en%2E2014-1079

Aberrant cerebellar development of transgenic miceexpressing dominant-negative thyroid hormonereceptor 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-lengthOriginal Research reports, Endocrinology

To study the role of the thyroid hormone (TH) in cerebellar development, we generated transgenicmice expressing a dominant-negative TH receptor (TR) in cerebellar Purkinje cells. A mutant humanTR�1 (G345R), which binds to the TH-response element but cannot bind to T3, was subcloned intoexon 4 of the full-length L7/ Pcp-2 gene, which is specifically expressed in Purkinje and retinal rodbipolar 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-inducedPurkinje 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 inositoltrisphosphate receptor type 1 and retinoic acid receptor-related orphan receptor � mRNAs, whichare mainly expressed in Purkinje cells, and brain-derived neurotrophic factor mRNA, which isexpressed in both Purkinje and granule cells were significantly decreased. The expression levels ofneurotrophin-3 and hairless mRNAs, which are mainly expressed in granule cells, and myelin basicprotein mRNA, which is mainly expressed in oligodendrocytes were also decreased. The motorcoordination of transgenic mice was significantly disrupted. These results indicate that TH actionthrough its binding to TR in Purkinje cells is required for the normal cerebellar development. THaction through TR in Purkinje cells is also important for the development of other subsets ofcerebellar cells such as granule cells and oligodendrocytes.

The thyroid hormone (TH; L-triiodothyronine, T3; L-tetraiodothyronine, thyroxine, T4) plays important

roles in the growth and development of many organs, in-cluding the central nervous system (CNS) (1, 2). TH de-ficiency during the perinatal period results in severe men-tal retardation, known as cretinism in humans (3, 4).However, the molecular mechanism underlying TH actionon neurodevelopment has not yet been fully understood.TH effects are mainly exerted through the nuclear THreceptor (TR; TR�1, TR�1, and TR�2), a ligand-depen-dent 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 ascoactivators and corepressors, in a ligand-dependent man-ner to activate or repress the transcription of associatedgenes (8, 9). TR is widely expressed both in adult anddeveloping brain (10, 11, 12). Various genes expressed inthe CNS are regulated by TH (13, 14). Most TH-respon-sive genes have a distinct critical period, during which THgreatly affects their expression.

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2015 by the Endocrine SocietyReceived January 28, 2014. Accepted January 16, 2015.

Abbreviations:

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

doi: 10.1210/en.2014-1079 Endocrinology endo.endojournals.org 1

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 March 2015. at 18:47 For personal use only. No other uses without permission. . All rights reserved.

Morphologically, TH deficiency during the perinatalperiod results in various disorders such as delayed neuro-nal migration and proliferation, and decreased synapseformation (1). Consequently, perinatal hypothyroidismcauses various neurological symptoms such as mental re-tardation, deafness, and disturbance of motor coordina-tion (15). These abnormal neurodevelopments cannot berescued unless TH is replaced before the termination of thecritical period.

To study TH actions on neurodevelopment, the rodentcerebellum has been considered as an excellent model, be-cause its structure is relatively simple and its developmen-tal progression has been precisely reported (16). Function-ally, the cerebellum is associated with the coordinating ofmotor behavior, emotion, memory and language process-ing (17, 18). The development of the rodent cerebellarcortex occurs largely postnatal, allowing to manipulateprecisely cerebellar TH status at various developmentalstages (16). Perinatal hypothyroidism dramatically affectsthe morphogenesis of this region. The growth, dendritearborization and dendrite spine number of Purkinje cellsare markedly decreased (16). Synaptic formation betweenPurkinje and granule cells in the molecular layer (ML) isalso dramatically repressed. The disappearance of the ex-ternal granule cell layer (EGL) is delayed as a result of thedelayed proliferation and migration of granule cells intothe internal granule cell layer (IGL) (19). TRs are widelyexpressed in the most subsets of cells in the rodent cere-bellum, during development and in adult (11, 20). TR�1is abundantly expressed in granule cells, whereas TR�1 ismainly expressed in Purkinje cells (12). The expression ofmany cerebellar genes is altered by perinatal hypothyroid-ism (21). Representative TH-responsive genes in the cer-ebellum include neurotrophins, such as the nerve growthfactor, brain-derived neurotrophic factor (BDNF), neu-rotrophin (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 expressionsof these genes are controlled by TH in a temporal andspatial manner (22, 23).

To study the role of TR in organ development and func-tional maintenance, several groups have artificially ma-nipulated TR expressions (24). Interestingly, TR� knock-out mice, TR� knock-out mice and TR�/TR� doubleknock-out mice do not display obvious cerebellar defects,suggesting that most of the consequences of congenitalhypothyroidism in the brain are due to the detrimentalactivity of unliganded TR. This hypothesis is supported bystudies of transgenic animals expressing mutant TR andshowing a severe neurodevelopmental defect and even sur-vival (25, 26, 27).

Although these animal models are useful in studying themechanisms of TR action, these may not sufficiently ad-dress the mechanisms of direct TH action in the CNS.Since TH acts not only in the brain but also in peripheralorgans, abnormal brain development may be inducedthrough the change in peripheral organ metabolism. Toexclude such a possibility, it is necessary to inhibit THaction specifically in the brain.

In the present study, we generated a line of transgenicmice expressing a dominant-negative TH receptor (Mf-1)specifically in cerebellar Purkinje cells.

Materials and Methods

AnimalsThe mice used in the present study were bred in the Animal

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

Transgene constructionFor the vector used to express the gene specifically in Purkinje

cells, a full- length L7/pcp2 gene, which is expressed specificallyin Purkinje cells and retinal bipolar neurons, was used. The vec-tor was kindly provided by Dr. John Oberdick, Ohio State Uni-versity. A previous study has shown that its promoter regionalone is not sufficient for Purkinje cell-specific expression (28);therefore we used the full length. Schematic figure showing thestructure of transgene is shown in Figure 1A. In each exon, allpotential in-frame translation start sites, ATG, were mutated sothat translation begins only from the inserted gene (28). It shouldbe noted that a TRE is located in the promoter region (29). Ofnote, however, although the expression level of the gene de-creased slightly in the hypothyroid animals, significant levels ofexpression was retained (23). Thus we considered it sufficient touse this gene as a vector.

To inhibit endogenous TR action, we utilized a human TR�1mutant, Mf-1, in which glycine at the amino terminus 345 isreplaced by arginine (30, 31). This mutant can bind with TREsbut not with TH. This mutant was subcloned into exon IV of thevector together with three copies of hemagglutinin (HA)-tag(amino acid sequence: YPYDVPDYA) (32) peptide nucleotideslocated upstream of Mf-1 cDNA (Figure 1A). Oocyte injection oftransgene and implantation of oocyte were performed at

2 Dominant-negative TR in Purkinje cells Endocrinology


Brigham and Women’s Hospital Core Transgenic facility, Bos-ton, USA.

