Establishment of TSH b real-time monitoring system inmammalian photoperiodism
Kaori Tsujino1,2a, Ryohei Narumi2,3, Koh-hei Masumoto1,4, Etsuo A. Susaki1,
Yuta Shinohara3,5, Takaya Abe6, Masayuki Iigo7,8,9, Atsushi Wada1b, Mamoru Nagano4,
Yasufumi Shigeyoshi4 and Hiroki R. Ueda1,2,3,5,10*
1

Laboratory for Systems Biology, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan
Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
3
Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe,
Hyogo 650-0047, Japan
4
Department of Anatomy and Neurobiology, Kinki University Faculty of Medicine, 377-2 Ohno-Higashi, Osakasayama City,
Osaka 589-8511, Japan
5
Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan
6
Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan
7
Department of Applied Biochemistry, Faculty of Agriculture, Utsunomiya University, 350 Mine-machi, Utsunomiya,Tochigi 321-8505, Japan
8
Center for Bioscience Research & Education (C-Bio), Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi 321-8505, Japan
9
Utsunomiya University Center for Optical Research & Education (CORE), 7-1-2 Yoto, Utsunomiya, Tochigi 321-8585, Japan10
Department of Systems Pharmacology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan
2

Organisms have seasonal physiological changes in response to day length. Long-day stimulation induces thyroid-stimulating hormone beta subunit (TSHb) in the pars tuberalis (PT),which mediates photoperiodic reactions like day-length measurement and physiological adaptation. However, the mechanism of TSHb induction for day-length measurement is largely
unknown. To screen candidate upstream molecules of TSHb, which convey light information
to the PT, we generated Luciferase knock-in mice, which quantitatively report the dynamics ofTSHb expression. We cultured brain slices containing the PT region from adult and neonatal
mice and measured the bioluminescence activities from each slice over several days. A
decrease in the bioluminescence activities was observed after melatonin treatment in adult andneonatal slices. These observations indicate that the experimental system possesses responsiveness of the TSHb expression to melatonin. Thus, we concluded that our experimental systemmonitors TSHb expression dynamics in response to external stimuli.

Introduction
Many organisms adapt their physiological functions
and behaviors to seasonal environmental changes by
Communicated by: Ryoichiro Kageyama*Correspondence: uedah-tky@umin.ac.jpaPresent address: Laboratory for Developmental Morphogeometry, RIKEN Center for Developmental Biology,
Kobe 650-0047, Japan.
bPresent address: Cell Analysis Center, Scientific Affairs,
Sysmex Corporation, 1-3-2 Murotani, Nishi-ku, Kobe
658-2241, Japan.

measuring day length, a biological process known as
photoperiodism (Dawson et al. 2001; Ebling & Barrett 2008; Revel et al. 2009). Integration of external
light information and endogenous time mediated bycircadian clock genes (clockwork genes) is important
for the photoperiodic responses (Hamner 1963; Pittendrigh & Minis 1964).
The pars tuberalis (PT) of the pituitary gland may
be responsible for the photoperiodic responses
because of the high expression levels of the clockwork genes and melatonin receptor 1a (MT1) in the
PT (Morgan & Williams 1996; Lincoln et al. 2003;

DOI: 10.1111/gtc.12063© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

Genes to Cells (2013) 18, 575–588

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Ikegami & Yoshimura 2012). In mammals, it is suggested that nocturnal melatonin conveys darkness
information to the PT, but the molecules conveying
light information remain unknown (Hoffman &Reiter 1965; Morgan & Williams 1996). In a recent
study, thyroid-stimulating hormone beta subunit (TSHb)
gene was found to be rapidly induced by long-day
stimulation in the PT and the product of TSHb, thyroid-stimulating hormone (TSH), has a functionalrole in seasonal testicular growth in birds (Yoshimura
et al. 2003; Nakao et al. 2008). This TSHb induction
is conserved even in mammals including melatoninproficient mice (Hanon et al. 2008, 2010; Ono et al.
2008; Dardente et al. 2010; Dupre et al. 2010;
Masumoto et al. 2010; Yasuo et al. 2010). Therefore,
TSHb is a key factor for day-length detection andcontrols seasonal physiological changes.
Delivery of light information from the retina to
the PT is an important pathway for inducing TSHb
expression and regulating photoperiodic responses.
Indeed, removal of the eyes, the suprachiasmatic
nucleus or the pineal gland disrupts testicularresponses in hamsters (Hoffman & Reiter 1965;
Rusak & Morin 1976; Stetson & Watson-Whitmyre1976). In the mouse PT, long-day stimulationinduces a transcription cofactor eyes absent 3 (Eya3)(Masumoto et al. 2010). This molecule regulates
TSHb expression with DNA-binding transcription
factor sine oculis-related homeobox 1 (Six1) and several
clockwork genes such as thyrotroph embryonic factor(Tef) and/or hepatic leukemia factor (Hlf ) (Dardente
et al. 2010; Masumoto et al. 2010). These molecularmechanisms have also been found in sheep (Dardente
et al. 2010; Dupre et al. 2010). On the other hand,darkness information is mediated by melatonin thatsuppresses Eya3 and TSHb expression in sheep andmice, respectively (Ono et al. 2008; Unfried et al.
2009; Dardente et al. 2010; Yasuo et al. 2010). Thus,
the regulation of Eya3 by external light conditions is
critical to control TSHb expression for photoperiodism. However, the intracellular and extracellular signals that transfer the external light information to the
PT to regulate Eya3 and TSHb expression remain
unknown.
In this study, we focused on how TSHb expression in the PT is regulated by long-day stimuli. To
accomplish this, we established a real-time monitoring
system for measuring TSHb expression dynamics forthe purpose of screening candidate light informationtransferring signals in the PT. We generated a genetic
mouse model in which TSHb expression dynamics in
the PT can be visualized by bioluminescence. Treat576

Genes to Cells (2013) 18, 575–588

ment of cultured PT slices with melatonin decreased
bioluminescence activities, and these results showedthat PT slices can response to melatonin in our culture condition. Thus, our mouse model could be
used to search for molecules that relay external lightsignals in the PT.

