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.
1.
2.

3.

4.
5.

6.

7.
8.

Dawson, A., King, V. M., Bentley, G. E. & Ball, G. F. Photoperiodic control ofseasonality in birds. J. Biol. Rhythms 16, 365–380 (2001).
Nicholls, T. J., Follett, B. K. & Robinson, J. E. A photoperiodic response ingonadectomised Japanese quail exposed to a single long day. J. Endocrinol. 97,
121–126 (1983).
Follett, B. K., King, V. M. & Meddle, S. L. in Biological Rhythms and Photoperiodism in
Plants (eds Lumsden, P. J. & Miller, A. J.) 231–242 (BIOS Scientific Publishers Ltd,
Oxford, 1998).
Silver, R. et al. Coexpression of opsin- and VIP-like-immunoreactivity in CSFcontacting neurons of the avian brain. Cell Tissue Res. 253, 189–198 (1988).
Yasuo, S., Watanabe, M., Okabayashi, N., Ebihara, S. & Yoshimura, T. Circadianclock genes and photoperiodism: comprehensive analysis of clock genes
expression in the mediobasal hypothalamus, the suprachiasmatic nucleus and thepineal gland of Japanese quail under various light schedules. Endocrinology 144,
3742–3748 (2003).
Sharp, P. J. & Follett, B. K. The effect of hypothalamic lesions on gonadotrophin
release in Japanese quail (Coturnix coturnix japonica). Neuroendocrinology 5,
205–218 (1969).
Juss, T. S. in Avian Endocrinology (ed. Sharp, P. J.) 47–60 (Society for
Endocrinology, Bristol, UK, 1993).
Yoshimura, T. et al. Light-induced hormone conversion of T4 to T3 regulates
photoperiodic response of gonads in birds. Nature 426, 178–181 (2003).

321©2008 Nature Publishing GroupARTICLES

9.

NATURE | Vol 452 | 20 March 2008

Yasuo, S. et al. The reciprocal switching of two thyroid hormone-activating and inactivating enzyme genes is involved in the photoperiodic gonadal response ofJapanese quail. Endocrinology 146, 2551–2554 (2005).
10. Follett, B. K. & Nicholls, T. J. Acute effect of thyroid hormones in mimicking
photoperiodically induced release of gonadotropins in Japanese quail. J. Comp.
Physiol. B 157, 837–843 (1988).
11. Wada, M. Photoperiodic control of LH secretion in Japanese quail with specialreference to the photoinducible phase. Gen. Comp. Endocrinol. 39, 141–149 (1979).
12. Ueda, H. R. et al. A transcription factor response element for gene expression
during circadian night. Nature 418, 534–539 (2002).
13. Ganong, W. F. Circumventricular organs: definition and role in the regulation of
endocrine and autonomic function. Clin. Exp. Pharmacol. Physiol. 27, 422–427(2000).
14. Grommen, S. V. H. et al. Molecular cloning, tissue distribution, and ontogenic
thyroidal expression of the chicken thyrotropin receptor. Endocrinology 147,
3943–3951 (2006).
15. Iwasawa, A. et al. Specific anti-peptide antibody to b subunit of chicken
thyrotropin: production and characterization. J. Reprod. Dev. 48, 197–204 (2002).
16. Turnbull, A. V. & Rivier, C. L. Intracerebroventricular passive immunization. II.
Intracerebroventricular infusion of neuropeptide antisera can inhibit
neuropeptide signalling in peripheral tissues. Endocrinology 139, 128–136 (1998).
17. Maruyama, K. & Sugano, S. Oligo-capping: a simple method to replace the cap
structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171–174(1994).
18. Murakami, M. et al. Expression and regulation of type II iodothyronine deiodinasein human thyroid gland. Endocrinology 142, 2961–2967 (2001).
19. Panda, S. et al. Coordinated transcription of key pathways in the mouse by thecircadian clock. Cell 109, 307–320 (2002).
20. Rebay, I., Silver, S. J. & Tootle, T. L. New vision from Eyes absent: transcription
factors as enzymes. Trends Genet. 21, 163–171 (2005).
21. Matzuk, M. M., Kornmeier, C. M., Whitfield, G. K., Kourides, I. A. & Boime, I. The
glycoprotein a-subunit is critical for secretion and stability of the human
thyrotropin b-subunit. Mol. Endocrinol. 2, 95–100 (1988).
22. Sharp, P. J. Tanycyte and vascular patterns in the basal hypothalamus of Coturnix
quail with reference to their possible neuroendocrine significance. Z. Zellforsch.
Mikrosk. Anat. 127, 552–569 (1972).
23. Murakami, M. et al. Thyrotropin receptors in brown adipose tissue: thyrotropin
stimulates type II iodothyroine deiodinase and uncoupling protein-1 in brown
adipocytes. Endocrinology 142, 1195–1201 (2001).
24. Takagi, T. et al. Involvement of transforming growth factor a in the photoperiodic
regulation of reproduction in birds. Endocrinology 148, 2788–2792 (2007).
25. Morgan, P. J. & Williams, L. M. The pars tuberalis of the pituitary: a gateway forneuroendocrine output. Rev. Reprod. 1, 153–161 (1996).
26. Wittkowski, W., Bockmann, J., Kreutz, M. R. & Bockers, T. M. Cell and molecularbiology of the pars tuberalis of the pituitary. Int. Rev. Cytol. 185, 157–194 (1999).
27. Klosen, P. et al. The mt1 melatonin receptor and RORb receptor are co-localized in
specific TSH-immunoreactive cells in the pars tuberalis of the rat pituitary.
J. Histochem. Cytochem. 50, 1647–1657 (2002).
28. Juss, T., Meddle, S. M., Servant, R. S. & King, V. M. Melatonin and photoperiodic
time measurement in the Japanese quail (Coturnix coturnix japonica). Proc. R. Soc.
Lond. B 254, 21–28 (1993).

29. Bockmann, J. et al. Thyrotropin expression in hypophyseal pars tuberalis-specificcells is 3,5,39-triiodothyronine, thyrotropin-releasing hormone, and Pit-1
independent. Endocrinology 138, 1019–1028 (1997).
30. Wittkowski, W., Bergmann, M., Hoffmann, K. & Pera, F. Photoperiod-dependentchanges in TSH-like immunoreactivity of cells in the hypophysial pars tuberalisof the Djungarian hamster, Phodopus sungorus. Cell Tissue Res. 251, 183–187(1988).
31. Schaub, C., Bluet-Pajot, M. T., Szikla, G., Lornet, C. & Talairach, J. Distribution of
growth hormone and thyroid-stimulating hormone in cerebrospinal fluid andpathological compartments in the central nervous system. J. Neurol. Sci. 31,
123–131 (1977).
32. Hojvat, S., Baker, G., Kirsteins, L. & Lawrence, A. M. TSH in the rat and monkey
brain: distribution, characterization and effect of hypophysectomy.
Neuroendocrinology 34, 327–332 (1982).
33. Bockmann, J., Winter, C., Wittkowski, W., Kreutz, M. R. & Bockers, T. M. Cloning
and expression of a brain-derived TSH receptor. Biochem. Biophys. Res. Commun.
238, 173–178 (1997).
34. Crisanti, P. et al. The expression of thyrotropin receptor in the brain. Endocrinology
142, 812–822 (2001).
35. Lincoln, G. A., Clarke, I. J., Hut, R. A. & Hazlerigg, D. G. Characterizing a
mammalian circannual pacemaker. Science 314, 1941–1944 (2006).

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).

©2008 Nature Publishing Groupdoi: 10.1038/nature06738

SUPPLEMENTARY INFORMATION

Supplementary Figure 1. Histogram showing hybridization signals for chicken(left) and quail (right) genomic DNA analyzed by Chicken GeneChip (n=3).

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Supplementary Figure 2. Independent verification of microarray quantification.
Relative mRNA levels of cycling clock genes and CGA (common pituitary
glycoprotein alpha subunit) were measured with Q-PCR assay in the same
samples that were used to prepare probes for microarray analysis. QuailGAPDH was used as the internal control.

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Supplementary Figure 3. Detailed temporal expression profile of 1st wave and2nd wave genes during the photoinduction process determined by microarray.
Data were normalized such that the median signal strength for each gene over
all time points was 1.0. The average signal strength at each point was thendisplayed as a ratio relative to the median signal strength of that gene.