Southern blot analysisTo confirm the transgene expression by southern blot anal-

ysis, mouse genomic DNA was extracted from tails using a tissueminiprep system (Promega, Madison, WI). Extracted DNA wasdigested with the restriction endonuclease SacI. The digestionsites are shown in Figure 1A (downward arrows). The expectedDNA fragment sizes of endogenous L7/pcp2 and the transgeneafter this digestion were 716 and 950 bases, respectively. Thedigests were electrophoresed on a 0.8% agarose gel at 20 Vovernight, and the separated products were transferred to a Hy-bond N� nylon membrane (Amersham, Little Chalfont, U.K.)by capillary blotting. A 479 base DNA fragment of L7/pcp2 gene(3157–3636) (28) containing parts of intron 3 and exon 4 wasamplified by PCR, purified by agarose gel electrophoresis, andlabeled with 32P by random priming (Rediprime II RandomPrime Labeling System; GE Healthcare, Little Chalfont, U.K.)

before hybridization. The primer sequences used in the PCR areas follows; forward, 5�-GTTTAGAGCTCCGGGCACACGTG-3�, reverse, 5�-CAGGATGGAATGCAGAAACGAC-3�. The po-sition of the primer is shown in the left panel of Figure 1A (lateralarrows). After autoradiography, the density of DNA bands wasdetermined using an automated densitometer.

Measurement of free T3 and free T4concentrations

To measure plasma hormone levels, male mice (n � 4/geno-type) were sacrificed on P60 under diethylether anesthesia.Trunk blood samples were then collected immediately. Bloodsamples were centrifuged (6,000 g, 15 minutes) to separateplasma. Free T3 and free T4 levels in plasma were analyzed usingautomated immunoassay analyzers Architect i2000 (Abbott Di-agnostics, Lake Forest, IL) and Architect FT3 kit or ArchitectFT4 kit (Abbott Diagnostics) according to manufacture’sinstruction.

Cell culture and transienttransfection-based reportergene assays

CV-1 cells were cultured in Dulbec-co’s Modified Eagle Medium (DMEM,Invitrogen, Grand Island, NY) supple-mented with 10% fetal bovine serum and100 U/ml penicillin and 100 �g/ml strep-tomycin, at 37°C with 5% CO2. The cellswere plated in 24-well culture plates 48hours before transfection. Transfectionwas performed using the TR�1-pcDNA3and/or Mf-1-pcDNA3 expression plas-mid (0.02 �g/well) and reporter plasmid(0.2 �g/well) by the calcium-phosphateprecipitation method (33). Mf-1-pcDNA3 was made by PCR fragment in-sertion at HindIII and EcoRI sites. Thecytomegalovirus (CMV)-�-galactosi-dase expression plasmid (0.1 �g/well)was used as an internal control. The me-dium was changed 16–24 hours aftertransfection and T3 was added to a finalconcentration of 10-7 M. After 24 hours,the cells were harvested to measure lu-ciferase and �-galactosidase activities.Luciferase activity was normalized to�-galactosidase activity and then calcu-lated as relative luciferase activity. Thetransfection studies were repeated atleast three times in triplicate.

ImmunohistochemistryMice were transcardially perfused on

P7, 15, and 30 with 4% paraformalde-hyde in 0.1 M phosphate buffer (pH 7.4).Cerebella were dissected, cryoprotectedin sucrose solution at 4°C, and embed-ded in an O.C.T. compound (Tissue-Tek, Torrance, CA). Frozen sagittal cer-ebellar sections were cut by a cryostat

A

B

6000

0

2000

4000

8000

10000

12000

14000

1

0

2

3

4

6

5

7

Rel

ativ

e Lu

cife

rase

Act

ivity

TRβ1

Mf-1

--- -

-++

++

+++ ++

** **

HA HAHA

ATG

1 8Kb

Mf-1

ATG1 1.6Kb

L7/pcp-2 gene

Tg/+ Tg/Tg +/+

→←

TransgeneEndogenouspcp2

Figure 1. Transgene structure and dominant-negative action of Mf-1 on TR action. A, Leftpanel: Structure of transgene used in the present study. A mutant TR� (Mf-1) cDNA ligated withthree copies of HA tag was subcloned into exon IV of full-length of L7/pcp2 gene. Arrowheadindicates endogenous L7/PCP-2 translation start site (ATG) that was mutated so that translationstarts from the transgene. Downward arrows indicate SacI restriction endonuclease site, whereaslateral 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 upperbands (closed arrow) show the transgene (716 bases), whereas the lower bands showendogenous L7/pcp2 (950 bases). Note that while the band densities of transgene L7/pcp2 andendogenous L7/pcp2 were identical in the Tg/� mice, the transgene band density is higher in theTg/Tg mice. B, Dominant-negative action of Mf-1, studied by reporter gene assay. Expressionplasmids encoding TR�1 (0.02 �g/well) and/or HA-tagged Mf-1 were cotransfected with F2-TK-LUC (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. Datarepresent mean � SEM of experiments performed in triplicate. **, P � .01 compared withTR�1(�), Mf-1(-),T3(�).

doi: 10.1210/en.2014-1079 endo.endojournals.org 3


(Thermo Fisher Scientific Inc, Waltham, MA) at a 10 �m thick-ness through the vermis and placed on slides. The sections werewashed with PBS and incubated with 5% normal horse serum in0.3% Triton X-100 in PBS for 30 minutes at room temperature.Then, the sections were incubated with a mouse anti-calbindin-D28K antibody (Sigma, St. Louis, MO) overnight at 4°C (1:3000dilution). Next, the sections were incubated with biotinylatedhorse antimouse IgG (1:200 dilution) for 45 minutes. After rins-ing in PBS, the sections were incubated with the avidin-biotincomplex (ABC) for 1 hour and visualized by 3,3�-diaminoben-zidine (0.5 mg/ml Tris-HCl containing 0.01% H2O2). The sec-tions were then immersed in 0.5% cresyl violet for 1–3 minutes,dehydrated by graded series of ethanol, cleared in xylene, andplaced under coverslips.

Western blot analysis for HA-Mf-1The dissected cerebella were weighted, homogenized in lysis

buffer containing 10 mm Tris-HCl (pH 7.8), 150 mm NaCl, 1mm EDTA, 1% Nonidet P40 and protease inhibitors. After cen-trifugation, protein samples were heat-denatured at 96°C for 4minutes. Samples (10 �g/lane) were separated by sodium dode-cylsulphate-polyacrylamide gel electrophoresis and were thentransferred to a nitrocellulose membrane (GE Healthcare) for 1hour at room temperature, and blocked overnight at 4°C in 2%blocking reagent (GE Healthcare) in Tris-buffered saline bufferwith 0.1% Tween 20. Immunoblotting was performed using amouse 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 enhancedchemiluminescence prime reagent (GE Healthcare) and imaged(ImageQuant LAS 4010, GE Healthcare). The band intensitieswere determined using ImageJ Software (NIH). Blots were re-probed with an anti-�-actin antibody (dilution 1:1000, Cell Sig-naling Technology Inc., Danvers, MA) to monitor the level ofprotein.