Results
Generation of TSHbLuc mice

To monitor TSHb expression in the PT, we applieda knock-in approach in which 5′ exon of TSHb-coding sequence was knocked out and replaced with afirefly Luciferase (Luc) gene (Fig. 1A). In this scheme,
the luc activity represents direct transcription fromthe endogenous TSHb promoter. Southern blot analysis confirmed that the desired mutation was successfully introduced (Fig. 1B, left), and genotypes ofoffspring were routinely analyzed by PCR (Fig. 1B,
right). Animals were backcrossed and maintained on
a CBA/N background because unlike the more common C57BL/6 strain, CBA/N mice maintain melatonin proficiency (Ebihara et al. 1986; Nakahara et al.
2003).
To verify the loss of TSHb transcript, we performed quantitative RT-PCR (qPCR) to measure
the expression levels of TSHb and its related genes in
the pituitary, where the highest TSHb expression
level in the brain was observed (Kasukawa et al.
2011). By using primers aligned within exon 4,
which was deleted by the targeting (Fig. 1A), TSHb
mRNA was undetectable in homozygous TSHbLuc
mice. On the other hand, only 12% decrease in
TSHb expression levels was observed in heterozygous
TSHbLuc mice compared with wild-type mice
(Fig. 1C). The expression levels of common glycoprotein
a-subunit (Cga), the alpha subunit of TSH, were notdifferent between wild-type and heterozygous
TSHbLuc mice, but there was a four-fold increase inhomozygous TSHbLuc mice (Fig. 1C), suggesting
antirepression of negative feedback loop due to thedisruption of the TSH heterodimer. Luc expression
levels were observed in heterozygous TSHbLuc mice,significantly increased in homozygous TSHbLuc miceand undetectable in wild-type mice, also suggesting
the disruption of the TSH heterodimer (Fig. 1C).
One hundred and thirty-seven offspring fromheterozygous matings were examined. 30 animals
were wild-type (21%), 85 heterozygous (53%) and 35
animals homozygous TSHbLuc mice (26%). These
results are consistent with single-gene Mendelian© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

Neo

5′ UTR

123

5
Neo

Luc
5′ probe

DTA

loxP

Luc

Avr

Targetedallele3′ probe

P1 P2Targeting
vector

1 kbp

Avr

5′ probe

4 5

Avr

123

loxP

WT
allele3′ UTRAvr(A)

3′ UTR

5′ UTRTSHb real-time monitoring system

P1P3 neo probe

3′ probe(Avr) MT: 18 kbp(Avr ) WT: 14.5 kbp

5′ probe

(B)

WT

+/L

WT +/L

L/L

MT

MT

WT

WT

TSHβ

Relative expression levels
to Actb

(C)
8

*

Cga

**

250

6

L/L

Luc

**

15

200

12

150

9

100

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50

3

**

in homozygous TSHbLuc mice was recovered by supplementation with 100 ppm thyroid powder, consistent with the observation in TSH receptor (TSHR)
knockout mice (Marians et al. 2002) (Fig. 1D). Fromthese observations, we concluded that phenotypes ofheterozygous TSHbLuc mice are comparable withthose of wild-type mice.

WT +/L L/L

Relationship between TSHb and Luc expression in
TSHbLuc mice

4
2
0

0

WT +/L L/L

WT +/L L/L

0**

Body weight (g)(D) 35
30
25
20
15
10
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0

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**
** ** **** ** WT
**
**
+/L
**
** **
L/L
**
L/L (Thyroid powder)
3

4

5

6

7

8

9

10

Figure 1 Generation of TSHbLuc mice. (A) Targeting schema.
Exons 1–5 are indicated by solid boxes. TSHb ORF is indicatedby filled boxes. Luc ORF sequence was inserted in frame to thefirst ATG of exon 4 of the TSHb gene. The hybridization
probes used for Southern blot analysis (5′, 3′ and Neo probes)
and PCR primers for routine genotyping (P1-P3) are indicatedby bold black lines and black arrowheads, respectively. (B)Southern blot analysis of the 5′ region (left) and genotyping
PCR (right). Wild-type allele, heterozygous and homozygous
mutant alleles are indicated as WT, +/L and L/L, respectively.
One extra band was appeared in the +/L lane in genotyping
PCR (right). (C) Expression levels of TSHb, Cga and Luc in thepituitary of TSHbLuc mice by using quantitative RT-PCR(qPCR). The statistical differences between wild-type andheterozygous or homozygous TSHbLuc mice were calculated.
*P < 0.05; **P < 0.01 (two-sample t-test, n = 6–8, average  SEM). (D) Growth curves in male TSHbLuc mice. The
statistical differences between wild-type and heterozygous or
homozygous TSHbLuc mice were calculated. **P < 0.01 (twosample t-test, n = 6–8, average  SEM).

11

12

Age (weeks)

inheritance, indicating that the TSHb null mutation
did not lead to fetal loss and TSHb was not necessaryfor embryonic growth or viability. However, maleand female homozygous TSHbLuc mice were sterile.
Weekly body weight results showed that homozygous
TSHbLuc mice display a low rate of growth, whereas
heterozygous TSHbLuc mice were indistinguishable
from wild-type mice (Fig. 1D). These results indicatethat slightly reduced TSHb expression and Luc
expression do not affect the physiology in heterozygous TSHbLuc mice. In addition, growth retardation

Next, we checked whether the knock-in Luc mice
respond to changes in day length similar to endogenous photoperiodic genes in the PT. TSHb, Luc andCga expressions in the PT of wild-type and heterozygous TSHbLuc mice under different day-length conditions were analyzed by radioisotope (RI) in situ
hybridization. After 3 weeks of exposure to short-day[light/dark = 8:16 hour (L8 : D16), zeitgeber time 0(ZT0; ZT0 was defined as the time of light-on)= lights-on, ZT8 = lights-off] or long-day (L16 : D8,
ZT0 = lights-on, ZT16 = lights-off) conditions,brain samples from each light condition were collected 4 h before light-off that corresponds to thesame circadian phase under both conditions (Masumoto et al. 2010) (Fig. 2A). TSHb, Luc and Cga weredetected only in the PT under both conditions, andthe TSHb expression increased 2.5-fold under longday conditions in wild-type and heterozygous
TSHbLuc mice. Although Luc expression was almost
undetectable in heterozygous TSHbLuc mice undershort-day conditions and not detected in wild-type© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

Genes to Cells (2013) 18, 575–588

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Sampling(4 h before light off)(A)

Short-day (SD)
L12:D12Long-day (LD)

Birth

WT
Luc

(B)

