Vol 452 | 20 March 2008 | doi:10.1038/nature06738

ARTICLES
Thyrotrophin in the pars tuberalis triggers
photoperiodic response
Nobuhiro Nakao1*, Hiroko Ono1*, Takashi Yamamura1, Tsubasa Anraku1, Tsuyoshi Takagi1, Kumiko Higashi1,
Shinobu Yasuo1, Yasuhiro Katou1, Saburo Kageyama1, Yumiko Uno1, Takeya Kasukawa2, Masayuki Iigo3,
Peter J. Sharp4, Atsushi Iwasawa5, Yutaka Suzuki6, Sumio Sugano6, Teruyuki Niimi7, Makoto Mizutani8,
Takao Namikawa8, Shizufumi Ebihara1,8, Hiroki R. Ueda2,9 & Takashi Yoshimura1,8,10
Molecular mechanisms regulating animal seasonal breeding in response to changing photoperiod are not well understood.
Rapid induction of gene expression of thyroid-hormone-activating enzyme (type 2 deiodinase, DIO2) in the mediobasal
hypothalamus (MBH) of the Japanese quail (Coturnix japonica) is the earliest event yet recorded in the photoperiodic signaltransduction pathway. Here we show cascades of gene expression in the quail MBH associated with the initiation ofphotoinduced secretion of luteinizing hormone. We identified two waves of gene expression. The first was initiated about
14 h after dawn of the first long day and included increased thyrotrophin (TSH) b-subunit expression in the pars tuberalis; thesecond occurred approximately 4 h later and included increased expression of DIO2. Intracerebroventricular (ICV)
administration of TSH to short-day quail stimulated gonadal growth and expression of DIO2 which was shown to be mediatedthrough a TSH receptor–cyclic AMP (cAMP) signalling pathway. Increased TSH in the pars tuberalis therefore seems totrigger long-day photoinduced seasonal breeding.
Animals living outside the tropics use changes in photoperiod toadapt to seasonal changes in environment, but the molecularmechanisms underlying photoperiodic time measurement are notfully understood1. The Japanese quail is a robust model for the studyof these mechanisms because of its rapid and dramatic response tochanges in photoperiod. When quail are transferred from short tolong days, plasma luteinizing hormone increases at the end of the firstlong day: this photoperiodic response is the core feature of the avian‘first day release model’ of reproductive photoperiodism2,3. In birds,
the components required for photoperiodic signal transduction arelocated in the mediobasal hypothalamus (MBH) and include a deepbrain photoreceptor4, a clock to measure daylength5, and output
pathways to regulate the secretion of gonadotrophin-releasing hormone (GnRH)6,7. Recently, we have reported that long-day-induced
local activation of thyroid hormone metabolism in the quail MBH isan early event in photoperiodic signal transduction8,9. Under shortday conditions, expression of type 2 deiodinase (DIO2), which converts the prohormone thyroxine (T4) to bioactive triiodothyronine(T3), is maintained at a low level, whereas expression of type 3 deiodinase (DIO3), which metabolizes T4 and T3 to reverse (r)T3 and T2,
respectively, is maintained at a high level. When quail are transferredfrom short to long days, rapid reciprocal switches in DIO2 and DIO3
expression occur at the end of the first long day, resulting in a local
increase in T3 concentration. This increase in MBH T3 concentration
precedes the first rise in the concentration of photoinduced plasma
luteinizing hormone and is causally related. Administration of T3
to short-day quail stimulates secretion of luteinizing hormone andtesticular growth, whereas conversely, administration of a DIO2
inhibitor inhibits photoinduced testicular growth8,10. The question
now is the identity of the photoperiodic transduction pathway regulating DIO2 expression in the MBH.
To address this, we have dissected the molecular dynamics of gene
expression regulating photoinduced thyroid hormone metabolism inthe quail MBH during the first day of photoinduced luteinizing
hormone secretion by using a chicken high-density oligonucleotide
microarray. Quail and chicken are both galliforms with predictedhigh interspecific DNA sequence conservation. To test this prediction, we applied biotinylated chicken and quail genomic DNA to thearray. Signals for 82.2% of the probes were statistically indistinguishable between the two species (Welch’s t-test, Benjamini and
Hochberg false discovery rate (FDR) multiple test, P . 0.05, n 5 3)
(Supplementary Fig. 1).
Genome-wide expression analysis
To study changes in gene expression during the first long day, eightweek-old male quail kept under short days (6/18 h light/dark cycle)
for four weeks were transferred to long days (20/4 h light/dark cycle).
Plasma samples and brains were collected from six birds every 4 h forthree days during this transition. In addition, samples were collected
every 2 h between 10 and 22 h after dawn of the first long day to cover
the period during the initiation of the photoperiodic response whenthe most rapid changes in gene expression were predicted to occur2,3.
The first increase in plasma luteinizing hormone was observed at 22 hafter dawn of the first long day as previously reported3,11 (Fig. 1a)

