Ablation of TR{alpha}2 and a Concomitant Overexpression of {alpha}1 Yields a Mixed Hypo- and Hyperthyroid Phenotype in Mice

Carmen Saltó1, Jenny M. Kindblom, Catarina Johansson, Zhendong Wang, Hjalmar Gullberg, Kristina Nordström, Anethe Mansén, Claes Ohlsson, Peter Thorén, Douglas Forrest and Björn Vennström

Department of Cell and Molecular Biology (B.V., C.S., A.M., H.G., K.N.), Department of Physiology (C.J., P.T.), Karolinska Institute, S-171 77 Stockholm, Sweden; Department of Human Genetics (Z.W., D.F.), Mount Sinai School of Medicine, New York, New York 10029; and Endocrine Division (J.M.K., C.O.), Department of Internal Medicine, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden

Address all correspondence and requests for reprints to: Dr. Björn Vennström, Department of Cell and Molecular Biology, Box 285, Karolinska Institute, S-171 77 Stockholm, Sweden. E-mail: Bjorn.Vennstrom{at}cmb.ki.se


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormone governs a diverse repertoire of physiological functions through receptors encoded in the receptor genes {alpha} and ß, which each generate variant proteins. In mammals, the {alpha} gene generates, in addition to the normal receptor TR{alpha}1, a non-hormone-binding variant TR{alpha}2 whose exact function is unclear. Here, we present the phenotype associated with the targeted ablation of TR{alpha}2 expression. Selective ablation of TR{alpha}2 resulted in an inevitable, concomitant overexpression of TR{alpha}1. Both TR{alpha}2 +/- and -/- mice show a complex phenotype with low levels of free T3 and free T4, and have inappropriately normal levels of TSH. The thyroid glands exhibit mild morphological signs of dysfunction and respond poorly to TSH, suggesting that the genetic changes affect the ability of the gland to release thyroid hormones. However, the phenotype of the mutant mice also has features of hyperthyroidism, including decreased body weight, elevated heart rate, and a raised body temperature. Furthermore, TR{alpha}2-/- and TR{alpha}2+/- mice are obese and exhibit skeletal alterations, associated with a late-onset growth retardation. The results thus suggest that the overexpression of TR{alpha}1 and the concomitant decrease in TR{alpha}2 expression lead to a mixed hyper- and hypothyroid phenotype, dependent on the tissue studied.

The phenotypes suggest that the balance of TR{alpha}1:TR{alpha}2 expressed from the TR{alpha} gene provides an additional level of tuning the control of growth and homeostasis in mammalian species.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE THYROID HORMONE (TH) T3 and its prohormone, T4 govern several physiological and developmental processes. TH is essential for amphibian metamorphosis (1), whereas in mammals hypothyroidism impairs neurogenesis (2) and many physiological parameters in the adult organism (3, 4).

The functions of TH are mediated by nuclear hormone receptors, which belong to a family of ligand-dependent transcription factors (5). The TRs are encoded by two distinct but closely related genes, TR{alpha} and TRß (6, 7). The TR{alpha} gene generates the TR{alpha}1 and TR{alpha}2 isoforms that are identical for the first 370 amino acids but differ as a consequence of differential splicing at their C terminus: the last 40 specific residues of TR{alpha}1 are replaced by 122 amino acids encoded by the TR{alpha}2-specific exon 10, the last exon in the TR{alpha} locus. As a consequence of the C-terminal change, the TR{alpha}2 protein is unable to bind TH, and no other ligand has been identified (8, 9, 10). TR{alpha}2 binds DNA weakly and only binds a subset of T3-responsive sites (T3REs). Furthermore, it dimerizes poorly with RXR and lacks the activating function domain 2 that interacts with coactivators (11, 12). Although TR{alpha}2 is highly conserved in man, rat, and mouse it appears to be absent in nonmammalian vertebrates (13, 14).

The TR{alpha}1 and TR{alpha}2 RNAs are widely coexpressed early in development (10) and in adult tissues (8, 15, 16, 17). TR{alpha}2 expression levels are generally 2- to 10-fold higher than those of TR{alpha}1. TR{alpha}2 has been suggested to exert a suppressive function on other TRs (18). Suggested mechanisms for suppression include competition for binding to thyroid hormone response elements (TREs) on target genes, formation of inactive heterodimers, or squelching (19, 20, 21). A dominant negative effect of TR{alpha}2 has also been reported to occur without binding to certain TREs (22). Recently, TR{alpha}2 was described to be only a weak antagonist of TH action due to its low affinity for several response elements and its failure to interact with corepressors (12). Dephosphorylation at the C terminus of TR{alpha}2 has been reported to increase its DNA binding affinity (23).