TSHβ
*

Relative mRNA
abundance

3
2
1
0

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1

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LD

Relative mRNA
abundance

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0

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ND

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LD

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0

+/L
Luc

1.5

10

1

5

0.5

SD

LD

LD

0

SD

LD

Neonatal PT

Bright field Luminescence

Bright field Luminescence

+/L

WT

Adult PT

mice, its expression was clearly detected in heterozygous TSHbLuc mice under long-day conditions(Fig. 2B). We also observed that Cga expression pattern was comparable in wild-type and heterozygous
TSHbLuc mice. These results indicate that the Luc
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Genes to Cells (2013) 18, 575–588

expression represents the physiological photoperiodic
responses of TSHb in the PT and does not affect theexpressions of endogenous TSHb and Cga.
PT-specific signals of the TSHb-Luc reporter

2

15

0

SD

Cga

*

20

*

Cga

2

TSHβ

(C)

6-week-old

3-week-old

Figure 2 Photoperiodic gene responses in TSHbLuc mice. (A)Experimental schema. 3-week-old mice were transferred toshort-day (SD; light/dark = 8:16 hour) or long-day (LD;light/dark = 16:8 h) conditions and maintained for 3 weeks.
Brain samples were collected 4 h before light-off (ZT4 of SD
and ZT12 of LD). (B) Expressions of TSHb, Luc and Cga
under SD and LD conditions in wild-type (WT) and heterozygous TSHbLuc mice (+/L) measured by radioisotope (RI) in
situ hybridization. The signal intensities were quantified andnormalized by the values in SD samples except Luc measurement in WT mice. Scale bar: 300 lm. ND, not detected.
*P < 0.05 (two-sample t-test, n = 3–5, average  SEM). (C)Bioluminescence imaging of the PT. Adult PT slices wereprepared from LD conditioned mice and neonatal PT slices
were prepared from L12 : D12 conditioned mice. Bright-fieldand corresponding bioluminescence CCD images captured
from the representative PT slices are shown. Scale bar:
100 lm (see also Fig. S1 and Video S1 in Supporting Information).

To detect bioluminescence derived from the TSHb
promoter in the PT region, we prepared brain slices
including regions from TSHbLuc mice. First, wemonitored the spatial pattern of the bioluminescence
activity in the slice with a macrozoom microscope
and a CCD camera. As expected, bioluminescence
activity was observed only in the putative PT
region surrounding the median eminence of theslices from 5- to 6-week-old (adult) heterozygous
TSHbLuc mice kept under long-day conditions for
2–3 weeks (Fig. 2C). These spatial bioluminescencepatterns are compatible to signals of Luc mRNA
(Fig. 2B,C). By contrast, no bioluminescence wasobserved in the slices from wild-type mice
(Fig. 2C). Therefore, bioluminescence activity measured in the slice can be attributed to luc activity
in the PT. In addition, similar bioluminescence pattern in the slices from adult mice was observed in
the slices from postnatal 6- to 8-day-old (neonate)
heterozygous TSHbLuc mice kept under L12 : D12(ZT0 = lights-on, ZT12 = lights-off) conditions(Fig. 2C). These observations are consistent with
the TSHb expression in vivo (Schulze-Bonhage &Wittkowski 1990; Japon et al. 1994) and thus imply
the possibility of slice usage from both adult andneonatal PT.

© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

TSHb real-time monitoring system

Real-time bioluminescence monitoring of theTSHb-Luc reporter

Next, we developed a real-time monitoring system
for TSHb-Luc bioluminescence by a photomultipliertube (PMT). Because Luc appears to be specifically
expressed in the PT of the slice (Fig. 2C), photon
counts by PMT measurements represent a mean bioluminescence of the tissue. First, the bioluminescencesignals of slices from adult heterozygous TSHbLuc
mice kept under short- or long-day conditions for
2–3 weeks (adult short- or long-day slices) were measured (Fig. 3A,B). Significantly higher bioluminescence signals were detected from adult long-day slices
than from adult short-day slices and wild-type slices.
We further checked whether the slices have in vivolike responsiveness to photoperiodic signals. We treated adult long-day slices with 10 nM melatonin fromsubjective ZT16 (ZT16 is the start of the culture andsubjective night) (Fig. 3C). In agreement with previous in vivo studies (Ono et al. 2008; Unfried et al.
2009; Yasuo et al. 2010), a decrease in luc activity
was observed by melatonin treatment. These results
suggest that the experimental system keeps TSHb
response to melatonin even in our culture condition.
One important application of this real-time monitoring system is to screen molecules that convey lightsignals under long-day conditions and induce theTSHb expression. Thus, we examined the possibilityof using of neonatal tissues instead of adult tissues
because of some advantages: (i) higher viability of tissues, (ii) ease of slice preparation and (iii) significant
signal levels even in L12 : D12 conditioned animals
rather than long-day conditioned animals (Fig. 2C).
To check whether neonatal tissues can be used forthe monitoring assay, luc activity in the PT slices ofneonatal heterozygous TSHbLuc mice kept under
L12 : D12 conditions (neonatal slices) was measured(Fig. 3A). The robust bioluminescence activities of
the neonatal slices were observed for over 2 days andgradually reached baseline levels after additional 2 or
3 days (Fig. 3D), with dynamics similar to those in
adult long-day slices. Continuous luminescence in the
PT for 2–4 days by imaging neonatal slices again
confirmed that whole-tissue bioluminescence approximates activity in the PT (Fig. S1A and Video S1 in
Supporting Information). Luminescence was also
observed in individual PT cells over 4 days, but withnoisy fluctuations (Fig. S1B and Video S1 in Supporting Information). Furthermore, the neonatal slices
were treated with various concentrations of melatonin, and dose-dependent repression was observed(Fig. 3E). These patterns of bioluminescence with orwithout melatonin were similar to the results in theadult long-day slices and in vivo.
To independently confirm that the observed
dynamics of bioluminescence is correlated withendogenous TSHb expressions in neonatal PT slices,
the expression levels of TSHb, Eya3 and Cga, which
are known to be decreased by melatonin (Ono et al.
2008; Unfried et al. 2009; Dardente et al. 2010; Yasuo et al. 2010), were measured by qPCR. Cultured
PT tissues with vehicle or 10 nM melatonin treatment
for 48 h after the start of the culture were collected
and subjected to qPCR analysis. The values of sine
oculis–related homeobox 5 (Six5) and potassium voltagegated channel, subfamily Q, member 5 (Kcnq5) were used
for measuring the relative PT amounts of the samples
and normalizing TSHb values and related genes,
because Six5 and Kcnq5 expression is specific to the
PT and does not respond to melatonin (Dupre et al.
2008; Masumoto et al. 2010). Consistent with in vivo
observations (Ono et al. 2008; Unfried et al. 2009;
Dardente et al. 2010; Yasuo et al. 2010) and bioluminescence results, the expression levels of endogenous
TSHb, Eya3 and Cga were significantly decreased
under melatonin treatment, excluding the possibility
that the decrease in the bioluminescence by melatonin might be an artifact (Fig. 3E,F and S2 in Supporting Information). In addition, there was a strong
positive correlation between TSHb mRNA levels by
qPCR analysis and bioluminescence counts of the PT
slices by PMT measurements (r2 = 0.71; Fig. 3F,
right). Furthermore, the culture medium of neonatal
PT slices in Fig. 3F and S2 in Supporting Information was collected, and secreted TSHb amounts were
measured by mass spectrometry. In the melatonintreated culture medium, secreted TSHb amounts
were 50% lower than vehicle-treated samples(Fig. 3G). There was a strong correlation between
secreted TSHb amounts and the bioluminescence
counts (r2 = 0.81; Fig. 3G, middle) as well as the
TSHb mRNA levels (r2 = 0.66; Fig. 3G, right).
These results indicate that the responsiveness of the
luc activity to an external signal reflects TSHb
mRNA expression and secreted TSHb protein.
Recently, it was reported that glutamine and glutamic acid induce the TSHb expression in the rat PT(Aizawa et al. 2012). To check whether the mouse
PT also responds to glutamine and glutamic acid, theslices were treated with 1 mM glutamine or 1 mM
glutamic acid after 2 h of glutamine and glutamic
acid starvation. Although no response was observed
by glutamine treatment, a transient increase in luc© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

