Expression of thyroid hormone receptors A and B in developing rat tissues; evidence for extensive...

523

Expression of thyroid hormone
receptors A and B indeveloping rat tissues; evidence for extensiveposttranscriptional regulation
Richard Keijzer1,*, Piet-Jan E Blommaart1,2,*, Wil T Labruyere2, Jacqueline L M Vermeulen2,Behrouz Zandieh Doulabi3, Onno Bakker3, Dick Tibboel1 and Wouter H Lamers2

1
Department of Pediatric Surgery, Erasmus MC-Sophia, Dr Molewaterplein 60, 3015 GJ Rotterdam, The Netherlands
2Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, Meibergdreef 69–71, 1105 AZ Amsterdam, The Netherlands

3Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

(Requests for offprints should be addressed to W H Lamers who is now at AMC Liver Center, Academic Medical Center, University of Amsterdam, Meibergdreef 69-71, 1105 BK,Amsterdam, The Netherlands; Email: [email protected])

(R Keijzer and P-J E Blommaart contributed equally to this study)

Abstract

The perinatal changes in the pattern of expression of the thyroid hormone receptor (TR) isoformsTRa1 TRa2, TRb1, and TRb2

were investigated using in situ hybridization and immunohistochemistry, and RT-PCR and western blotting as visualization

and quantification techniques respectively. In liver, lung, and kidney, TRa mRNA was expressed in the stromal and TRb

mRNA in the parenchymal component of the tissues. When compared with liver, TRa mRNA concentrations were tenfold

higher in lung, kidney, and intestine, and 100-foldhigher inbrain,withTRa2 mRNAconcentrations exceeding those of TRa1 5-

to 10-fold. Tissue TRb1 mRNA concentrations were similar in liver, lung, and brain, and 3- to 5-fold higher in kidney and

intestine. None of the TRb2 mRNA could be detected outside the pituitary. Tissue TRa2 and TRb1 protein levels reached adult

levels at 5 days before birth, whereas TRa1 protein peaked after birth. Because of the distinct time-course of thyroid hormone-

binding receptors TRa1 and TRb1, we speculate that an initiating, TRb1-mediated signaling from the parenchyma is followed

by a TRa1-mediated response in the stroma. When compared with organs with a complementary parenchymal–stromal

expression pattern, organs with extensive cellular co-expression of TRa and TRb (brain and intestinal epithelium) were

characterized by a very low TRa protein: mRNA ratio, implying a low translational efficiency of TR mRNA or a high turnover of

TR protein. The data indicate that the TR-dependent regulatory cascades are controlled differently in organs with a

complementary tissue expression pattern and in those with cellular co-expression of the TRa and TRb genes.

Journal of Molecular Endocrinology (2007) 38, 523–535

Introduction

Since it was discovered that metamorphosis can beinduced in amphibians by feeding them thyroid glandextract (Gudernatsch 1912), it has become generallyaccepted that thyroid hormones play a crucial role incontrolling growth, development, differentiation, andmetabolism of virtually all tissues of vertebrates. Thebeneficial effects of thyroid hormones onperinatal organdevelopment and maturation are most readily recogniz-able in brain (Oppenheimer & Schwartz 1997, Morrealede Escobar et al. 2004). However, since thyroid hormonescan pass the placenta and accumulate in embryonic cellsto functionally relevant concentrations well before theembryonic thyroid starts to functionon its own (Morrealede Escobar et al. 1990), thyroid hormone-dependentinstructive effects in earlier phases of organ developmentare likely (Glinoer 2001, van Tuyl et al. 2004).

Journal of Molecular Endocrinology (2007) 38, 523–5350952–5041/07/038–523 q 2007 Society for Endocrinology Printed in Great Britain

The effects of thyroid hormones on gene expressionare mediated via the thyroid hormone receptor (TR)genes a (NR1A1) and b (NR1A2), which are both themembers of the steroid/TR superfamily of ligand-dependent transcription factors (Zhang & Lazar 2000,Yen 2001, Yen et al. 2006). The TRa and TRb genesgenerate, via alternative splicing, four thyroidhormone-binding receptors, namely TRa1 and TRb1–3(Williams 2000, Chassande 2003). In addition, severalproteins, including TRa2, which do not bind thyroidhormone-response elements and/or thyroidhormones, are formed. Presumably, these proteinsmodulate thyroid hormone-mediated gene expression(Cheng 2000, Williams 2000, Plateroti et al. 2001,Yen 2001).

In rat, northern blot analysis showed that TRa mRNAconcentration increased in brain, liver, and brownadipose tissue from 18 days of development onwards to

DOI: 10.1677/jme.1.02125Online version via http://www.endocrinology-journals.org

R KEIJZER, P-J E BLOMMAART and others . Expression of TRa and -b in developing tissues524

reach its highest level perinatally (Strait et al. 1990, Tuca etal. 1993). TRbmRNA also increased perinatally in brain,but was reported to reach its peak only in the secondpostnatal week (Strait et al. 1990). In contrast, TRbmRNAdecreased perinatally in liver and brown adipose tissue(Strait et al. 1990, Tuca et al. 1993). In the developingnervous system of the rat, the highest cellular concen-tration of TRamRNAswas found in the areas of neuronaldifferentiation such as the fetal neocortical plate, whereasTRb mRNAs were largely restricted to the zones withneuroblast proliferation, such as the germinal trigoneand the cortical ventricular layer (Bradley et al. 1992).Although these findings suggested complementary and,perhaps, mutually exclusive roles of the TRa and TRbgenes in organogenesis, they did not result in a specifichypothesis for the mechanism of action of thyroidhormones in development. Furthermore, these studiesdid not address TR expression at the protein level. We,therefore, studied the spatial and the temporal distri-bution of the mRNAs and proteins encoding TRa andTRb by in situ hybridization and immunohistochemistry.Since our in situ probes did not differentiate between therespective splice variants, quantitative PCR techniqueswere used to determine the tissue levels of TRa1, TRa2,TRb1, and TRb2 mRNA. Since tissue TRmRNA level andnuclear thyroid hormone-binding capacity (i.e.,functional TR protein) do not always correlate (Strait etal. 1990, Tuca et al. 1993), we also investigated tissue TRprotein level by western blot analysis and immunohis-tochemistry, using our recently described antisera(Zandieh Doulabi et al. 2002, 2003). We report a highlyspecific, but heterogeneous spatial and temporalexpression pattern of the TRa and TRb mRNAs, andreport evidence for extensive posttranscriptionalregulation in the perinatal period.

Materials and methods

Animals

Female Wistar rats in heat were selected and mated inthe morning, the next day being designated embryonicday 1 (E1). Fetuses were harvested by caesarian sectionat E13, E14, E15, E16, E17, E18, E19, and E20; or wereborn naturally and killed 1 (P1) or 4 days (P4) later, oras adults. All experiments were performed underprotocols approved by the University of AmsterdamCommittee on Animal Research.