To understand the role of TRs in development, mice deficient in the expression of one or several TR isoforms have been developed. We previously showed (24) that mice deficient in TR{alpha}1 but retaining TR{alpha}2 are fully viable although they exhibit lower heart rate, lower body temperature, and slower ventricular repolarization as compared with normal mice.

The TRß-deficient mice (25) have severely impaired hearing, have high serum levels of TH and TSH, and have goiter (25, 26, 27). They also exhibit a slightly increased heart rate that is nonresponsive to T3 (28). Mice lacking all known receptors for TH (TR{alpha}1-/-ß-/- mice) are viable, suggesting that the T3 receptors encoded in the TR{alpha} and TRß loci are dispensable for life. These mice exhibit growth retardation, delay in bone maturation, and poor female reproduction (29). Studies of heart function and control of body temperature revealed defects similar to those found in TR{alpha}1-/- mice (28), suggesting that TR{alpha}1 has the primary role in these processes. Surprisingly, the disruption of one of the first exons on the TR{alpha} locus (30), resulting in a lack of TR{alpha}1 and TR{alpha}2 expression, showed a dramatic phenotype in which pups die shortly after weaning unless treated with T3. In these mice the thyroid gland fails to develop properly, and both bone and small intestine maturation is delayed. It was suggested that the lethality of the TR{alpha}-/- mice was caused by residual expression of short variants of TR{alpha} proteins that lack DNA-binding domains. Alternatively, the TR{alpha}2 protein could have vitally important functions in mammals, or the TR{alpha}2 protein may have compensatory functions that overlap those of TR{alpha}1 instead of having the antagonistic role that had been suggested.

To address this issue, mice with a selective disruption of TR{alpha}2 were generated by gene targeting in embryonic stem cells. The mice overexpress TR{alpha}1 as an inevitable consequence of the targeting event and are viable. The levels of free T3 (FT3) and free T4 (FT4) are significantly reduced, and their thyroid glands show features of dysfunction. The mice also exhibit other signs of hypothyroidism, such as a retarded growth spurt, increased fat content, decreased cortical bone dimensions, and decreased trabecular bone mineral density. TSH serum levels, however, are inappropriately normal, suggesting an alteration in the pituitary-thyroid axis. In addition, when compared with wild-type (wt) mice, the mutants have a higher heart rate and body temperature, parameters usually associated with elevated TH levels. The results indicate that the loss of TR{alpha}2 or the changed balance of TR{alpha}1/TR{alpha}2 perturbs a range of functions, suggesting a role for TR{alpha}2 in fine tuning mammalian growth and homeostasis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of the TR{alpha}2 Mutant Mice
For abrogation of TR{alpha}2 expression, we decided to block transcription of the TR{alpha}2-specific exon 10. For this purpose, we introduced a strong polyadenylation site and transcriptional stop in the 3'-untranslated region of the TR{alpha}1 mRNA, thus preventing transcription into the TR{alpha}2-specific exon 10. This approach was necessary, since the splice donor site for exon 10 is located within exon 9, and its alteration would have generated a mutant TR{alpha}1 protein (Fig. 1Go). The procedure used also avoided generation of truncated TR{alpha}1 proteins and left the overlapping rev-erbA{alpha} gene intact (31, 32). This approach was expected to raise the level of TR{alpha}1 expression in mutant cells equal to that found for TR{alpha}2 in wt tissues. This consequence, however, cannot be avoided in any exclusive ablation of TR{alpha}2 expression.



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Figure 1. Targeting the TR{alpha}2 Gene

A, 3'-End of the TR{alpha} and Rev-erbA{alpha} loci. The targeted allele is shown in the middle. The dotted lines indicate the extent of the targeting vector. Broken horizontal arrows indicate splicing effects. Details about restriction sites and probes were described in Ref. 24 . The two TR{alpha} proteins are depicted at the bottom. B, Southern blot analysis of a wt (right lanes) and a targeted (left lanes) embryonic stem cell colony. DNA was digested with the indicated enzymes, and the filters were hybridized against several probes (5' p, 3' p, ex10p, ex8–9p). C, Southern blot analysis of BamHI-digested DNA prepared from the tails of the progeny of one representative litter from a heterozygote intercross. The white arrow indicates the wt allele, and the black arrow shows the mutated gene.