1
Division of Biomodelling, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. 2Functional Genomics Subunit, Centre forDevelopmental Biology, RIKEN, 2-3-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan. 3Department of Applied Biochemistry, Faculty of Agriculture, Utsunomiya
University, 350 Mine-machi, Utsunomiya, 321-8505, Japan. 4Division of Genetics and Genomics, Roslin Institute and Royal (Dick) School of Veterinary Studies, University of
Edinburgh, Roslin, Midlothian EH25 9PS, UK. 5Laboratory of Comparative Biochemistry, Graduate School of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan.
6
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. 7Division of
Biofunctions Development, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. 8Avian Bioscience Research Centre,
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. 9Laboratory for Systems Biology, Centre for Developmental Biology,
RIKEN, 2-3-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan. 10Institute for Advanced Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
*These authors contributed equally to this work.

317©2008 Nature Publishing GroupARTICLES

NATURE | Vol 452 | 20 March 2008

that encoding the TSH-b suggests that the proteins encoded by thesegenes associate in the pars tuberalis to form TSH. This view is supported by the observation that TSH-b protein occurs in pars tuberalis
cells (Fig. 2a). We therefore deduced that increased TSH in the pars
tuberalis may be functionally significant for photoperiodic signaltransduction. To test this hypothesis, we first determined whether
TSH receptor (TSHR) gene expression occurs in the MBH.

a

Plasma luteinizing hormone(ng ml–1)(one-way analysis of variance (ANOVA), F20,105 5 5.860, P , 0.0001,
Fisher’s least significant difference (LSD) post-hoc test, P , 0.05,
n 5 6).
For each time point, biotinylated antisense RNAs (cRNAs) prepared from pooled MBH were hybridized to duplicate sets of arrays
to minimize experimental error. Using the RMA algorithm andstatistical cosine filters12, we identified 77 cycling genes which weredisqualified from consideration as long-day-induced genes (Fig. 1band Supplementary Table 1). These genes included eight circadianclock genes (Supplementary Fig. 2). We next focused on genes showing 1.5-fold or more changes in expression during the first long day,and found two waves of expression initiated at around 14 h (peaktime 16.46 h, Fig. 1a) and the other initiated at around 18 h after
dawn (peak time 21.15 h, Fig. 1a) (Supplementary Table 2 andSupplementary Fig. 3) (Welch’s one-way ANOVA, FDR P , 0.01).
The first wave comprised two genes encoding thyrotrophin-b(TSH-b) and eyes absent 3 (EYA3); the second wave comprised 11
genes including DIO2 and DIO3 which showed inversely relatedchanges in expression (Supplementary Fig. 4). Using in situ hybridization, the expression of the two first-wave genes (TSHB and EYA3)
was observed in the pars tuberalis of the pituitary gland, whereas
expression of six of the second-wave genes including DIO2 andDIO3 was observed in the ependymal cells lining the ventro-lateralwalls of third ventricle and in the adjacent infundibular nucleus(Fig. 1c).
We also noted rhythmic expression of the gene encoding common
pituitary glycoprotein alpha subunit (CGA) in the pars tuberalis(Fig. 1c and Supplementary Fig. 2). The expression of this gene with
b
8

*
*

4

*

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–2SD

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2
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0

12

24

36

48

EYA3

12
First wave
8

4
0
–24
4.5

–12

0

12

ICER
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PLSCR1
LOC415818

24

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48

SOCS3
RASD1
EST
LOC421368

NR4A3
DIO3

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3

1.5

0
–24

–12

0

12
24
Time (h)36cNormalized signal intensity

6

TSHB

Normalized signal intensity

TSH receptor in the MBH
Strong expression of TSHR was observed in the ependymal cells andpars tuberalis; weak expression was observed in the infundibular
nucleus (Fig. 2b). TSHR expression in the pars tuberalis was detected
at 6 and 22 h after dawn of the first long day but not at 14 h, whereas it
occurred in the ependymal cells all the times examined (n 5 2). To
verify these results, we further performed a 125I-labelled TSH binding
assay. We first demonstrated specific binding of 125I-labelled TSH in
the thyroid gland of quail as a positive control (Fig. 2c). Specificity ofthe binding assay was also confirmed by radioreceptor assay (Supple
mentary Fig. 5). We then observed specific binding of 125I-labelled
TSH in the ependymal cells, infundibular nucleus and the pars tuberalis. This observation is consistent with expression sites of TSHR
mRNA at these loci. Although TSH binding in the ependymal cells
was observed at all the times examined, that in the pars tuberalis
was undetectable at time 22 h (Fig. 2d). Because the median eminence
is one of the circumventricular organs and is outside the blood–brain
barrier13, long-day-induced TSH in the pars tuberalis has thepotential to enter the brain to interact with TSHR in the ependymal
cells and infundibular nucleus. We therefore predicted that the