Genes to Cells (2013) 18, 575–588

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Adult PT

(A)

Neonatal PT
Short-day (SD)
L12:D12Long-day (LD)P6 – 8Birth

3-week-old

5 – 6-week-old

40 000+/L, LD+/L, SD
WT, LD
WT, SDAdult

30 000Fold activity

20 000
10 000
0
2
Day

3

4

0

2
Day

TSHβ
30

*

25
20
15

TSHβ amounts (fmol)

10
5
0

Veh

20

Mel

**

15
10
5
0

Veh

Mel

8
7
6
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Fold activity

+/L
WT

1

4

Eya3
**

**

25
20
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5

Veh

Mel

0

Veh

3

4

Melatonin at the start of the culture

ZT12
6 Neonate
5
4
3
2
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0
1

Cga
30

2
Day

Mel

Veh
1 pM Mel
10 pM Mel
100 pM Mel
10 nM Mel

2
Day

3

60 0004

r2 = 0.71

40 000
20 000
0

Veh
Mel
0

200

400

600

800

TSHβ mRNA levels
TSHβ amounts (fmol)

Bioluminescence (cpm)1

Bioluminescence (cpm)

1

60 000 Neonate
50 00040 00030 000
20 000
10 0000
01

Relative expresion levels
to Six5

2

(E)

(F)

Veh
10 nM Mel

ZT16
3 Adult

0
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(D)

(G)

Melatonin at the start of the culture30

r2 = 0.81

20
10
0

Veh
Mel
0

20 000 40 000 60 000

Bioluminescence (cpm)

TSHβ amounts (fmol)

Bioluminescence (cpm)

(C)
(B)

30

r2 = 0.66

20
10
0

Veh
Mel
0

200

400

600

800

TSHβ mRNA levels

Figure 3 Real-time monitoring of the TSHb expression by bioluminescence. (A) Experimental schema. Neonatal PTs were collected at postnatal 6–8 (P6–8) under L12 : D12 conditions, and 5- to 6-week-old mice were collected after being maintained in
short-day (SD) or long-day (LD) conditions for 2–3 weeks. (B and D) Real-time bioluminescence recording in adult or neonatal
PT slices of indicated genotypes. White and black bars on the graphs indicate the subjective day and night on the following days
after sampling, respectively [n = 2, wild-type (WT) mice or n = 6, heterozygous TSHbLuc mice (+/L) in B; n = 5, WT andn = 17, +/L in D, average  SEM]. (C and E) Real-time monitoring of the luciferase (luc) reporter activities in adult or neonatal
PT slices after the addition of vehicle (Veh) or melatonin (Mel). The PT slices were prepared from animals kept under LD (adult,C) or L12 : D12 (neonate, E) conditions. Vehicle or melatonin was applied at the start of the culture. Normalized luc reporter
activities of PT slices from +/L mice are shown (n = 4 in C and n = 3–4 in E, average  SEM). (F) Confirmation of expression
level changes in TSHb, Eya3 and Cga in the cultured PT by qPCR analysis. Neonatal PT slices were treated with vehicle or
10 nM melatonin 48 h with PMT monitoring. Expression levels of TSHb, Eya3 and Cga were normalized by the value of Six5,
which was specifically expressed in the PT. *P < 0.05; **P < 0.01 (two-sample t-test, n = 9, average  SEM). Correlation
between TSHb mRNA levels and bioluminescence counts (right). White circles (Veh) and gray circles (Mel) indicate data points.
The line is regression: r2 = 0.71. (G) Quantification of secreted amounts of TSHb into the culture medium of the PT slices in 3F
and S2 by mass spectrometry analysis. **P < 0.01 (two-sample t-test, n = 9, average  SEM). Correlation between secreted
TSHb amounts (middle) and TSHb mRNA levels or bioluminescence counts (right). White circles (Veh) and gray circles (Mel)indicate data points. The line is regression: r2 = 0.81 (middle), r2 = 0.66 (right).

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Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

TSHb real-time monitoring system

activity was observed by glutamic acid treatment.
These results suggest that the slices can also respondto an inducer of TSHb (Fig. S3 in Supporting Information).
In summary, we visualized the TSHb expression
dynamics in the PT by using adult and neonatal
TSHbLuc mice and observed responsiveness of the
TSHb expression to an external signal.