 
The screening identified three independent embryonic stem cell clones, containing the desired homologous recombination. Southern blot analysis of one of them, clone 165, is shown in Fig. 1BGo. The DNA fragments obtained after the BamHI digestion had the expected size, 23 kb from the wt allele and 13 kb or 9.9 kb from the mutated allele, when 5'-probe or 3'-probe were used, respectively. An internal probe, ex10p, excluded the random integration of the targeting vector in this clone. Two other independent digestions of the DNA from the 165 clone (XbaI, StuI) confirm that homologous recombination occurred as intended.

Cells from clone 165 were injected into blastocysts of C57BL/6 mice, and several chimeric mice were generated. Mating of heterozygous offspring resulted in a non-Mendelian distribution of surviving pups: in 231 mice genotyped from heterozygote (+/-) intercrosses, we obtained 33% wt, 56% TR{alpha}2+/-, and 11% TR{alpha}2-/- mice. The ratio between male and female offspring was 1:1. However, in a different facility, numbers of TR{alpha}2-/- progeny increased to near Mendelian ratios: 23% wt, 58% TR{alpha}2+/-, and 21% TR{alpha}2-/- (total progeny, n = 102). This suggested that survival of TR{alpha}2-/- mice was susceptible to environmental factors. Female TR{alpha}2-/- mice generally failed to conceive, or if pups were born, to rear their offspring. The +/- and -/- females had extended estrous cycles: 4.9 ± 0.2 d (n = 25) and 5.4 ± 0.1 d (n = 20), respectively, as compared with wt, 4.3 ± 0.2 d (n = 22) (P < 0.05). Thus, the mutation impaired reproduction.

Abrogation of TR{alpha}2 RNA Expression
Next, we verified that expression of TR{alpha}2 RNA in the mutant mice was abrogated. RNA from different tissues of wt, TR{alpha}2+/-, and TR{alpha}2-/- mice were analyzed. Northern blots of brain RNA (Fig. 2AGo) showed the different wt and mutant TR{alpha} RNAs in wt, TR{alpha}2+/-, and TR{alpha}2-/- mice after hybridization with probes that recognized both TR{alpha}1 and TR{alpha}2, or with specific probes that recognize either of the specific RNAs isoforms. As expected, no TR{alpha}2 RNA was found in the TR{alpha}2-/- mice, as the wt 2.6-kb band was undetectable in the -/- samples analyzed. The TR{alpha}1 RNA from the targeted allele, TR{alpha}11, was shorter (2.2 kb) than the wt RNA (5.8 kb) as a consequence of the targeting event and was present in both TR{alpha}2+/- and -/- mice. In the latter mice the normal TR{alpha}1 RNA was undetectable. TRß1 and rev-erbA{alpha} levels of expressions were unaltered in the TR{alpha}2+/- and -/- mice (Fig. 2AGo, left panel).



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Figure 2. No TR{alpha}2 RNA Generated from the Targeted Allele

A, Northern blot analysis of poly(A)-selected RNA from brain tissue of +/+, +/-, and -/- mice. The images to the left show hybridization against three different probes: a TR{alpha}-cDNA probe detecting TR{alpha}1 and TR{alpha}2, a probe specific to TR{alpha}1, and a probe specific to TR{alpha}2. The TR{alpha}1 transcript from the targeted allele (TR{alpha}1*) migrates faster as compared with the wt TR{alpha}1. The images to the right depict hybridization of the same filter to TRß and rev-erb{alpha} probes, respectively. B, RT-PCR analysis with RNA from total embryo and several tissues of +/+, +/-, and -/- mice. The primers for detection of total TR{alpha}1 or TR{alpha}1* mRNAs gave the expected band in all the samples analyzed (upper bands in lanes 1–12); the TR{alpha}2- specific primers were only detected in +/- and +/+ animal tissues (lower bands in lanes 5–12). C, RT-PCR control made with and without reverse transcriptase on RNA from TR{alpha}2-/- (lanes 5–8) and wt mice (lanes 13–16) to exclude DNA contamination in the RNA preparation.

 
To confirm the lack of TR{alpha}2 expression, we performed RT-PCR with RNA obtained from 13–15-d-old embryos (E13–E15) and several tissues from newborn and adult mice: heart, liver, and brain. Figure 2BGo (lanes 1–4, lower panel) shows that primers specific for TR{alpha}2 failed to amplify any transcript in the homozygous mice, whereas samples from heterozygous and wt animals scored positive as expected (lanes 5–12, lower panel). The primers common for wt TR{alpha}1 and TR{alpha}11 sequences generated the expected amplified fragments (lanes 1–12, upper panel). To control for any possible DNA contamination, the RNA samples were used in amplification experiments in which reverse transcriptase had been omitted, and no DNA fragments were detected (Fig. 2CGo, lanes 5–8 and 13–16). We therefore conclude that expression of the TR{alpha}2 product had been efficiently abrogated, as no detectable expression of TR{alpha}2 could be observed in any of the different tissues analyzed from the mutant mice.