48

0

+2SD
CGA

PER2
CRY1
DLG5
E4BP4
CR388823
HTRA3
BX936168
FGD3
CR385166
SGPL1
LOC422242
CD764248
PHLPP
CGA
LOC416774
BU353686
SEPP1
PAPSS1
BMAL1
CR386060
LOC425395
FGFR2
TARDBP
NCAM1
BU415550
LOC424441
LOC419338
ACVR1
APOH
LOC424308
CRYAB
CR387761
ENSGALT00000003108
COMT
CR524314
CR388514
LOC396473
ENSGALT00000022222
LOC427675
CR524099
LOC424570
LOC416371
IPO13
PKP4
LOC425380
LOC418178
SCN5A
BU379404
LOC426871
KIF20AREV- ERBB
DEC1
BX932362
BX932098
ENSGALT00000016696
BU118866
LOC417217
DEC2
CR523575
CADPS
BX275469
HCRT
IGSF4DSNCB
BU477621
MDH1
SNCA
CR387467
BU312867
PER3
AL588323
BU354163
ALC
BX273662
TPI1
LOC421058
ZA20D2

TSHB

EYA3

DIO2

ICER

NR4A3

CEBPB

SOCS3

DIO3
6

–24

–12

Figure 1 | Plasma luteinizing hormone and genome-wide analysis of genes
expressed in the quail MBH during the first day of photostimulation (time0 h is dawn of the first long day). a, Changes in plasma luteinizing hormone(mean 6 s.e.m., n 5 6, *P , 0.05 versus the value at time 222 h); and timingof first- and second-wave gene expression. Data are normalized such that themedian signal strength for each gene over all time points was 1.0. The average
signal strength at each point was then displayed as a ratio relative to themedian signal strength of that gene. b, Organization of 77 genes showing

0

12Time (h)

24

36

48

14
Time (h)2224 h rhythmic changes in expression. Data are normalized such that themean and s.d. of log expression values over all time points for each gene are 0
and 1, respectively. Italic script, gene identities and accession numbers.
c, Spatio-temporal expression of common glycoprotein hormone subunit(CGA), first-wave (red bar) and second-wave genes (blue bar). Expression of
CGA and first-wave genes was observed in the pars tuberalis, whereas that ofsecond-wave genes was observed in the ependymal cells and the infundibular
nucleus.

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NATURE | Vol 452 | 20 March 2008

photoinduced increase in pars tuberalis TSH may function to stimulate the expression of DIO2 and possibly other second-wave genes.
TSH regulation of DIO2 geneTo test this prediction, a range of doses (0.01, 0.1, 1.0 mIU) of bovine
TSH14 dissolved in 10 ml saline was administered 16 h after dawn toshort-day quail to correspond with the time that TSHB expression isat its highest in the pars tuberalis after dawn of a first long day. Brains
were collected 4 h after the injection when the induction of secondwave genes was predicted to be maximal. As shown in Fig. 3a, b,
intracerebroventricular (ICV) TSH injection induced the expressionof the DIO2 and three other second-wave genes in a dose-dependent
manner (one-way ANOVA, Fisher’s LSD post-hoc test, P , 0.05,
n 5 3–6). Induction of these genes was observed in the dorsal andventrolateral ependymal cells and in the infundibular nucleus (Figs 3a
and 5a) and was more prominent in the ependymal cells than inphotostimulated birds (Fig. 1c and Supplementary Fig. 6). This is
likely to be a consequence of the periventricular ependymal cells
being more assessable to ICV TSH than to TSH originating fromthe pars tuberalis. We further confirmed this physiological effect of
TSH by ICV injection of TSH-b antibody to long-day quail. Antichicken/quail TSH-b IgG15 or pre-immune serum IgG (1 mg per
10 ml) was administered every 2 h (ref. 16) from 12 h to 18 h after
dawn of the first long day, and brains were collected 2 h after the last
injection. As shown in Fig. 3c, d, anti-TSH-b IgG injections suppressed the expression of the four second-wave genes, including
DIO2, shown to be induced by ICV TSH.