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Supplementary Figure 4. Independent verification of microarray quantification.
Relative mRNA levels of 1st wave (red characters) and 2nd wave genes (bluecharacters) were measured with Q-PCR assay in the same samples that wereused to prepare probes for microarray analysis. Quail GAPDH was used as theinternal control.

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Supplementary Figure 5. Competitive inhibition by cold bovine TSH of 125I-TSH
binding to quail thyroid membranes. Specificity of 125I-TSH binding was tested byradioreceptor assay (RRA) using quail thyroid membranes. Thyroid glands ofquail were homogenized in ten volumes of ice-cold 50 mM Tris-HCl buffer (pH7.4) with a teflon-glass homogenizer (3,000 rpm x 5 strokes). The homogenate
was centrifuged at 40,000 g for 20 min at 4 ˚C. The pellet was washed,resuspended in the Tris-HCl buffer, and centrifuged for a second time under thesame conditions. The crude membrane pellet was finally resuspended in
Tris-HCl buffer (pH 7.5) and used for RRA. Binding was initiated by the additionof 100 μl aliquots of membranes to tubes containing 125I-bovine TSH(Bolton-Hunter labelled, 17,500 cpm/50 μl in 50 mM Tris HCl (pH 7.4) containing
0.1 % BSA) in the presence of various concentrations of TSH (50 μl). Afterincubation at 37 ˚C for 1 h, tubes were centrifuged at 20,000 g for 2 min at 4 ˚Cand the supernatants were aspirated. Then, the radioactivity of the pellet wascounted with a γ-counter. Non-specific binding was defined as the binding in thepresence of 550 μg/ml cold bovine TSH and the data was expressed as relative
binding. Cold bovine TSH dose-dependently inhibited the binding of 125I-TSH to
the quail thyroid membranes.

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Supplementary Figure 6. Induction of four 2nd wave genes by ICV TSH injection
was prominent in periventricular ependymal cells. Dark field photomicrographsof in situ hybridization are shown.

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Supplementary Figure 7. Comparison of quail and chicken 2nd wave gene 5’
upstream sequences. a. Conserved segments are boxed and putative CRE sites
are shown as solid ovals. open oval: E and E’-boxes. b. Dot plot analysis of quailand chicken 5’ upstream regions using BLASTN, with greater than 80% identity.

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Supplementary Figure 8. Induction of DIO2 mRNA by TSH requires TSHR. The
transcriptional activation of a luciferase reporter containing a 1,642 bp fragmentof the 5’-flanking region of the quail DIO2 gene was assessed by co-transfection
with quail TSHR expression vectors or empty vector in cultured 293 cells.
Twenty four hours after transfection, the cells were incubated with bovine TSH (0,0.1, 1, 10, 100 mIU/ml). The luciferase activity obtained from the reporter was
normalized to the positive control Renilla luciferase reporter. Asterisks, P<0.01,
t-test TSHR (+) versus TSHR (-).

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Supplementary Figure 9. Identification and localization in the MBH ofdifferentially expressed genes over a 24 h period after chronic exposure to shortor long day conditions (left and right hand sides of the panels respectively).
Identification of 124 long day-up-regulated genes (a) and 59 down-regulated
genes (b) and localization of expression of up-regulated (red bar) anddown-regulated (blue bar) named genes (c).

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Supplementary Figure 10. Independent verification of microarray quantification.
Relative mRNA levels of differentially expressed genes under short days (SD)and long days (LD) were measured with Q-PCR in the same samples that wereused to prepare probes for GeneChip analysis. Quail GAPDH was used as theinternal control.

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Supplementary Figure 11. Spatio-temporal expression profile of EYA (eyesabsent), SIX (sine oculis), DACH (dachshund) family in the MBH of quail during
the photoinduction process.

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Supplementary Figure 12. Effect of TSH administration on TGFα mRNA andtesticular growth. a. ICV injection of TSH induced TGFα expression in a dose
dependent manner. Different characters indicate statistically significant
differences (ANOVA, F3,12=16.856, P<0.0001, Fisher’s LSD post-hoc test
P<0.05, n=3-5) b,c. Effect of chronic ICV infusion of TSH on MBH TGFα
expression (b, *P<0.05, t-test, n=4,5) and testicular growth (c, Different
characters indicate statistically significant differences. ANOVA, F3,12=192.40,
p<0.0001, Fisher’s post-hoc test, p<0.001, n=3-5). SD: intact birds kept undershort days; LD intact birds kept under long days for 2 weeks.

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Supplementary Figure 13. Model of the mechanisms regulating photoperiodic
time measurement in birds. Light information received by the deep brain
photoreceptors induces expression of TSHB in the pars tuberalis (PT) of thepituitary gland. Long day-induced TSHβ and cycling CGA (common pituitary
glycoprotein alpha subunit) form TSH in the PT and act on TSH receptor (TSHR)
localized in ependymal cells lining the ventrolateral walls of third ventricle.
Expression of 2nd wave genes includes DIO2 is induced by TSH through
TSHR-cAMP signalling pathway.

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13

S U P P L E M E N TA RY I N FO R M AT I O N

doi: 10.1038/nature06738
Supplementary Table 1

Supplementary table 1. Cycling genes in the MBH of the Japanese quail. Red characters indicates the circadian clock genes.
Affymetrix ID
Gene Symbol
Accession
Description
RefSeqGga.1014.1.S1_at
PER2
CD732610
circadian clock protein
NM_204245
Gga.4107.1.S1_at
CRY1
NM_204245
cryptochrome 1 (photolyase-like)
XM_421604
GgaAffx.20805.1.S1_s_at DLG5
CR523810
discs, large homolog 5 (Drosophila)
NM_204618
Gga.956.1.S1_at
NFIL3(E4BP4)
NM_204618
nuclear factor, interleukin 3 regulated
Gga.17109.1.S1_at
CR388823
Finished cDNA, clone ChEST690b10
XM_420813
Gga.5710.1.S1_a_at
HTRA3
BX934040
HtrA serine peptidase 3
Gga.5520.1.S1_at
BX936168
Finished cDNA, clone ChEST146b12
XM_414331
GgaAffx.21135.1.S1_s_at FGD3
CR523480
FYVE, RhoGEF and PH domain containing 3
Gga.15288.1.S1_at
CR385166
Finished cDNA, clone ChEST543o8
NM_00100794
Gga.16071.1.S1_s_at
SGPL1
BU446083
sphingosine-1-phosphate lyase 1
XM_420230
GgaAffx.3894.1.S1_s_at
LOC422242
ENSGALT00000010111 similar to Mtap7 protein
Gga.8797.1.S1_at
CD764248
Finished cDNA, clone ChEST189k11
GgaAffx.21243.1.S1_s_at PHLPP; PLEKHE1 CR523372
PH domain and leucine rich repeat protein phosphatase XM_418990
GgaAffx.10071.1.S1_at
CGA
ENSGALT00000025514 LOC421829
XM_429886
similar to Yippee-like protein 1 (DiGeorge syndromeXM_415068
GgaAffx.21354.1.S1_at
LOC416774
CR523261
related protein FKSG3)
Gga.15971.1.S1_at
BU353686
Finished cDNA, clone ChEST136j22
NM_00103160
Gga.7044.3.S1_s_at
SEPP1
BU244903
selenoprotein P, plasma, 1
XM_420493
Gga.7016.1.S1_at
PAPSS1
BU138060
3'-phosphoadenosine 5'-phosphosulfate synthase 1
NM_00100146
Gga.1329.1.S1_at
ARNTL; BMAL1
AF246957
aryl hydrocarbon receptor nuclear translocator-like
Gga.16254.1.S1_at
CR386060
Finished cDNA, clone ChEST237h3
similar to 1-phosphatidylinositol-4,5-bisphosphate
phosphodiesterase gamma 2 (Phosphoinositide
XM_423160
GgaAffx.22378.1.S1_s_at LOC425395
ENSGALT00000012718
phospholipase C) (PLC-gamma-2) (Phospholipase Cgamma-2) (PLC-IV)
fibroblast growth factor receptor 2 (bacteria-expressedkinase, keratinocyte growth factor receptor, craniofacial
NM_205319
Gga.3477.1.S1_at
FGFR2; BEK; cek3 NM_205319
dysostosis 1, Crouzon syndrome, Pfeiffer syndrome,
Jackson-Weiss syndrome)
NM_00103087
GgaAffx.12938.9.S1_at
TARDBP
AJ719699
TAR DNA binding protein
GgaAffx.22381.3.S1_at
NCAM1
ENSGALT00000012725 neural cell adhesion molecule 1
XM_425812
Gga.6073.1.S1_at
BU415550
Finished cDNA, clone ChEST152d3
similar to heme binding protein 2; putative heme-binding
XM_422283
GgaAffx.2851.1.S1_at
LOC424441
ENSGALT00000007329
protein
Gga.3242.1.S1_at
LOC419338
AL587491
similar to novel amplified in breast cancer-1
XM_417504
NM_204560
Gga.2875.1.S1_at
ACVR1
NM_204560
activin A receptor, type I
XM_415683
Gga.1609.1.S1_at
APOH
BX932102
apolipoprotein H (beta-2-glycoprotein I)
XM_422153
GgaAffx.7899.1.S1_at
LOC424308
ENSGALT00000020351 similar to KIAA1461 protein
NM_205176
Gga.1999.1.S1_a_at
CRYAB
U26661crystallin, alpha B
Gga.16213.1.S1_at
CR387761
Finished cDNA, clone ChEST532c9
GgaAffx.25359.2.A1_at
ENSGALT00000003108
XM_415077
GgaAffx.25365.1.S1_at
COMT
ENSGALT00000003148 catechol-O-methyltransferase
GgaAffx.20301.1.S1_at
CR524314
Finished cDNA, clone ChEST819a3
Gga.17320.1.S1_at
CR388514
Finished cDNA, clone ChEST1018l10