Discussion
In the present study, we established a TSHb realtime monitoring system in which the TSHb expression dynamics can be monitored as real-time bioluminescence activities. The difference in luc activity
of PT slices derived from adult mice under short- or
long-day conditions was consistent with in vivo
TSHb expression levels in response to each daylength condition (Figs 2 and 3B). Moreover, the luc
activities of adult and neonatal PT slices were suppressed by melatonin treatment (Fig. 3C,E). We also
observed dose-dependent suppression of TSHb inneonatal PT slices by melatonin (Fig. 3E) at physiological levels (Kennaway et al. 2002), which is consistent with the results using ovine PT cell cultures(Morgan et al. 1989). Furthermore, the luc activities
were transiently up-regulated by glutamic acid treatment as seen in the rat PT (Aizawa et al. 2012) (Fig.
S3 in Supporting Information). These observations
suggest that PT slices possess at least TSHb responsiveness to melatonin and glutamic acid, and changes
in TSHb expression dynamics under other stimuli arealso expected to be observed by using our monitoring system.
Chronic melatonin treatment in this monitoring
system may not necessarily the same as physiologicalconditions, comparing with nocturnal melatoninsecretion in vivo. However, because TSHb was
induced within several hours after long-day stimulation in vivo, this monitoring system is at least available
for screening of candidate molecules upstream of
TSHb, by observing luc activity during the same period. Transiently increased luc activity by glutamic
acid treatment supports this idea (Fig. S3 in Supporting Information).
Several studies show that melatonin implantation(constant release of melatonin) has no effect on testicular regression in hamsters (Goldman et al. 1979).
Furthermore, melatonin injection under long-day
conditions does not suppress TSHb expression for atleast 4 days in hamster (Yasuo et al. 2010), which isinconsistent with the immediate suppression pre-sented in Fig. 3C. We speculate that difference
between these in vivo observations and our in vitro
results may be due to experimental conditions such as
existence of TSHb-inducible signals. Therefore, careful design of experiments will be necessary in eachpurpose.
One possible application of our experimental system is to screen upstream light-information signals of
the TSHb induction. Our system has several advantages for this purpose: (i) The PT slices can be usedand easily treated with drugs compared with previous
methods such as subcutaneous injection (Yasuo et al.
2007), intracerebroventricular injection (Ono et al.
2008) or dissociated PT cells (Morgan et al. 1989),(ii) availability of neonatal tissues increases thethroughput of the experiment and viability of theslices. Previous reports support that neonatal PT has
the similar photoperiodic properties to adult PT.
First, high expression levels of TSHb (Schulze-Bonhage & Wittkowski 1990; Japon et al. 1994), MT1
receptor (Williams et al. 1991; Johnston et al. 2003)
and rhythmic expression of the clockwork genes(Ansari et al. 2009) were observed in both embryonicand neonatal PT. Second, fetal and neonatal lambs
showed changes in prolactin concentration in
response to day length (Ebling et al. 1988, 1989).
We consider that neurotransmitters or humoral
factors are possible candidates involving the extracellular upstream signals of TSHb. For example, there
are several known neurotransmitters of deep brain
photoreceptor–expressing cells in birds (Halford et al.
2009; Nakane et al. 2010). The afferent signals mediated by these neurotransmitters might transfer lightstimuli to the PT. Additionally, receptors expressedin PT cells like Gs-coupled receptors and downstream cAMP signaling are possible candidates
because these signals can antagonize decreases in
TSHb by melatonin and the Gi-coupled MT1 receptor (Morgan et al. 1995).
Our system is also applicable to investigate themechanism of photoinducible phase, by which the
response of the PT to a light signal including TSHb
induction is gated during the late night (Masumoto
et al. 2010). Internal circadian time is probably
involved in the determination of the gate timing,
because photoperiodic responses can be induced bylight stimuli at specific time (Ravault & Ortavant
1997). Our previous study showed that the brainslices of mPer2Luc mice containing PT exhibited circadian oscillations of luc (Masumoto et al. 2010).
Also, it was reported that PER2::LUC rhythms inslices were detected in the median eminence/pars© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

Genes to Cells (2013) 18, 575–588

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K Tsujino et al.

tuberalis (Guilding et al. 2009). These reports support
that the PT region contains its intrinsic circadianoscillator. Analyzing circadian time dependency ofTSHb expression after chemical/neurotransmitter
stimulation in the PT may facilitate reconstitution of
the in vivo phenomenon and help to show the detailsof gating.
The TSHbLuc mice generated here are the firstreport of TSHb null mice. TSHbLuc mice enabled us
to study the photoperiodic roles of TSHb. Although
TSHb is also expressed in the pituitary and functions
as a part of the hypothalamus–pituitary–thyroid(HPT) axis, it is believed that the molecule in the PT
acts differently from the HPT axis. This idea is supported by the results that TSHb expression in ovine
PT did not change with thyroid-releasing hormone(TRH) and thyroid hormone (TH) treatment,
because the lack of TRH and TH receptor or transcription factor Pit-1 induces TSHb in the pituitary(Bockmann et al. 1997). Growth retardation observedin our homozygous TSHbLuc mice (Fig. 1D) is consistent with previous observations in hypothyroid
mice, such as TSHR knockout mice (Marians et al.
2002), the Snell dwarf mice (Cordier et al. 1976), thecog mice (Taylor & Rowe 1987) and the hyt mice(Beamer et al. 1981), and could be recovered by supplementation with thyroid powder (Fig. 1D). Thus,
photoperiodic phenotypes in the TSHbLuc mice, ifany, can be discriminated from the effects of theimpaired HPT axis by supplementation with thyroid
powder. Because no obvious photoperiodic phenotype has not been identified in laboratory mice, suchas testicular growth, which is a typical photoperiodic
phenotype (Ono et al. 2008), it will be intriguing tofurther explore photoperiodic phenotypes in laboratory mice with reference to known seasonal physiological changes such as gonad growth in female
European hamsters (Hanon et al. 2010), metabolicchanges such as adipose depot mass and serum leptin(Atcha et al. 2000) and immune system such as NK
cell cytolytic activity and phagocytosis in Siberian
hamsters (Yellon et al. 1999; Martin et al. 2008).
Even if no obvious photoperiodic phenotype can be
identified in laboratory mice, molecular mechanisms
underlying the induction of TSHb by long-day light
exposure are evolutionary conserved (Hanon et al.
2008, 2010; Nakao et al. 2008; Ono et al. 2008; Dardente et al. 2010; Dupre et al. 2010; Masumoto et al.
2010; Yasuo et al. 2010). Therefore, TSHbLuc mice,
established in this study, will provide useful insights
into the evolutionary conserved molecular mechanism of photoperiodism.
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Genes to Cells (2013) 18, 575–588