Overexpression of TR{alpha}1
Because the alteration in the TR{alpha} locus was expected to increase the expression of TR{alpha}1, we determined TR{alpha}1 RNA and protein levels in different tissues. The novel TR{alpha}11 transcript was expressed at higher levels than the wt TR{alpha}1 RNA in brain tissue from heterozygous (3- to 5-fold) and homozygous animals (6- to 10-fold), as revealed by phosphoimager quantification (Fig. 2AGo). The overexpression was detected in all the tissues analyzed: brain, pituitary (Fig. 3AGo), eye, white adipose tissue, heart, and muscle, with values that ranged from 3- to 10-fold (data not shown). No significant alteration in the expression of TRß2 RNA was observed (Fig. 3AGo).



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Figure 3. Overexpression of TR{alpha}1

A, Northern blot with RNA from pituitaries of wt and -/- mice (15 pituitaries for each group), hybridized with TR{alpha}- and TRß2-specific probes. The bottom of the panel displays the ratio of the different RNAs as quantified by PhosphorImager analysis standardized against hybridization with a G3PDH probe. The gel retardation assay in B shows increased TR binding to a TRE in tissue extracts from TR{alpha}2-deficient mice as compared with wt mice. The T3 binding assay in C shows increased specific T3 binding capacity in nuclear extracts from liver and brain of TR{alpha}2-/- mice compared with wt, TR{alpha}1-/-, and TRß-/- mice. A 125I-T3 saturation binding experiment is shown in D; nuclear extract from brain of wt and TR{alpha}2-/- mice was used. Open symbols represent wt; gray symbols depict mutant.

 
Next, we studied whether the overexpressed, mutant TR{alpha}11 RNA gave rise to an overproduction of functional TR{alpha}1 receptor in the homozygous mice. The ability of the receptor to bind DNA and T3 was examined in gel mobility shift and T3 binding assays, respectively, in a comparison with the receptor from wt mice. Nuclear extracts from wt and TR{alpha}2-/- mice were incubated with and without TR- or RXR-specific antibodies that facilitated detection of the retarded DNA bands. The results in Fig. 3BGo show that the TR{alpha}2 extract contains more TR{alpha}1 protein than the control extract and that the overexpressed protein had retained the ability to bind DNA. Ligand binding experiments with 125I-T3 and nuclear extract from liver and brain of TR{alpha}2-/- mice showed, as expected, a markedly increased hormone binding as compared with extracts from wt, TR{alpha}1-/-, and TRß-/- mice (Fig. 3CGo). Brain nuclear extracts from wt and homozygous animals were furthermore used in T3 saturation binding assays to allow a better estimation of the TR{alpha}1 receptor levels. The results show that extracts from TR{alpha}2-/- mice contained about 3.7 more T3 binding receptors than wt controls (Fig. 3DGo). The data confirm that the recombination event that abrogated expression of TR{alpha}2 resulted in a concomitant increased expression of TR{alpha}1 mRNA and receptor protein.

Function of the Pituitary-Thyroid Axis
To determine the hormonal status of the mutant mice, the concentrations of FT3 and FT4 in serum were measured (Fig. 4AGo). The results show that the FT3 and FT4 levels were reduced in the male TR{alpha}2-/- mice, and that the TR{alpha}2+/- values were intermediate between that of the wt and -/- mice. Similar changes were detected in females. All the differences in FT3 and FT4 were statistically significant (see legend to Fig. 4AGo). Total T3 (TT3) levels showed a similar trend although the changes were significant only in females (76.6 ± 2.7, 55.3 ± 2.0 and 61.0 ± 10.3 for wt, +/-, and -/- mice, respectively). The data thus show that the genetic alteration in the TR{alpha} locus led to significantly decreased serum levels of THs in both TR{alpha}2-/+ and -/- animals.