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UniGene Phase
P-valueFDR
gga.1014
0.38
00
gga.4107
6.57
1.28E-07 9.68E-04
gga.20315
7.08
1.54E-05 2.47E-02
gga.956
7.14
2.23E-06 7.17E-03
gga.17109
7.19
2.05E-07 1.03E-03
gga.5710
7.19
6.44E-05 4.86E-02
gga.5520
7.44
8.19E-05 5.42E-02
gga.7018
7.51
1.12E-04 5.81E-02
gga.15288
7.53
6.20E-05 4.86E-02
gga.16071
7.62
3.07E-05 3.32E-02
7.79
2.04E-05 2.81E-02
gga.8797
7.83
5.60E-05 4.50E-02
gga.20702
7.96
1.74E-04 7.78E-02
7.97
2.39E-05 3.07E-02
8.07

8.85E-05 5.50E-02

8.08
8.23
8.31
8.52
8.64

1.07E-04 5.77E-02
3.06E-06 8.42E-03
2.21E-04 8.43E-02
1.51E-07 9.68E-04
2.11E-04 8.39E-02

8.73

9.61E-05 5.70E-02

gga.3477

8.82

9.90E-08 9.54E-04

gga.16753

8.84
8.84
9.02

7.37E-05 5.17E-02
3.53E-05 3.58E-02
1.43E-05 2.47E-02

9.10

2.32E-04 8.67E-02

9.35
9.40
9.64
9.65
9.67
9.72
9.72
9.83
9.85
9.93

1.38E-04 6.81E-02
1.06E-04 5.77E-02
9.77E-05 5.70E-02
9.08E-05 5.56E-02
9.37E-05 5.64E-02
2.36E-04 8.76E-02
2.55E-04 9.10E-02
2.97E-04 9.95E-02
1.86E-05 2.65E-02
8.31E-05 5.42E-02

gga.15971
gga.7044
gga.10727
gga.1329
gga.16254

gga.6073

gga.2875
gga.1609
gga.1999
gga.16213
gga.7199
gga.20550
gga.17320

1

S U P P L E M E N TA RY I N FO R M AT I O N

doi: 10.1038/nature06738
Supplementary Table 1
Affymetrix ID
Gga.4058.1.S1_at
GgaAffx.8704.1.S1_at
Gga.1026.2.S1_a_at
GgaAffx.20516.1.S1_s_at
Gga.5178.1.S1_at
GgaAffx.2789.1.S1_at
GgaAffx.12959.1.S1_at
GgaAffx.12308.1.S1_s_at
GgaAffx.4781.1.S1_at
GgaAffx.24104.1.S1_at
GgaAffx.3833.5.S1_at
Gga.2154.1.S1_at
Gga.1527.1.S1_at
Gga.1308.2.S1_at
Gga.4315.1.S2_at
Gga.15700.1.S1_at
Gga.10816.1.S1_at
Gga.10636.1.S1_at
GgaAffx.6473.1.A1_at
Gga.14127.1.S1_at
Gga.9491.1.S1_at
Gga.1229.1.S1_at
GgaAffx.21040.1.S1_s_at
GgaAffx.22193.1.S1_s_at
Gga.8026.1.S1_at
Gga.11.1.S1_at
Gga.10186.1.S1_at
Gga.203.1.S1_at
Gga.10505.1.S1_at
GgaAffx.11545.1.S1_s_at
Gga.2726.1.S1_at
Gga.4730.1.S1_at
Gga.17835.1.S1_at
GgaAffx.349.4.S1_s_at
Gga.1673.1.A1_at
Gga.5353.1.S1_at
Gga.5129.1.S1_at
Gga.14806.1.S1_at
Gga.4148.1.S1_at
GgaAffx.9414.1.S1_at
GgaAffx.12473.1.S1_s_at

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Gene Symbol
LOC396473

Accession
Description
RefSeq
NM_205480
myristoylated alanine-rich C kinase substrate (MARCKS) NM_205480
ENSGALT00000022222
XM_425249
LOC427675
BU104660
similar to Rps15a protein
CR524099
Finished cDNA, clone ChEST262g11
XM_422409
LOC424570
BU485658
similar to receptor protein tyrosine phosphatase LAR
XM_414685
LOC416371
ENSGALT00000007194 similar to betaine homocysteine methyl transferase
NM_00100653
IPO13
AJ720856
importin 13
plakophilin 4
NM_00100652
PKP4
AJ720205
XM_423148
LOC425380
ENSGALT00000012398 similar to protein kinase C-theta
XM_416409
LOC418178
ENSGALT00000021384 similar to glutathione S-transferase
XM_418535 ;SCN5A; LOC430306 ENSGALT00000009919 oltage-gated sodium channel type V alphaXM_427864
BU379404
Finished cDNA, clone ChEST794n20
XM_424479
LOC426871
BX931252
similar to DKFZP586A0522 protein
NM_00101278
KIF20A
CR388986
Kinesin family member 20A
NM_205205
NR1D2; REV-ERBB BU434806
nuclear receptor subfamily 1, group D, member 2
bHLHB2(DEC1)
BU144794
Finished cDNA, clone ChEST258o16
BX932362
Finished cDNA, clone ChEST355i12
BX932098
Finished cDNA, clone ChEST38a21
ENSGALT00000016696
BU118866
Finished cDNA, clone ChEST140e9
XM_415501
LOC417217
BU228626
similar to Dynamin-1
bHLHB3(DEC2)
BU244306
Finished cDNA, clone ChEST132b22
CR523575
Finished cDNA, clone ChEST537j12
XM_414412CADPS
ENSGALT00000011770 Ca2+-dependent secretion activator
BX275469
Transcribed locus
NM_204185
HCRT
NM_204185
hypocretin (orexin) neuropeptide precursor
XM_416672
IGSF4D
BX950409
immunoglobulin superfamily, member 4D
NM_204671
SNCB
NM_204671
synuclein, beta
BU477621
Finished cDNA, clone ChEST53b5
NM_00100639
MDH1
AJ719442
malate dehydrogenase 1, NAD (soluble)
NM_204673
SNCA
NM_204673
synuclein, alpha (non A4 component of amyloid
CR387467
Finished cDNA, clone ChEST490o21
BU312867
Finished cDNA, clone ChEST198e11
XM_417527
PER3
ENSGALT00000000799 circadian clock protein
AL588323
Finished cDNA, clone ChEST883o18
Transcribed locus, weakly similar to XP_425603.1
BU354163
PREDICTED: similar to ORF2 [Gallus gallus]
NM_204440
ALC
NM_204440
ALC protein
BX273662
Finished cDNA, clone ChEST39h14
NM_205451
TPI1
NM_205451
triosephosphate isomerase 1
similar to elastin microfibril interfacer 2; extracellular
XM_419145
LOC421058
ENSGALT00000023921
glycoprotein EMILIN-2 precursor
ZA20D2
AJ720370
zinc finger, A20 domain containing 2
NM_00103142