Experimental procedures
Generation of TSHbLuc mice
TSHbLuc mice [Tshb(Luc); Acc. No. CDB0713K: http://www.cdb.riken.jp/arg/mutant%20mice%20list.html] were generated as follows: a 1.9 kbp of Luc cassette that contains Luciferase (Luc)-coding region and simian virus 40 polyadenylation
signal was amplified by PCR from pGL4.10 (Promega, Madison, WI, USA) using Luc-F-EcoRI (5′-AGAATTCTACT
GTTGGTAAAGCCACCATGGAAGAT-3′) and Luc-RNotI (5′-CGGAGCGGCCGCGATTTTACCACATTTGTAGAGGTTTTACTTGC-3′) primers (Hokkaido System Science, Sapporo, Japan). The amplified product was ligated into
the EcoRI/NotI site of pBluescript SK ( ) (Stratagene, La Jolla, CA, USA). The 6.3-kb 5′-arm was PCR-amplified from abacterial artificial chromosome (BAC) clone containing TSHb
(RP24-230F23; BACPAC Resources, Oakland, CA, USA)using 5-F-SalI (5′-GCAGGTCGACCAATAGAGGGAACAGAATAGTCCCAAACG-3′) and 5-R-BsmBI (5′-ACGGCG
TCTCTCATGCTGAATCAGAGAGAAACATCAAAGAGC
TC-3′) primers, and the amplicon was ligated into the SalI/
NcoI site upstream of the Luc of pBluescript SK ( ) to makethe 5′ arm-Luc cassette, in which the Luc cassette was connected in frame to the first ATG of the TSHb gene. The 5′
arm-Luc cassette was inserted into the SalI/NotI site of the
PGK-Neo-pA/DT-A vector (detailed descriptions of the vector are available from http://www.cdb.riken.go.jp/arg/cassette.html). The 2.8-kbp 3′-arm was also PCR-amplified fromthe BAC clone using 3-F-AvrII (5′-GACCCTAGGATGTTGTTCAATGCATTTCTTTTAGCTGTAA-3′) and 3-RSalI (5′-ATTAGTCGACGTACCATGCTATGCTGTTAACCTGCAATAC-3′) primers, and the amplicon was ligated into
the XbaI/XhoI site of the PGK-Neo-pA/DT-A vector. The
resultant targeting vector was linearized with the AscI site andintroduced into TT2 embryonic stem cells by electroporation(Yagi et al. 1993; Murata et al. 2004). Screening of homologous recombinant embryonic stem cells and production of chimera mice are described elsewhere (http://www.cdb.riken.jp/
arg/Methods.html). PCR primers used to routinely identify
the wild-type allele are P1 (5′-CGCAGGGCCCAGGGATAAGTAACCAGTCAG-3′) and P2 (5′-ACCCGTGTCATACAATACCCAGCACAGATGGTG-3′); those to identify the
mutant allele are P1 (5′-CGCAGGGCCCAGGGATAAGTAACCAGTCAG-3′) and P3 (5′-ACAGCCACACCGATGAACAGGGCACCCAAC-3′) (Fig. 1A). PCR products of 305
and 446 bp were derived from wild-type and mutant alleles,
respectively. For Southern blot analysis, genomic DNA
extracted from ES cells or a mouse tail was digested with AvrII, and resulted bands were detected with radioactive probes(14.5- and 18-kbp bands from wild-type and mutant alleles,
respectively, in Fig. 1A). Many laboratory mouse strains suchas C57BL/6 lack melatonin production ability because of natural knockdown of the serotonin N-acetyltransferase(AANAT) activity (Roseboom et al. 1998) and low expression
levels of hydroxyindole O-methyltransferase (HIOMT) protein(Kasahara et al. 2010; Shimomura et al. 2010), which are rate-© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

TSHb real-time monitoring system
limiting enzymes for melatonin synthesis. By contrast, CBA/N
mice are melatonin proficient and they show significant TSHb
induction after long-day stimulation (Ono et al. 2008; Masumoto et al. 2010). Thus, TSHbLuc mice were backcrossed formore than 10 generations and maintained on a CBA/N background for all experiments except the measurement of growthin Fig. 1D (more than four generations).

Animals
Mice were carefully kept and handled according to the RIKEN Regulations for Animal Experiments. For in situ hybridization experiments, L12 : D12 conditioned male 3-week-old
wild-type and heterozygous TSHbLuc mice were housed undershort-day conditions (L8 : D16, 400 lux) or under long-day
conditions (L16 : D8, 400 lux) for 3 weeks.
For neonatal PT slice culture experiments, neonatal wildtype and heterozygous TSHbLuc mice were generated by overnight breeding of heterozygous TSHbLuc and CBA/N mice
(Japan SLC, Hamamatsu, Japan). Pregnant mice and pups werehoused under L12 : D12 conditions (400 lux) because it wasreported that exposure to different light/dark conditions frombirth affects circadian properties (Ciarleglio et al. 2011). For
adult PT slice culture experiments, L12 : D12 conditioned 3week-old wild-type and heterozygous TSHbLuc mice werehoused under short- or long-day conditions for 2 to 3 weeks.
For other experiments, TSHbLuc mice were housed under
L12 : D12 conditions. All mice were given commercial chow
and water ad libitum except the supplementation of thyroid
powder experiment. Homozygous TSHbLuc mice were given
commercial chow with 100 ppm thyroid powder (Sigma, St.
Louis, MO, USA) and water ad libitum (Fig. 1D).

In situ hybridization
Mice were deeply anesthetized with isoflurane (Mylan, Tokyo,
Japan) and intracardially perfused with 10 mL saline and
20 mL of a fixative containing 4% paraformaldehyde in 0.1 M
phosphate buffer (PB), pH 7.4. Mouse brain samples were
post-fixed in the same fixative for 24 h at 4 °C, soaked in PB
containing 20% sucrose for several days and stored frozen at
80 °C for further use. In situ hybridization was carried out
as previously described (Shigeyoshi et al. 1997; Masumoto
et al. 2010). Serial coronal sections (40 lm thick) of themouse brain were prepared using a cryostat (CM 1850; Leica
Microsystems, Wetzlar, Germany). To prepare probes, fragments of cDNA were obtained by PCR and subcloned into
the pGEM-T easy vector (Promega). Radiolabeled probes
were generated using 35S-UTP (PerkinElmer, Norwalk, CT,
USA) via a standard protocol for cRNA synthesis. The primers used in the construction of in situ hybridization cRNA
probes were as follows:
TSHb cRNA probe:
Forward primer: 5′-TGGGTGGAGAAGAGTGAGCG-3′
Reverse primer: 5′-ACCAGATTGCACTGCTATTG-3′