Primary cerebellar culture and semiquantitativeanalysis of Purkinje cell dendrite arborization

Newborn mice were decapitated under diethylether anesthe-sia on the first day of birth. Details of the culture methods werepreviously described (34). Briefly, cerebella were digested in 0.2U/ml of papain (Worthington, Lakewood, NJ) in PBS containing0.2 mg/ml L-cysteine, 0.2 mg/ml bovine serum albumin (Inter-gen, Purchase, NY), 5 mg/ml glucose and 0.02 mg/ml DNase I(Sigma-Aldrich, 400–600 U/mg) for 25 minutes at 36.5°C. Dis-sociated cells were suspended in a serum-free medium withoutTHs and plated at a density of 2.0 � 105 cells/0.2 ml in wells ofchamber slides (Lab-Tek 8-mm-diameter wells, Nalge Nunc In-ternational, Rochester, NY), precoated with 0.1 mg/ml poly-L-lysine (Sigma-Aldrich). Sixteen to 24 hours after plating, theindicated amount of TH was added to the culture medium andone-half of the medium was replaced with fresh medium every3–4 days. Cells were culutured in a 5% CO2 incubator for 17days.

Immunocytochemistry of the cultured cells was describedpreviously (34). Briefly, Purkinje cells were immunostained witha mouse monoclonal anticalbindin-28K antibody (1:1000; Sig-ma-Aldrich) and a fluorescein isothiocyanate (FITC)-labeleddonkey antimouse IgG antibody (1:200; Molecular Probes, Eu-

gene, OR) and inspected under a laser confocal scanning micro-scope (FV1000D spectral type inverted microscope IX81, Olym-pus, Tokyo, Japan). To quantify dendrite arborization, the totalarea covered by the dendrite tree of randomly selected Purkinjecells (n � 10) in each experiment was determined by tracing theoutline of a cell and its dendrite branches and computing the areausing ImageJ software (NIH). Ten randomly selected Purkinjecells were used for each experiment because of the limitations ofphoto bleaching associated with the use of the laser conforcalmicroscope. More than three independent experiments wereperformed.

Quantitative realtime RT-PCRTotal RNA was extracted from P2, 7, 15, and 30 cerebella

using RNeasy (QIAGEN, Hilden, Germany). Two �g of totalRNA were used for cDNA synthesis using High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA). Specificprimers for BDNF, NT-3, ROR�, IP3 receptor, hairless, MBP,and GAPDH and cyclophilin as internal controls were used (Ap-plied Biosystems, Mm04230607 s1, Mm00435413 s1,Mm01296312 m1, Mm00439907 m1,Mm00498963 m1, Mm01266402 m1, Mm99999915 g1,Mm02342430 g1). Specific primers for TR�1 (sense: 5�-GT-CAGAGGGAATGCCAGTACA-3�, antisense: 5�- GGC-CATTTTCTGTCATACTGTTAGGA-3�) were used. RealtimeRT-PCR was carried out as described in the instruction manualof the TaqMan Fats Advanced Master mix kit (Applied Biosys-tems) and the StepOne Real-Time PCR System (Applied Biosys-tems). The realtime RT-PCR protocol for all genes was 95°C for20 seconds, followed by amplification using 95°C for 1 second,60°C for 20 seconds (40 cycles). All experiments were repeatedthree times (n � 4), with independent RNA preparations to con-firm the consistency of results. Levels of mRNA shown abovewere normalized by GAPDH mRNA level. We confirmed thatthe levels of mRNA normalized by cyclophilin were essentiallysame as those by GAPDH (data not shown).

Rotarod testsOn P15 and 30, the accelerating rotarod test was performed

(LSI-Letica Scientific Instruments, Barcelona, Spain) to assessmotor coordination. The apparatus was started at an initialspeed 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 onthe device for each genotype was recorded.

Statistical AnalysisTreatment effects were analyzed using ANOVA. Post hoc

comparison was made using Bonferroni’s test. P values � 0.05were considered to be significant.

Results

Effect of Mf-1 on TR-mediated transcriptionThe effect of Mf-1 on TR-mediated transcription was

examined using a transient transfection-based reportergene assay (Figure 1B). Mf-1 effectively suppressed theTR�1-mediated transcription on F2-TRE with T3. Thisresult indicates that Mf-1 functions as a dominant-nega-



tive inhibitor. TR�1-mediated transcription was also sup-pressed by Mf-1 (Supplemental Figure S1).

Generation of transgenic miceInitially, several transgenic mouse lines were isolated.

Purkinje cell-specific transgene expression was confirmedby 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 amongthe transgenic lines. One line containing 2 copies of the

transgene was selected and used throughout the experi-ment (Figure 1A).

The transgenic mice were fertile. The mating of Tg/�offspring resulted in a Mendelian distribution of pups. Theratio of male to female offspring was 1:1. There was nosignificant difference in body and cerebellar weightsamong Tg/Tg, Tg/� and �/� mice during development(Supplemental Figure S2). Mf-1 protein was detected fromP2. Its levels were significantly increased with age (Figure2B).

Effect of Mf-1 on theexpression of endogenous TR�1

The expression of endogenousTR�1 mRNA was examined usingrealtime RT-PCR. We designed Taq-Man sense primer at nontranslatedregion of TR�1 mRNA so that onlyendogenous TR mRNA was ampli-fied. Interestingly, a significant in-crease in endogenous TR�1 mRNAlevels in the Tg/� and Tg/Tg micewere observed on P15 (Figure 2C).On P30, the expression levels be-came the same among three groups.

Effect of transgene expressionon Thyroid hormone levels

As shown in Figure 2D, plasmafree T3 and free T4 levels at P60 weremeasured in male mice (Figure 2D).Free T3 and free T4 levels were notsignificantly different among 3groups, indicating that expression ofMf-1 in Purkinje cells did not alterplasma TH levels.

Aberrant cerebellar morphologyin transgenic mice

To examine the morphologicalchanges in the developing transgenicmouse cerebellum, immunohisto-chemistry for calbindin-D28k,which is specifically expressed inPurkinje cells, and Cresyl Violetstaining were performed (Figure 3).Morphological alterations of thePurkinje and granule cell layers in thetransgenic mice became evident onP7. The Purkinje cell dendrite poorlydeveloped and showed misalign-ment in the transgenic mice. The den-

A+/+ Tg/TgTg/+

ML

PCL

IGL

B

C

P2

P7

P15

P30

β-actin (43KDa)HA-Mf-1 (55.5KDa)

β-actinHA-Mf-1

β-actinHA-Mf-1

β-actinHA-Mf-1

+/+ Tg/+ Tg/Tg

0

0.4

0.8

1.2

1.6

P2 P7 P15 P30

He

Ho2

3

4

0

1

**

*

*

Tg/+

Tg/Tg

Rel

ativ

e In

tens

ity

P2 P7 P15 P30

Rel

ativ

e m

RN

A Le

vels TRβ1

**

1

2

3

45

0

**

D

pg/m

l

0

1

2

+/+ Tg/+ Tg/Tg

Free T3

ng/d

l

0

0.5

1

+/+ Tg/+ Tg/Tg

Free T4

Figure 2. Charactrization of transgene expression and thyroid hormone levels.A, The transgene expression in Pukinje cell was confirmed by immunohistochemistry. Sagittalsections 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 withthe Tg/� mice. (C) Effect of Mf-1 on mRNA expression of wild type TR�1. The mRNA expressionlevels of wild type TR�1 in the cerebella of the �/� (�), Tg/� (), and Tg/Tg (Œ) mice wereanalyzed by quantitative realtime RT-PCR. The mRNA expression level was normalized to theGAPDH mRNA expression level. Data are expressed as mean � SEM (n � 4). **, p� 0.01compared with the �/� mice by Bonferroni’s test. D, Plasma free T3 and free T4 levels measuredby ELISA. Blood sample of each group were taken from male mice at P60. Data are expressed asmean � SEM (n � 4) compared with the �/� mice. No significant difference was observed byBonferroni’s test.