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Figure 4. Analysis of Pituitary and Thyroid Hormones

A, Levels of FT3 and FT4 in serum from males and females wt ({circ}), TR{alpha}2+/- ({triangleup}), TR{alpha}2-/- ({blacktriangleup}). Mean values are indicated by bars. All the differences are significant, P < 0.001, except for FT3 in females +/- vs. -/- and for FT4 in males and females +/- compared with +/+ or -/- mice, where P < 0.05. B, TSH levels in serum from control, TR{alpha}2+/-, and TR{alpha}2 -/- mice. The serum used for TSH, FT3, and FT4 was obtained from tail bleedings of mice between 2 and 4 months of age. C, Northern blot analysis of pituitary mRNAs; each lane contains poly(A)- selected RNA from four pituitaries from 4- to 9-month-old mice. To detect TSH{alpha} and TSHß mRNAs, we used specific cDNA probes. Quantification was done with a PhosphorImager (Molecular Dynamics, Inc.), and a G3PDH probe was used as a control and for normalizing the results. D, Immunostaining analysis with a TSHß antibody of pituitaries from wt, +/-, and -/- mice; E, Western blot analysis performed on pituitaries from wt, +/-, and -/- mice showing an increase of TSHß in the -/- mice.

 
Since TSH regulates T3 and T4 production and T3, in turn, feedback regulates TSH, we examined the serum levels of TSH in adult mice (6–20 wk old). The results show no difference between TR{alpha}2+/+, +/-, and -/- mice of either sex (Fig. 4AGo).

We also determined pituitary expression levels for TSH{alpha} and TSHß RNAs in male mutant mice (Fig. 4BGo). Northern blot analysis showed no significant differences between any of the genotypes for TSH{alpha}, and a slight increase for TSHß in TR{alpha}2+/- and -/- mice (1.3- and 1.5-fold, respectively) as compared with +/+ mice. Increased levels of TSHß but not TSH{alpha} protein were also found by Western blotting of pituitary extracts in the TR{alpha}2-/- mice (Fig. 4EGo). However, immunostaining histological sections of pituitaries with TSHß antibodies revealed a normal gland morphology (Fig. 4EGo) and no apparent increase in the number of TSH producing cells. In conclusion, the data suggest that the low serum levels of THs increase TSHß RNA and intracellular protein levels. As a corresponding increase of serum TSH could not be demonstrated, other mechanisms must compensate to bring serum TSH levels to normal.

Morphological and Functional Changes in Thyroid Glands
The thyroid glands of adult mice showed that the overall size of the mutant glands was normal when compared with wt mice. In agreement with this observation, no goiter was detected under the period of observation (>18 months). Histological analyses of the thyroid gland showed a general disorganization of the follicles, accompanied by a flattening of the epithelium, as exemplified by uneven follicle sizes and reduced interstitial space (Fig. 5AGo).



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Figure 5. Histological Analysis of the Thyroid Gland of the Mutant Mice

A, Hematoxylin and eosin sections of the thyroid glands from normal and TR{alpha}2-/- male mice (4 months old). Upper panels show the overall structure and morphology of the glands. The bottom panels show the thyroid follicles from wt (left) and -/- mice (right); note in this last panel the flattened aspect of the follicle cells, the larger size of the colloid, and the interstitial space among follicles that are abnormal compared with wt mice. B, Reduced increase of FT3 and FT4 after TSH injections in homozygous mice as compared with wt animals.

 
To test whether the morphological alterations correlate with a dysfunction in the response to TSH and therefore caused reduced levels of TH in serum, we studied the response of the thyroid glands to injected TSH. Accordingly, recombinant bovine TSH was injected into 15 wt and TR{alpha}2-/- mice. Serum samples for determination of FT3 and FT4 were collected 1 wk before and 6 h after the injections. Figure 5BGo shows that whereas the wt mice released serum T3 and T4 by 48% and 75%, respectively, the levels for TR{alpha}2-/- mice increased only by 29 and 33%. These data corroborate the histological analyses and suggest that the thyroid glands of the mutant mice are dysfunctional.

Decreased Growth Rate and Adult Body Weight
Since TH disorders are associated with growth abnormalities, we determined the growth rate of newborn pups as well as adult body weight, a physiological parameter under the control of T3. The results show that both female and male mutant mice have a slightly reduced body weight from birth to adult life. The growth spurt (Fig. 6AGo) during the first 9 postnatal weeks, for both female and male mutant mice, was slightly delayed when compared with wt. At 5 wk the body weight of wt female mice was 16.9 ± 1.3 g compared with 13.5 ± 1.2 g for -/- female mice (P < 0.001). The mean weight for 25- to 35-wk-old male wt mice was 38.1 ± 3.4 g while TR{alpha}2+/- and -/- mice weighed 34.8 ± 1.5 g and 27.6 ± 1.0 g, respectively (Fig. 6BGo). Similar results were obtained for female mice of the same interval of age, with values of 34.0 ± 3.2, 31.1 ± 2.3 g, and 23.8 ± 3.0 g for wt, TR{alpha}2+/-, and TR{alpha}2-/- mice, respectively. The differences in body weight among the groups where all significant (P < 0.05 for wt to +/- and P < 0.001 for wt or +/- to -/-). However, GH RNA and protein levels in the pituitary were normal in TR{alpha}2+/- and -/- mice (Fig. 6Go, C and D). This suggests that a defect in other intermediary factors rather than GH accounted for the growth deficiencies (see below).