a

Involvement of cyclic AMP signalling pathway
To further address the mechanism through which TSH might regulate the expression of DIO2 and three other second-wave genes wefirst determined the transcriptional start sites using the oligo-capping
method17 and mapped them to quail and chicken genome sequences(Fig. 4a, Supplementary Fig. 7a). It is reported that the expressionof DIO2 in human thyroid gland is regulated through a TSHR-GsacAMP regulatory cascade18. We found several putative cAMP
responsive elements (CREs) in the 1.5 kilobase (kb) 59 upstream
regions of DIO2, in quail and chicken (Fig. 4a) and in the three othersecond-wave genes (Supplementary Fig. 7a). Conservation of CREs
between the two species suggests the functional significance of thiselement (Fig. 4b and Supplementary Fig. 7b). To validate whether
these CREs are involved in the regulation of DIO2 gene by TSH, weanalysed the promoter activity of the DIO2 gene transfected into
the 293 cell line. TSH administration induced expression of DIO2
reporter activity in a dose-dependent manner only when TSHR was
co-transfected (Supplementary Fig. 8). However, when CREs weremutated, induction by TSH was not observed (Fig. 4c). These results
demonstrate that induction of the DIO2 gene by TSH involves acAMP signalling pathway through TSHR.
Photoperiodically regulated output genes
We next performed a microarray analysis on quail kept under shortand long-day conditions for two weeks to assess the chronic effects ofphotostimulation on MBH gene expression. MBH samples were collected from six birds every 4 h during a 24 h lighting cycle. This
analysis identified 183 differentially expressed genes (Welch’s twoway ANOVA, FDR P , 0.05) (Supplementary Fig. 9a, b and Supplementary Table 3). Among these genes, 124 were upregulated and
59 were downregulated under long-day conditions (Supplementary

18
SD

12

18

c
DIO2

DIO2

ICER

ICER

NR4A3

NR4A3

TSHR

Specific

CEBPB

CEBPB

c
Nonspecific

Specific22

1.0

TSH (mIU)
EC
PT

0.04

0.02

0
6

14
Time (h)22

Figure 2 | Localization of TSH-b and TSHR in the pars tuberalis and MBH.
a, Positive immunolabelling for TSH-b in the pars tuberalis (arrowhead)
induced by the long-day stimulus was eliminated by pre-adsorption of theanti-TSH-b antibody with the synthetic TSH-b peptide sequence used to
produce the antibody. Scale bars: left, 100 mm; right, 10 mm. SD, short day;LD, long day. b, Expression of TSHR mRNA in the ependymal cells wasobserved at all the times examined, whereas that in the pars tuberalis(arrowhead) was not observed at time 14 h (n 5 2). c, d, Binding of 125Ilabelled TSH to quail thyroid gland (c) and the MBH (d). Although TSH
binding in the ependymal cells and the infundibular nucleus was observed at
all the times examined, that in the pars tuberalis was not observed at time
22 h (n 5 2).

b

d 0.5
0.3

DIO2
ICER
NR4A3
CEBPB

**

0.2
**
**
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0.4
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Saline10–2

10–1
TSH (mIU)

100

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ICER
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Ig -β
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0.04

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EC
PT

0.06

(nCi)

0.08

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TSHR mRNA level (nCi)d14Time (h)

-β

LD

b

6

SH

m
m

Time (h)P
Ig reG iaAdsorptionA
Ig nti
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un

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TSH-β

Figure 3 | Induction of the expression of DIO2 and three other second-wave
genes by ICV injection of TSH and inhibition by ICV injection of anti-TSH-b
IgG. ICV injection of TSH (a, b) and anti-TSH-b IgG (c, d). Representative
autoradiograms (a, c) and densitometric quantification (b, d) showing theeffect of ICV injections of TSH/anti-TSH-b (b, *P , 0.05, **P , 0.01,
ANOVA, Fisher’s LSD post-hoc test, n 5 3–6; d, *P , 0.05, **P , 0.01,
t-test, mean 1 s.e.m., n 5 3).
319©2008 Nature Publishing GroupARTICLES

NATURE | Vol 452 | 20 March 2008ba

Kilobase
1.5

Base pair–1,500–1,000

–500

Quail
0

0

Chicken

Quail
Chicken

1.5
Kilobasec–1,504–601–332 –95–1

M1
WT –336 CCACGTCA –329
M1
CC----CA

M2

WT –99 TGACGTCA –92
M2
TG----CA

M1 M2
pGL3-basic

TSH (+)TSH (−)
0

1

2
3
4
5
Relative luciferase activity

6

7

Figure 4 | Involvement of a cAMP signalling pathway in TSH induction of
DIO2 gene expression. a, Comparison of quail and chicken DIO2 gene 59
upstream sequences. Conserved segments are boxed. Filled ovals, putative
CRE sites. b, Dot plot analysis of 59 upstream regions using BLASTN, withgreater than 85% identity. c, Promoter activity of quail DIO2. Wild-type anddeletion/mutant reporters fused to the luciferase gene were assayed for theiractivities in response to TSH. Each value represents the mean 6 s.e.m. of
three replicates for a single assay.