UniGenegga.4058

Phase
P-valueFDR
10.14
1.97E-04 8.34E-02
10.18
6.51E-06 1.39E-02
10.52
1.45E-04 6.95E-02
gga.13061
10.69
2.78E-05 3.32E-02
10.95
4.35E-05 3.81E-02
11.14
8.91E-07 3.43E-03
gga.22612
11.24
3.95E-05 3.80E-02
gga.5277
11.38
3.09E-05 3.32E-02
11.90
8.44E-05 5.42E-02
12.12
1.62E-04 7.61E-02
gga.564

12.43

2.66E-04 9.25E-02

gga.2154
gga.1527
gga.1308
gga.4315
gga.15700
gga.10816
gga.10636

gga.1673

13.05
14.33
18.38
19.47
19.65
20.04
20.07
20.31
20.39
20.62
20.64
20.73
20.83
20.91
21.00
21.05
21.11
21.17
21.20
21.21
21.21
21.30
21.54
22.07

3.78E-05 3.73E-02
4.75E-06 1.22E-02
2.14E-04 8.39E-02
2.14E-07 1.03E-03
5.91E-06 1.37E-02
1.70E-04 7.78E-02
2.17E-04 8.39E-02
1.07E-04 5.77E-02
4.68E-05 4.01E-02
7.56E-06 1.53E-02
1.37E-06 4.80E-03
1.21E-04 6.21E-02
4.17E-05 3.80E-02
2.91E-04 9.83E-02
2.66E-04 9.20E-02
6.81E-05 4.86E-02
3.11E-05 3.32E-02
2.40E-04 8.79E-02
1.84E-04 8.05E-02
9.91E-05 5.70E-02
1.06E-05 2.05E-02
2.49E-04 8.96E-02
1.52E-05 2.47E-02
2.48E-04 8.96E-02

gga.5353

22.20

2.16E-04 8.39E-02

gga.5129
gga.14806
gga.4148

22.38
22.50
22.81

1.75E-04 7.78E-02
1.82E-05 2.65E-02
2.73E-04 9.38E-02

23.18

6.31E-05 4.86E-02

23.29

1.78E-05 2.65E-02

gga.14127
gga.1229
gga.9927
gga.16126
gga.8026
gga.11
gga.10186
gga.203
gga.10505
gga.1141
gga.2726
gga.4730
gga.17835

gga.117162doi: 10.1038/nature06738

S U P P L E M E N TA RY I N FO R M AT I O N

Supplementary Table 2. Photoperiodically induced genes during the photoinduction process in the MBH of the Japanese quail.
Red characters and blue characters indicate 1st wave and 2nd wave genes, respectively.
Affymetrix ID
Gene Symbol Accession
Description
RefSeqGga.551.1.S1_at
TSHB; TSH-B NM_205063
thyroid stimulating hormone, beta
NM_205063
GgaAffx.508.1.S1_at
EYA3
ENSGALT00000001127 Eya3 protein
XM_417715
Gga.1819.1.S1_at
DIO2
NM_204114
deiodinase, iodothyronine, type II
NM_204114
Gga.6220.1.S1_a_at
ICER
NM_204387
ICER protein
NM_204387
Gga.4285.1.S1_at
CEBPB; NF-M NM_205253
CCAAT/enhancer binding protein (C/EBP), beta NM_205253
Gga.13328.1.S1_at
NR4A3
BU436288
nuclear receptor subfamily 4 group A member 3 XM_419081
Gga.915.1.S1_at
SOCS3
NM_204600
suppressor of cytokine signaling 3
NM_204600
GgaAffx.12426.1.S1_s_at PLSCR1
AJ720323
phospholipid scramblase 1
XM_422696
Gga.9972.1.S1_at
RASD1
BX930456
RAS, dexamethasone-induced 1
XM_414811
GgaAffx.3568.1.S1_at
LOC415818 ENSGALT00000009128 similar to hypothetical protein DKFZp434B044 XM_414180
Gga.2461.1.S1_at
BU264985
Transcribed locus
Gga.10787.1.S1_at
LOC421368 BX932508
similar to Jun dimerization protein p21SNFT
XM_419428
Gga.552.1.S1_at
DIO3
Y11273
deiodinase, iodothyronine, type III
XM_426465

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UniGenegga.551
gga.1018
gga.1819
gga.6220
gga.4285
gga.33742
gga.915
gga.7604
gga.9972
gga.2461
gga.10787
gga.552

3

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doi: 10.1038/nature06738

Supplementary Table 3
Supplementary Table 3. Differentially expressed genes between short and long day conditions. Black and blue characters indicate long day-up-regulated genes and -down-regulated genes, respectively.
Affymetrix ID
P-value
Gene Symbol
Accession
Description
RefSeqUniGenegga.17403
Gga.17403.1.S1_at
1.50E-07
BU140017
Finished cDNA, clone ChEST724o21
XM_426465
gga.552
Gga.552.1.S1_at
1.63E-07 DIO3
Y11273
deiodinase, iodothyronine, type III
NM_001012945 gga.5644
Gga.5644.1.S1_s_at
7.69E-07 DNAJA1
BX271179
DnaJ (Hsp40) homolog, subfamily A, member 1
gga.2461
Gga.2461.1.S1_at
9.58E-07
BU264985
Transcribed locus
NM_204114
gga.1819
Gga.1819.1.S1_at
1.03E-06 DIO2
NM_204114
deiodinase, iodothyronine, type II
NM_205063
gga.551
Gga.551.1.S1_at
2.69E-06 TSHB; TSH-B
NM_205063
thyroid stimulating hormone, beta
NM_204412
gga.488
Gga.488.1.S1_at
2.76E-06 ASCL1
NM_204412
achaete-scute complex-like 1(Drosophila)
NM_001001741 gga.2441
GgaAffx.21834.1.S1_s_at
4.22E-06 CCK
NM_001001741
cholecystokinin
Gga.9368.1.S1_at
1.45E-05
BU281873
NM_204807
gga.3035
Gga.3035.1.S1_at
1.84E-05 EPAS1
NM_204807
endothelial PAS domain protein 1
gga.19300
Gga.19300.1.S1_at
1.84E-05
BM426102
Finished cDNA, clone ChEST556c20
XM_419156
GgaAffx.9485.1.S1_at
2.81E-05 LOC421068
ENSGALT00000024095 similar to lung alpha/beta hydrolase fold protein 3
GgaAffx.10071.1.S1_at
4.65E-05 LOC421829(CGA)ENSGALT00000025514 LOC421829
XM_429886
gga.7364
Gga.7364.1.S1_a_at
4.65E-05
BX931037
Finished cDNA, clone ChEST446b11
similar to GREB1 protein isoform a; gene regulated by
XM_419956
GgaAffx.24962.1.S1_s_at
4.77E-05 LOC421944
ENSGALT00000026546
estrogen in breast cancer protein
proopiomelanocortin (adrenocorticotropin/ beta-lipotropin/
Gga.6271.1.S1_at
5.11E-05 POMC
BX540524
NM_001031098 gga.6271
alpha-melanocyte stimulating hormone/ beta-melanocyte
stimulating hormone/ beta-endorphin)
GgaAffx.11648.1.S1_at
5.63E-05 BCL6
AJ719545
B-cell CLL/lymphoma 6 (zinc finger protein 51)
NM_001012930 gga.4457
Gga.12627.1.S1_at
0.000184
BU461086
XM_426301
GgaAffx.23246.2.S1_s_at
0.000184 LOC428743
ENSGALT00000017135 similar to alanine-glyoxylate aminotransferase 2-like 1
XM_423073
GgaAffx.3940.1.S1_at
0.000184 LOC425295
ENSGALT00000010212 similar to Chloride channel protein 2 (ClC-2)
NM_001005431 gga.4750
GgaAffx.22637.1.S1_s_at
0.000184 FKBP5; FKBP51; PPIase ENSGALT00000001399 FK506 binding protein 5
NM_205308
gga.4318
Gga.4318.1.S1_at
0.00025 FABP7
NM_205308
fatty acid binding protein 7, brain
XM_424810
Gga.12454.1.S1_at
0.000628 LOC427223; RLN3
BU435007
similar to relaxin 3 preproprotein; insulin-like 7
NM_204669
gga.3807
Gga.3807.1.S2_s_at
0.000706 ALDH1A3; ALDH6
NM_204669
aldehyde dehydrogenase 1 family, member A3
gga.4865
GgaAffx.24555.1.S1_s_at
0.000778
ENSGALT00000024134 Finished cDNA, clone ChEST284i20
gga.20399
GgaAffx.20969.1.S1_at
0.000954
CR523646
Finished cDNA, clone ChEST322a24
XM_415014
gga.12773
Gga.12773.1.S1_at
0.00107 LOC416717
BX950498
similar to chromosome 20 open reading frame 39
gga.3138
Gga.3138.1.S1_a_at
0.00108 LOC395858
NM_205007
histone macroH2A1.2
NM_205007
gga.16384
Gga.16384.1.S1_at
0.00109
BU236248
Finished cDNA, clone ChEST229g1
similar to solute carrier family 13 (sodium-dependent citrate
XM_415923
GgaAffx.26692.1.S1_at
0.00144 LOC417678
ENSGALT00000009642
transporter), member 5; sodium-coupled citrate transporter
NM_001001761 gga.859
GgaAffx.21816.1.S1_s_at
0.00201 CYP19A1
NM_001001761
cytochrome P450, family 19, subfamily A, polypeptide 1
Gga.9233.1.S1_at
0.00203 GHR
BU231940
Growth hormone receptor precursor
gga.9233
GgaAffx.23689.1.S1_at
0.00203 LOC423515
ENSGALT00000019348 LOC423515
XM_430062
XM_430126
gga.18427
Gga.18427.1.S1_at
0.00246 LOC424098
BU213226
hypothetical gene supported by CR390562
gga.12774
Gga.12774.1.S1_at
0.00252
BX950493
Finished cDNA, clone ChEST523o19
XM_418737
GgaAffx.7118.1.S1_s_at
0.00252 LOC420638
ENSGALT00000018264 similar to 2-hydroxyphytanoyl-CoA lyase (2-HPCL)
XM_414289
GgaAffx.26220.2.S1_s_at
0.00264 LOC415945
ENSGALT00000007128 similar to semaphorin sem2
phosphorylase kinase, gamma 1 (muscle)
NM_001006217 gga.6022
Gga.6022.1.S1_s_at
0.00278 PHKG1
BU106819
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4