Tissue isolation and preparation

All tissues were fixed in phosphate-buffered 4%formaldehyde for in situ hybridization and in the ratioof methanol:acetone:water (2:2:1 (v/v)) for immuno-histochemistry as described (van Tuyl et al. 2004).


In situ hybridization

In situ hybridization was carried out exactly as described(Moorman et al. 2000). In situ hybridization carried outaccording to this protocol produces semiquantitativedata, that is, differences in absorbance betweendifferent tissues or cells within one section reflectdifferences in mRNA concentration between thesetissues or cells (Jonker et al. 1997). This protocol isincluded in the method paper referenced to Moormanet al. (2000). The EcoRI–HindIII cDNA fragments ofthe clones rc-erbA-a (nt K29 to C1821) and rc-erbA-b(nt K37 to C2165) were used as probes to detect TRaand TRbmRNA respectively (Bradley et al. 1992). Thesefragments do not discriminate between TR isoforms.[a-35S]UTP-labeled antisense and sense probes weregenerated with Sp6 and T7 RNA polymerase respect-ively. We opted to use almost the entire TR mRNAsequence to make the in situ assay as sensitive aspossible, because the hybridization signal is dependenton the length of the probe (before fragmentation). Wethen used PCR to quantify subtypes.

Quantitative PCR

At E15, E18, P1, and in the adult, TR mRNA levels werequantified in brain, pituitary gland, lung, liver, kidney,and intestines. For each stage, minimally three rats wereassayed. TR mRNAs were quantified by establishing theefficiency of both the reverse-transcription and poly-merase chain reaction steps of the assay. Large-scalemRNA synthesis from the respective TR cDNA fragmentswas performed with the MEGAshortscript T7 Kit(Ambion, Nieuwerkerk a/d IJssel, The Netherlands)and quantified by including a known amount of[a-35S]CTP. Reverse-transcriptase efficiency was esti-mated by comparing mRNA input (different amounts ofthe RNA transcripts) and DNA output, and amounted to80–100%. TRa cDNA amplification was estimated bycompetitive RT-PCR as described (Moller & Jansson1997), whereas TRb cDNA amplification was measuredby real-time PCR (Lightcycler, Roche).

TRa

The sense primer was chosen in the common part ofthe TRa1 and TRa2 sequences, whereas the antisenseprimers were in the specific parts. For TRa1, a 343 bpfragment was formed (nt 1219–1562 of the mRNA) andfor TRa2 a 242 bp fragment (nt 1411–1653). Afterreverse transcription (RT) and PCR amplification, theconcentration of TRa1 and TRa2 cDNA in the samplewas estimated by titration of 8–5000!10K4 amol(TRa1) or 4–2500!10K3 amol (TRa2) of a ‘compe-timer’ fragment that differed from the parent frag-ments by missing an identical 82 bp 5 0 HincII fragment.

www.endocrinology-journals.org

Expression of TRa and -b in developing tissues . R KEIJZER, P-J E BLOMMAART and others 525

The amount of competimer added was determined byincluding a known amount of [a-35S]dATP duringlarge-scale PCR synthesis. TRa mRNA concentration inthe sample was calculated from a double logarithmicplot of the TRa:competimer ratio and the competimerconcentration (Moller & Jansson 1997).

TRb

The antisense primer was chosen in the common part ofTRb1 and TRb2, whereas the sense primers were in thespecific part. For TRb1, a 185 bp fragment was formed (nt437–622 of the mRNA) and for TRb2, a 244 bp fragment(nt 343–587 of the mRNA). TRb cDNA concentration inthe sample was deduced from a standard curve with5–50 000!10K4 amol TRb1 or TRb2 DNA fragment. TheTRb1 and TRb2 fragments were made by large scale PCRsynthesis and quantified by including a known amount of[a-32P]dATP. TRb2 levels were also quantified withfluorescence resonance energy transfer of hybridizationprobes nt 264–290 (red640-labeled) and nt 292–316(fluorescein-labeled) on amplified fragment nt 241–556.Dilutions of pituitary gland mRNA with a predeterminedTRb2 mRNA concentration were used to generate astandard curve.

Western blotting and immunohistochemistry

Extracts of brain, pituitary gland, thyroid, trachea, lung,heart, muscle, liver, intestines, kidneys, adrenals, testis,epididymal fat pad, bladder, and spleen from 3 E17, P4,

Table 1 Developmental changes in the expression of thyroid hormoneadrenal, and intestines

Stage ofdevelopment TRa1 TR

m mRNA Protein m

OrganCentral Early fetal K K Cnervous system Late fetal K K C

Neonatal K K CLung Early fetal K K C

Late fetal K C CNeonatal K C C

Liver Early fetal K K CLate fetal C C CNeonatal K C C

Kidney Early fetal K K CLate fetal K C CNeonatal K C C

Adrenal Early fetal C K CLate fetal C C CNeonatal K C K

Intestines Early fetal K K KLate fetal K C CNeonatal K K C

Early fetal, E12–E16; late fetal, E17–E22; neonatal, P0–P4. K, no expression


and adult rats were analyzed in triplicate by westernblotting, using antisera specifically identifying TRa1,TRa2, and TRb1. The specificity of the antibodies usedwas described previously (Zandieh Doulabi et al. 2002,2003). The extracts were prepared in 0.25 M sucrose,40 mM dithiothreitol, 2% SDS, 1 mM EDTA, 62.5 mMTris–HCl (pH 7.6), and 10% glycerol, separated on 10%(w/v) polyacrylamide gels, and blotted onto an Immo-bilon-P (Millipore)membrane. After staining with amidoblack to verify similar protein loading, themembrane wasincubated in 10 mM Tris–HCl (pH 8.0), 150 mM NaCl,0.5% Tween20, and 5% nonfat dried milk powder(Natrinon, Nutricia) for 6 h, followed by an overnightincubation in a ratio of 1:1000 dilution of the respectiveantisera. Antibody binding was quantified using chemi-luminescence (CDP-Star, Roche), in combination withthe LUMI-imager F1 (Roche; Bakker 1998). In theabsenceofprimaryantisera,nostainingwas seen,whereasin their presence, bands at the anticipated Mr of thecorresponding receptors appeared. However, in liver andkidney extracts, extra bands at both higher and lowerMr

than found in other organs were observed for TRa1 andTRb1. These patterns were also found in organs that wererapidly isolated and analyzed immediately.

Immunoperoxidase staining was performed ondewaxed tissue sections. Antibody binding onsections was visualized by the indirect unlabeledantibody peroxidase anti-peroxidase (PAP) method(Sternberger et al. 1970). PAP immunocomplexeswere purchased from Nordic (Tilburg, TheNetherlands). If care is taken not to overdevelop

receptors (TRs) in the central nervous tissue, lung, liver, kidney,

a2 TRb1 TRb2

RNA Protein mRNA Protein MRNA Protein

K K K K KK K CC K K

C K CC CC K KC C C K K KC K CC C K K

K C C K KC K K K K K

C CC CC K KC C C K KK K K K KK C C K KK C C K KK K K K KCC K C K KCC K K K KK K K K K

C K C C K KC C C K K

detected; C, well detectable expression; CC, strong expression.



the enzymic staining, local differences in immuno-histochemical staining intensity represent differencesin protein concentration between these cells ortissues (van Straaten et al. 2006).