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Figure 6. Weight Gain and Growth Curves

A, Weight gain during the first 10 wk for TR{alpha}2-/-, ({bullet}) TR{alpha}2 +/- ({diamond}), and wt ({triangleup}) female mice. Each point represents the average weight of between 5 and 11 animals. B, Weight gain curves of male mice TR{alpha}2+/- (gray diamond), TR{alpha}2-/- ({bullet}), and wt ({triangleup}) male mice over 56 wk. C, Northern blot analysis of GH poly(A) RNA (four pituitaries per lane); D, Western blot with extracts from pituitaries from each genotype, one pituitary extract per lane.

 
Increased Fat Content
As TH influences lipid metabolism and the content of adipose tissue in the body, we measured the amount of fat using dual x-ray absorptiometry (DXA). We found an increase in fat content (121%, P < 0.05) in TR{alpha}2-/- as compared with wt mice. The TR{alpha}2+/- were similar to TR{alpha}2-/- mice (data not shown). Metabolic serum parameters including serum levels of corticosterone and leptin were not significantly altered in the TR{alpha}2-/- mice compared with wt mice (Table 2Go).


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Table 2. Serum Parameters

 
Bone Abnormalities
Since TH has an effect in bone development, we examined whether the mutant mice presented any skeletal alteration. Table 1Go shows that no differences were found in the lengths of femurs and tibia from the wt, TR{alpha}2+/-, and TR{alpha}2-/- mice. Normal longitudinal growth was further supported by unaltered proximal tibial growth plate width in the TR{alpha}2-/- mice compared with wt (data not shown). However, serum levels of IGF-I, an important factor involved in growth, were clearly reduced in both TR{alpha}2-/- (268 ± 28 ng/ml) and TR{alpha}2+/- (308 ±17) mice compared with wt (401 ± 22) (P < 0.05). Levels of tartrate-resistant acid phosphatase (TRAP) 5b activity, a marker of osteoclast activity, were reduced by 57% in the TR{alpha}2-/- mice compared with wt, whereas serum levels of osteocalcin, a marker of bone formation, remained the same in both groups (Table 2Go).


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Table 1. Dimensions of Bones

 
As the bone formation was unaffected in the mutant mice but osteoclast activity was reduced, we decided to study whether this imbalance would result in an altered bone density.

DXA measurements were performed to determine the areal bone mineral density (BMD) and the bone mineral content (BMC). The areal BMDs and BMCs of the femur and vertebrae L6 were significantly reduced in the TR{alpha}2-/- mice compared with wt (Table 3Go). In contrast, no effect was seen on these parameters in the cranium, which consists of intramembranous bone (Table 3Go). Hypothyroid rodents have a delayed mineralization of the epiphysis. However, the epiphysis was fully mineralized in the adult TR{alpha}2-/- mice (data not shown). Furthermore, peripheral quantitative computerized tomography (pQCT) measurements were performed to distinguish between effects on cortical and trabecular bone. The cortical bone was analyzed by a middiaphyseal tibial scan, which revealed decreased cortical BMC, periosteal, and endosteal circumferences and cortical area in both TR{alpha}2+/- and -/- mice compared with wt (Table 4Go). The trabecular BMD was measured in a metaphyseal pQCT scan of the proximal tibia and revealed a reduction with 24% in the TR{alpha}2-/- mice compared with wt, and intermediate values for the TR{alpha}2+/- mice (Table 4Go).


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Table 3. DXA Measurements

 

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Table 4. pQCT Measurements of the Tibia

 
Alteration in Heart Rate and Body Temperature
In humans, hypothyroidism is associated with low heart rate, while hyperthyroidism correlates with increased heart rate (tachycardia). Because mice lacking TR{alpha}1 have a decreased heart rate (24), we examined whether the TR{alpha}2-/- mice had heart function alterations. Telemetric analyses show (Fig. 7Go) that both the TR{alpha}2+/- and -/- mice have an increased heart rate (544 ± 5 and 522 ± 18 beats/min, respectively) as compared with control mice (491 ± 6.5 beats/min). Daily administration of T3 for 4 d (as indicated by arrows) resulted in a similar and parallel increase in heart rate (about 50 beats/min) in the three groups, showing that the mice respond to T3.