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doi: 10.1038/nature06738

Supplementary Table 3
Affymetrix ID
Gga.17706.1.S1_at
Gga.7143.2.S1_a_at
Gga.18324.1.S1_s_at

P-value
Gene Symbol
0.003310.00354 LOC424614
0.00386

GgaAffx.23445.1.S1_s_at

0.0041 LOC422591

GgaAffx.2984.1.S1_s_at

0.00414 LOC422181

Gga.4285.1.S1_at
Gga.6052.3.S1_a_at
Gga.14762.1.S1_at
GgaAffx.21240.1.S1_at
Gga.2608.1.S1_at
GgaAffx.7332.1.S1_at
Gga.14263.1.S1_at
Gga.4003.1.S1_at

0.00414 CEBPB; NF-M
0.00431 LOC422598
0.00455
0.00466
0.00548 DCX; 18C15.5
0.00558 LOC423488
0.00558
0.00593 LOC423209

Gga.16760.1.S1_at

0.006 LOC416710

Gga.19895.1.S1_at
Gga.11081.1.S1_s_at
Gga.9734.1.S1_at
GgaAffx.8162.1.S1_at

0.006
0.006 LOC427493
0.006
0.00613 MC4R

Gga.11300.1.S1_at

0.0063 LOC422439

GgaAffx.1176.1.S1_at
Gga.13008.1.S1_at
Gga.16449.1.S1_at
GgaAffx.2068.1.S1_at
Gga.2606.1.S1_at
Gga.5461.1.S1_at

0.00696 LOC427031
0.00732
0.00734
0.00795 LOC415705
0.00808
0.00865

Gga.5353.1.S1_at

0.00926

Gga.11231.1.S1_at
GgaAffx.21413.1.S1_s_at
GgaAffx.9726.1.S1_at
Gga.2283.1.S2_at
Gga.15955.1.S1_s_at
Gga.6220.2.S1_a_at
Gga.11996.1.S1_at
GgaAffx.24735.1.S1_at
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0.01 LOC419178
0.01080.0113 LOC428533, OPRL1
0.0125 DLL1
0.01260.0127 ICER
0.0127 RASD2
0.0133 LOC420181

Accession
CR388473
BU386059
BU391693

Description
RefSeqUniGenegga.17706
Finished cDNA, clone ChEST591g11
XM_422450
similar to fatty acid amide hydrolase
gga.18324
Finished cDNA, clone ChEST228a16
similar to protein tyrosine phosphatase, non-receptor type 13
isoform 2; protein tyrosine phosphatase, nonreceptor typeENSGALT00000018050
XM_420550
13; protein-tyrosine phosphatase PTPL1; protein tyrosine
phosphatase 1E; Fas-associated phosphatase-1
similar to Phosphorylase B kinase alpha regulatory chain,
XM_420177
ENSGALT00000007661
skeletal muscle isoform (Phosphorylase kinase alpha M
NM_205253
gga.4285
NM_205253
CCAAT/enhancer binding protein (C/EBP), beta
XM_420557
BU300073
similar to acyl-CoA-desaturase
gga.14762
BU202866
Finished cDNA, clone ChEST103n10
gga.16698
CR523375
Finished cDNA, clone ChEST853e7
NM_204335
gga.2608
NM_205204
doublecortex; lissencephaly, X-linked (doublecortin)
XM_421393
ENSGALT00000018877 similar to hypothetical protein
gga.14263
CR352731
Finished cDNA, clone ChEST39a12
XM_421133
gga.4003
BU143709
similar to hypothetical protein MGC4504
similar to RNA polymerase I polypeptide B; DNA-directed
XM_415007
BU326734
RNA polymerase I 135kDa polypeptide; RNA polymerase I
gga.19895
CR407144
Finished cDNA, clone ChEST749n19
CF255496
similar to RANTES factor of late activated T lymphocytes-1 XM_425065
gga.9734
BU416270
Finished cDNA, clone ChEST612l15
NM_001031514 gga.32096
ENSGALT00000021044 melanocortin 4 receptor
similar to E74-like factor 2 (ets domain transcription factor)
XM_420406
BU348384
isoform 2; new Ets-related factor
XM_424632
ENSGALT00000002758 similar to HECT domain containing 2
gga.13008
CR353534
Finished cDNA, clone ChEST11k17
gga.16449
BU455377
Finished cDNA, clone ChEST1038b12
XM_414072
ENSGALT00000005243 similar to neuronal calcium binding protein 2
gga.2606
BU320234
Finished cDNA, clone ChEST380k16
gga.5461
BU118178
Finished cDNA, clone ChEST938n22
Transcribed locus, weakly similar to XP_425603.1
gga.5353
BU354163
PREDICTED: similar to ORF2 [Gallus gallus]
similar to growth hormone-releasing hormone/pituitary
BX929984
XM_417357
gga.11231
adenylate cyclase-activating polypeptide precursor
gga.17584
CR523202
Finished cDNA, clone ChEST629c13
ENSGALT00000024646 similar to opioid receptor, kappa 1; Opiate receptor, kappa-1 XM_426087
NM_204973
gga.2283
BU227707
delta-like 1 (Drosophila)
gga.1750
BU131661
Finished cDNA, clone ChEST147b19
NM_204387
gga.6220
BX931695
ICER protein
XM_416293
gga.11996
BX933905
RASD family, member 2
ENSGALT00000025208 LOC420181
XM_429743
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doi: 10.1038/nature06738