Cga cRNA probe:
Forward primer: 5′-GCAGGCACTGAAAAATCCAGAGACATTGTTC-3′
Reverse primer: 5′-ACACACAGCGCCATTGAATGGCTC-3′
Luc cRNA probe:
Forward primer: 5′-CACCGGTAAGACACTGGGTGTGAACCAGC-3′
Reverse primer: 5′-CACGGCGATCTTGCCGCCCTTCTTGGC-3′

PT slice culture
TSHbLuc mice were decapitated 1–4 h before light-off. PT
slices were prepared from neonatal pups (6–8 days old) or
adult mice (5 to 6 weeks old). For neonatal PT slice culture
experiments, brains were rapidly removed and were cut by
a tissue chopper (McIlwain Tissue Chopper; McIlwain Laboratory Engineering, Gomshall, Surrey, UK) to a thicknessof 350 lm. For adult PT slice culture experiments, brains
were rapidly removed and were cut by a vibratome typelinearslicer (PRO7; Dosaka EM, Kyoto, Japan) to a thickness of 300 lm. The PT region slices were dissected using
a surgical knife. The slices were then placed on a culture
membrane (MilliCell-CM; Millipore, Bedford, MA, USA)and set on a dish with 1.2 mL culture medium containing
DMEM/F12 (Invitrogen, Carlsbad, CA, USA) supplemented
with 300 mg/L NaHCO3 (Sigma), 20 mg/L kanamycin (Invitrogen), 100 mg/L apo-transferrin (Sigma), 100 lM putrescine (Sigma), 20 nM progesterone (Sigma), 30 nM sodium
selenite (Sigma) and 1 mM luciferin (Biosynth, Staad, Switzerland). The dish was sealed with silicone grease (HVG;
Dow Corning-Toray, Tokyo, Japan). Explants from different
pups were cultured separately, and only those of appropriate
genotypes were chosen for further experiments. Slices weretreated with vehicle [ethanol (Sigma)] or melatonin (Sigma)(Fig. 3 and S2 in Supporting Information). For glutamineand glutamic acid starvation, culture medium with DMEM/
F12 without glutamine and glutamic acid (Cell Science &Technology Institute, Sendai, Japan) was used. For glutamine or glutamic acid treatments, the glutamine- and glutamic acid–starved culture medium was replaced with theabove fresh culture medium or with added glutamine(Sigma) or glutamic acid (Sigma) (Fig. S3 in Supporting
Information).

Quantitative RT-PCR (qPCR)
For qPCR experiments of the pituitary, TSHbLuc mice were
decapitated at ZT2 to 6 under L12 : D12 conditions to collect the part of the tissue embedded in the sella turcica (pituitary fossa) from 12-week-old male TSHbLuc mice andtemporarily stocked at 80 °C for further use. The total
RNA was prepared from each pituitary, using RNeasy microkit (Qiagen, Valencia, CA, USA). The cDNAs were synthe-© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

Genes to Cells (2013) 18, 575–588

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K Tsujino et al.
sized from 0.25 lg of the total RNA using Superscript III
transcriptase (Invitrogen). qPCR was performed using SYBRGreen PCR Master Mix (Applied Biosystems, Foster City,
CA, USA).
For qPCR experiments of the cultured PT samples, thecultured PT samples were collected 48 h after the start of theculture. The total RNA was prepared from each cultured PT,
using NucleoSpin RNA XS kit (Takara, Otsu, Japan). The
cDNAs were synthesized from 0.05 lg of the total RNA of
the cultured PT using Superscript Vilo transcriptase (Invitrogen). qPCR was performed using Quantitect SYBR GreenPCR mastermix (Qiagen).
qPCR was performed using the ABI PRISM 7900
(Applied Biosystems) at the following conditions: Samples
contained 1 9 SYBR Green PCR Master Mix or QuantiTectSYBR Green PCR Master Mix, 0.8 lM primers and 1/50
synthesized cDNA in a 10-lL volume. The PCR conditions
were as follows: 10 min at 95 °C, then 45 cycles of 15 s at
94 °C, 1 min at 59 °C. The absolute cDNA abundance wascalculated using a standard curve obtained from murine genomic DNAs. We used TATA box-binding protein (Tbp) or actin,
beta (Actb) as the internal controls. Primer information is as follows:

PMT bioluminescence measurements
The time-course bioluminescence results were obtained asreported previously (Isojima et al. 2009). PMT measurementswith a high-sensitivity bioluminescence detection system (LM2400; Hamamatsu Photonics, Hamamatsu, Japan) were started
immediately after the start of PT slice culture at the time oflight-off (ZT12 for neonatal PT slices, ZT8 or 16 for adultshort- or long-day slices). For the data analysis in Fig. 3B,D,
raw photon counts were plotted. For the data analysis in
Figs 3C,E and S3 in Supporting Information, photon countsof the PT slice from heterozygous TSHbLuc mice were calculated by subtracting background photon counts of PT slice
from wild-type mice. The resultant values were further normalized by photon counts at ZT4.75, which is 12.75 h after
the start of the culture in adult long-day slices (Fig. 3C), or at
ZT23, which is 11 h after the start of the culture in neonatal
PT slices (Fig. 3E), which were the median time for the lowest bioluminescence values from all vehicle-treated samples inadult and neonatal slices, respectively (Fig. S4 in Supporting
Information). The resultant values were normalized by photon
counts at ZT13.45, which is just before glutamine and glutamic acid treatment in neonatal PT slices (Fig. S3 in Supporting Information).

TSHb mRNA (exon 4):
Forward primer: 5′-GTGGGCAAGCAGCATCCTTTTG-3′
Reverse primer: 5′-GCACACTCTCTCCTATCCACGTAC-3′
Cga mRNA:
Forward primer: 5′-TGCTGAGCCGAGCCATTCAATG-3′
Reverse primer: 5′-GAAGTCTGGTAGGGAGGAGGTGG-3′
Luc mRNA:
Forward primer: 5′-TCCTCAACGTGCAAAAGAAGCTACC-3′
Reverse primer: 5′-GTCGGTCTTGCTATCCATGATGATGATC-3′
Actb mRNA:
Forward primer: 5′-TTGTCCCCCCAACTTGATGT-3′
Reverse primer: 5′-CCTGGCTGCCTCAACACCT-3′
TSHb mRNA:
Forward primer: 5′-CTGCATACACGAGGCTGTCAG-3′
oReverse primer: 5′-CCCCAGATAGAAAGACTGCGG-3′