Results

Distribution of TRa and TRb

We describe the expression patterns TRa and TRb foreach organ separately, going from mRNA to proteinand from early fetal to postnatal. An overview of ourfindings is provided in Table 1.

Central nervous system

The staining pattern of TRs in the central nervoussystem has been described (Bradley et al. 1992). Inagreement, TRa mRNA expression in the basal plate ofthe neural tube was already pronounced at E15(Fig. 1A) and very strong in the cortical plate of thebrain at E16 (Fig. 1C). In contrast, TRb mRNAexpression was undetectable (Fig. 1B) or much weaker(Fig. 1D). Since the TRa2 mRNA concentrationexceeded that of TRa1 more than 20-fold (Fig. 2A), itwas responsible for the observed TRamRNA expressionpattern. The expression of TRa2 and TRb1 mRNA

TRα TRβ

E15

E16

E16

AABB

CC DD

EE FF

GG

HHCo

B

CP

C

Figure 1 Thyroid hormone receptor expression in perinatal central nespinal cord (A) well before TRb mRNA (B). In the E16 brain, TRa mRNexpression (D). In contrast, the TRb signal is intense in the cochlear scochlear nerves (E). At E18 and P4, protein expression of TRa1 and Texpressed in various structures of the brain (I and L). B, basal nucleinose. Bar, 100 mm.


followed a similar temporal pattern and was highest atP1 (Fig. 2A). TRb2 mRNA was not detectable. Bothbefore and shortly after birth, the TRa1 and TRa2protein concentration in the brain (Fig. 1G, H, J and K)was very low when compared with that in the otherorgans (Fig. 3A–F). In contrast, TRb1 protein wasalready present in fetal brain (E17) and abundant atP4 (Figs 1I, L and 3). These data show that thedeveloping brain was characterized by a very lowprotein: mRNA ratio for TRa1 and, in particular TRa2,whereas this ratio was much higher for TRb1. Asummary of these findings can be found in Table 1.

Other intracranial organs

TRa and TRb expression colocalized at many sites in thecentral nervous tissue. In the inner ear, however, thestrong signal of TRbmRNA in the sensory epithelium ofthe cochlea and the very weak signal in the cochlearnerve(Fig. 1F) contrasts with the opposite staining pattern TRamRNA (Fig. 1E) and demonstrates the specificity of thein situ hybridization procedure. We have included thisresult as an example of the sometimes highly contrastingTR expression patterns in organs.

Protein for all the three receptors was observed in theextracts of adult pituitary glands (Fig. 3A, D and I).TRb1 protein was present in E18 and P4 pituitary

E18 P4

TRα2

TRα1

TRβ1

II

JJ

KK

LL

Co

CeCe

N

CP

rvous system. TRa mRNA appears in the basal plate of the E15A expression in the cortical plate (C) is much stronger than TRb

ensory epithelium (F), whereas TRa expression dominates in theRa2 is very low (G, H, J and K). In contrast, TRb1 protein is highly; C, cochlea; Ce, cerebellum; Co, cortex; CP, choroid plexus; N,


0

2

4

0

10

20

A B

C D

E F

0·0

0·1

0·2

0·3

0

4

8

E15 E18 ND1 Ad E15 E18 ND1 Ad

0

4

8

0

2

4

12

0

2

4

6

0

4

8

0

2

4

6

8

0

10

20

0

40

80

0

2

4

amol

TR

mR

NA

/µg

poly

A+ R

NA

Figure 2 Quantification of thyroid hormone receptor mRNA levels during perinatal development. A, brain; B, lung; C, liver; D, kidney; E,small intestine; F, colon. TRa1 (triangles), TRa2 (squares), and TRb1 (dots) mRNA concentrations are depicted GS.E.M. The age ofthe animals is indicated on the X-axis, while the concentration of the mRNAs is shown on the Y-axis (left, TRa1 (C–F) and TRa2, andright, TRa1 (A and B) and TRb1 concentration). Note that the scales of the Y-axes differ in the respective panels.


glands, with the highest concentration in the posteriorlobe (Fig. 4C and F). Both TRa isoforms only becamedetectable after birth in the anterior and middle lobes(Fig. 4A, B, D and E).

Lung

The pulmonary epithelium is initially surrounded by aconspicuous mass of mesenchyme. Expression of TRamRNA in this mesenchyme was first observed at E13and became pronounced at E15 (Fig. 5A). TRb mRNAexpression in the epithelium became more intenseduring the subsequent ‘canalicular’ phase of the lungdevelopment (E16–E18; Fig. 5B and D), when TRamRNA expression remained distinctly present in themesenchyme (Fig. 5C). Owing to the attenuation of thewalls of the airways during the ‘alveolar’ phase of lungdevelopment (OE19), the complementary expressionpattern of TRa and TRb became more and more


difficult to discern (Fig. 5E and F). TRb mRNAremained present in the wall of the pulmonary arteries(arrows Fig. 5D and F) and TRa mRNA in themyocardium of the pulmonary veins (arrows Fig. 5C).TRa2 mRNA levels were approx. tenfold higher thanTRa1 mRNA levels and peaked at E18 and P1 (Fig. 2B),indicating that the TRa in situ hybridization signalactually represented TRa2 mRNA. The TRb signal inthe in situ hybridizations represented TRb1, because noTRb2 mRNA could be detected. TRb1 mRNA levelspeaked at E18 (Fig. 2B).

Western blot analysis revealed the presence of TRa1protein at E17 and a marked increase in theconcentration by P4 (Fig. 3A and B). At the sametime points, the intensity of the TRa2-protein band wasrelatively weak and even appeared to decrease withdevelopment (Fig. 3D and E). TRb1 protein levels weresimilar at E17 and P4 (Fig. 3G and H). Despite thepositive western blots, we were not able to demonstrate


TRα1 TRα2 TRβ1

A D

B E

C F

G

H

I

M

Tr

Te Th Fp Je II BI Te Th Fp Je II BI Te Th Fp Je II Pg Pg LiBI

Lu Li K A M He Sp B Tr Lu Li K A M He Sp B Tr Lu Li K A M He Sp B

B K Li LiAd

MWPgInHe Lu M B K Li Li

Ad Ad

Ad

1758362

25

47·5

32·5

1758362

25

47·5

32·5

1758362

25

47·5

32·5

1758362

25

47·5

32·5

1758362

25

47·5

32·5

1758362

25

47·5

32·5

MWPgInHe Lu M B A KK LiInHe Lu

Figure 3 Western blot of thyroid hormone receptor protein levels during perinatal development. Representative example of threeextracts assayed in triplicate. A–C, TRa1; D–F, TRa2; G–I, TRb1; A, D and G: E17; B, C, E, F, H and I: P4. Ad, adult; MW, molecularweight. Organ abbreviations: A, adrenal gland; Bl, bladder; B, brain; Fp, epididymal fat pad; He, heart; In, intestine; Il, ileum; Je,jejunum; K, kidneys; Li, liver; Lu, lung; M, muscle; Pg, pituitary gland; Sp, spleen; Te, testis; Th, thyroid gland; Tr, trachea.


either TRa1 or TRa2 protein by immunohistochemistry(Fig. 5G, H, J and K), indicating that the proteins werepresent at low levels in many cells. TRb1 protein waspredominantly expressed in lung epithelium (Fig. 5Jand L). The protein: mRNA ratio for TRa1 increased indevelopment, and remained more or less constant forTRa2 and TRb1.