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 thePurkinje cells, the disappearance of the EGL was delayed.On P15, the EGL was still observed in the Tg/� and Tg/Tgmice. It was thicker in the Tg/Tg mice. On P30, no signif-icant morphological difference among 3 groups wasobserved.

Inhibition of T4-mediated Purkinje cell dendritearborization in transgenic mice

To examine further whether TH-medicated Purkinjecell development is suppressed in transgenic mice, a pri-mary culture of newborn mice cerebellum was performed.Dispersed newborn pup cerebella were cultured for 17days, fixed and stained with the anticalbindin-28k anti-body (Figure 4A). Without T4 treatment, the Purkinje celldendrite poorly developed. However, the dendritic area ofthe Tg/Tg mice was significantly smaller (Figure 4B). Withthe increase in T4 concentration, dendritic area signifi-cantly increased in a dose-dependent manner (Figure 4A).The dendritic area of the transgenic mice was significantlysmaller, particularly in the Tg/Tg mice (Figure 4B). Wehave also used T3 and performed the same experiment.The result is essentially the same (data not shown). Thesedata indicate that TH-induced dendrite arborization ispartially suppressed by Mf-1.

Change in TH-responsive gene expression intransgenic mice

To examine the changes in the TH- responsive geneexpression in Purkinje cells, the expression levels of IP3

receptor and ROR� mRNAs, which are dominantly ex-pressed in the Purkinje cells in the cerebellum (35, 36) andare regulated by TH (23, 37), were studied by quantitativerealtime RT-PCR study. The IP3 receptor mRNA expres-sion level in the �/� mice started to increase from P7(Figure 5A). The increase in IP3 receptor mRNA expres-sion levels on P7 and P15 were smaller in the Tg/Tg andTg/� mice. However, the expression levels became thesame as those in the �/� mice on P30. The ROR� mRNAexpression level peaked on P15 (Figure 5B). The increasein ROR� mRNA expression levels on P7 was smaller in theTg/Tg and Tg/� than those in the �/� mice. Thesechanges are essentially the same as those observed in theperinatal hypothyroid animals (23, 37).

Next, we examined the expression of neurotrophinmRNAs including BDNF and NT-3, which play an im-portant role in cerebellar development (38, 39). BDNFmRNA is expressed mainly in granule cells, but is alsoweakly expressed in Purkinje cells, whereas NT-3 mRNAis predominantly expressed in granule cells (40). TheBDNF mRNA expression level in the �/� mice started toincrease from P15 and increased further on P30 (Figure6A). The transgenic mice also showed the same tendencyof 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 onP7 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/Tgmice were the same as those in the �/� mice. A significantdifference in NT-3 mRNA expression level between the�/� and Tg/Tg mice was observed on P30. HairlessmRNA expression started to increase on P7 in �/� mice.Such significant increase was not observed in Tg/� andTg/Tg mice on P7. By P15, however, the expression levelretuned to be the same among 3 groups (Figure 6C).

We also examined the changes in the expression level ofMBP mRNA, which is predominantly expressed in oligo-dendrocytes (41). The MBP mRNA expression levelpeaked on P15 and decreased on P30 (Figure 7). The in-crease in MBP mRNA expression level on P7 was smallerin the Tg/Tg and Tg/� mice. No significant difference inMBP mRNA level was observed among the three groupson P15 and P30.

Alteration of motor coordination in transgenicmice

To examine the changes in motor coordination func-tion, the rotarod test was performed using P15 and P30mice. On both P15 and P30, the latency on the rotarod wassignificantly shorter in the Tg/Tg male mice than in the

+/+ Tg/+ Tg/Tg

P7

P15

P30

EGLML

PCL

IGL

EGL

ML

PCLIGL

ML

PCLIGL

Figure 3. Morphological alterations in the postnatalcerebellum. 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.



�/� mice (Figure 8). The female mice showed similar re-sults to the male group (Supplemental Figure S3).

Discussion

In the present study, we established a transgenic mouseline expressing dominant-negative mutant TR�1 (G345R)specifically in the cerebellar Purkinje cells. Although thetransgenic mice showed no significant retardation in gen-eral growth or cerebellar weight, cerebellar morphogen-esis was widely disrupted. Not only Purkinje cell dendritegrowth, but also granule cell migration from the EGL tothe IGL were retarded. The mRNA expression levels ofTH-responsive genes not only in Purkinje cells but also ingranule cells and oligodendrocytes decreased in a devel-

opmental stage-specific manner. Furthermore, althoughcerebellar morphology became almost identical amongthe three groups at P30, the motor coordination of trans-genic mice was disrupted. These results indicate that TR-mediated gene expression in Purkinje cells plays a criticalrole in the entire cerebellar development.

Compared with a drug-induced hypothyroid model ora mutant TR� knock-in model, there is a great advantagein the use of this transgenic mouse line in the study of THaction in a developing cerebellum. Since mice are generallyeuthyroid, the effect of general hypothyroidism, whichmay cause various metabolic changes, can be excluded.The influence of maternal hypothyroidism, which mayalter nursing behavior, can also be excluded. Thus, we caninvestigate the effect of the inhibition of TR action spe-

cifically in Purkinje cells, withoutconsidering the general metabolicalteration.

In the present study, endogenousTR�1 expression was transiently in-creased on P7 and then decreasedthereafter in the �/� mice. This isessentially the same as those of pre-vious study showing a transient in-crease in TR�1 expression in the cer-ebellum (11). Interestingly, theexpression was significantly in-creased on P15 in both Tg/� andTg/Tg mice. Although the mecha-nism of the increase cannot be clar-ified, such increase may be the reasonfor milder cerebellar phenotype thanthose expected by the reporter geneassay results.

Since we specifically inhibitedTR-mediated transcription only inPurkinje cells, we initially hypothe-sized that the Purkinje cell morpho-genesis is specifically altered. As ex-pected, Purkinje cell developmentwas affected both in vivo (Figure 3)and in vitro (Figure 4). Particularly inthe primary culture of the cerebel-lum, T4-induced Purkinje cell den-drite arborization was significantlysuppressed by transgene expression.Since the suppression was greater inthe Tg/Tg than in the Tg/� mice,suppression may be induced in atransgene dose-dependent manner.The expression of TH-responsivegenes such as those encoding the IP3

+/+

Tg/+

Tg/T

g

T4 (M): - 10-10 10-9 10-8

0

9000

18000

27000

36000

45000

con -10 -9 -8

WTHEHO

T4 (M): - 10-10 10-9 10-80

1

2

3

4

5

Rel

ativ

e de

ndrit

ic a

rea

+/+Tg/+Tg/Tg

*

***

****

*

A

B

Figure 4. Changes in Purkinje cell dendrite arborization in primary cerebellarculture. (A) Photomicrographs showing the effect of T4 on Purkinje cell morphology. Primaryculture of cerebellum contains the whole set of cerebellar cells including astrocytes, in which T4is 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 antibodyto visualize Purkinje cells. Bars, 50 �m. (B) Changes in dendritic areas of Purkinje cells. Dendriticarea was quantified using Object-Image 2.11 software (NIH). Data are expressed as mean �SEM. (n � 10 determinations). Data shown are representative of at least three independentexperiments. *, p�0.05 and **, p�0.01 compared with the �/� mice by Bonferroni’s test.



receptor and ROR�, which are predominantly expressedin Purkinje cells (35, 36), was also altered. These resultsclearly indicate that endogenous TR-mediated transcrip-tion was suppressed by mutant TR, leading to aberrantPurkinje cell development.

Recently, Fauquier T et al has reported the transgenicmice that express a mutant TR�1 specifically in Purkinjecells using Cre/loxP technology (42). They used severalCre lines including L7-Cre mice, in which L7/Pcp2 pro-moter was used to express CRE recombinase, which in-duce Purkinje cell-specific mutant TR�1 expressionthrough loxP sequence recombination. This mouseshowed limited alterations in cerebellar morphogenesiscompared with those of wild type. Although it is difficultto clarify the mechanisms inducing difference in mopho-genesis in the present study, one possible reason may be thedifference in the timing of transgene expression. By L7-Crerecombination, the transgene expression was obserbed af-

ter P8 (42), whereas the expression of mutant TR�1 wasobserved as early as P2 in the present study (Figure 2B). Onthe other hand, by Ptf1a-Cre recombination, mutantTR�1 in Purkine cells and GABAergic interneurons wasexpressed from prenatal stage, showing the alteration ofPurkinje cell morphogenesis (42). Thus, the earlier expres-sion of mutant TR in our animal model may induce more

*

0

0.8

1.6

2.4

3.2

4

P2 P7 P15 P30

Rel

ativ

e m

RN

A Le

vels RORα Male

B

1

2

3

4

5

0

**

**

0

1.2

2.4

3.6

4.8

6

P2 P7 P15 P30

Rel

ativ

e m

RN

A Le

vels IP3 receptor Male

A

1

2

3

4

5

0**

**

*

Figure 5. Expression of IP3 receptor and ROR� mRNAsduring postnatal development of the male micecerebellum. The mRNA expression levels of IP3 receptor (A) andROR� (B) in the cerebella of the �/� (�), Tg/� (), and Tg/Tg (Œ)mice were analyzed by quantitative realtime RT-PCR. The mRNAexpression level was normalized to the GAPDH mRNA expression level.Data are expressed as mean � SEM (n � 4). *, p� 0.05 and **,p�0.01 compared with the �/� mice by Bonferroni’s test.

0

10

20

30

40

50

P2 P7 P15 P30

BNDF Male

**

A

**R

elat

ive

mR

NA

Lev

els 5

4

3

2

1

0

0

0.5

1

1.5

2

2.5

P2 P7 P15 P30

NT-3 Male

***

B

**

**

**

5

4

3

2

1

0

Rel

ativ

e m

RN

A L

evel

s

56

84

112

140

P2 P7 P15 P30

Hairless Male

C

****

5

4

3

2

1

0

Rel

ativ

e m

RN

A L

evel

s

Figure 6. Expression of BDNF, NT-3 and hairless mRNAsduring postnatal development of male mice cerebellum.The mRNA expression levels of BDNF, NT-3 and hairless in the cerebellaof the �/� (�), Tg/� (), and Tg/Tg (Œ) mice were analyzed byquantitative realtime RT-PCR. The mRNA expression level wasnormalized to the GAPDH mRNA expression level. Data are expressedas mean � SEM (n � 4). *, p � 0.05 and **, p�0.01 compared withthe �/� mice by Bonferroni’s test.



sever phenotype than those induced by L7-Crerecombination.

To our surprise, granule cell migration was also af-fected (Figure 3) on P7 and P15. The disappearance of theEGL was delayed in the Tg/� and Tg/Tg mice. Such de-layed migration was also observed in antithyroid drug-induced hypothyroid mice (21, 43, 44). Furthermore, theexpression of several TH-responsive genes such as thoseencoding BDNF, NT-3 and hairless, which are predomi-nantly expressed in granule cells (40) was altered (Figure6). The expression level of NT-3 in the �/� mice peakedon P7, whereas those in the Tg/� and Tg/Tg peaked onP15. These results indicate the delayed expression bytransgene. The expression levels of hairless mRNA, whichis directly regulated by TR (45), was also decreased on P7,indicating that additional factors other than TR may reg-ulate hairless expression. In addition, the expression ofMBP, which is predominantly expressed in oligodendro-cytes (41) was also altered. As shown by immunohisto-chemistry (Figure 2A), since the transgene was expressedspecifically in the Purkinje cells in the cerebellum, othersubsets of cell in the cerebellum including the granule cellsand oligodendrocytes should be under the euthroid status.

Thus, it is not likely that such effects are induced by thedirect inhibition of TH action in granule cells or oligo-dendrocytes. Rather, the disruption of Purkinje cell func-tion may indirectly alter the normal granule cell and oli-godendrocyte development. Since TR�1 is abundantlyexpressed in granule cells (11, 21), the change in granulecell development in hypothyroid animals or mutant TRknock-in mice have been considered as a consequence ofthe direct action of TH. The results of the present studyhave demonstrated that abnormal granule cell develop-ment in the perinatal hypothyroidism may be, at least inpart, exerted by the indirect action of TH through Purkinjecells.

On the other hand, mechanisms inducing abnormalgranule cell development in the present study have not yetbeen clarified. Purkinje cells may regulate granule cell de-velopment through several mechanisms. One possiblepathway may be mediated by sonic hedgehog (Shh) (46,47), which is a secretory glycoprotein that plays an im-portant role in cell proliferation and cell fate determina-tion (48, 49). Thus, Purkinje cells may regulate the pro-liferation of granule cells through Shh secretion. Inaddition, several proteins such as vitronectin and laminininteract with Shh and take part in granule cell migration(50). A previous study has shown that Shh expression isdisrupted by perinatal hypothyroidism (51). Thus, al-though TH-responsive genes in the granule cells are acci-dental in transgenic animals, TH may indirectly regulatethe expression of these genes through Purkinje cell-derivedtrophic factors.