Supplementary Table 3
Affymetrix ID
GgaAffx.751.1.S1_s_at
GgaAffx.8173.1.S1_at
Gga.4888.1.S1_s_at
GgaAffx.6911.1.A1_at
Gga.4038.1.S1_at
Gga.15280.1.S1_s_at
GgaAffx.22776.2.S1_s_at
Gga.4120.1.S1_at
Gga.7088.1.S1_at
GgaAffx.26392.1.S1_at
Gga.7283.1.S1_at
Gga.8506.1.S2_s_at
GgaAffx.4097.1.S1_at
Gga.571.1.S1_at
Gga.17278.1.S1_at
Gga.16331.1.S1_at
GgaAffx.21237.1.S1_at
GgaAffx.7307.1.S1_at
Gga.10041.1.S1_a_at
GgaAffx.5888.1.S1_at
GgaAffx.2936.1.S1_at
GgaAffx.22126.1.S1_at
GgaAffx.10913.1.S1_at
Gga.2396.1.S1_at
GgaAffx.21945.1.S1_at
Gga.8797.1.S1_at
GgaAffx.6689.1.S1_at
GgaAffx.22642.1.S1_at
Gga.6972.2.S1_at
Gga.4737.1.S1_at
Gga.5058.1.S1_s_at
Gga.13363.1.S1_at
Gga.12437.2.S1_at
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P-value

Gene Symbol

Accession

Description
RefSeqUniGenesimilar to membrane metallo-endopeptidase-like 2; zinc
XM_425737
0.0145 LOC428179
ENSGALT00000001684
metallopeptidase
XM_416361
0.0145 LOC418129
ENSGALT00000021057 similar to synaptotagmin 10
protein tyrosine phosphatase type IVA, member 1
NM_001008461 gga.4888
0.0146 PTP4A1
BU124448
0.0146ENSGALT00000017767
gga.4038
0.0151
BU202624
Transcribed locus
gga.14583
0.0155CN223021
Finished cDNA, clone ChEST491d14
NM_205422
gga.7390.0155 P20K; EXFABP; EX-FABP ENSGALT00000014676 quiescence-specific protein
XM_422941
gga.41200.0155 DUSP1
CR387287
dual specificity phosphatase 1
gga.7088
0.0165
BX259751
Finished cDNA, clone ChEST695o3
XM_415871
0.0165 LOC417627
ENSGALT00000008115 similar to protein phosphatase 1E; partner of PIX 1
gga.7283
0.0165
BU452897
Finished cDNA, clone ChEST462c20
NM_204401
gga.8506
0.017 TRIB2; TRB2
BU410444
tribbles homolog 2 (Drosophila)
XM_416842
0.0176 LOC418644
ENSGALT00000010624 similar to Metabotropic glutamate receptor 3 precursor
NM_205077
gga.5710.0176 NR5A1; SF-1; Ad4BP
NM_205077
nuclear receptor subfamily 5, group A, member 1
XM_414649
gga.17278
0.018 ANKRD43
BU294824
ankyrin repeat domain 43
gga.16331
0.018
BU339029
Finished cDNA, clone ChEST209l4
gga.20704
0.018
CR523378
Finished cDNA, clone ChEST576k23
similar to 25-hydroxyvitamin D-1 alpha hydroxylase,
mitochondrial precursor (Calcidiol 1-monooxygenase) (250.0185 LOC424227
ENSGALT00000018803
XM_422077
gga.10648
OHD-1 alpha-hydroxylase) (25-hydroxyvitamin D(3) 1-alphahydroxylase) (VD3 1A hydroxylase) (P450C1 alpha)prepronociceptin
XM_420026
gga.10041
0.0188 PNOC
BU392271
similar to zinc finger, FYVE domain containing 1 isoform 1;
tandem FYVE fingers-1 protein; zinc finger protein, subfamily
0.0188 LOC423252
ENSGALT00000015206
XM_421172
2A, member 1; double FYVE-containing protein 1;
phosphoinositide-binding protein SR3; zinc finger protein,
XM_414136
0.0188 LOC415774
ENSGALT00000007532 similar to system asc amino acid transporter Asc-1
similar to immunoglobulin superfamily, member 4A isoform a;
XM_417901
gga.5291
0.019 LOC419762
ENSGALT00000011374
immunosuperfamily protein Bl2
similar to stomatin-like 3; erythrocyte band 7 integral
0.019 LOC428074
ENSGALT00000027529
XM_425632
membrane protein; protein 7.2B
NM_205350
gga.2396
0.0195 BTG1
NM_205350
B-cell translocation gene 1, anti-proliferative
XM_414633
0.0195 LOC416316
ENSGALT00000010386 similar to ubiquitin conjugating enzyme
gga.8797
0.0195
CD764248
Finished cDNA, clone ChEST189k11
0.0197 LOC423390
ENSGALT00000017215 similar to RIKEN cDNA 4933401N24
XM_421302
XM_418616
gga.13510
0.0197 SLC39A12ENSGALT00000014008 solute carrier family 39 (zinc transporter), member 12
Transcribed locus, strongly similar to XP_418110.1
gga.6972
0.0207
BM426736
PREDICTED: similar to ABI gene family, member 3 [Gallus
NM_001007824 gga.4520
0.0207 LOC415296
BU119431
actin, cytoplasmic, type 5
NM_205500
gga.5058
0.0207 CST3
NM_205500
cystatin C (amyloid angiopathy and cerebral hemorrhage)
XM_419531
gga.13363
0.0212 LOC421483
CR353569
C21orf19-like protein
XM_421330
0.0214 LOC423420
BU118220
similar to chromogranin A precursor - mouse
6

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doi: 10.1038/nature06738

Supplementary Table 3
Affymetrix ID
Gga.11323.1.S1_at
GgaAffx.20609.1.S1_s_at
Gga.9122.1.S1_at
GgaAffx.4102.1.S1_at
GgaAffx.10053.1.S1_at
Gga.13507.1.S1_at
GgaAffx.20301.1.S1_at
Gga.1980.1.S1_s_at
Gga.11835.1.S1_at
GgaAffx.4634.4.S1_at

P-value
Gene Symbol
0.0214 CRH
0.02140.02160.02270.0227 LOC421816
0.02270.02270.0232 THBS4
0.0246 LOC419492
0.0247 LOC416083

GgaAffx.25755.1.S1_s_at

0.0254 LOC416815

Gga.1840.1.S2_at
Gga.12904.1.S1_at
Gga.8081.1.S1_at

0.0257 NEUROD1; NEUROD
0.0258 LOC416438
0.0264 FSTL4

GgaAffx.4468.2.S1_s_at

0.0274 LOC416068

Gga.7020.1.S1_at
GgaAffx.21710.1.S1_s_at
Gga.2200.1.S1_at
GgaAffx.3409.1.S1_at
Gga.15700.1.S1_at
Gga.2353.1.S1_at
GgaAffx.3860.2.S1_s_at