Fig. 9a, b). We found long-day-induced expression of DIO2 andreduced expression of DIO3, as previously reported8,9 (Supplementary Fig. 10). In situ analysis of genes with known functions andshowing differences in expression between long and short days, inthe MBH, other than DIO2 and DIO3, confirmed the microarray
analysis (Supplementary Fig. 9c). In addition, we found a set of genes
encoding various hormones and hormone receptors, which included
TSHB and CGA (Supplementary Fig. 10 and Supplementary Table 3).
Because high expression of TSHB and CGA was observed underchronic long-day conditions, we deduced that increased pars tuberalis TSH may not only play a role in initiating photoinduced secretion of luteinizing hormone, but may also be necessary to maintainthe expression of other genes required to support a full reproductive
response. We therefore investigated this possibility by prolonged ICV
infusion of TSH (1.2 mIU per day) in short-day quail between 8 and
10 weeks of age. This treatment simulated MBH DIO2 expression andgonadal development (Fig. 5).

in DIO2, a key element in the photoperiodic signal transduction
pathway8. EYA3 is a transcriptional co-activator involved in thedevelopment of the eye and forms a nuclear complex with SIX (sineoculis) DNA-binding homeodomain factor and DACH (dachshund)nuclear cofactors20; we considered the possibility that the photoinduction of EYA3 may induce expression of second-wave genes.
However, this is unlikely because if EYA3 is involved in second-wave
gene expression it would need to be co-localized with these genes toexert its function. In the present study, EYA3 was expressed in thepars tuberalis whereas second-wave genes were expressed in theependymal cells and infundibular nucleus. Several SIX genes wereobserved in the pars tuberalis (Supplementary Fig. 11), suggestingthat these may interact with long-day-induced EYA3 to regulate theexpression of genes, the identity of which remains to be established.
We therefore focused on the possibility that TSHB in the pars tuberalis might be involved in the initiation of DIO2 and the expression ofother second-wave genes. Among the various cycling genes, we foundrhythmic expression of CGA, and the peak of CGA preceded that ofba8
Testicular growth (mm)0.16
DIO2 mRNA level (nCi)

Discussion
We have used the ‘first day release model’ of photoinduced luteinizing hormone release in quail to dissect the temporal pattern ofchanges in gene expression in the MBH associated with the initiationof photoinduced reproductive function. We found 77 genes that
displayed a temporal pattern of expression under short and long days
that would be expected of clock genes or clock-driven genes. Because
most cycling genes are tissue specific12,19, future analyses of the relations between the functions of these genes are likely to reveal further
details of the molecular basis of the photoperiodic response. Our
most important observation was the photoinduction of a first wave
of gene expression initiated about 14 h after dawn of the first longday, comprising TSHB and EYA3 in the pars tuberalis. These changes
in gene expression are the earliest yet reported, to our knowledge, forthe photoperiodic signal transduction pathway. This was followed
approximately 4 h later by a second wave of gene expression in theependymal cells and infundibular nucleus and included an increase

0.12
0.08

*

0.04
0

Saline

TSH

6

4

2

**
0

SalineTSH

Figure 5 | Effect of chronic ICV infusion of TSH on MBH DIO2 expression
and testicular growth under short-day conditions. a, MBH DIO2 expression.
b, Testicular growth. (*P , 0.05, **P , 0.01, t-test, mean 1 s.e.m. n 5 5).