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Figure 7. Telemetry Analyses for Heart Rate and Body Temperature

Telemeric devices were implanted in the abdomens of adult male mice. After 48 h of baseline records T3 was injected for 4 consecutive days at 1300 h (as indicated by arrows). The values are presented as (mean ± SEM). A, Heart rate; B, body temperature. Symbols: TR{alpha}2 +/+ ({bullet}), TR{alpha}2 +/- ({triangleup}), and TR{alpha}2 -/-mice ({circ}).

 
We also determined by telemetry whether elevated levels of TR{alpha}1 expression or the abrogation of TR{alpha}2 affected the body temperature of the mutants. Our results show that -/- (36.9 ± 0.1 C) and +/- (36.7 ± 0.1 C) mice had an increased body temperature as compared with wt controls (36.5 ± 0.1 C). Moreover, T3 injection resulted in an increase of 0.5 C in all the three genotypes. Our telemetry studies thus revealed unexpectedly that the +/- and -/- mice exhibited features usually associated with an hyperthyroid phenotype, despite their low levels of FT3 and FT4.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of TR{alpha}2-Deficient Mice
The introduction of a strong polyadenylation site in the untranslated region of exon 9 not only abrogated the production of a TR{alpha}2 RNA, but also generated a shorter TR{alpha}1 transcript expressed at markedly higher levels than the normal TR{alpha}1 RNA in all the tissues analyzed. Because the shorter TR{alpha}1 RNA resulted in a receptor with unaffected ability to transactivate (data not shown) and to bind T3 and DNA (Fig. 3Go), the mutant mice can be considered to lack detectable TR{alpha}2 expression while overproducing the TR{alpha}1 protein.

The alteration in the TR{alpha} locus resulted in breeding abnormalities. TR{alpha}2-/- intercrosses rarely generated litters, a problem that was exacerbated after the mutation was backcrossed for two generations onto the C57BL/6J background (Forrest, D., data not shown). TR{alpha}2-/- males were fertile while females showed partial fertility but only under optimized animal care conditions (Vennström, B., unpublished); thus, there is no absolute requirement for TR{alpha}2 in reproduction. TR{alpha}2-/- females had a somewhat prolonged estrous cycle, suggesting that the reduced fertility reflected irregular ovulation. More severe estrous cycling defects have been found in mice lacking all known T3 receptors (TR{alpha}1-/-ß-/-; Wang, Z., and D. Forrest, unpublished data). Our results suggest that TR{alpha}2 is dispensable for reproduction and indicate that the overexpression of TR{alpha}1, as exemplified by the TR{alpha}2+/- females, may be a major interfering factor in reproduction.

Function of the Pituitary-Thyroid Axis
During normal conditions, a reduction of T3 in serum causes an increase in serum TSH. The lower T3 and T4 levels in the hetero- and homozygous mice, however, were not reflected by elevated TSH levels. Thus, defects in TSH regulation cannot be the sole cause of the low TH levels in the mutant mice. Our results suggest a double impairment in the pituitary-thyroid axis: an inability of the thyroid gland to produce hormone, and an alteration in the negative feedback at the hypothalamic-pituitary level, which may also include a defect in TRH response.

A deficiency in thyroid gland function is supported by the mild morphological changes and the decreased response to TSH (Fig. 5Go). The underlying defect is at present unclear, but could entail a subtle misdevelopment of the gland, decreased ability to bind TSH, or defects in the pathways of hormone synthesis or release.

The failure to elevate serum TSH in response to the slightly lowered T3 and T4 levels indicates another defect: in TSH regulation at the hypothalamic-pituitary level. As may be predicted, TSHß mRNA and protein levels within the pituitary were somewhat up-regulated (Fig. 3AGo). However, the lack of an accompanying increase in serum TSH levels suggested a possible defect in the regulation of the assembly or secretion of mature TSH by the pituitary thyrotropes (33). Such a subtle defect could reside within the pituitary or potentially at the level of hypothalamic TRH. TR{alpha}2 is coexpressed with the TR{alpha}1 and TRß T3 receptors in the hypothalamus and pituitary (34), and abnormalities in TRH (35) and TSH expression occur in the absence of TR{alpha}1 and TRß (29, 36). It is also possible that the defects in regulation of pituitary TSH result in secretion of TSH with reduced biological activity, as was suggested for mice with a deletion of the TRH gene (37).

The observation that TR{alpha}2+/- animals had normal serum TSH and TH levels intermediate between those of wt and -/- mice (Fig. 4Go) indicates that elevation of TR{alpha}1 expression impedes the function of the pituitary-thyroid axis, although an effect of the reduction in TR{alpha}2 expression cannot be completely ruled out. That TR{alpha}1 can act in regulation of TSH synthesis is supported by comparing mice that lack TR{alpha}1, TRß, or both receptors: only the total lack of T3 binding receptors resulted in serum TSH levels equal to those found in severely hypothyroid wt mice (29, 30, 36).