Liver

Expression of TRamRNA in the liver was first observedat E15 (Fig. 6A) and could be located in the stromalcomponent, including the wall of the veins, from E17onwards (Fig. 6C and E). Expression of TRbmRNA wasalso observed at E15, declined during the next 2 days toundetectable levels, but reappeared in the parenchymaltissue at E19 (Fig. 6B, D and F). At E20, thecomplementary expression of TRa in the stromal andof TRb in the parenchymal tissue had become very


striking (Fig. 6E and F). The concentration of TRa1and, in particular, TRa2 mRNA, was very low whencompared with other tissues during all ages investigated(Fig. 2C). As a result, the TRa1: TRa2 ratio was thehighest observed in any of the tissues investigated andwas more than five times higher than that in the brain.TRb1 mRNA levels peaked at E18 (Fig. 2C), whereasTRb2 mRNA levels were undetectable.

In liver extracts, the TRa1 antibody detected not onlya band at the expected size (47 kDa) but also bands athigher and lower Mrs. TRa1 levels increased betweenE17 and P4, when concentrations reached adult values(Fig. 3A and B), whereas the TRa2 protein did notchange (Fig. 3D and E). Similarly, the 57 kDa TRb1band did not change in development, but bands of 30and 110 kDa became increasingly prominent (Fig. 3Gand H). Immunohistochemical staining revealed weakstaining of TRa1 protein in parenchymal cells sur-rounding the central veins, but after birth only (Fig. 6G


E18 P4

TRα2

TRβ1

TRα1

DD

EE

FF

AA

BB

CC

APAPP I

Figure 4 Thyroid hormone receptor expression in the pituitarygland. TRa1 and TRa2 proteins are not detectable in the E18pituitary (A and B) and begin to accumulate postnatally (P4) in theanterior lobe (D and E). In contrast, TRb1 protein was highlyexpressed at E18 (C) and P4 (F), in particular, in the middle lobe.A, anterior; I, intermediate; and P, posterior lobe of pituitary. Bar,100 mm.


and J; cf. (Zandieh-Doulabi et al. 2003), whereas TRa2protein had then become detectable in isolated livercells (Fig. 6H and K). In fetal rat liver, TRb1 protein wasobserved faintly in the parenchyma of the liver (Fig. 6I).After birth, expression had become much stronger inthe hepatocytes surrounding the central veins (Fig. 6L;cf. (Zandieh Doulabi et al. 2002)). The protein: mRNAratios for TRa1, TRa2, and TRb1 did not changeappreciably with development.

Kidney

The development of the definitive kidney is charac-terized by the penetration of the ureteric bud into themetanephric mass. The metanephric tubules expressedTRb mRNA (Fig. 7B, D and F), whereas the uretericbuds and surrounding mesenchyme expressed TRamRNA (Fig. 7A, C and E). TRa2 mRNA levels were morethan ten times higher than TRa1 levels (Fig. 2D). BothTRa1 and TRa2 mRNA concentrations peaked perina-tally, whereas TRb1 mRNA peaked just after birth. TRb2mRNA was undetectable.

TRa1 protein levels increased perinatally, with a bandof 30 kDa, which is also present in liver extracts,becoming more prominent with age (Fig. 3A and B).At E18, TRa1 protein was weakly present in the nephrictubules (Fig. 7G), but had disappeared at P4 (Fig. 7J).


TRa2 protein was not detectable in any cell type in thekidney (Fig. 7H and K). TRb1 protein was detected inthe glomeruli and tubules at E18 (Fig. 7I). After birth,the glomeruli were no longer positive (Fig. 7L). Theprotein:mRNA ratio for TRa1 and TRa2 did not changeappreciably in development. If, however, the 30 kDaband is a proper TRa1 gene product, the protein:mRNA ratio for TRa1 does increase with development.The protein:mRNA ratio for TRb1 declined withdevelopment, but in the adult, a 30 kDa band that wasalso seen in liver, had become very prominent. If this isa bona fide TRb1 gene product, the ratio is substantiallyhigher in the adult.

Adrenals

TRa mRNA, which was still found in the entire adrenalat E16 (Fig. 7A), had become confined to dispersedislands of cells in the medulla and the capsule at E18(Fig. 7C). TRb mRNA levels were low and confined tothe capsule (Fig. 7B and D). PCR analysis was notperformed. Western blot analysis showed prominentbands for both TRa1 and TRa2 after birth (Fig. 3B andE). The TRa2 antibody produced an additional 100 kDaband. This band was also observed in human (notshown), but not in rat pituitary gland. Only one bandwas observed for TRb1 protein (Fig. 3F). The TRa1protein was diffusely distributed in the adrenal at E18(Fig. 8A), but had become confined to the adrenalmedulla at P4 (Fig. 8D). The distribution of TRa2protein was confined to well-defined islets of cells at E18(Fig. 8B) and to the medulla after birth (Fig. 8E). TheTRb1 protein was diffusely distributed in the adrenaltissue and capsule at E18 (Fig. 8C), but becamelocalized to the adrenal cortex after birth (Fig. 8F).The morphological data indicate that the TRa mRNAin the adrenal capsule does not generate detectableprotein. In contrast, the low abundance of TRb mRNAand the readily detectable TRb1 protein in sectionsindicate a high protein:mRNA ratio.

Intestines

The epithelial layer of the intestines (Fig. 9A–F) and theparenchymal cells of the pancreas (Fig. 9C and D)co-expressed TRa and TRb mRNA from E16 onward(Fig. 9A–F). Between E18 and E20, both TRa and TRbmRNAs became confined to the crypts. TRa1, TRa2,and TRb1 mRNA levels followed a similar develop-mental pattern and peaked perinatally (Fig. 2E and F).