320©2008 Nature Publishing GroupARTICLES

NATURE | Vol 452 | 20 March 2008

long-day-induced TSHB. The biological activity of TSH requires anon-covalent association of CGA and TSH-b21. Thus it appears thatthe cycling CGA is available for dimerization with long-day-induced
TSH-b to form bioactive TSH. It is also of note that translation of
TSH occurs at least within 20 min21. In addition, unlike luteinizing
hormone, dimerization of TSH-b and CGA, and secretion of TSH isvery rapid and efficient, with a value of t1/2 for the intracellular
disappearance (that is secretion) of about 1 h (ref. 21). The expression of CGA and TSHB in the pars tuberalis therefore indicates that
this is a source of biologically active TSH which may be transported
into the third ventricle, possibly through tanycytes which abut thepars tuberalis22. The target site for pars tuberalis TSH was suggestedby the presence of TSHR gene expression in the ependymal cells,
infundibular nucleus and the pars tuberalis. This observation was
supported by a 125I-labelled TSH binding assay which showed specific TSH binding in these loci. Observations on the effects of ICV
injection of TSH and anti-TSH-b IgG on DIO2 and three othersecond-wave genes demonstrated that TSH triggers the expressionof these genes in the ependymal cells. Furthermore, promoter analysis indicated that the induction of DIO2 is likely to be mediated by
the cAMP-signalling pathway. This observation is consistent with theaction of TSH on human thyroid gland and rat brown adipose tissue,
where DIO2 expression is regulated by a TSHR–cAMP mediatedmechanism18,23. Our study revealed a similar TSHR–cAMP mediatedmechanism in the quail MBH. In addition to the acute effect of ICV
TSH, chronic administration of TSH maintained increased DIO2
expression and induced testicular growth under short-day conditions. This suggests that elevated TSH in the pars tuberalis may berequired to maintain photoinduced reproductive function.
Although it is known that several species become photoperiodically
blind after thyroidectomy, quail can respond to photoperiod evenafter thyroidectomy1. Recently, we have reported the involvement of
TGF-a in the photoperiodism, and that the TGF-a signalling pathway
is not dependent on thyroid hormone activity24. Interestingly, thesimilarity in expression profile between DIO2 and TGF-a suggested
that these two genes share the same transcriptional regulation.
Although we failed to detect TGF-a gene expression in the present
microarray analysis, we found TSH induced expression of TGF-a(Supplementary Fig. 12a, b). It is, therefore, possible that pars tuberalis TSH may signal photoperiodic information through both DIO2
and TGF-a. The magnitude of testicular growth induced by ICV TSH
administration in short-day quail was indistinguishable from that of
intact birds kept under long-day conditions (Supplementary Fig. 12c).
This suggests that TSH is important not only for triggering photoperiodic responses, but also for the maintaining photoperiodicallyinduced reproductive neuroendocrine function.
Since the discovery of dense melatonin receptors in the pars tuberalis in most mammalian species, but not birds, the pars tuberalis in
mammals is considered to be involved in the transmission of photoperiodic stimuli to endocrine outputs through melatonin25,26.
Further, the thyrotrophe cell type in the mammalian pars tuberalis
expresses a high density of melatonin receptors and may regulateseasonal prolactin secretion27. However, unlike mammals, there isno evidence that circulating melatonin plays a role in photoperiodictransduction in birds28. Consequently, the mechanism transducing
photoperiodic information to the avian pars tuberalis remains to bediscovered. However, pars tuberalis TSH may be an evolutionarily
conserved element of a photoperiodic signal transduction pathway inbirds and mammals. This view is consistent with an earlier finding insheep that expression of TSH-b in the pars tuberalis is not regulated
by classical thyrotrophe receptors and their intracellular pathways,
but through a novel, photoperiod-dependent mechanism29.
Our view that photoinduced pars tuberalis TSH in the quail enters
the cerebrospinal fluid to induce a photoperiodic response is consistent with the finding in the hamster that photoperiod-dependentchanges in TSH-like immunoreactivity occur in the pars tuberalis30
whereas TSH is found in the cerebrospinal fluid and central nervous