0.02740.0276 IFRD1; IFR1
0.0276 CYR61
0.0282 LOC428401
0.02850.0285 TIPARP
0.0285 LOC428711

GgaAffx.10313.1.S1_at

0.0285 LOC428381

GgaAffx.3458.1.S1_s_at
Gga.3950.1.S1_at

0.0291 DPYD
0.0315 BMP2

GgaAffx.6812.1.S1_at

0.032 LOC429108

GgaAffx.25940.1.S1_at

0.0339 LOC423677

GgaAffx.24643.1.S1_at
Gga.1750.1.S1_at
GgaAffx.6904.1.S1_s_at

0.0339 LOC421766
0.03390.0339 LOC428753

Gga.7979.1.S1_at

0.034

GgaAffx.11584.1.S1_s_at
Gga.6002.1.S1_a_at

0.034 LOC420846
0.034 DNMT3A

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Accession
Description
RefSeqUniGene
XM_418279
gga.11323
AJ621492
corticotropin releasing hormone
gga.9871
CR524006
Finished cDNA, clone ChEST701p4
gga.9122
CF253290
Transcribed locus
ENSGALT00000010639
XM_419840
ENSGALT00000025446 similar to ACY1L2 protein
gga.13507
CR385142
Finished cDNA, clone ChEST406g11
gga.20550
CR524314
Finished cDNA, clone ChEST819a3
XM_424763
gga.1980
BU388187
thrombospondin 4
gga.11835
BU118597
similar to AGPA3119
XM_417647
XM_414418
ENSGALT00000012023 similar to MAGI1c alpha beta2 gamma
similar to silencing mediator of retinoic acid and thyroid
XM_415107
ENSGALT00000004933
hormone receptor extended isoform
NM_204920
gga.1840
BU199562
neurogenic differentiation 1
XM_414750
gga.12904
BX950566
similar to Neuronal pentraxin II precursor (NP-II) (NP2)
gga.8081
NM_204502
follistatin-like 4
NM_204502
similar to Acyl-coenzyme A oxidase 2, peroxisomal
XM_414406
ENSGALT00000011554 (Branched-chain acyl-CoA oxidase) (BRCACox)(Trihydroxycoprostanoyl-CoA oxidase) (THCCox) (THCABU349489
Finished cDNA, clone ChEST299j12
gga.7020
NM_001001468 gga.2458
CR522905interferon-related developmental regulator 1
NM_001031563 gga.2200
J04496cysteine-rich, angiogenic inducer, 61
XM_425962
ENSGALT00000008721 similar to hypothetical protein LOC259173 isoform 1
gga.15700
BU144794
Finished cDNA, clone ChEST258o16
XM_422828
gga.2353
BU123098
TCDD-inducible poly(ADP-ribose) polymerase
XM_426269
ENSGALT00000009987 similar to four and a half LIM domains 1 protein, isoform C
similar to G protein-coupled receptor 20; G protein-coupled
XM_425941
ENSGALT00000026044
receptor 5-1
XM_426639
gga.20542
ENSGALT00000008842 dihydropyrimidine dehydrogenase
NM_204358
gga.3950
NM_204358
bone morphogenetic protein 2similar to thioesterase, adipose associated isoform BFIT2;ENSGALT00000017540 brown fat inducible thioesterase; START domain containing XM_426664
14; thioesterase superfamily member 1
similar to Bifunctional 3-phosphoadenosine 5-phosphosulfate
ENSGALT00000005844 synthethase 2 (PAPS synthethase 2) (PAPSS 2) (Sulfurylase XM_421557
kinase 2) (SK2) (SK 2)
ENSGALT00000024672 similar to Sestrin 1 (p53-regulated protein PA26)
XM_419796
gga.1750
BU315258
Finished cDNA, clone ChEST147b19
XM_426309
ENSGALT00000017742 similar to protein kinase, cGMP-dependent, type II; cGKIITranscribed locus, strongly similar to XP_418130.1
BX270512
gga.7979
PREDICTED: similar to Vesicle amine transport protein 1
homolog (T californica) [Gallus gallus]
AJ719481
similar to Hypothetical protein MGC75808
NM_001012863 gga.9773
NM_001024832 gga.6002
BU201511
DNA (cytosine-5)-methyltransferase 3A
7

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doi: 10.1038/nature06738

Supplementary Table 3
Affymetrix ID
GgaAffx.1849.2.S1_s_at
Gga.2166.1.S1_at
Gga.11766.2.S1_a_at
Gga.5095.1.S1_at
Gga.4922.1.S2_at
Gga.19368.1.S1_at
GgaAffx.8374.1.S1_s_at
GgaAffx.12984.1.S1_s_at
GgaAffx.21555.1.S1_s_at

P-value
Gene Symbol
0.0345 LOC416208, STC2
0.03450.0345 NDP
0.0345 CHRNA3
0.0348 EGR1
0.03480.0359 LOC418190
0.036 SNX3
0.0361 LOC420944

Gga.10364.1.S1_at

0.0361 LOC422064

GgaAffx.25620.1.S1_s_at
Gga.7640.2.S1_at
GgaAffx.4941.1.S1_at
Gga.2105.1.S1_at
Gga.16273.1.S1_at
GgaAffx.5015.1.S1_at
Gga.3676.1.S1_at

0.0361 AHCYL1
0.0365 NXT2
0.0369 LOC423158
0.0376 M-RIP
0.0376
0.0376 ITGA11
0.0378 LOC423952

Gga.12183.3.S1_s_at

0.0388 LOC428007

Gga.1014.1.S1_at

0.0394

Gga.2827.2.S1_a_at

0.0394 TIMP3; IMP-3

Gga.12811.1.S1_at
Gga.520.1.S1_at
Gga.3640.1.S1_at
GgaAffx.21459.1.S1_s_at
Gga.20018.1.S1_at
Gga.3575.1.S1_at

0.0411 NUAK2; 1200013B22RIK
0.0412 LOC396126
0.0412 LOC396335
0.04120.04120.0412 LOC422926

GgaAffx.24012.1.S1_at

0.0412 LOC418109

GgaAffx.5182.1.S1_at
Gga.261.3.S1_a_at
Gga.19107.1.S1_at
GgaAffx.5983.1.S1_at
GgaAffx.26575.1.S1_at
GgaAffx.4754.1.S1_s_at
Gga.17110.1.S1_at
GgaAffx.21308.1.S1_s_at
Gga.2182.1.S1_at
Gga.3624.1.S1_at

0.0412 LOC431262
0.0412 PRLR; PRL-R
0.04230.0423 LOC423936
0.04290.045 SLIT1
0.04580.0461
0.0461 IGF1R; IGF-1R
0.0468 PDE3B

www.nature.com/nature

Accession
Description
RefSeqUniGeneENSGALT00000004562 similar to stanniocalcin
XM_414534
gga.2166
BU376245
Finished cDNA, clone ChEST732g16
XM_416765
gga.11766
BX935162
Norrie disease (pseudoglioma)
NM_204416
gga.5095
M37336cholinergic receptor, nicotinic, alpha polypeptide 3
NM_204136
gga.4922
NM_204136
early growth response 1
gga.19368
CR406662
Finished cDNA, clone ChEST290o16
ENSGALT00000021500 similar to organic anion transporting polypeptide 1a2 variant XM_416421
NM_001006408 gga.15744
AJ720881
sorting nexin 3
gga.11980
CR523060
similar to PSST739
XM_419028
similar to dJ402H5.2 (novel protein similar to worm and fly
XM_420069
gga.10364
BX934281
proteins)
NM_001030913 gga.5821
ENSGALT00000000438 S-adenosylhomocysteine hydrolase-like 1
NM_001006436 gga.3568
BU342317
Nuclear transport factor 2-like export factor 2
ENSGALT00000012806 similar to ELGC699
XM_421084
XM_414806
gga.20857
BU435267
myosin phosphatase-Rho interacting protein
gga.16273
BU141803
Finished cDNA, clone ChEST118g23
XM_413930
ENSGALT00000012998 integrin, alpha 11
XM_421811
gga.3676
AJ441642
N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase
similar to dehydrogenase/reductase (SDR family) X-linked;
XM_425577
BX934631
dehydrogenase/reductase (SDR family) X chromosome
Clone C1 putative apoptosis associated protein mRNA, 3'
CD732610
gga.1014
UTR sequence
TIMP metallopeptidase inhibitor 3 (Sorsby fundus dystrophy,
CR386544
NM_205487
gga.2827
pseudoinflammatory)
XM_417962
gga.12811
BX950299
NUAK family, SNF1-like kinase, 2
gga.520
AF055478
activin beta B
NM_205206
gga.3640
NM_205376
kinectin
NM_205376
gga.18074
CR523156
Finished cDNA, clone ChEST1028m18
gga.20018
BU290246
thrombomucin
XM_420865
gga.3575
BX950350
similar to Gag-Pol polyprotein
similar to Cystine/glutamate transporter (Amino acid
ENSGALT00000020949 transport system xc-) (xCT) (Calcium channel blocker
XM_416343
resistance protein CCBR1)ENSGALT00000013361 similar to feminization 1 homolog a
XM_428816
prolactin receptor
NM_204854
gga.261
AF051808
gga.19107
CR406474
Finished cDNA, clone ChEST959a8
ENSGALT00000015465 similar to arginine-tRNA-protein transferase 1-2p; ATE1-2p XM_421796
XM_421637
gga.22925
ENSGALT00000009181 SEC31-like 2 (S. cerevisiae)
XM_421715
gga.2156
ENSGALT00000012327 slit homolog 1 (Drosophila)
gga.17110
CR391266
Finished cDNA, clone ChEST701k15
gga.5874
CR523307
Finished cDNA, clone ChEST858c10
NM_205032
gga.2182
NM_205032
insulin-like growth factor 1 receptor
phosphodiesterase 3B, cGMP-inhibited
NM_001031182 gga.3624
BU247139
8

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doi: 10.1038/nature06738

Supplementary Table 3
Affymetrix ID
Gga.8052.1.S1_at
GgaAffx.7851.2.S1_s_at
Gga.3986.2.S1_a_at
Gga.12107.1.S1_s_at

P-value
Gene Symbol
0.04690.0474 LOC423583
0.0476 CA2
0.0476 GDPD4

Gga.2070.2.S1_a_at

0.0484 ID4Gga.13798.2.S1_s_at
Gga.16263.1.S1_at
Gga.25.1.S1_at

0.0484 CRYBB2
0.04850.0494 SH3GL2; SH3P4

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Accession
Description
RefSeq
BX275092
Transcribed locus
XM_421471
ENSGALT00000020239 similar to osteonidogen
NM_205317
CN217291
carbonic anhydrase II
glycerophosphodiester phosphodiesterase domain
XM_417276
CD727469
inhibitor of DNA binding 4, dominant negative helix-loop-helix
NM_204282
AY040529
protein
NM_205175
AY539830
crystallin, beta B2
BU442145
Finished cDNA, clone ChEST532h13
NM_204530
SH3-domain GRB2-like 2
NM_204530

UniGenegga.8052
gga.3986
gga.12107
gga.2070
gga.646
gga.16263
gga.259doi: 10.1038/nature06738

SUPPLEMENTARY INFORMATION

Supplementary Table 4. Probe sequences used in the in situ hybridization.
Gene symbol Probe sequence (sense sequence)
TSHB
5'- GAGTGTGCCTACTGCCTGGCCATCAACACCACCATCTGCGCTGGA -3'
EYA3
5'- CGTTGGAGGCCTTCTCAGCCCACAGAAGAGGGAAGCTCTGCAGCG -3'
DIO25'- GATGGTTCAGCCTCAATGAATATCAAGACGGAAATACATTCTGTA -3'
ICER
5'- CTCAGCAACTGGCAGAAGAGGCAACGCGCAAGAGAGAGCTGCGAC -3'
NR4A3
5'- GCAGGAACCTTCGCAGCCCTCCCCGCCTTCTCCTCCCATCAGCAT -3'
CEBPB
5'- GAAGGTGGAGCAGCTCTCTCGGGAGCTGAGCACCCTCAGGAACTT -3'
SOCS3
5'- CGCATCCAGTGCGAGGGCGGCAGCTTCTCTCTGCAGAGCGACCCG -3'
DIO35'- CCTACGGCGCCTACTTCGAGCGGCTCTACGTCATCCAGGAGGAGA -3'
CGA
5'- TCTGGGCAAGACACCGAGTAGACTTGTGAATGAGATGGATGGCTG -3'
FSTL45'- GGCCACACTCAGACCTGGGGAGGCTGCACCAAGAGGTATCCGGAA -3'
RLN35'- ACGGGAGTCTCTGGGTTTGGCAGGAATGTGTTGCAAGTGGGGCTG -3'
STC2
5'- TTCTGAGCCCCGAGAAGAAGACGGGCGAAGCCAGCAAAGCTGCTG -3'
PNOC
5'- TTCGACCTCCTTGTCTGCATCCTGGAGTGCGAAGGCGAACCCGTG -3'
POMC
5'- CGCTGTGCCACAGCCTGCCCGTGGTGCTGGGGCTGCTGCTGTGTC -3'
GHR
5'- CTCTCCACAAGGCCTTGTGCTCAATGCAACTGCACTGCCTGTGCC -3'
OPRL1
5'- CGCTGTGTCTGATGACCTTACCCTTCCAGGGTACAGACACGTTCC -3'
MC4R
5'- GCGAAGGGCCACTCCTCGGGAGGCTGCTATGAACAACTCTTTGTA -3'
CCK
5'- TACGGCGGCATCTGCATCTGCGTGCTCCTCGCTGCGCTGTCGGTG -3'
CRH
5'- AACTCTGCCCGTCCTGCTCCGCATGGGAGAGGAGTACTTCCTGCG -3'
PRLR
5'- CAGACAGTGACTCAGGACGAGGGAGCTGTGATAGCCCTTCTCTGC -3'
EYA1
5'- CCAGGGAGCGCTTACTTGAGGCAGCCCTGGGCTGTCGTGCAGTCT -3'
EYA2
5'- CACTGCCTACCCTCCTCCAGCGCAGCCCTACGGCATACCTTCCTA -3'
EYA45'- GAGGGCGGAAGAATAATCCATCACCCCCTCCGGACAGTGACTTGG -3'SIX15'- GCCACCAACAAACCCAACCAGCTCTCCCCCCTGGACGGGAGCAAA -3'
SIX25'- GACAGTTCTGGGGAGCTCGGAGGACGAGAAGACGCCGTCAGGGAC -3'SIX35'- CCCGACCACCAGTGTTTCCAGTTTGACAGAAAGAGCCGAGACGGG -3'
SIX65'- CTCCAGCCCAGCCGTCAGCCTCTCCAGCAAGGCGGCCACTTCAGC -3'DACH15'- GGGCCGAAGACCTGGCAGCCACCCATCCTCTCATCGAAGCAGCAG -3'
DACH25'- AGCCCTTCTCCAGCTCCATCCCTAGAAGACAGTCAGCGGCCTGGG -3'PAX65'- GAGTCCTCCGCAACCTGGCTAGCGAAAAGCAACAGATGGGTGCCG -3'
TSHR
5'- TGTGCAGCCAGGGCACGGAAAGGTGCCCGTCAGCCTTCTGCGAGT -3'
TGFA
5'- CGGTCACACTTCAATGAGTGCCCCGACTCCCACCGGCAGTTCTGC -3'

www.nature.com/nature10doi: 10.1038/nature06738

SUPPLEMENTARY INFORMATION

Supplementary Table 5. Primer sequences used in the Q-PCR.
Gene Symbol
Primer sequence
sense: 5’-CGCCGGATTCTGCATGAC-3'
TSHB
antisense: 5’-GTGCACACGTTTTGGGACAG-3’
sense: 5’-CCGAACTCCAGTGTAATCCACATAG-3’,
DIO2
antisense: 5’-CACACTTGCCACCAACACTCTT-3’
sense: 5’-AACAGCTGCCACTGGAGACAT-3’
ICER
antisense: 5’-CCATTACCACTCCCTGAGGTAAGT-3’
sense: 5’-ATGAGACCATCATAATTTTCCATTCA-3’
EYA3
antisense: 5’-GAGCCAATCACCAGAGTTGGAT-3’
sense: 5’-AGAAGGTGATGTACCAGGGC-3’
DIO3
antisense: 5’-TTACACTTGGATGACCACCG-3’
sense: 5’-GATCTCTTCGCCGAGGACTATAAA-3’
CEBPB
antisense: 5’-CGGGTGAGGCTGATGTAGGT-3’
sense: 5’-TTTCTCCCGGGCCTATCC-3’
CGA
antisense: 5’-GCTTCCGATGTAATGTTCTTTGG-3’
sense: 5’-GATTCCTGTCACAGAGACACGAAA-3’
PER2
antisense: 5’-CGGCGAAGCCTGGTCTT-3’
sense: 5’-CTCATGGAGACAATCAGCAATCA-3’
CRY1
antisense: 5’-TTTCCCGGCGCTAATGC-3’
sense: 5’-AACGACGACTGAATGACCTTGTC-3’
E4BP4
antisense: 5’-AGCAGCTCGGCCTTCAAA-3’
sense: 5’-ATGCAGCCTTCCCACAACTC-3’
BMAL1
antisense: 5’-TCCTTTTTGGGCCACCTTCT-3’
sense: 5’-CCATAGTTGCATTTGGGTGTGT-3’
NR1D2
antisense: 5’-CATTTAGCGATGAAGCAACAAAA-3’
sense: 5’-CCTGTTGCCCCAGCTGTT-3’
DEC1
antisense: 5’-AAACCAACCCAATGAAATAAACGT-3’
sense: 5’-CGCTTCGGTGCATTGCT-3’
DEC2
antisense: 5’-GTAGGCCGCTAAGGTCTGGAA-3’
sense: 5’-AGCCATTGTGCCTCTAGTGAATTTAC-3’
PER3antisense: 5’-GCCAATCCACTGCCTGATG-3’
sense: 5’-AACCCCCAATGTCTCTGTTGTT-3’
GAPDH
antisense: 5’-TCACTACCCTCTTGATGTCATCATATT-3’

www.nature.com/nature11