Western blotting showed that TRa1 protein levelsdecreased between 5 days before birth and 4 days afterbirth (Fig. 3A and C). The opposite was true for TRa2protein (Fig. 3D and F). Relatively low levels of TRb1protein were found both pre- and postnatally (Fig. 3Gand I). The TRa1 antiserum generated staining at the


TRα

TRα1

TRα2

TRβ1

TRβ

E15

E18

E20

E18 P4

GG

HH

II

JJ

KK

LL

AA BB

EE

CC DD

FF

Figure 5 Thyroid hormone receptor expression in perinatal lung. TRa mRNA was prominently present in the lung mesenchyme atE15 (A) and E18 (C). TRb mRNA in the pulmonary epithelium is visible at E15 (B) and pronounced at E18 (D). Note thecomplementarity of TRa and TRb mRNA expression (C and D). Thereafter (E and F: E20), the complementary distribution was nolonger demonstrable. The pulmonary vein is identifiable by its expression of TRa (C), whereas the pulmonary artery ischaracterized by TRb expression in its myocardial wall (D and F; arrowheads). TRa1 (G and J) and TRa2 proteins (H and K) wereundetectable at E18 (G and H) and P4 (J and K). TRb1 protein was largely confined to the epithelium and detectable at both E18 (I)and P4 (L). Arrows, terminal bronchioli (‘canaliculi’); asterisk, terminal bronchioli and alveoli. Bar, 100 mm.


apical surface of the enterocytes at E18, but not at P4(Fig. 9G and J). TRa2 protein could not be detected(Fig. 9H and K). In contrast, strong staining for TRb1protein was observed in the smooth-muscle layer of theintestine at E18 (Fig. 9I) and P4 (Fig. 9L), as opposed toa relatively weak staining in the mucosa. The TRamRNAs, therefore, generated very little protein in theepithelium. The protein:mRNA ratio for TRb1 in theepithelium was also low. At the same time, high levels ofTRb1 protein were present in the smooth muscle layer,where the corresponding mRNA was barely detectable.

Comparison of TR protein in organs

We demonstrated the presence of TRa1 and TRa2proteins on western blots of several other organs (Fig. 3,Table 1). Quantification showed that the concen-trations of TRa1 protein in lung, muscle, heart, kidney,adrenals, pituitary gland, spinal cord, testis, and fat padwere similar, that in brain, liver, and small intestine wereapproximately threefold lower, and that in trachea andred blood cells were twofold higher than average.Average concentrations of TRa2 were found in liver,small intestine, and kidney, a very low concentration inred blood cells, approximately twofold lower thanaverage concentrations in lung, trachea, muscle,


heart, spinal cord, and brain, and twofold higher thanaverage in the fat pad, testis, and adrenals. Theresulting TRa1:TRa2 ratio, a parameter for tissuethyroid hormone responsiveness, was 3- to 10-foldlower than average in brain, liver, prenatal heart,lung, postnatal small intestine, adrenals, and fat pad;2- to 10-fold higher than average in postnatal lung,trachea, muscle, and pituitary gland, and extremelyhigh for blood. The TRa1:TRa2 ratio declined with thedevelopment in liver and small intestine, and increasedin lung, muscle, heart, and brain. The TRb1 concen-tration was remarkably similar in all analyzed organsbefore birth and, after birth, relatively low in muscle,bone, fat pad, small intestine, and red blood cells, andrelatively high in brain and spinal cord.

Comparison of the protein:mRNA ratio betweenorgans

Protein synthesis is determined by the correspondingsteady-state mRNA level and translational efficiency,whereas the steady-state protein level is determined byits synthesis (translation) and degradation. If weassume that no major changes occurred in the tissueconcentration of polyAC mRNA or total protein withtime and that TR mRNAs are not sequestered from


TRα

TRα1

TRα2

TRβ1

TRβ

E15

E17

E20

A B

C D

E F

E18 P4

GG

HH

II

JJ

KK

LL

LiGLu

PC

P

C

Figure 6 Thyroid hormone receptor expression in perinatal liver. TRa mRNA was readily detectable in prenatal liver mesenchyme(A, E15; C, E17; E, E20). Expression declined in liver stroma, but became more prominent in the developing perivascularmesenchyme. TRb mRNA was also readily detectable at E15 (B), temporarily declined (D; E17), but had reappeared in thehepatocytes at E20 (F). Note the complementarities of TRa and TRb mRNA expressions (E and F). At E18 (G), TRa1 proteinconcentration was barely detectable, but at P4 (J), it had accumulated in the pericentral hepatocytes. Similarly, TRa2 protein wasundetectable before birth (H, E18), but was observed in isolated liver cells after birth (K, P4). TRb1 protein was present in mosthepatocytes before birth (I), but had become confined to the pericentral hepatocytes by P4 (L). Li, liver; Lu, lung; G, gonad; P,portal vein; C, central vein. Bar, 100 mm.


the translational machinery at a specific time point,protein:mRNA ratios inform us about the presence ofa posttranscriptional level of regulation of geneexpression (cf. Table 1). During lung development,the protein:mRNA ratio of TRa1 increased, but theratios of TRa2 and TRb1 remained more or lessconstant. Similarly, the protein:mRNA ratios for TRa1,TRa2, and TRb1 did not change appreciably with liverand kidney development. During intestinal develop-ment, the TRa and TRb mRNAs generated very littleprotein in the epithelium, but the reverse was true forthe smooth muscle layer, where a barely detectableTRb1 mRNA generated a high concentration ofprotein. In the brain, the protein:mRNA ratio wasvery low for TRa1 and TRa2, whereas that for TRb1was highest.

Discussion

Despite the detailed insights into the molecularmechanisms underlying the thyroid hormone- andTR-dependent modulation of gene expression that arenow available (Cheng 2000, Zhang & Lazar 2000,Baxter et al. 2001, Yen 2001, O’Shea & Williams 2002,


Chassande 2003, Flamant & Samarut 2003) and theundisputed role of thyroid hormone in organogenesis(Oppenheimer & Schwartz 1997, Tata 1999), themechanistic aspects of thyroid hormone action indevelopment are still debated. Nevertheless, it is likelythat epithelial–mesenchymal interactions play a role,because mesenchymal cells are thought to be primarydeterminants of epithelial cell fate during organogen-esis (Brard 1990). Using in situ hybridization andimmunohistochemistry as visualization techniques,and RT-PCR and western blotting as quantificationtechniques, we indeed observed a highly specificdistribution pattern of the TR isoforms that appearsto support such a mechanism of action for thyroidhormones. Expression of the TRa isoform wasobserved in the mesenchymal tissues and expressionof the TRb isoform in the epithelial tissues of lung,liver, kidney, sensory system of the inner ear, and bone.As far as we know, this generalization has not beenmade so far. However, this generalization did not applyto the central nervous system and the intestine. Inaddition, the relation was obscured by an extensiveposttranscriptional regulation of the expression of theTRs, with differences between organs and betweendifferent developmental stages of the same organ


TRα

TRα1

TRα2

TRβ1

TRα

E16

E18

E20

A B

C D

EE F

E18 P4

GG

HH

II

JJ

KK

LL

CT

G

Ad

K

G

Ad

U

U

U

C

Figure 7 Thyroid hormone receptor expression in perinatal kidney. TRb mRNA was present in the developing nephrons from E16onwards (B, E16; D, E18; F, E20). TRamRNA was present in the surrounding mesenchyme, but declined with development (A, E16;C, E18; E, E20). TRa1 protein was seen in the collecting tubules at E18 (G), but had disappeared at P4 (J). TRa2 protein was notdetectable at either E18 (H) or P4 (K). At E18, TRb1 protein was present in the glomeruli and, more abundantly, in the collectingtubules (I). After birth, TRb1 protein had disappeared from the glomeruli (L). At E16, TRa mRNA was weakly expressed in the entireadrenal (A), but had become largely confined to the capsule at E18 (C). A weak staining for TRb mRNA was seen in the capsule ofthe adrenals (B and D). Ad, adrenal; K, kidney with U, ureter; C, cortex; G, glomerulus; and CT, collecting tubules. Bar, 100 mm.