system (CNS) of mammals31,32. The expression of TSHR has beenreported in the mammalian brain33,34, but no function has been proposed in relation to the control of photoperiodic responsiveness. In
the present study, we show that long-day-induced TSH in the pars
tuberalis triggers the expression of DIO2 in the ependymal cells(Supplementary Fig. 13). To our knowledge, this is the first demonstration of the likely functional significance of pars-tuberalis-derived
TSH in the CNS. Recently, it has been proposed that the pars tuberalis
in sheep may be the circannual pacemaker for seasonal prolactin
secretion35. Thus, the pars tuberalis appears to be the locus for thecontrol of seasonality both in birds and other vertebrates.
In conclusion, one of the most important questions in photoperiodism is the identity of the molecular basis of the mechanism underlying the photoinducible phase that in the quail occurs 12–16 h after
dawn2. Because photoinduction of TSHB expression was observed
from about 12 h after dawn of the first long day, increased TSHB
expression in the pars tuberalis may be the key molecular event
defining the onset of the photoinducible phase. Our study presents
the first comprehensive analysis of changes in hypothalamic gene
expression likely to be involved in the regulation of the long-day
reproductive photoperiodic response, and identifies pars tuberalis
TSH as a key factor controlling photoperiodic signal transduction.
The identification of a key role for TSHB expression in the pars
tuberalis in reproductive photoperiodic time measurement marks amajor advance in our knowledge of molecular mechanisms controlling seasonal breeding.
METHODS SUMMARY
Animals. We used Japanese quail (C. japonica) obtained from a local dealer andchicken (Gallus domesticus) (WL-G) kept in our colony. Because female birds
have ZW chromosomes, they were used for genomic DNA analysis. In all otherexperiments, male quail were used. The present study was approved by theCommittee on Animal Experiments of the Graduate School of Bioagricultural
Sciences, Nagoya University.
Microarray experiments. We used Affymetrix Chicken Genome Array. This
array contains over 38,000 probe sets representing 32,773 transcripts.
Genomic DNA individually extracted from liver using DNeasy tissue kit(QIAGEN) was labelled by BioPrime DNA labelling system (Invitrogen). The
MBH was punched out (2.5 mm diameter) from 3 mm quail brain slices generated using a mouse brain matrix. Total RNA was prepared from two pools of
three MBH at each time point to duplicate our observations on two arrays, using
Trizol reagent (Invitrogen); cDNA synthesis and cRNA labelling reactions
were performed with One-Cycle Target Labelling and Control Reagents Kit(Affymetrix). Hybridization, wash and stain protocols and scanning were performed using standard Affymetrix protocols. Data were analysed by using
GeneSpring GX7.3 software (Agilent Technologies).
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 22 July 2007; accepted 25 January 2008.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank T. Kondo and the Nagoya University Radioisotope
Centre for use of facilities. We also thank J. A. Proudman and A. F. Parlow forproviding the chicken luteinizing hormone radioimmunoassay kit and the bovine
TSH, respectively. This work was done as a part of the Program for Promotion of
Basic Research Activities for Innovative Biosciences (PROBRAIN) and, in part, by
Grant-in-Aid for Scientific Research (S), (B), Grant-in-Aid for Young Scientists (S),
Grant-in-Aid for JSPS Fellows from the Japanese Society for the Promotion of
Science, and a grant of the Genome Network Project from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
Author Contributions T.Yo. conceived and directed the work. N.N., H.O., T.Ya.,
T.A., T.T., K.H., S.Y., Y.K., S.K., Y.U. and T.Yo. performed the microarray analysis
and in situ hybridization. N.N., T.K., H.R.U. and T.Yo. analysed the microarray data.
N.N. performed the quantitative PCR and promoter assay. M.I. and P.J.S.
determined the luteinizing hormone assay. H.O. and M.I. performed the125
I-labelled TSH binding assay. T.Ya. and A.I. performed theimmunocytochemistry. T.Ya., T.A. and A.I. examined the ICV injection andinfusion. N.N., H.O., Y.S. and S.S. determined transcriptional start sites andgenomic DNA sequences. T.Ni. cloned EYA, SIX and DACH family. M.M., T.Na. and
S.E. provided laboratory facilities and new materials. All authors discussed theresults and commented on the manuscript. T.Yo. and P.J.S. wrote the paper.
Author Information The microarray data and DNA sequence information havebeen deposited in NCBI Gene Expression Omnibus (GEO) (GSE8016–GSE8018)
and DDBJ/EMBL/GenBank (AB307676–AB307681), respectively. Reprints andpermissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to T.Yo.
(takashiy@agr.nagoya-u.ac.jp).

