Sci U S A 104:
6406–6411.

PLoS ONE | www.plosone.org

10

January 2009 | Volume 4 | Issue 1 | e4301

Wild-Derived Arrhythmic Mice

37. Preitner N, Damiola F, Lopez-Molina L, Zakany J, Duboule D, et al. (2002) The
orphan nuclear receptor REV-ERBalpha controls circadian transcription within
the positive limb of the mammalian circadian oscillator. Cell 110: 251–260.
38. Lowrey PL, Shimomura K, Antoch MP, Yamazaki S, Zemenides PD, et al.
(2000) Positional syntenic cloning and functional characterization of the
mammalian circadian mutation tau. Science 288: 483–492.
39. Shearman LP, Jin X, Lee C, Reppert SM, Weaver DR (2000) Targeted
disruption of the mPer3 gene: subtle effects on circadian clock function. Mol Cell
Biol 20: 6269–6275.
40. Jin X, Shearman LP, Weaver DR, Zylka MJ, de Vries GJ, et al. (1999) A
molecular mechanism regulating rhythmic output from the suprachiasmatic
circadian clock. Cell 96: 57–68.
41. van der Horst GT, Muijtjens M, Kobayashi K, Takano R, Kanno S, et al. (1999)
Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms.
Nature 398: 627–630.
42. Zheng B, Larkin DW, Albrecht U, Sun ZS, Sage M, et al. (1999) The mPer2
gene encodes a functional component of the mammalian circadian clock. Nature
400: 169–173.
43. Bunger MK, Wilsbacher LD, Moran SM, Clendenin C, Radcliffe LA, et al.
(2000) Mop3 is an essential component of the master circadian pacemaker inmammals. Cell 103: 1009–1017.
44. Cermakian N, Monaco L, Pando MP, Dierich A, Sassone-Corsi P (2001) Alteredbehavioral rhythms and clock gene expression in mice with a targeted mutationin the Period1 gene. EMBO J 20: 3967–3974.
45. Takumi T, Taguchi K, Miyake S, Sakakida Y, Takashima N, et al. (1998) A
light-independent oscillatory gene mPer3 in mouse SCN and OVLT. EMBO J
17: 4753–4759.
46. Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, et al. (2000) Resetting
central and peripheral circadian oscillators in transgenic rats. Science 288:
682–685.
47. Yamaguchi S, Isejima H, Matsuo T, Okura R, Yagita K, et al. (2003)
Synchronization of cellular clocks in the suprachiasmatic nucleus. Science 302:
1408–1412.
48. Vitaterna MH, King DP, Chang AM, Kornhauser JM, Lowrey PL, et al. (1994)
Mutagenesis and mapping of a mouse gene, Clock, essential for circadianbehavior. Science 264: 719–725.
49. Yoshikawa T, Yamazaki S, Menaker M (2005) Effects of preparation time onphase of cultured tissues reveal complexity of circadian organization. J Biol
Rhythms 20: 500–512.

24. Yoshimura T, Suzuki Y, Makino E, Suzuki T, Kuroiwa A, et al. (2000)
Molecular analysis of avian circadian clock genes. Brain Res Mol Brain Res 78:
207–215.
25. Herzog ED, Aton SJ, Numano R, Sakaki Y, Tei H (2004) Temporal precision in
the mammalian circadian system: a reliable clock from less reliable neurons.
J Biol Rhythms 19: 35–46.
26. Watanabe T, Naito E, Nakao N, Tei H, Yoshimura T, et al. (2007) Bimodal
clock gene expression in mouse suprachiasmatic nucleus and peripheral tissues
under a 7-hour light and 5-hour dark schedule. J Biol Rhythms 22: 58–
68.
27. Dietrich W, Katz H, Lincoln SE, Shin HS, Friedman J, et al. (1992) A genetic
map of the mouse suitable for typing intraspecific crosses. Genetics 131:
423–447.
28. King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, et al. (1997)
Positional cloning of the mouse circadian clock gene. Cell 89: 641–653.
29. Vadasz C (2000) Analysis of complex traits: mutagenesis versus QTLs. Nat
Genet 26: 395.
30. Kapfhamer D, Valladares O, Sun Y, Nolan PM, Rux JJ, et al. (2002) Mutationsin Rab3a alter circadian period and homeostatic response to sleep loss in themouse. Nat Genet 32: 290–295.
31. Pedrazzini T, Seydoux J, Kunstner P, Aubert JF, Grouzmann E, et al. (1998)
Cardiovascular response, feeding behavior and locomotor activity in micelacking the NPY Y1 receptor. Nat Med 4: 722–726.
32. Lall GS, Biello SM (2003) Attenuation of circadian light induced phase advancesand delays by neuropeptide Y and a neuropeptide Y Y1/Y5 receptor agonist.
Neuroscience 119: 611–618.
33. Soscia SJ, Harrington ME (2004) Neuropeptide Y attenuates NMDA-induced
phase shifts in the SCN of NPY Y1 receptor knockout mice in vitro. Brain Res
1023: 148–153.
34. Rankin CT, Bunton T, Lawler AM, Lee SJ (2000) Regulation of left-rightpatterning in mice by growth/differentiation factor-1. Nat Genet 24: 262–265.
35. Cheng MY, Bullock CM, Li C, Lee AG, Bermak JC, et al. (2002) Prokineticin 2transmits the behavioural circadian rhythm of the suprachiasmatic nucleus.
Nature 417: 405–410.
36. Kramer A, Yang FC, Snodgrass P, Li X, Scammell TE, et al. (2001) Regulationof daily locomotor activity and sleep by hypothalamic EGF receptor signaling.
Science 294: 2511–2515.

PLoS ONE | www.plosone.org

11

January 2009 | Volume 4 | Issue 1 | e4301