(cf. also (Strait et al. 1990, Lane et al. 1991, Rodd et al.1992, Schwartz et al. 1992, Weiss et al. 1998)). In thisrespect, it is of interest that the protein:mRNA ratiosthat we derived for brain, liver, and kidney resemblethose derived by Oppenheimer and Schwartz (Oppen-heimer & Schwartz 1997) with respect to the TRa1,but, in contrast to their findings, we concluded thatthe TRb1 protein:mRNA ratio is higher in brain thanin liver.

The complementary expression pattern of TRa andTRb was most pronounced in developing lung, liver,and kidney, that is, in organs with distinct epithelial andmesenchymal components. Since most epithelia cannotdifferentiate when separated from their associatedmesenchyme (Birchmeier & Birchmeier 1993), it isthought that epithelial differentiation is under controlof its underlying mesenchyme. The respectiveexpression patterns were much more pronounced atthe mRNA than at the protein level, implying importantposttranscriptional control mechanisms. Althoughboth mRNA and protein levels in the organs mentionedreached maximal levels perinatally, it is of interest thatfor those organs, for which paired samples wereavailable, tissue TRa2 and TRb1 protein peaked at5 days before birth. In contrast, TRa1 protein follows asimilar time course as circulating thyroid hormones


(Dubois & Dussault 1977) to reach its highest level afterbirth. This latter temporal association is in line with thefinding that unliganded TRa1 is detrimental topostnatal development (Chassande 2003). Our data,therefore, suggest that an initiating, (unliganded) TRb-mediated role of the epithelium induces a TRa-mediated response of the mesenchyme.

On the other hand, extensive co-expression of TRaand TRb was observed in the developing brain andintestinal epithelium. Despite the high level of TRa1and particularly TRa2 mRNA in these organs, TRa1protein levels were much lower, and TRa2 protein levelswere similar to those in the other organs investigated.In other words, organs with co-expression of TRa andTRb in the same cells seem to be characterized by a verylow TR protein:mRNA ratio in the perinatal period.Thus, the protein:mRNA ratio for TRa1 was more thantenfold lower and that of TRa2 15- to 50-fold lower inperinatal brain than in perinatal liver, lung, or kidney.In contrast, the protein:mRNA ratio for TRb1 wassimilar in these four organs in this period, but was verylow in intestinal epithelium, especially before birth.This implies that in organs with co-expression of TRaand TRb, the translational efficiency of one of the TRmRNAs is much lower, and/or that the turnover of TRprotein is much higher than that in cells without


TRα TRβ

E16

E18

E20

AA BB

CC DD

EE FF

GG

HH

II

PP

SMC

V

Figure 9 Thyroid hormone receptor expression in perinatal intestepithelium of the intestines at E16 (A and B), E18 (C and D), and Econcentrated in the crypts. Also note the expressions of TRa and Tstaining was present in the apical part of the villar epithelium befor(HandK). TRb1 proteinwasseen predominantly in thesmoothmuscE18 (I), and equally at both the locations after birth (L). C, crypt; V,

E18 P4

AA

BB

CC

DD

EE

FF

TRα2

TRα1

TRβ1

C

M

C

M

Figure 8 Thyroid hormone receptor expression in the adrenalglands. TRa1 protein was present in most parenchymal cells of theadrenal at E18 (A), but had become confined to the adrenalmedulla at P4 (D). TRa2 protein was confined to islets of cells atE18 (B) and to the adrenal medulla after birth (E). TRb1 proteinwas seen to be present in the adrenal and its capsule at E18 (C),but became concentrated in the adrenal cortex after birth (F). C,cortex and M, medulla of adrenal gland. Bar, 100 mm.



co-expression. These data indicate that the TR-depen-dent regulatory cascades function differently in thoseorgans in which the TRa and TRb genes are charac-terized by a complementary expression pattern andthose in which they are co-expressed. Intriguingly, theseposttranscriptional regulatory mechanisms, therefore,largely annul the apparent dominance of TRa2

expression in the central nervous system and that ofTRb1 expression in the intestine.

We observed an apparent discrepancy between therelatively weak staining of TRa proteins in histologicalsections of all organs except the adrenal, and theirstrong presence, in particularly TRa1, on western blots.Although one explanation would be that the TRas werenot well accessible in the tissue sections of most organs,the use of a denaturing rather than a cross-linkingfixative and absence of an effect of preprocessing thesections with antigen-retrieval techniques shows that,more likely, they are distributed over many more cells,hence that the cellular concentration remains too lowto strongly stain these tissues. This interpretationsuggests a more homogeneous distribution of TRasthan TRb1 in brain, lung, bone, and kidney. Inagreement, the TRa1 and TRa2 proteins stainedhomogeneously in the anterior pituitary gland(Fig. 4), while the TRa1 protein had a much widerpericentral expression pattern in the liver than the

TRα1

TRα2

TRβ1

E18 P4

JJ

KK

LL SM

ine. Note that TRa and TRb mRNAs are co-expressed in the20 (E and F). From E18 onwards, both mRNAs became

RbmRNA in the pancreatic epithelium (C and D). TRa1 proteine (G), but not after birth (J). TRa2 protein was not detectablele layerand toa lesserextent in theepitheliumof the intestineatvillus; P, pancreas; SM, smooth-muscle layer. Bar, 100 mm.



TRb1 protein (Fig. 6; cf. also Zandieh Doulabi et al.2002, 2003).

We detected 8.9G2.4 amol TRb2 mRNA in adult ratpituitary gland, but were unable to detect this TRbisoform outside the pituitary, implying that TRb2mRNA concentrations were more than 1000-foldlower. Nevertheless, we detected abundant stainingin many organs with four rabbit antisera that wereraised against a TRb2-specific oligopeptide (data notshown). In the pituitary, staining was most pro-nounced in the intermediate lobe of the pituitary,as was reported earlier (Li & Boyages 1997). Theputative TRb2 protein was 47 kDa, i.e., 11 kDa lessthan predicted by the reported open reading frame(Hodin et al. 1989). Although the size of this proteincorresponds with the TRb3 isoform (Williams 2000),the oligopeptide used for immunization is notpresent in TRb3. Possibly, TRb2 uses a more down-stream translation initiation site (cf. (Wood et al.1994)). The pI of the putative TRb2 protein was 7.6in liver (corresponding with that of the unmodifiedTRb2 protein) and 5.0 in the pituitary gland.Extremely low cellular levels of TRb2 mRNA inassociation with easily detectable TRb2 protein levelsoutside the pituitary gland were reported before(Lechan et al. 1993, Schwartz et al. 1994, Ercan-Fanget al. 1996, Oppenheimer & Schwartz 1997).