Bioluminescence imaging
Samples were prepared as described above. Sealed 35-mm culture dishes were placed on the stage of a macrozoom microscope (MVX10; Olympus, Tokyo, Japan) in a dark hood. The
culture dishes were kept at approximately 37 °C in a heatedchamber (Tokai Hit, Shizuoka, Japan) on the microscopestage. Bioluminescence was imaged using a 1.6 9 Plan Apochromat objective (NA 0.24; Olympus) with 6.3 9 zoom
and transmitted to an electron-multiplying charge-coupleddevice (EM-CCD) camera cooled to
80 °C (ImagEM;
Hamamatsu Photonics). The dimension of an image is
512 9 512 pixels, and each pixel corresponds to the size of
1.587 9 1.587 lm. Exposure time was 29 min with EM gainof 200 9 for snapshot images, and exposure time was 14 minwith EM gain of 300 9 for time-lapse images. Images weretransferred at 690 KHz to minimize readout noise, and analyzed using MetaMorph software (Molecular device, Sunnyvale, CA, USA). Single-cell tracking was performed aspreviously described (Ukai et al. 2007).

Mass spectrometry
Eya3 mRNA:
Forward primer: 5′-TTCACAGCTCCAAGTAGAATCTGACT-3′
Reverse primer: 5′-TATGGAAGCGCCATGAGCTT-3′
Tbp mRNA:
Forward primer: 5′-CCCCCTCTGCACTGAAATCA-3′
Reverse primer: 5′-GTAGCAGCACAGAGCAAGCAA-3′

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Genes to Cells (2013) 18, 575–588

The YALSQDVCTYR peptide, which was the uniquesequence for TSHb, was synthesized on a peptide synthesizer(Syro Wave; Biotage, Uppsala, Sweden) using Fmoc solidphase chemistry. The peptide was dimethyl-labeled with formaldehyde (CD2O) and desalted by C18 StageTips according topreviously described method (Boersema et al. 2009). The
dimethyl labeling of the peptide was checked by liquid chromatography coupled with tandem mass spectrometry (LC-MS/© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

TSHb real-time monitoring systemMS) with an LTQ orbitrap velos mass spectrometer (Thermo
scientific, Waltham, MA, USA) coupled to a nano-Advance
UHPLC system (Bruker Daltonics, Leipzig, Germany).
The culture medium of the PT slices was collected andtemporarily stocked at 80 °C for further use. Proteolytic
digestion was performed using a phase-transfer surfactant protocol (Masuda et al. 2008; Narumi et al. 2012). The resultant
digest was dimethyl-labeled with formaldehyde (CH2O).
For quantification by mass spectrometry, a triple quadrupole mass spectrometer (TSQ Vantage EMR; Thermo scientific) was used to design a selected reaction monitoring (SRM)
method. LC-SRM/MS was performed by a TSQ Vantage
EMR mass spectrometer equipped with a nanoLC interface(AMR), a nano-Advance UHPLC system (Bruker Daltonics)
and an HTC-PAL autosampler (CTC Analytics, Basingstoke,
UK). The parameters of the mass spectrometer were set as follows: 0.002 m/z scan width, 40 msec scan time, 0.7 fwhm
Q1 and Q3 resolution and 1.8 mTorr gas pressure.
For data analysis of the peak area in the chromatogram ofeach SRM transition, the peak area was extracted by using Pinpoint software (Thermo scientific). To confirm whether the
peaks of SRM chromatogram were derived from the endogenous YALSQDVCTYR peptide, we checked that ratios among
the peak areas of 6 SRM transitions were comparable withthose of the standard YALSQDVCTYR peptide. Three of six
SRM transitions, which had a lower variability in the ratio
between the 18 runs of LC-SRM/MS analysis, were used forthe quantification of the endogenous YALSQDVCTYR peptide. Chromatograms of SRM transition are shown in Fig. S5
in Supporting Information. Interassay coefficient of variation
was 7.9%. The lower limit of sensitivity was 13.27 attomole.

Statistical analyses
All data are shown as means  SEM. Comparison of twogroups was made using two-sample t-test. A value of P < 0.05
was considered as statistically significant. The analyses were
performed by Microsoft Excel (Microsoft, Redmond, WA,USA) or R software (see The Foundation for Statistical Computing, http://www.R-project.org).

Acknowledgements
We thank Shigehiro Kuraku for the ABI prism 7900 equipment and Kenichi Inoue, Hiroshi Kiyonari and Kazuki Nakao
for providing us TSHbLuc mice with IVF technique. We are
grateful to the Laboratory for Animal Resources and Genetic
Engineering for housing the mice. We also thank Arthur Millius for proof reading of the manuscript. This work was supported by a Grant-in-Aid for Scientific Research on
Innovative Areas ‘Spying minority in biological phenomena(No.3306)’ (23115006) of MEXT, Japan, by Research Program of Innovative Cell Biology by Innovative Technology of
MEXT, Japan (to H. R. Ueda), by RIKEN Junior Research
Associate program for graduate students (to K. Tsujino), by an
intramural Grant-in-Aid from the RIKEN Quantitative Biol-

ogy Center (to H. R. Ueda), by an intramural Grant-in-Aid
from the RIKEN Center for Developmental Biology (toH. R. Ueda), by Intramural Grant-in-Aid of Utsunomiya
University Center for Optical Research & Education (to M.
Iigo) and by the Special Postdoctoral Researchers Program at
RIKEN (to E. A. Susaki).

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Received: 1 March 2013
Accepted: 23 March 2013

Supporting Information
Additional Supporting Information may be found in the
online version of this article at the publisher’s web site:
Figure S1 (A) Time-lapse images of neonatal PT slice fromheterozygous TSHbLuc mice kept under L12:D12 conditions.
Scale bar: 100 lm. (see also Video S1 in Supporting Information) (B) Representative bioluminescence patterns in individual neonatal PT cells in Fig. S1A. Scale bar: 100 lm.
Figure S2 Confirmation of expression level changes of TSHb,
Eya3, and Cga in the cultured PT by qPCR analysis.

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K Tsujino et al.

Figure S3 Real-time monitoring of the luciferase reporter
activities in neonatal PT slices with glutamine or glutamic acidtreatment.
Figure S4 Histograms of the bottom time of bioluminescenceof adult long-day PT samples (n = 19, upper) and neonatal PT
samples (n = 157, bottom) that were treated with vehicle.

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Figure S5 The chromatograms of each selected reaction monitoring (SRM) transition.
Video S1 Real-time imaging of bioluminescence in a neonatal PT slice for 4 days.

© 2013 The Authors
Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd