T H Y R O I D - T R H - T S H

Aberrant cerebellar development of transgenic mice

expressing dominant-negative thyroid hormone
receptor in cerebellar Purkinje cells
Lu Yu1#, Toshiharu Iwasaki1#, Ming Xu1,2, Ronny Lesmana1,3, Yu Xiong4,
Noriaki Shimokawa1, William W. Chin5, Noriyuki Koibuchi1,5
1. Department of Integrative Physiology, Gunma University Graduate School of Medicine, Maebashi,
Gunma 371 8511, Japan; 2. Department of Neuropsychiatry, Keio University School of Medicine,
Shinjuku Tokyo 160 8582, Japan; 3. Department of Physiology, Universitas Padjadjaran, Bandung,
Indonesia; 4. Department of Laboratory Sciences, Gunma University Graduate School of Health Sciences,
Maebashi, Gunma 371 8511, Japan; 5. Harvard Medical School, Boston, MA 02115, USA Full-length
Original Research reports, Endocrinology

To study the role of the thyroid hormone (TH) in cerebellar development, we generated transgenic
mice expressing a dominant-negative TH receptor (TR) in cerebellar Purkinje cells. A mutant human
TR1 (G345R), which binds to the TH-response element but cannot bind to T3, was subcloned into
exon 4 of the full-length L7/ Pcp-2 gene, which is specifically expressed in Purkinje and retinal rod
bipolar cells. The transgene was specifically expressed in Purkinje cells in the postnatal cerebellum.
Purkinje cell dendrite arborization was significantly delayed in the transgenic mice. Surprisingly,
granule cell migration was also significantly delayed. In the primary cerebellar culture, TH-induced
Purkinje cell dendrite arborization was also suppressed. In quantitative realtime RT-PCR analysis,
the expression levels of several TH-responsive genes were altered. The expression levels of inositol
trisphosphate receptor type 1 and retinoic acid receptor-related orphan receptor mRNAs, which
are mainly expressed in Purkinje cells, and brain-derived neurotrophic factor mRNA, which is
expressed in both Purkinje and granule cells were significantly decreased. The expression levels of
neurotrophin-3 and hairless mRNAs, which are mainly expressed in granule cells, and myelin basic
protein mRNA, which is mainly expressed in oligodendrocytes were also decreased. The motor
coordination of transgenic mice was significantly disrupted. These results indicate that TH action
through its binding to TR in Purkinje cells is required for the normal cerebellar development. TH
action through TR in Purkinje cells is also important for the development of other subsets of
cerebellar cells such as granule cells and oligodendrocytes.

he thyroid hormone (TH; L-triiodothyronine, T3; Ltetraiodothyronine, thyroxine, T4) plays important
roles in the growth and development of many organs, including the central nervous system (CNS) (1, 2). TH deficiency during the perinatal period results in severe mental retardation, known as cretinism in humans (3, 4).
However, the molecular mechanism underlying TH action
on neurodevelopment has not yet been fully understood.
TH effects are mainly exerted through the nuclear TH
receptor (TR; TR1, TR1, and TR2), a ligand-dependent transcription factor (5). TR binds to specific DNA

enhancer sequences known as the TH-response elements

(TREs) located in the promoter region of target genes (6,
7). TR also interacts with various coregulators, such as
coactivators and corepressors, in a ligand-dependent manner to activate or repress the transcription of associated
genes (8, 9). TR is widely expressed both in adult and
developing brain (10, 11, 12). Various genes expressed in
the CNS are regulated by TH (13, 14). Most TH-responsive genes have a distinct critical period, during which TH
greatly affects their expression.

ISSN Print 0013-7227 ISSN Online 1945-7170

Printed in U.S.A.
Copyright 2015 by the Endocrine Society
Received January 28, 2014. Accepted January 16, 2015.

Abbreviations:

doi: 10.1210/en.2014-1079

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Dominant-negative TR in Purkinje cells

Morphologically, TH deficiency during the perinatal

period results in various disorders such as delayed neuronal migration and proliferation, and decreased synapse
formation (1). Consequently, perinatal hypothyroidism
causes various neurological symptoms such as mental retardation, deafness, and disturbance of motor coordination (15). These abnormal neurodevelopments cannot be
rescued unless TH is replaced before the termination of the
critical period.
To study TH actions on neurodevelopment, the rodent
cerebellum has been considered as an excellent model, because its structure is relatively simple and its developmental progression has been precisely reported (16). Functionally, the cerebellum is associated with the coordinating of
motor behavior, emotion, memory and language processing (17, 18). The development of the rodent cerebellar
cortex occurs largely postnatal, allowing to manipulate
precisely cerebellar TH status at various developmental
stages (16). Perinatal hypothyroidism dramatically affects
the morphogenesis of this region. The growth, dendrite
arborization and dendrite spine number of Purkinje cells
are markedly decreased (16). Synaptic formation between
Purkinje and granule cells in the molecular layer (ML) is
also dramatically repressed. The disappearance of the external granule cell layer (EGL) is delayed as a result of the
delayed proliferation and migration of granule cells into
the internal granule cell layer (IGL) (19). TRs are widely
expressed in the most subsets of cells in the rodent cerebellum, during development and in adult (11, 20). TR1
is abundantly expressed in granule cells, whereas TR1 is
mainly expressed in Purkinje cells (12). The expression of
many cerebellar genes is altered by perinatal hypothyroidism (21). Representative TH-responsive genes in the cerebellum include neurotrophins, such as the nerve growth
factor, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5, and inositol trisphosphate
(IP) 3 receptor, retinoic acid receptor (ROR) , hairless,
and myelin basic protein (MBP) (22, 23). The expressions
of these genes are controlled by TH in a temporal and
spatial manner (22, 23).
To study the role of TR in organ development and functional maintenance, several groups have artificially manipulated TR expressions (24). Interestingly, TR knockout mice, TR knock-out mice and TR/TR double
knock-out mice do not display obvious cerebellar defects,
suggesting that most of the consequences of congenital
hypothyroidism in the brain are due to the detrimental
activity of unliganded TR. This hypothesis is supported by
studies of transgenic animals expressing mutant TR and
showing a severe neurodevelopmental defect and even survival (25, 26, 27).

Endocrinology

Although these animal models are useful in studying the

mechanisms of TR action, these may not sufficiently address the mechanisms of direct TH action in the CNS.
Since TH acts not only in the brain but also in peripheral
organs, abnormal brain development may be induced
through the change in peripheral organ metabolism. To
exclude such a possibility, it is necessary to inhibit TH
action specifically in the brain.
In the present study, we generated a line of transgenic
mice expressing a dominant-negative TH receptor (Mf-1)
specifically in cerebellar Purkinje cells.

Materials and Methods

Animals
The mice used in the present study were bred in the Animal
Facility of Gunma University Graduate School of Medicine. The
animal experimentation protocol in the present study was approved by the Animal Care and Experimentation Committee,
Gunma University Showa Campus, and Harvard Medical Area
Standing Committee On Animals. The mice were kept at 24C
under a 12 hours light- 12 hours dark cycle (light on: 06:00
18:00), and 55% relative humidity, with food available ad libitum. Wild-type FVB mice were purchased from Japan Clea, Inc.
(Tokyo, Japan). Adult heterozygotes were mated for a suitable
reproduction period. Male and female pups were weighed and
sacrificed on postnatal days (P) 2, 7, 15, and 30 by decapitation
under diethyl ether anesthesia. The cerebella were dissected out,
weighed, and rapidly frozen in liquid nitrogen and stored at
80C until use. For immunohistochemistry, some brains were
fixed in 4% paraformaldehyde in phosphate buffered saline
(PBS) (0.2 M, pH 7.40) by transcardial perfusion (see below).

Transgene construction
For the vector used to express the gene specifically in Purkinje
cells, a full- length L7/pcp2 gene, which is expressed specifically
in Purkinje cells and retinal bipolar neurons, was used. The vector was kindly provided by Dr. John Oberdick, Ohio State University. A previous study has shown that its promoter region
alone is not sufficient for Purkinje cell-specific expression (28);
therefore we used the full length. Schematic figure showing the
structure of transgene is shown in Figure 1A. In each exon, all
potential in-frame translation start sites, ATG, were mutated so
that translation begins only from the inserted gene (28). It should
be noted that a TRE is located in the promoter region (29). Of
note, however, although the expression level of the gene decreased slightly in the hypothyroid animals, significant levels of
expression was retained (23). Thus we considered it sufficient to
use this gene as a vector.
To inhibit endogenous TR action, we utilized a human TR1
mutant, Mf-1, in which glycine at the amino terminus 345 is
replaced by arginine (30, 31). This mutant can bind with TREs
but not with TH. This mutant was subcloned into exon IV of the
vector together with three copies of hemagglutinin (HA)-tag
(amino acid sequence: YPYDVPDYA) (32) peptide nucleotides
located upstream of Mf-1 cDNA (Figure 1A). Oocyte injection of
transgene and implantation of oocyte were performed at

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doi: 10.1210/en.2014-1079

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Brigham and Womens Hospital Core Transgenic facility, Boston, USA.

before hybridization. The primer sequences used in the PCR are

as follows; forward, 5-GTTTAGAGCTCCGGGCACACGTG3, reverse, 5-CAGGATGGAATGCAGAAACGAC-3. The position of the primer is shown in the left panel of Figure 1A (lateral
arrows). After autoradiography, the density of DNA bands was
determined using an automated densitometer.

Southern blot analysis

To confirm the transgene expression by southern blot analysis, mouse genomic DNA was extracted from tails using a tissue
miniprep system (Promega, Madison, WI). Extracted DNA was
digested with the restriction endonuclease SacI. The digestion
sites are shown in Figure 1A (downward arrows). The expected
DNA fragment sizes of endogenous L7/pcp2 and the transgene
after this digestion were 716 and 950 bases, respectively. The
digests were electrophoresed on a 0.8% agarose gel at 20 V
overnight, and the separated products were transferred to a Hybond N nylon membrane (Amersham, Little Chalfont, U.K.)
by capillary blotting. A 479 base DNA fragment of L7/pcp2 gene
(31573636) (28) containing parts of intron 3 and exon 4 was
amplified by PCR, purified by agarose gel electrophoresis, and
labeled with 32P by random priming (Rediprime II Random
Prime Labeling System; GE Healthcare, Little Chalfont, U.K.)

ATG

1
HA HA HA

Measurement of free T3 and free T4

concentrations
To measure plasma hormone levels, male mice (n 4/genotype) were sacrificed on P60 under diethylether anesthesia.
Trunk blood samples were then collected immediately. Blood
samples were centrifuged (6,000 g, 15 minutes) to separate
plasma. Free T3 and free T4 levels in plasma were analyzed using
automated immunoassay analyzers Architect i2000 (Abbott Diagnostics, Lake Forest, IL) and Architect FT3 kit or Architect
FT4 kit (Abbott Diagnostics) according to manufactures
instruction.

Tg/+ Tg/Tg

1.6Kb

+/+

Mf-1

Transgene
Endogenous
pcp2

ATG

8Kb

L7/pcp-2 gene

B
Relative Luciferase Activity

14000
7
12000
6
10000
5
8000
4
6000
3

4000
2

**

2000
1

**

00

TR1
Mf-1

+
-

++

++

Figure 1. Transgene structure and dominant-negative action of Mf-1 on TR action. A, Left

panel: Structure of transgene used in the present study. A mutant TR (Mf-1) cDNA ligated with
three copies of HA tag was subcloned into exon IV of full-length of L7/pcp2 gene. Arrowhead
indicates endogenous L7/PCP-2 translation start site (ATG) that was mutated so that translation
starts from the transgene. Downward arrows indicate SacI restriction endonuclease site, whereas
lateral arrows indicate the position of primers used to generate probe for southern blot analysis.
SacI digestion produces 716 and 950 bases bands for endogenous L7/pcp2 and the transgene,
respectively. Right panel: confirmation of genotype by genomic Southern blot analysis. The upper
bands (closed arrow) show the transgene (716 bases), whereas the lower bands show
endogenous L7/pcp2 (950 bases). Note that while the band densities of transgene L7/pcp2 and
endogenous L7/pcp2 were identical in the Tg/ mice, the transgene band density is higher in the
Tg/Tg mice. B, Dominant-negative action of Mf-1, studied by reporter gene assay. Expression
plasmids encoding TR1 (0.02 g/well) and/or HA-tagged Mf-1 were cotransfected with F2-TKLUC (0.2 g/well) into CV-1 cells. Cells were cultured in the presence or absence of 10-7 M T3.
Total amounts of DNA for each well were balanced by adding the pcDNA3 vector. Data
represent mean SEM of experiments performed in triplicate. **, P .01 compared with
TR1(), Mf-1(-),T3().

Cell culture and transient

transfection-based reporter
gene assays
CV-1 cells were cultured in Dulbeccos Modified Eagle Medium (DMEM,
Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum and
100 U/ml penicillin and 100 g/ml streptomycin, at 37C with 5% CO2. The cells
were plated in 24-well culture plates 48
hours before transfection. Transfection
was performed using the TR1-pcDNA3
and/or Mf-1-pcDNA3 expression plasmid (0.02 g/well) and reporter plasmid
(0.2 g/well) by the calcium-phosphate
precipitation method (33). Mf-1pcDNA3 was made by PCR fragment insertion at HindIII and EcoRI sites. The
cytomegalovirus
(CMV)--galactosidase expression plasmid (0.1 g/well)
was used as an internal control. The medium was changed 16 24 hours after
transfection and T3 was added to a final
concentration of 10-7 M. After 24 hours,
the cells were harvested to measure luciferase and -galactosidase activities.
Luciferase activity was normalized to
-galactosidase activity and then calculated as relative luciferase activity. The
transfection studies were repeated at
least three times in triplicate.

Immunohistochemistry
Mice were transcardially perfused on
P7, 15, and 30 with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4).
Cerebella were dissected, cryoprotected
in sucrose solution at 4C, and embedded in an O.C.T. compound (TissueTek, Torrance, CA). Frozen sagittal cerebellar sections were cut by a cryostat

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Dominant-negative TR in Purkinje cells

(Thermo Fisher Scientific Inc, Waltham, MA) at a 10 m thickness through the vermis and placed on slides. The sections were
washed with PBS and incubated with 5% normal horse serum in
0.3% Triton X-100 in PBS for 30 minutes at room temperature.
Then, the sections were incubated with a mouse anti-calbindinD28K antibody (Sigma, St. Louis, MO) overnight at 4C (1:3000
dilution). Next, the sections were incubated with biotinylated
horse antimouse IgG (1:200 dilution) for 45 minutes. After rinsing in PBS, the sections were incubated with the avidin-biotin
complex (ABC) for 1 hour and visualized by 3,3-diaminobenzidine (0.5 mg/ml Tris-HCl containing 0.01% H2O2). The sections were then immersed in 0.5% cresyl violet for 13 minutes,
dehydrated by graded series of ethanol, cleared in xylene, and
placed under coverslips.

Western blot analysis for HA-Mf-1

The dissected cerebella were weighted, homogenized in lysis
buffer containing 10 mm Tris-HCl (pH 7.8), 150 mm NaCl, 1
mm EDTA, 1% Nonidet P40 and protease inhibitors. After centrifugation, protein samples were heat-denatured at 96C for 4
minutes. Samples (10 g/lane) were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis and were then
transferred to a nitrocellulose membrane (GE Healthcare) for 1
hour at room temperature, and blocked overnight at 4C in 2%
blocking reagent (GE Healthcare) in Tris-buffered saline buffer
with 0.1% Tween 20. Immunoblotting was performed using a
mouse monoclonal anti-HA-tag antibody (dilution 1 : 10 000,
M180; Medical & Biological Laboratories co., Ltd, Nagoya,
Japan) and antimouse IgG coupled to horseradish peroxidase
(GE Healthcare). The signals were developed using enhanced
chemiluminescence prime reagent (GE Healthcare) and imaged
(ImageQuant LAS 4010, GE Healthcare). The band intensities
were determined using ImageJ Software (NIH). Blots were reprobed with an anti--actin antibody (dilution 1:1000, Cell Signaling Technology Inc., Danvers, MA) to monitor the level of
protein.

Primary cerebellar culture and semiquantitative

analysis of Purkinje cell dendrite arborization
Newborn mice were decapitated under diethylether anesthesia on the first day of birth. Details of the culture methods were
previously described (34). Briefly, cerebella were digested in 0.2
U/ml of papain (Worthington, Lakewood, NJ) in PBS containing
0.2 mg/ml L-cysteine, 0.2 mg/ml bovine serum albumin (Intergen, Purchase, NY), 5 mg/ml glucose and 0.02 mg/ml DNase I
(Sigma-Aldrich, 400 600 U/mg) for 25 minutes at 36.5C. Dissociated cells were suspended in a serum-free medium without
THs and plated at a density of 2.0 105 cells/0.2 ml in wells of
chamber slides (Lab-Tek 8-mm-diameter wells, Nalge Nunc International, Rochester, NY), precoated with 0.1 mg/ml poly-Llysine (Sigma-Aldrich). Sixteen to 24 hours after plating, the
indicated amount of TH was added to the culture medium and
one-half of the medium was replaced with fresh medium every
3 4 days. Cells were culutured in a 5% CO2 incubator for 17
days.
Immunocytochemistry of the cultured cells was described
previously (34). Briefly, Purkinje cells were immunostained with
a mouse monoclonal anticalbindin-28K antibody (1:1000; Sigma-Aldrich) and a fluorescein isothiocyanate (FITC)-labeled
donkey antimouse IgG antibody (1:200; Molecular Probes, Eu-

Endocrinology

gene, OR) and inspected under a laser confocal scanning microscope (FV1000D spectral type inverted microscope IX81, Olympus, Tokyo, Japan). To quantify dendrite arborization, the total
area covered by the dendrite tree of randomly selected Purkinje
cells (n 10) in each experiment was determined by tracing the
outline of a cell and its dendrite branches and computing the area
using ImageJ software (NIH). Ten randomly selected Purkinje
cells were used for each experiment because of the limitations of
photo bleaching associated with the use of the laser conforcal
microscope. More than three independent experiments were
performed.

Quantitative realtime RT-PCR

Total RNA was extracted from P2, 7, 15, and 30 cerebella
using RNeasy (QIAGEN, Hilden, Germany). Two g of total
RNA were used for cDNA synthesis using High Capacity RNAto-cDNA kit (Applied Biosystems, Foster City, CA). Specific
primers for BDNF, NT-3, ROR, IP3 receptor, hairless, MBP,
and GAPDH and cyclophilin as internal controls were used (Applied Biosystems, Mm04230607 s1, Mm00435413 s1,
Mm01296312 m1,
Mm00439907 m1,
Mm00498963 m1, Mm01266402 m1, Mm99999915 g1,
Mm02342430 g1). Specific primers for TR1 (sense: 5-GTCAGAGGGAATGCCAGTACA-3, antisense: 5- GGCCATTTTCTGTCATACTGTTAGGA-3) were used. Realtime
RT-PCR was carried out as described in the instruction manual
of the TaqMan Fats Advanced Master mix kit (Applied Biosystems) and the StepOne Real-Time PCR System (Applied Biosystems). The realtime RT-PCR protocol for all genes was 95C for
20 seconds, followed by amplification using 95C for 1 second,
60C for 20 seconds (40 cycles). All experiments were repeated
three times (n 4), with independent RNA preparations to confirm the consistency of results. Levels of mRNA shown above
were normalized by GAPDH mRNA level. We confirmed that
the levels of mRNA normalized by cyclophilin were essentially
same as those by GAPDH (data not shown).

Rotarod tests
On P15 and 30, the accelerating rotarod test was performed
(LSI-Letica Scientific Instruments, Barcelona, Spain) to assess
motor coordination. The apparatus was started at an initial
speed of 4 rpm and gradually accelerated at a rate of 0.2 rpm/s.
The mice were tested in three trials, and the average latency on
the device for each genotype was recorded.

Statistical Analysis
Treatment effects were analyzed using ANOVA. Post hoc
comparison was made using Bonferronis test. P values 0.05
were considered to be significant.

Results
Effect of Mf-1 on TR-mediated transcription
The effect of Mf-1 on TR-mediated transcription was
examined using a transient transfection-based reporter
gene assay (Figure 1B). Mf-1 effectively suppressed the
TR1-mediated transcription on F2-TRE with T3. This
result indicates that Mf-1 functions as a dominant-nega-

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tive inhibitor. TR1-mediated transcription was also suppressed by Mf-1 (Supplemental Figure S1).

transgene was selected and used throughout the experiment (Figure 1A).
The transgenic mice were fertile. The mating of Tg/
offspring resulted in a Mendelian distribution of pups. The
ratio of male to female offspring was 1:1. There was no
significant difference in body and cerebellar weights
among Tg/Tg, Tg/ and / mice during development
(Supplemental Figure S2). Mf-1 protein was detected from
P2. Its levels were significantly increased with age (Figure
2B).

Generation of transgenic mice

Initially, several transgenic mouse lines were isolated.
Purkinje cell-specific transgene expression was confirmed
by the immunohistochemistry of the HA-tag (Figure 2A).
We also confirmed the protein expression levels on P2,
7, 15, and 30 by the Western blot analysis (Figure 2B).
Essentially, the cerebellar phenotype was the same among
the transgenic lines. One line containing 2 copies of the

A
+/+

Tg/+

Tg/Tg
ML
PCL
IGL

1.64

Tg/+ Tg/Tg

P2

HA-Mf-1 (55.5KDa)
-actin (43KDa)

P7

HA-Mf-1
-actin

P15

HA-Mf-1
-actin

P30

HA-Mf-1
-actin

Relative Intensity

+/+

1.23

0.82

Tg/Tg
Ho

0.41
0

P2

P7

pg/ml

Relative mRNA Levels

TR1

**
**

P15

P30

Free T3

Tg/+
He

+/+

Tg/+

Tg/Tg

Effect of Mf-1 on the

expression of endogenous TR1
The expression of endogenous
TR1 mRNA was examined using
realtime RT-PCR. We designed TaqMan sense primer at nontranslated
region of TR1 mRNA so that only
endogenous TR mRNA was amplified. Interestingly, a significant increase in endogenous TR1 mRNA
levels in the Tg/ and Tg/Tg mice
were observed on P15 (Figure 2C).
On P30, the expression levels became the same among three groups.
Effect of transgene expression
on Thyroid hormone levels
As shown in Figure 2D, plasma
free T3 and free T4 levels at P60 were
measured in male mice (Figure 2D).
Free T3 and free T4 levels were not
significantly different among 3
groups, indicating that expression of
Mf-1 in Purkinje cells did not alter
plasma TH levels.

0
P7

P15

Free T4

P30
ng/dl

P2

0.5

0
+/+

Tg/+

Tg/Tg

Figure 2. Charactrization of transgene expression and thyroid hormone levels.

A, The transgene expression in Pukinje cell was confirmed by immunohistochemistry. Sagittal
sections of the cerebellum were stained with the rabbit anti-HA antibody (1:1000). Bar, 20 m.
B, Ontogenic expression of Mf-1 protein in the developing cerebellum. *, P .05 compared with
the Tg/ mice. (C) Effect of Mf-1 on mRNA expression of wild type TR1. The mRNA expression
levels of wild type TR1 in the cerebella of the / (), Tg/ (), and Tg/Tg () mice were
analyzed by quantitative realtime RT-PCR. The mRNA expression level was normalized to the
GAPDH mRNA expression level. Data are expressed as mean SEM (n 4). **, p 0.01
compared with the / mice by Bonferronis test. D, Plasma free T3 and free T4 levels measured
by ELISA. Blood sample of each group were taken from male mice at P60. Data are expressed as
mean SEM (n 4) compared with the / mice. No significant difference was observed by
Bonferronis test.

Aberrant cerebellar morphology

in transgenic mice
To examine the morphological
changes in the developing transgenic
mouse cerebellum, immunohistochemistry for calbindin-D28k,
which is specifically expressed in
Purkinje cells, and Cresyl Violet
staining were performed (Figure 3).
Morphological alterations of the
Purkinje and granule cell layers in the
transgenic mice became evident on
P7. The Purkinje cell dendrite poorly
developed and showed misalignment in the transgenic mice. The den-

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Dominant-negative TR in Purkinje cells

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drite growth was more severely affected in the Tg/Tg mice.

The alignment of a Purkinje cell was also disrupted on P7.
Although the transgene was specifically expressed in the
Purkinje cells, the disappearance of the EGL was delayed.
On P15, the EGL was still observed in the Tg/ and Tg/Tg
mice. It was thicker in the Tg/Tg mice. On P30, no significant morphological difference among 3 groups was
observed.
Inhibition of T4-mediated Purkinje cell dendrite
arborization in transgenic mice
To examine further whether TH-medicated Purkinje
cell development is suppressed in transgenic mice, a primary culture of newborn mice cerebellum was performed.
Dispersed newborn pup cerebella were cultured for 17
days, fixed and stained with the anticalbindin-28k antibody (Figure 4A). Without T4 treatment, the Purkinje cell
dendrite poorly developed. However, the dendritic area of
the Tg/Tg mice was significantly smaller (Figure 4B). With
the increase in T4 concentration, dendritic area significantly increased in a dose-dependent manner (Figure 4A).
The dendritic area of the transgenic mice was significantly
smaller, particularly in the Tg/Tg mice (Figure 4B). We
have also used T3 and performed the same experiment.
The result is essentially the same (data not shown). These
data indicate that TH-induced dendrite arborization is
partially suppressed by Mf-1.
Change in TH-responsive gene expression in
transgenic mice
To examine the changes in the TH- responsive gene
expression in Purkinje cells, the expression levels of IP3
+/+

Tg/+

Tg/Tg
EGL

P7

ML
PCL
IGL
EGL

P15

ML
PCL
IGL

P30

ML
PCL
IGL

Figure 3. Morphological alterations in the postnatal

cerebellum. The sagittal sections of the cerebellum (vermis) at P7,
P15, and P30 were stained with mouse anti-calbindin-D28K (1:1000)
and Cresyl Violet. Bar, 50 m.

receptor and ROR mRNAs, which are dominantly expressed in the Purkinje cells in the cerebellum (35, 36) and
are regulated by TH (23, 37), were studied by quantitative
realtime RT-PCR study. The IP3 receptor mRNA expression level in the / mice started to increase from P7
(Figure 5A). The increase in IP3 receptor mRNA expression levels on P7 and P15 were smaller in the Tg/Tg and
Tg/ mice. However, the expression levels became the
same as those in the / mice on P30. The ROR mRNA
expression level peaked on P15 (Figure 5B). The increase
in ROR mRNA expression levels on P7 was smaller in the
Tg/Tg and Tg/ than those in the / mice. These
changes are essentially the same as those observed in the
perinatal hypothyroid animals (23, 37).
Next, we examined the expression of neurotrophin
mRNAs including BDNF and NT-3, which play an important role in cerebellar development (38, 39). BDNF
mRNA is expressed mainly in granule cells, but is also
weakly expressed in Purkinje cells, whereas NT-3 mRNA
is predominantly expressed in granule cells (40). The
BDNF mRNA expression level in the / mice started to
increase from P15 and increased further on P30 (Figure
6A). The transgenic mice also showed the same tendency
of increase in BDNF mRNA expression level. However,
the magnitude of increase was not as large as those in the
/ mice. The NT-3 mRNA expression level peaked on
P7 in the /, but on P15 in the Tg/ and Tg/Tg mice
(Figure 6B). On P15, the expression level of NT-3 in the
/ was decreased. The peak levels in the Tg/ and Tg/Tg
mice were the same as those in the / mice. A significant
difference in NT-3 mRNA expression level between the
/ and Tg/Tg mice was observed on P30. Hairless
mRNA expression started to increase on P7 in / mice.
Such significant increase was not observed in Tg/ and
Tg/Tg mice on P7. By P15, however, the expression level
retuned to be the same among 3 groups (Figure 6C).
We also examined the changes in the expression level of
MBP mRNA, which is predominantly expressed in oligodendrocytes (41). The MBP mRNA expression level
peaked on P15 and decreased on P30 (Figure 7). The increase in MBP mRNA expression level on P7 was smaller
in the Tg/Tg and Tg/ mice. No significant difference in
MBP mRNA level was observed among the three groups
on P15 and P30.
Alteration of motor coordination in transgenic
mice
To examine the changes in motor coordination function, the rotarod test was performed using P15 and P30
mice. On both P15 and P30, the latency on the rotarod was
significantly shorter in the Tg/Tg male mice than in the

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doi: 10.1210/en.2014-1079

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/ mice (Figure 8). The female mice showed similar results to the male group (Supplemental Figure S3).

Relative dendritic area

Tg/Tg

Tg/+

+/+

opmental stage-specific manner. Furthermore, although

cerebellar morphology became almost identical among
the three groups at P30, the motor coordination of transgenic mice was disrupted. These results indicate that TRmediated gene expression in Purkinje cells plays a critical
Discussion
role in the entire cerebellar development.
In the present study, we established a transgenic mouse
Compared with a drug-induced hypothyroid model or
line expressing dominant-negative mutant TR1 (G345R) a mutant TR knock-in model, there is a great advantage
specifically in the cerebellar Purkinje cells. Although the in the use of this transgenic mouse line in the study of TH
transgenic mice showed no significant retardation in gen- action in a developing cerebellum. Since mice are generally
eral growth or cerebellar weight, cerebellar morphogen- euthyroid, the effect of general hypothyroidism, which
esis was widely disrupted. Not only Purkinje cell dendrite may cause various metabolic changes, can be excluded.
growth, but also granule cell migration from the EGL to The influence of maternal hypothyroidism, which may
the IGL were retarded. The mRNA expression levels of alter nursing behavior, can also be excluded. Thus, we can
TH-responsive genes not only in Purkinje cells but also in investigate the effect of the inhibition of TR action spegranule cells and oligodendrocytes decreased in a develcifically in Purkinje cells, without
considering the general metabolic
A
alteration.
In the present study, endogenous
TR1 expression was transiently increased on P7 and then decreased
thereafter in the / mice. This is
essentially the same as those of previous study showing a transient increase in TR1 expression in the cerebellum (11). Interestingly, the
expression was significantly increased on P15 in both Tg/ and
Tg/Tg mice. Although the mechanism of the increase cannot be clarified, such increase may be the reason
for milder cerebellar phenotype than
T4 (M):
10-10
10-9
10-8
those expected by the reporter gene
assay results.
Since we specifically inhibited
B 450005
TR-mediated transcription only in
360004
Purkinje cells, we initially hypothesized that the Purkinje cell morpho270003
+/+
WT
**
genesis is specifically altered. As exTg/+
HE
pected, Purkinje cell development
180002
**
*
was affected both in vivo (Figure 3)
Tg/Tg
HO
**
90001
and in vitro (Figure 4). Particularly in
*
the primary culture of the cerebel*
00
lum, T4-induced Purkinje cell dencon
-10-10
-9 -9
-8 -8
T4 (M):
10
10
10
drite arborization was significantly
Figure 4. Changes in Purkinje cell dendrite arborization in primary cerebellar
suppressed by transgene expression.
culture. (A) Photomicrographs showing the effect of T4 on Purkinje cell morphology. Primary
Since the suppression was greater in
culture of cerebellum contains the whole set of cerebellar cells including astrocytes, in which T4
the Tg/Tg than in the Tg/ mice,
is converted to T3 by type 2 deiodenase under physiological conditions. After 17 days in vitro,
the cells were fixed and immunocytochemistry was carried out using the anticalbindin antibody
suppression may be induced in a
to visualize Purkinje cells. Bars, 50 m. (B) Changes in dendritic areas of Purkinje cells. Dendritic
transgene dose-dependent manner.
area was quantified using Object-Image 2.11 software (NIH). Data are expressed as mean
The expression of TH-responsive
SEM. (n 10 determinations). Data shown are representative of at least three independent
experiments. *, p0.05 and **, p0.01 compared with the / mice by Bonferronis test.
genes such as those encoding the IP3

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Dominant-negative TR in Purkinje cells

Endocrinology

receptor and ROR, which are predominantly expressed

in Purkinje cells (35, 36), was also altered. These results
clearly indicate that endogenous TR-mediated transcription was suppressed by mutant TR, leading to aberrant
Purkinje cell development.
Recently, Fauquier T et al has reported the transgenic
mice that express a mutant TR1 specifically in Purkinje
cells using Cre/loxP technology (42). They used several
Cre lines including L7-Cre mice, in which L7/Pcp2 promoter was used to express CRE recombinase, which induce Purkinje cell-specific mutant TR1 expression
through loxP sequence recombination. This mouse
showed limited alterations in cerebellar morphogenesis
compared with those of wild type. Although it is difficult
to clarify the mechanisms inducing difference in mophogenesis in the present study, one possible reason may be the
difference in the timing of transgene expression. By L7-Cre
recombination, the transgene expression was obserbed af-

ter P8 (42), whereas the expression of mutant TR1 was

observed as early as P2 in the present study (Figure 2B). On
the other hand, by Ptf1a-Cre recombination, mutant
TR1 in Purkine cells and GABAergic interneurons was
expressed from prenatal stage, showing the alteration of
Purkinje cell morphogenesis (42). Thus, the earlier expression of mutant TR in our animal model may induce more

A
Relative mRNA Levels

50
5

BNDF Male

40
4
30
3
20
2
10
1

**
**

00

P2

Relative mRNA Levels

A
65

P7

P15

P30

B
IP3 receptor Male

2.55

3.63

Relative mRNA Levels

4.84

**
**

2.42

**
**

1.21
00

P2

P7

NT-3 Male

**

24

1.53

**

12

**

**

0.51

P15

P30

0
P2

P7

P15

P30

P15

P30

45

140
5

ROR Male

Relative mRNA Levels

Relative mRNA Levels

C
3.2
4

Hairless Male

112
4

2.4
3

84
3

1.6
2

0.8
1

56
2

00

P2

P7

P15

P30

Figure 5. Expression of IP3 receptor and ROR mRNAs

during postnatal development of the male mice
cerebellum. The mRNA expression levels of IP3 receptor (A) and
ROR (B) in the cerebella of the / (), Tg/ (), and Tg/Tg ()
mice were analyzed by quantitative realtime RT-PCR. The mRNA
expression level was normalized to the GAPDH mRNA expression level.
Data are expressed as mean SEM (n 4). *, p 0.05 and **,
p0.01 compared with the / mice by Bonferronis test.

**
**

P2

P7

Figure 6. Expression of BDNF, NT-3 and hairless mRNAs

during postnatal development of male mice cerebellum.
The mRNA expression levels of BDNF, NT-3 and hairless in the cerebella
of the / (), Tg/ (), and Tg/Tg () mice were analyzed by
quantitative realtime RT-PCR. The mRNA expression level was
normalized to the GAPDH mRNA expression level. Data are expressed
as mean SEM (n 4). *, p 0.05 and **, p0.01 compared with
the / mice by Bonferronis test.

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doi: 10.1210/en.2014-1079

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60
5

MBP Male

48
4
36
3
24
2

**

12
1

**

0
0
P2

P7

P15

P30

Time on Rotarod (sec)

Figure 7. Expression of MBP mRNAs during postnatal

development of male mice cerebellum. The mRNA expression
levels of IP3 receptor and ROR in the cerebella of the / (), Tg/
(), and Tg/Tg () mice were analyzed by quantitative realtime RTPCR. The mRNA expression level was normalized to the GAPDH mRNA
expression level. Data are expressed as mean SEM (n 4). **,
p0.01 compared with the / mice by Bonferronis test.
120

Thus, it is not likely that such effects are induced by the

direct inhibition of TH action in granule cells or oligodendrocytes. Rather, the disruption of Purkinje cell function may indirectly alter the normal granule cell and oligodendrocyte development. Since TR1 is abundantly
expressed in granule cells (11, 21), the change in granule
cell development in hypothyroid animals or mutant TR
knock-in mice have been considered as a consequence of
the direct action of TH. The results of the present study
have demonstrated that abnormal granule cell development in the perinatal hypothyroidism may be, at least in
part, exerted by the indirect action of TH through Purkinje
cells.
On the other hand, mechanisms inducing abnormal
granule cell development in the present study have not yet
been clarified. Purkinje cells may regulate granule cell development through several mechanisms. One possible
pathway may be mediated by sonic hedgehog (Shh) (46,
47), which is a secretory glycoprotein that plays an important role in cell proliferation and cell fate determination (48, 49). Thus, Purkinje cells may regulate the proliferation of granule cells through Shh secretion. In
addition, several proteins such as vitronectin and laminin
interact with Shh and take part in granule cell migration
(50). A previous study has shown that Shh expression is
disrupted by perinatal hypothyroidism (51). Thus, although TH-responsive genes in the granule cells are accidental in transgenic animals, TH may indirectly regulate
the expression of these genes through Purkinje cell-derived
trophic factors.
The mechanism underlying alternation of MBP mRNA
expression in oligodendrocytes have also not yet been clarified. Myelination is a highly regulated timed event that
starts a few days after birth and depends on the proper
differentiation of oligodendrocytes (52). The expression
of each oligodendrocyte/myelin gene may be affected by
TH. Hypothyroidism impairs transiently the 23cyclic
nucleotide 3phosphodiesterase (E.C. 3.1.4.37-CNPase)
gene expression, that precedes the myelin basic protein
(MBP), the proteolipidic protein (PLP) and the myelin associated glycoprotein (MAG) gene transcription (53).

P15 Male

120

100
80

**

60
40
20
0

Time on Rotarod (sec)

Relative mRNA Levels

sever phenotype than those induced by L7-Cre

recombination.
To our surprise, granule cell migration was also affected (Figure 3) on P7 and P15. The disappearance of the
EGL was delayed in the Tg/ and Tg/Tg mice. Such delayed migration was also observed in antithyroid druginduced hypothyroid mice (21, 43, 44). Furthermore, the
expression of several TH-responsive genes such as those
encoding BDNF, NT-3 and hairless, which are predominantly expressed in granule cells (40) was altered (Figure
6). The expression level of NT-3 in the / mice peaked
on P7, whereas those in the Tg/ and Tg/Tg peaked on
P15. These results indicate the delayed expression by
transgene. The expression levels of hairless mRNA, which
is directly regulated by TR (45), was also decreased on P7,
indicating that additional factors other than TR may regulate hairless expression. In addition, the expression of
MBP, which is predominantly expressed in oligodendrocytes (41) was also altered. As shown by immunohistochemistry (Figure 2A), since the transgene was expressed
specifically in the Purkinje cells in the cerebellum, other
subsets of cell in the cerebellum including the granule cells
and oligodendrocytes should be under the euthroid status.

P30 Male

100
80

60
40
20
0

+/+
Wild

Tg/+
Hetero

Tg/Tg
Homo

+/+
Wild

Tg/+
Hetero

Tg/Tg
Homo

Figure 8. Changes in motor coordination, studied by rotarod test of male mice. Male /, Tg/, and Tg/Tg mice were trained on
the rotarod at P15 and P30. The time until they fell off the accelerating rod was determined. Data are expressed as mean SEM (n 10). *, p
0.05 and **, p0.01 compared with the / mice by Bonferronis test.

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10

Dominant-negative TR in Purkinje cells

These effects were observed only in the differentiated oligodendrocyte, suggesting a role for TH in the regulation of
the oligodendrocyte differentiation. Thus, as similar to
granule cells, although TH-responsive genes in the oligodendrocytes are accidental in transgenic animals, TH may
indirectly regulate the expression of these genes through
Purkinje cell-derived trophic factors. On the other hand,
Picou F, et al (54) reported the effect of TR1 in oligodendrocyte precursor cells (OPC) using Cre/loxP-based
transgenic mice. They concluded that TR1 acts on OPC
through two mechanisms. At early postnatal stage, TH
promotes the secretion of neurotrophic factor in Purkinje
cells and granule cells inducing differentiation of OPC;
Later TH mainly affects OPC differentiation in a cell-autonomous manner. In the present study, simultaneous decreases in both NT-3 and MBP was observed on P7. This
result may support their hypothesis that a decrease in neurotrophin secretion may affect the OPC differentiation at
early postnatal stage.
The time on the rotarod was decreased in transgenic
mice, suggesting that the motor coordination was disrupted (Figure 8). The defect was observed not only on
P15, but also on P30, when cerebellar morphologies become almost identical. This finding is consistent with previous studies using a drug-induced hypothyroid animal
model showing that, although cerebellar morphologies
become indistinguishable from euthyroid animal on P30
(55, 56), the motor coordination defect was still observed
(57). Similar symptoms were also observed in a human
study showing that, if hypothyroidism is not normalized
within the first 3 months of life, patients may show permanent cerebellar symptoms (58). The present study also
confirmed that the inhibition of TR action in Purkinje cells
during the critical development period may produce a permanent cerebellar defect. Although many reports showed
that TR1 may be important for the normal cerebellar
development (42, 59 63), the molecular mechanisms inducing cerebellar ataxia in hypothyroidism have not yet
been fully clarified. Since thyroid hormone directly or indirectly regulates a large number of genes in a temporallyand spatially- dependent manner, multiple genes may be
involved. Together with previous study, we consider that
both TR1 and 1 may be important for normal cerebellar
development. Indeed, we showed that TR1-mediated
transcription was also suppressed by Mf-1 (Supplemental
Figure S1). Coordination of TR1 and 1 may be complicated. In addition, complicated regulation of cell-cell
interactions including neuron-neuron or neuron-glia interactions that are also directly and/or indirectly regulated
by TR1 and 1. Thus, to differentiate the role of TR and
TR, many additional studies are required. Further study

Endocrinology

may be also required to clarify involvement of TRs on

formation and regulation of a neuronal or gene network.
In summary, we established a line of transgenic mice
expressing mutant TR specifically in Purkinje cells. These
mice showed a hypothyroid phenotype in the cerebellum
not only in Purkinje cells but also in granule cells and
oligodendrocytes. As a consequence, these mice showed a
motor coordination defect. These results indicate that TH
action through its binding to TR expressed in Purkinje
cells is required for the normal cerebellar development,
although factors transmitting the TH influence in Purkinje
cell to other subset of cells have not yet been clarified. TH
action through TR in Purkinje cells is also important for
the development of other subsets of cerebellar cells such as
granule cells and oligodendrocytes.

Acknowledgments
We would like to thank Dr. John Oberdick for kindly preparing
the construct, Drs. Yusuke Takatsuru and Asahi Haijima for
helpful discussion, and Ms. Misae Ohta, Ms. Hiroko Masuda,
and Drs. Izuki Amano, Winda Ariyani, Chun-Hong Qiu, Kingsley Ibhazehiebo, Junko Kimura-Kuroda, Hideki Yokoo and
Masami Murakami for technical assistance and advice.
Address all correspondence and requests for reprints to: Noriyuki Koibuchi, M.D., Ph.D., Department of Integrative Physiology, Gunma University Graduate School of Medicine,
339 22 Showa-machi, Maebashi, Gunma 371 8511, Japan,
Tel: 8127220 7923; Fax: 8127220 7926; E-mail:
nkoibuch@gunma-u.ac.jp.
This work was supported by a Grant-in-Aid for Scientific
Research (No. 21 390 065, 25 281 024) to N.K, T.I, N.S, (No.
20 510 061, 23 510 072) to T.I and N.K from the Japanese Ministry of Education, Culture, Sports, Science and Technology
(MEXT), and Cosmic Research Award to T.I from Japan Thyroid Association.
# These authors contributed equally.
Conflict of Interest: There is no conflict of interest in this
manuscript.

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