Presently, gene targeting is one of the most powerfulapproaches to establish causal relations between geneexpression and phenotype. The analysis of single andcompound mutations of the Thra and Thrb loci hasrevealed that TRa1 and TRb1 cooperate and canpartially substitute for each other, and that theconsequences of thyroid hormone deficiency differfrom those of TR deficiency (O’Shea & Williams 2002).TRb1 expression appears to be preferentially associatedwith the development of the cochlea and liver geneexpression (Forrest et al. 1996, Weiss et al. 1998; cf. Figs1 and 6), whereas TRb2 appears to be responsible forthe development and maintenance of the hypo-thalamic–pituitary axis (Abel et al. 1999). As suggestedby the pronounced phenotype of hypothyroid neo-nates, unoccupied TRbs can interfere with normaldevelopment as well (Hashimoto et al. 2001). TRa1expression appears to be more closely associated withintestinal, cardiac, and bone development (Wikstromet al. 1998, Plateroti et al. 1999, 2001). In agreementwith our immunohistochemical data (cf. Figs 4 and 6with Fig. 9), the effects of TRa deficiency becomeevident at weaning, that is, substantially later than thoseof TRb deficiency. However, the expression andfunction of TRa1 is materially complicated by theother products of the Thra locus, TRa2, and the TRDaisoforms (O’Shea & Williams 2002, Flamant & Samarut2003). Thus, TRa1 deficiency differs from TRa1/2deficiency in the more severe phenotype of the latter,


with hypothyroidism, runting, and maturational delaysin bone and intestinal development that becomemanifest at weaning (Fraichard et al. 1997). In contrast,TRa0/0 mice that lack all products from the Thra locus,including TRDa, have a milder phenotype than TRa1/2-deficient mice (Macchia et al. 2001), due to the absenceof the effects of TRDa (Plateroti et al. 2001). Irrespectiveof these complexities, our study has shown that thedevelopmental appearance of the respective TRproteins corresponds nicely with the phenotypes ofmutations of the corresponding genes.

Acknowledgements

The authors declare that there is no conflict of interestthat would prejudice the impartiality of this scientificwork.

References

Abel ED, Boers ME, Pazos-Moura C, Moura E, Kaulbach H, Zakaria M,Lowell B, Radovick S, LibermanMC&Wondisford F 1999 Divergentroles for thyroid hormone receptor b isoforms in the endocrine axisand auditory system. Journal of Clinical Investigation 104 291–300.

Bakker O 1998 LUMI-imager F1 Lab Protocols. Berlin, Heidelberg, NewYork: Springer Verlag.

Baxter JD, Dillmann WH, West BL, Huber R, Furlow JD, Fletterick RJ,Webb P, Apriletti JW & Scanlan TS 2001 Selective modulation ofthyroid hormone receptor action. Journal of Steroid Biochemistry andMolecular Biology 76 31–42.

Birchmeier C & Birchmeier W 1993 Molecular aspects of mesenchy-mal-epithelial interactions. Annual Review of Cell Biology 9 511–540.

Bradley DJ, Towle HC & Young WSd 1992 Spatial and temporalexpression of a- and b-thyroid hormone receptor mRNAs,including the b2-subtype, in the developing mammalian nervoussystem. Journal of Neuroscience 12 2288–2302.

Brard J 1990. Morphogenesis, the Cellular and Molecular Processes ofDevelopmental Anatomy, Cambridge: Cambridge University Press.

Chassande O 2003 Do unliganded thyroid hormone receptors havephysiological functions? Journal of Molecular Endocrinology 31 9–20.

Cheng SY 2000 Multiple mechanisms for regulation of the transcrip-tional activity of thyroid hormone receptors. Reviews in Endocrineand Metabolic Disorders 1 9–18.

Dubois JD & Dussault JH 1977 Ontogenesis of thyroid function in theneonatal rat. Thyroxine (T4) and triiodothyronine (T3) pro-duction rates. Endocrinology 101 435–441.

Ercan-Fang S, Schwartz HL & Oppenheimer JH 1996 Isoform-specific3,5,3 0-triiodothyronine receptor binding capacity and mRNAcontent in rat adenohypophysis: effect of thyroidal state andcomparison with extrapituitary tissues. Endocrinology 137 3228–3233.

Flamant F & Samarut J 2003 Thyroid hormone receptors: lessons fromknockout and knock-in mutant mice. Trends in Endocrinology andMetabolism 14 85–90.

Forrest D, Hanebuth E, Smeyne RJ, Everds N, Stewart CL, Wehner JM& Curran T 1996 Recessive resistance to thyroid hormone in micelacking thyroid hormone receptor b: evidence for tissue-specificmodulation of receptor function. EMBO Journal 15 3006–3015.

Fraichard A, Chassande O, Plateroti M, Roux JP, Trouillas J, Dehay C,Legrand C, Gauthier K, Kedinger M, Malaval L et al. 1997 The T3Ragene encoding a thyroid hormone receptor is essential for postnataldevelopment and thyroid hormone production. EMBO Journal 164412–4420.



Glinoer D 2001 Potential consequences of maternal hypothyroidismon the offspring: evidence and implications. Hormone Research 55109–114.

Gudernatsch JF 1912 Feeding experiments on tadpoles I. Theinfluence of specific organs given as food on growth anddifferentiation. Archiv fr Entwicklungsmechanik der Organismen 35 457.

Hashimoto K, Curty FH, Borges PP, Lee CE, Abel ED, Elmquist JK,Cohen RN &Wondisford FE 2001 An unliganded thyroid hormonereceptor causes severe neurological dysfunction. PNAS 983998–4003.

Hodin RA, Lazar MA, Wintman BJ, Darling DS, Koenig RJ, Larsen PR,Moore DD & Chin WW 1989 Identification of a thyroid hormonereceptor that is pituitary-specific. Science 244 76–79.

Jonker A, de Boer PA, van den Hoff MJ, Lamers WH & Moorman AF1997 Towards quantitative in situ hybridization. Journal of Histo-chemistry and Cytochemistry 45 413–423.

Lane JT, Godbole M, Strait KA, Schwartz HL & Oppenheimer JH 1991Prolonged fasting reduces rat hepatic b1 thyroid hormone receptorprotein without changing the level of its messenger ribonucleicacid. Endocrinology 129 2881–2885.

Lechan RM, Qi Y, Berrodin TJ, Davis KD, Schwartz HL, Strait KA,Oppenheimer JH & Lazar MA 1993 Immunocytochemicaldelineation of thyroid hormone receptor b2-like immunoreactivityin the rat central nervous system. Endocrinology 132 2461–2469.