322©2008 Nature Publishing Groupdoi:10.1038/nature06738

METHODS
Luteinizing hormone radioimmunoassay. Plasma luteinizing hormone concentrations of quail were determined by radioimmunoassay (RIA) as previously
described24,36.
Discrimination of long-day waves of gene expression. Pearson’s correlation
analysis was used to calculate the timing of the first and second waves of photoinduced gene expression. Correlation values were statistically tested and the peaktime of the expression of each wave was determined by fitting a quadratic function to expression values of 66 h around the time points with the maximum
expression values.
In situ hybridization. In situ hybridization was performed by using antisense andsense 45-nucleotide probes (Supplementary Table 4) as previously described37.
No hybridization signal was observed in sense controls (data not shown).
Quantitative PCR. Reverse transcription was performed on total RNA (0.5 mg)
using ReverTra Ace (Toyobo) and oligo-dT primers. Samples contained 13
SYBR Premix Ex Taq (Takara), 0.3 mM gene-specific primers (Supplementary
Table 5) and 1/20 synthesized cDNA in a 25 ml volume. Quantitative PCR was
performed in duplicates by using ABI Prism 7000 (Applied Biosystems) as follows: 95 uC for 10 s, then 40 cycles of 95 uC for 5 s, 60 uC for 30 s. We used
GAPDH as an internal control.
Immunocytochemistry. Coronal frozen sections (20 mm) were fixed by 4%paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 for 10 min at room temperature. Immunocytochemistry for the anti-chicken/quail TSH-b (1:5,000) was
performed using Vectastain Elite ABC rabbit IgG kit (Vector Laboratories) witha standard protocol.
TSH binding assay. Bovine TSH (AFP8755, NIDDK) was labelled with 125I by
use of [125I]Bolton-Hunter reagent (NEX120H, PerkinElmer) and purified bygel filtration using PD-10 column (GE Healthcare). Frozen sections were airdried for 15 min, preincubated in the binding buffer (50 mM Tris-HCl buffer(pH 7.4) containing 0.1% BSA) at 37 uC for 1 h, and then incubated with 125Ilabelled TSH (78,000 c.p.m. per millilitre) in the binding buffer with (nonspecific binding) or without (specific binding) cold bovine TSH (550 mg ml21,
Sigma) at 37 uC for 1 h. Slides were rinsed in the ice-cold buffer without BSA(twice, 5 min each) followed by a rapid rinse in ice-cold distilled water to remove
buffer salts. Labelled sections were apposed to BioMax MR (Kodak).
ICV TSH and TSH-b antibody administration. One week after the cannula
implantation into the third ventricle of seven-week-old quail, we injected bovine

TSH (T8931, Sigma) or TSH-b antibody through a guide cannula (24-gauge,
6 mm) and measured resulting changes in gene expression. We used bovine TSH,
because avian TSH is unavailable and bovine TSH is known to activate avian
TSHR14. An infrared viewer (NVR 2015, NEC) was used to facilitate TSH injection in darkness. An Alzet 2002 osmotic minipump was used for the prolonged
ICV infusion of TSH, as previously reported8.
Constructs. The 59-flanking region of quail DIO2 was subcloned into pGL3basic vector (Promega) using 59-ttgctgcctctcttctgccggatgaattca-39 and 59tgaaagctctctcaatgcctcaaggtctg-39. Deletion constructs and a mutated CRE sitewere created by PCR-based site-directed mutagenesis38. Mutation in the CRE
site was generated by deletion of the central four nucleotides (299: TGACGTCAR TGCA; 2336: CCACGTCA R CCCA), as previously reported39. Quail TSHR
cDNA was subcloned into pcDNA 3.1 vector (Invitrogen) using 59catgctgtggctgcctgtcgcct-39, 59-tcacagctcagtttgcctgc-39. Constructs were verified
by sequencing.
Transfection and luciferase assay. The 293 cells (RIKEN BRC Cell Bank) wereplated in 24-well plates at a density of about 2 3 105 cells per well in 1 ml MEM
supplemented with 10% fetal calf serum and 0.1 mM NEAA. DIO2 promoter–luciferase construct (400 ng) was co-transfected with TSHR expression vector(400 ng) and the Renilla luciferase (phRL-TK, 2 ng, Promega) (for an internal
control for transfection efficiency) using LipofectAMINE 2000 reagent(Invitrogen). After 24 h, cells were washed by PBS and treated with or without
1 mIU ml21 bovine TSH (Sigma) in MEM medium. Five hours after this treatment, these media were washed with ice-cold PBS and transcriptional activity
was determined using the dual-luciferase assay system (Promega) with Lumat
LB950 (Berthold) according to the manufacturer’s protocols. Firefly relative
luciferase unit (RLU) measurements were normalized to Renilla RLU.
36. Krishnan, K. A., Proudman, J. A. & Bahr, J. M. Purification and partial
characterization of isoforms of luteinizing hormone from the chicken pituitary
gland. Comp. Biochem. Physiol. B 108, 253–264 (2004).
37. Yoshimura, T. et al. Molecular analysis of avian circadian clock genes. Brain Res.
Mol. Brain Res. 78, 207–215 (2000).
38. Imai, Y., Matsushima, Y., Sugimura, T. & Terada, M. A simple and rapid method
for generating a deletion by PCR. Nucleic Acids Res. 19, 2785 (1991).
39. Travnickova-Bendova, Z., Cermakian, N., Reppert, S. M. & Sassone-Corsi, P.
Bimodal regulation of mPeriod promoters by CREB-dependent signalling and
CLOCK/BMAL1 activity. Proc. Natl Acad. Sci. USA 99, 7728–7733 (2002).

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