The mechanism underlying alternation of MBP mRNAexpression in oligodendrocytes have also not yet been clar-ified. Myelination is a highly regulated timed event thatstarts a few days after birth and depends on the properdifferentiation of oligodendrocytes (52). The expressionof each oligodendrocyte/myelin gene may be affected byTH. Hypothyroidism impairs transiently the 2�3�cyclicnucleotide 3�phosphodiesterase (E.C. 3.1.4.37-CNPase)gene expression, that precedes the myelin basic protein(MBP), the proteolipidic protein (PLP) and the myelin as-sociated glycoprotein (MAG) gene transcription (53).

**

0

12

24

36

48

60

P2 P7 P15 P30

Rel

ativ

e m

RN

A Le

vels MBP Male

1

2

3

4

5

0**

Figure 7. Expression of MBP mRNAs during postnataldevelopment of male mice cerebellum. The mRNA expressionlevels of IP3 receptor and ROR� in the cerebella of the �/� (�), Tg/�(), and Tg/Tg (Œ) mice were analyzed by quantitative realtime RT-PCR. The mRNA expression level was normalized to the GAPDH mRNAexpression level. Data are expressed as mean � SEM (n � 4). **,p�0.01 compared with the �/� mice by Bonferroni’s test.

0

20

40

60

80

100

120

Wild Hetero Homo

Tim

e on

Rot

arod

(sec

)

P15 Male

**

+/+ Tg/+ Tg/Tg0

20

40

60

80

100

120

Wild Hetero Homo

Tim

e on

Rot

arod

(sec

)

P30 Male

*

+/+ Tg/+ Tg/Tg

Figure 8. Changes in motor coordination, studied by rotarod test of male mice. Male �/�, Tg/�, and Tg/Tg mice were trained onthe 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 **, p�0.01 compared with the �/� mice by Bonferroni’s test.



These effects were observed only in the differentiated oli-godendrocyte, suggesting a role for TH in the regulation ofthe oligodendrocyte differentiation. Thus, as similar togranule cells, although TH-responsive genes in the oligo-dendrocytes are accidental in transgenic animals, TH mayindirectly regulate the expression of these genes throughPurkinje cell-derived trophic factors. On the other hand,Picou F, et al (54) reported the effect of TR�1 in oligo-dendrocyte precursor cells (OPC) using Cre/loxP-basedtransgenic mice. They concluded that TR�1 acts on OPCthrough two mechanisms. At early postnatal stage, THpromotes the secretion of neurotrophic factor in Purkinjecells and granule cells inducing differentiation of OPC;Later TH mainly affects OPC differentiation in a cell-au-tonomous manner. In the present study, simultaneous de-creases in both NT-3 and MBP was observed on P7. Thisresult may support their hypothesis that a decrease in neu-rotrophin secretion may affect the OPC differentiation atearly postnatal stage.

The time on the rotarod was decreased in transgenicmice, suggesting that the motor coordination was dis-rupted (Figure 8). The defect was observed not only onP15, but also on P30, when cerebellar morphologies be-come almost identical. This finding is consistent with pre-vious studies using a drug-induced hypothyroid animalmodel showing that, although cerebellar morphologiesbecome indistinguishable from euthyroid animal on P30(55, 56), the motor coordination defect was still observed(57). Similar symptoms were also observed in a humanstudy showing that, if hypothyroidism is not normalizedwithin the first 3 months of life, patients may show per-manent cerebellar symptoms (58). The present study alsoconfirmed that the inhibition of TR action in Purkinje cellsduring the critical development period may produce a per-manent cerebellar defect. Although many reports showedthat TR�1 may be important for the normal cerebellardevelopment (42, 59–63), the molecular mechanisms in-ducing cerebellar ataxia in hypothyroidism have not yetbeen fully clarified. Since thyroid hormone directly or in-directly regulates a large number of genes in a temporally-and spatially- dependent manner, multiple genes may beinvolved. Together with previous study, we consider thatboth TR�1 and �1 may be important for normal cerebellardevelopment. Indeed, we showed that TR�1-mediatedtranscription was also suppressed by Mf-1 (SupplementalFigure S1). Coordination of TR�1 and �1 may be com-plicated. In addition, complicated regulation of cell-cellinteractions including neuron-neuron or neuron-glia in-teractions that are also directly and/or indirectly regulatedby TR�1 and �1. Thus, to differentiate the role of TR� andTR�, many additional studies are required. Further study

may be also required to clarify involvement of TRs onformation and regulation of a neuronal or gene network.

In summary, we established a line of transgenic miceexpressing mutant TR specifically in Purkinje cells. Thesemice showed a hypothyroid phenotype in the cerebellumnot only in Purkinje cells but also in granule cells andoligodendrocytes. As a consequence, these mice showed amotor coordination defect. These results indicate that THaction through its binding to TR expressed in Purkinjecells is required for the normal cerebellar development,although factors transmitting the TH influence in Purkinjecell to other subset of cells have not yet been clarified. THaction through TR in Purkinje cells is also important forthe development of other subsets of cerebellar cells such asgranule cells and oligodendrocytes.

Acknowledgments

We would like to thank Dr. John Oberdick for kindly preparingthe construct, Drs. Yusuke Takatsuru and Asahi Haijima forhelpful discussion, and Ms. Misae Ohta, Ms. Hiroko Masuda,and Drs. Izuki Amano, Winda Ariyani, Chun-Hong Qiu, King-sley Ibhazehiebo, Junko Kimura-Kuroda, Hideki Yokoo andMasami Murakami for technical assistance and advice.

Address all correspondence and requests for reprints to: Nori-yuki Koibuchi, M.D., Ph.D., Department of Integrative Physi-ology, Gunma University Graduate School of Medicine,3–39–22 Showa-machi, Maebashi, Gunma 371–8511, Japan,Tel: �81–27–220–7923; Fax: �81–27–220–7926; E-mail:[email protected].

This work was supported by a Grant-in-Aid for ScientificResearch (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 Min-istry of Education, Culture, Sports, Science and Technology(MEXT), and Cosmic Research Award to T.I from Japan Thy-roid Association.

# These authors contributed equally.Conflict of Interest: There is no conflict of interest in this

manuscript.

References

1. Bernal J. Thyroid hormone receptors in brain development and func-tion. Nat Clin Pract Endocrinol Metab. 2007;3:249–259.

2. Oppenheimer JH, Schwartz HL. Molecular basis of thyroid hor-mone-dependent brain development. Endocr Rev. 1997;18:462–475.

3. Calvo R, Obregón MJ, Ruiz de Oña C, Escobar del Rey F, Morrealede Escobar G. Congenital hypothyroidism, as studied in rats. Crucialrole of maternal thyroxine but not of 3,5,3�-triiodothyronine in theprotection of the fetal brain. J Clin Invest. 1990;86:889–899.

4. Porterfield SP, Hendrich CE. The role of thyroid hormones in pre-natal and neonatal neurological development–current perspectives.Endocr Rev. 1993;14:94–106.



mailto:[email protected]

5. Lazar MA. Thyroid hormone receptors: multiple forms, multiplepossibilities. Endocr Rev. 1993;14:184–193.

6. Samuels HH, Forman BM, Horowitz ZD, Ye ZS. Regulation of geneexpression by thyroid hormone. J Clin Invest. 1988;81:957–967.

7. Bigler J, Eisenman RN. Novel location and function of a thyroidhormone response element. EMBO J. 1995;14:5710–5723.

8. Koenig RJ. Thyroid hormone receptor coactivators and corepres-sors. Thyroid. 1998;8:703–713.

9. Ramos HE, Weiss RE. Regulation of nuclear coactivator and core-pressor expression in mouse cerebellum by thyroid hormone. Thy-roid. 2006;16:211–216.

10. Forrest D, Hallböök F, Persson H, Vennström B. Distinct functionsfor thyroid hormone receptors alpha and beta in brain developmentindicated by differential expression of receptor genes. EMBO J.1991;10:269–275.

11. Bradley DJ, Towle HC, Young WS 3rd. Spatial and temporal ex-pression of alpha- and beta-thyroid hormone receptor mRNAs, in-cluding the beta 2-subtype, in the developing mammalian nervoussystem. J Neurosci. 1992;12:2288–2302.

12. Mellström B, Naranjo JR, Santos A, Gonzalez AM, Bernal J. Inde-pendent expression of the alpha and beta c-erbA genes in developingrat brain. Mol Endocrinol. 1991;5:1339–1350.

13. Tagami T, Gu WX, Peairs PT, West BL, Jameson JL. A novel naturalmutation in the thyroid hormone receptor defines a dual functionaldomain that exchanges nuclear receptor corepressors and coactiva-tors. Mol Endocrinol. 1998;12:1888–1902.

14. Xu L, Glass CK, Rosenfeld MG. Coactivator and corepressor com-plexes in nuclear receptor function. Curr Opin Genet Dev. 1999;9:140–147.

15. Rovet JF. Congenital hypothyroidism: an analysis of persisting def-icits and associated factors. Child Neuropsychol. 2002;8:150–162.

16. Koibuchi N, Chin WW. Thyroid hormone action and brain devel-opment. Trends Endocrinol Metab. 2000;11:123–128.

17. O’Halloran CJ, Kinsella GJ, Storey E. The cerebellum and neuro-psychological functioning: a critical review. J Clin Exp Neuropsy-chol. 2012;34:35–56.

18. De Zeeuw CI, Yeo CH. Time and tide in cerebellar memory forma-tion. Curr Opin Neurobiol. 2005;15:667–674.

19. Lauder JM, Altman J, Krebs H. Some mechanisms of cerebellarfoliation: effects of early hypo- and hyperthyroidism. Brain Res.1974;76:33–40.

20. Strait KA, Schwartz HL, Seybold VS, Ling NC, Oppenheimer JH.Immunofluorescence localization of thyroid hormone receptor pro-tein beta 1 and variant alpha 2 in selected tissues: cerebellar Purkinjecells as a model for beta 1 receptor-mediated developmental effectsof thyroid hormone in brain. Proc Natl Acad Sci U S A. 1991;88:3887–3891.

21. Koibuchi N, Jingu H, Iwasaki T, Chin WW. Current perspectives onthe role of thyroid hormone in growth and development of cerebel-lum. Cerebellum. 2003;2:279–289.

22. Koibuchi N, Yamaoka S, Chin WW. Effect of altered thyroid statuson neurotrophin gene expression during postnatal development ofthe mouse cerebellum. Thyroid. 2001;11:205–210.

23. Strait KA, Zou L, Oppenheimer JH. Beta 1 isoform-specific regu-lation of a triiodothyronine-induced gene during cerebellar devel-opment. Mol Endocrinol. 1992;6:1874–1880.

24. O’Shea PJ, Williams GR. Insight into the physiological actions ofthyroid hormone receptors from genetically modified mice. J En-docrinol. 2002;175:553–570.

25. Portella AC, Carvalho F, Faustino L, Wondisford FE, Ortiga-Car-valho TM, Gomes FC. Thyroid hormone receptor beta mutationcauses severe impairment of cerebellar development. Mol Cell Neu-rosci. 2010;44:68–77.

26. Hashimoto K, Curty FH, Borges PP, Lee CE, Abel ED, Elmquist JK,Cohen RN, Wondisford FE. An unliganded thyroid hormone re-ceptor causes severe neurological dysfunction. Proc Natl Acad Sci US A. 2001;98:3998–4003.

27. Venero C, Guadaño-Ferraz A, Herrero AI, Nordström K, ManzanoJ, de Escobar GM, Bernal J, Vennström B. Anxiety, memory im-pairment, and locomotor dysfunction caused by a mutant thyroidhormone receptor alpha1 can be ameliorated by T3 treatment.Genes Dev. 2005;19:2152–2163.

28. Smeyne RJ, Chu T, Lewin A, Bian F, Sanlioglu S, Kunsch C, Lira SA,Oberdick J. Local control of granule cell generation by cerebellarPurkinje cells. Mol Cell Neurosci. 1995;6:230–251.

29. Zou L, Hagen SG, Strait KA, Oppenheimer JH. Identification ofthyroid hormone response elements in rodent Pcp-2, a developmen-tally regulated gene of cerebellar Purkinje cells. J Biol Chem. 1994;269:13346–13352.

30. Sakurai A1, Takeda K, Ain K, Ceccarelli P, Nakai A, Seino S, BellGI, Refetoff S, DeGroot LJ. Generalized resistance to thyroid hor-mone associated with a mutation in the ligand-binding domain ofthe human thyroid hormone receptor beta. Proc Natl Acad Sci U SA. 1989;86:8977–8981.

31. Liu Y1, Takeshita A, Misiti S, Chin WW, Yen PM. Lack of coacti-vator interaction can be a mechanism for dominant negative activityby mutant thyroid hormone receptors. Endocrinology. 1998;139:4197–4204.

32. Field J, Nikawa J, Broek D, MacDonald B, Rodgers L, Wilson IA,Lerner RA, Wigler M. Purification of a RAS-responsive adenylylcyclase complex from Saccharomyces cerevisiae by use of an epitopeaddition method. Mol Cell Biol. 1988;8:2159–2165.

33. Qiu CH, Shimokawa N, Iwasaki T, Parhar IS, Koibuchi N. Alter-ation of cerebellar neurotropin messenger ribonucleic acids and thelack of thyroid hormone receptor augmentation by staggerer-typeretinoic acid receptor-related orphan receptor-alpha mutation. En-docrinology. 2007;148:1745–1753.

34. Kimura-Kuroda J, Nagata I, Negishi-Kato M, Kuroda Y. Thyroidhormone-dependent development of mouse cerebellar Purkinje cellsin vitro. Brain Res Dev Brain Res. 2002;137:55–65.

35. Nakagawa T, Okano H, Furuichi T, Aruga J, Mikoshiba K. Thesubtypes of the mouse inositol 1,4,5-trisphosphate receptor are ex-pressed in a tissue-specific and developmentally specific manner.Proc Natl Acad Sci U S A. 1991;88:6244–6248.

36. Ino H. Immunohistochemical characterization of the orphan nu-clear receptor ROR alpha in the mouse nervous system. J HistochemCytochem. 2004;52:311–323.

37. Koibuchi N, Chin WW. ROR alpha gene expression in the perinatalrat cerebellum: ontogeny and thyroid hormone regulation. Endo-crinology. 1998;139:2335–2341.

38. Koibuchi N, Fukuda H, Chin WW. Promoter-specific regulation ofthe brain-derived neurotropic factor gene by thyroid hormone in thedeveloping rat cerebellum. Endocrinology. 1999;140:3955–3961.

39. Barbacid M. The Trk family of neurotrophin receptors. J Neurobiol.1994;25:1386–1403.

40. Rocamora N, García-Ladona FJ, Palacios JM, Mengod G. Differ-ential expression of brain-derived neurotrophic factor, neurotro-phin-3, and low-affinity nerve growth factor receptor during thepostnatal development of the rat cerebellar system. Brain Res MolBrain Res. 1993;17:1–8.

41. Monge M, Kadiiski D, Jacque CM, Zalc B. Oligodendroglial ex-pression and deposition of four major myelin constituents in themyelin sheath during development. An in vivo study. Dev Neurosci.1986;8:222–235.

42. Fauquier T, Chatonnet F, Picou F, Richard S, Fossat N, Aguilera N,Lamonerie T, Flamant F. Purkinje cells and Bergmann glia are pri-mary targets of the TR�1 thyroid hormone receptor during mousecerebellum postnatal development. Development. 2014;141:166–175.

43. Lauder JM. The effects of early hypo- and hyperthyroidism on thedevelopment of rat cerebellar cortex. III. Kinetics of cell prolifera-tion in the external granular layer. Brain Res. 1977;126:31–51.

44. Nicholson JL, Altman J. The effects of early hypo- and hyperthy-



roidism on the development of rat cerebellar cortex. I. Cell prolif-eration and differentiation. Brain Res. 1972;44:13–23.

45. Potter GB1, Zarach JM, Sisk JM, Thompson CC. The thyroid hor-mone-regulated corepressor hairless associates with histonedeacetylases in neonatal rat brain. Mol Endocrinol. 2002;16:2547–2560.

46. Traiffort E, Charytoniuk DA, Faure H, Ruat M. Regional distribu-tion of Sonic Hedgehog, patched, and smoothened mRNA in theadult rat brain. J Neurochem. 1998;70:1327–1330.

47. Traiffort E, Charytoniuk D, Watroba L, Faure H, Sales N, Ruat M.Discrete localizations of hedgehog signalling components in the de-veloping and adult rat nervous system. Eur J Neurosci. 1999;11:3199–3214.

48. Roelink H, Porter JA, Chiang C, Tanabe Y, Chang DT, Beachy PA,Jessell TM. Floor plate and motor neuron induction by differentconcentrations of the amino-terminal cleavage product of sonichedgehog autoproteolysis. Cell. 1995;81:445–455.

49. Jensen AM, Wallace VA. Expression of Sonic hedgehog and its pu-tative role as a precursor cell mitogen in the developing mouse retina.Development. 1997;124:363–371.

50. Sotelo C. Cellular and genetic regulation of the development of thecerebellar system. Prog Neurobiol. 2004;72:295–339.

51. Hasebe M, Ohta E, Imagawa T, Uehara M. Expression of sonichedgehog regulates morphological changes of rat developing cere-bellum in hypothyroidism. J Toxicol Sci. 2008;33:473–477.

52. Roots B. The evolution of myelinating cells. In: Vernadakis A, RootsB, eds. Neuron-Glia Interrelations During Phylogeny: I. Phylogenyand Ontogeny of Glial Cells. Totowa; Humana Press. 1995;223–248.

53. Kanfer J, ParentyM, Goujct-Zalc C, Munge M, Bernier L, Campa-gnoni AT, Dautigny A, Zalc B. Developmental expression of myelinproteolipid protein, myelin basic protein, and 2�,3�-cyclic nucleotide3�-phosphodiesterase transcripts in different brain regions. J MolNeurosci. 1989;1:39–46.

54. Picou F, Fauquier T, Chatonnet F, Flamant F. A Bimodal Influence

of Thyroid Hormone on Cerebellum Oligodendrocyte Differentia-tion. Mol Endocrinol. 2012;26:608–618.

55. Nicholson JL, Altman J. The effects of early hypo- and hyperthy-roidism on the development of the rat cerebellar cortex. II. Synap-togenesis in the molecular layer. Brain Res. 1972;44:25–36.

56. Lauder JM. Effects of early hypo- and hyperthyroidism on devel-opment of rat cerebellar cortex. IV. The parallel fibers. Brain Res.1978;142:25–39.

57. Hasebe M, Matsumoto I, Imagawa T, Uehara M. Effects of ananti-thyroid drug, methimazole, administration to rat dams on thecerebellar cortex development in their pups. Int J Dev Neurosci.2008;26:409–414.

58. Wiebel J. Cerebellar-ataxic syndrome in children and adolescentswith hypothyroidism under treatment. Acta Paediatr Scand. 1976;65:201–205.

59. Fauquier T. Romero E, Picou F, Chatonnet F, Nguyen XN,Quignodon L, Flaman F. Severe impairment of cerebellum devel-opment in mice expressing a dominant-negative mutation inactivat-ing thyroid hormone receptor alpha1 isoform. Dev Biol. 2011;356:350–358.

60. Morte B, Beatriz Morte B, Manzano J, Scanlan T, Vennström B,Bernal J. Deletion of the thyroid hormone receptor alpha 1 preventsthe structural alterations of the cerebellum induced by hypothyroid-ism. Proc Natl Acad Sci U S A. 2002;99:3985–3989.

61. Heuer H, Mason CA. Thyroid hormone induces cerebellar Purkinjecell dendritic development via the thyroid hormone receptor alpha1.J Neurosci. 2003;23:10604–10612.

62. Peeters RP, Hernandez A, Ng L, Ma M, Sharlin DS, Pandey M,Simonds WF, St. Germain DL, Forrest D. Cerebellar abnormalitiesin mice lacking type 3 deiodinase and partial reversal of phenotypeby deletion of thyroid hormone receptor �1. Endocrinology. 2013;154:550–561.

63. Avci HX, Lebrun C, Wehrlé R, Doulazmi M, Chatonnet F, MorelMP, Ema M, Vodjdani G, Sotelo C, Flamant F, Dusart I. Thyroidhormone triggers the developmental loss of axonal regenerative ca-pacity via thyroid hormone receptor a1 and krüppel-like factor 9 inPurkinje cells. Proc Natl Acad Sci U S A. 2012;109:14206–14211.



Documents

en%2E2014-1079