Li M& Boyages SC 1997 Expression of b2-thyroid hormone receptor ineuthyroid and hypothyroid rat pituitary gland: an in situ hybrid-ization and immunocytochemical study. Brain Research 773 125–131.

Macchia PE, Takeuchi Y, Kawai T, Cua K, Gauthier K, ChassandeO, SeoH, Hayashi Y, Samarut J, Murata Y et al. 2001 Increased sensitivity tothyroid hormone in mice with complete deficiency of thyroidhormone receptor a. PNAS 98 349–354.

Moller A & Jansson JK 1997 Quantification of genetically taggedcyanobacteria in Baltic Sea sediment by competitive PCR.BioTechniques 22 512–518.

Moorman AFM, de Boer PAJ, Ruijter JM, Hagoort J, Franco D &Lamers WH 2000 Radio-isotopic in situ Hybridization on tissuesections: practical aspects and quantification. In DevelopmentalBiology Protocols, vol 137, pp 97–115. Eds RS Tuan & CW Lo. Totowa,NJ: Humana Press Inc. (vol 3, ch 11).

Morreale de Escobar G, Calvo R, Obregon MJ & Escobar del Rey F1990 Contribution of maternal thyroxine to fetal thyroxine pools innormal rats near term. Endocrinology 126 2765–2767.

Morreale de Escobar G, Obregon MJ & Escobar del Rey F 2004 Role ofthyroid hormone during early brain development. EuropeanJournal of Endocrinology 151 25–37.

Oppenheimer JH & Schwartz HL 1997 Molecular basis of thyroidhormone-dependent brain development. Endocrine Reviews 18462–475.

O’Shea PJ &Williams GR 2002 Insight into the physiological actions ofthyroid hormone receptors from genetically modified mice.Journal of Endocrinology 175 553–570.

Plateroti M, Chassande O, Fraichard A, Gauthier K, Freund JN,Samarut J & Kedinger M 1999 Involvement of T3Ralpha- and beta-receptor subtypes in mediation of T3 functions during postnatalmurine intestinal development. Gastroenterology 116 1367–1378.

Plateroti M, Gauthier K, Domon-Dell C, Freund JN, Samarut J &Chassande O 2001 Functional interference between thyroidhormone receptor alpha (TRa) and natural truncated TRDaisoforms in the control of intestine development. Molecular andCellular Biology 21 4761–4772.

Rodd C, Schwartz HL, Strait KA &Oppenheimer JH 1992 Ontogeny ofhepatic nuclear triiodothyronine receptor isoforms in the rat.Endocrinology 131 2559–2564.

Schwartz HL, Strait KA, Ling NC & Oppenheimer JH 1992Quantitation of rat tissue thyroid hormone binding receptorisoforms by immunoprecipitation of nuclear triiodothyroninebinding capacity. Journal of Biological Chemistry 267 11794–11799.


Schwartz HL, Lazar MA & Oppenheimer JH 1994 Widespreaddistribution of immunoreactive thyroid hormone b2 receptor(TRb2) in the nuclei of extrapituitary rat tissues. Journal of BiologicalChemistry 269 24777–24782.

Sternberger LA, Hardy PH Jr, Cuculis JJ & Meyer HG 1970 Theunlabeled antibody enzyme method of immunohistochemistry:preparation and properties of soluble antigen-antibody complex(horseradish peroxidase-antihorseradish peroxidase) and its usein identification of spirochetes. Journal of Histochemistry andCytochemistry 18 315–333.

van Straaten HWM, He Y, van Duist MM, Labruyere WT, VermeulenJLM, van Dijk PJ, Ruijter JM, Lamers WH & Hakvoort TBM 2006Cellular concentrations of glutamine synthetase in murine organs.Biochemistry and Cell Biology 84 215–231.

Strait KA, Schwartz HL, Perez-Castillo A & Oppenheimer JH 1990Relationship of c-erbA mRNA content to tissue triiodothyroninenuclear binding capacity and function in developing and adult rats.Journal of Biological Chemistry 265 10514–10521.

Tata JR 1999 Amphibian metamorphosis as a model for studying thedevelopmental actions of thyroid hormone. Biochimie 81 359–366.

Tuca A, Giralt M, Villarroya F, Vinas O, Mampel T & Iglesias R1993 Ontogeny of thyroid hormone receptors and c-erbAexpression during brown adipose tissue development: evidenceof fetal acquisition of the mature thyroid status. Endocrinology 1321913–1920.

van Tuyl M, Blommaart PE, de Boer PA, Wert SE, Ruijter JM, Islam S,Schnitzer J, Ellison AR, Tibboel D, Moorman AF et al. 2004 Prenatalexposure to thyroid hormone is necessary for normal postnataldevelopment of murine heart and lungs. Developmental Biology 272104–117.

Weiss RE, Murata Y, Cua K, Hayashi Y, Seo H & Refetoff S 1998 Thyroidhormone action on liver, heart, and energy expenditure in thyroidhormone receptor b-deficient mice. Endocrinology 139 4945–4952.

Wikstrom L, Johansson C, Salto C, Barlow C, Campos Barros A, Baas F,Forrest D, Thoren P & Vennstrom B 1998 Abnormal heart rate andbody temperature in mice lacking thyroid hormone receptor a1.EMBO Journal 17 455–461.

Williams GR 2000 Cloning and characterization of two novel thyroidhormone receptor b isoforms. Molecular and Cellular Biology 208329–8342.

Wood WM, Dowding JM, Haugen BR, Bright TM, Gordon DF &Ridgway EC 1994 Structural and functional characterization of thegenomic locus encoding the murine b2 thyroid hormone receptor.Molecular Endocrinology 8 1605–1617.

Yen PM 2001 Physiological and molecular basis of thyroid hormoneaction. Physiological Reviews 81 1097–1142.

Yen PM, Ando S, Feng X, Liu Y, Maruvada P & Xia X 2006 Thyroidhormone action at the cellular, genomic and target gene levels.Molecular and Cellular Endocrinology 121–127.

Zandieh Doulabi B, Platvoet-terSchiphorst M, van Beeren HC,Labruyere WT, Lamers WH, Fliers E, Bakker O & Wiersinga WM2002 TRb1 protein is preferentially expressed in the pericentralzone of rat liver and exhibits marked diurnal variation. Endocrinology143 979–984.

Zandieh-Doulabi B, Dop E, Schneiders M, Schiphorst MP, Mansen A,Vennstrom B, Dijkstra CD, Bakker O & Wiersinga WM 2003 Zonalexpression of the thyroid hormone receptor a isoforms in rodentliver. Journal of Endocrinology 179 379–385.

Zhang J & Lazar MA 2000 The mechanism of action of thyroidhormones. Annual Review of Physiology 62 439–466.

Received in final form 13 February 2007Accepted 16 February 2007Made available online as an Accepted Preprint 28 February 2007


Expression of thyroid hormone receptors A and B in developing rat tissues; evidence for extensive...

Documents

Transcript of Expression of thyroid hormone receptors A and B in developing rat tissues; evidence for extensive...