Skeletal Properties
TR{alpha}2-/- mice showed slightly reduced body weight and decreased serum levels of IGF-I. In contrast, no significant reduction in longitudinal bone growth was detected. However, the mice showed decreased cortical BMC and reduced dimensions of the tibial cortical bone. These parameters are associated with decreased adult periosteal growth of bones and indicate late-onset growth retardation in the mice. Interestingly, the heterozygous mice displayed a similar phenotype of the cortical bone as the TR{alpha}2-/- mice, caused by either overexpression of TR{alpha}1 or the reduction in TR{alpha}2 expression, or both. It has previously been reported that TR{alpha}1-/- and TRß-/- mice lack overt growth phenotype (24, 25). In contrast, TR{alpha}-/- mice (that express neither TR{alpha}1 nor TR{alpha}2 but instead truncated versions thereof) show growth inhibition from 2 wk of age (30, 35). Moreover, TR{alpha}1-/-ß-/- mice exhibit pre- and postnatal growth retardation as a consequence of GH/IGF-I deficiency and exhibit compensatory growth in response to GH substitution (Refs. 29 37A ).

The fact that TR{alpha}2+/- and -/- mice had femurs and tibias of normal length but reduced cortical dimensions suggest that the inhibition of growth occurred after the main part of longitudinal bone growth had taken place. The observation of growth retardation affecting the cortical dimensions of bones but not longitudinal growth is further strengthened by the finding that the homozygous mice had normal total growth plate width as well as width of the individual hypertrophic and proliferative layers. The effects of GH on bone include increased longitudinal bone growth in young mice and periosteal bone formation in adult mice (38). Serum bone markers, including serum levels of osteoclast-derived TRAP 5b activity and osteoblast-derived osteocalcin, are often used as indicators of acute changes in bone metabolism. The BMD was decreased in the TR{alpha}2-/- mice, but the bone metabolism had probably reached a new steady state in these mice as the osteocalcin levels were unchanged. We have previously seen that TRAP 5b activity is well correlated to the trabecular bone mineral density when the bone metabolism has reached a steady state (Ohlsson, C., unpublished). Thus, the decreased TRAP 5b activity in the TR{alpha}2-/- mice might be due to the decreased trabecular bone mineral density associated with a decreased total number of osteoclasts.

A plausible explanation for the adult-onset growth retardation in the mice is that either the lack of TR{alpha}2 or the increased TR{alpha}1 expression results in a late-onset IGF-I deficiency. That both hetero- and homozygous mice exhibit similar alterations of the tibial cortical bone excludes deficiency of the TR{alpha}2 isoform alone as the direct cause of these alterations. In contrast, trabecular BMD was clearly decreased in homozygotes while only a tendency to decrease was seen in heterozygous mice, indicating that the TR{alpha}2 isoform may be of importance for trabecular bone. Interestingly, the IGF-I reduction is probably GH independent since no major changes in GH were detected.

T3 is required for a normal terminal differentiation of chondrocytes (39), which is necessary for a proper mineralization. Accordingly, bone maturation is inhibited and there is a mineralization defect in hypothyroid rats (40, 41) as well as in TR{alpha}-/- and TR{alpha}1-/-ß-/- mice, but not in TR{alpha}1-/- or TRß-/- mice. Adult TR{alpha}2-/- mice have low T3 but overexpress the {alpha}1 isoform, and they exhibit normal mineralization of the epiphysis. These results indicate that TR{alpha}2 alone is not essential for skeletal mineralization.

TH is known to affect basal metabolism of almost all cells, and pathological conditions of the thyroid axis often affect body weight. The increased fat content in both TR{alpha}2-deficient mouse strains contrasts with the lower overall body weight and normal cortical bone length. The increased fat content is a feature of hypothyroidism (42), but the lower body weight is a feature of hyperthyroidism. This is likely to reflect distinct physiological mechanisms governing the development of the concerned tissues.

Heart Rate and Body Temperature
The heart is a major target for TH. Deficiency in TR isoforms causes discrete alterations in heart rate and ventricular function (24, 28, 43, 44). Notably, loss of TR{alpha}1 in mice causes an approximately 20% reduction in basal heart rate while still allowing T3 to increase the heart rate. This contrasts with the cardiac effects of TRß deficiency: a minor increase in basal heart rate (likely to be due to slightly increased serum levels of T3) and an impaired heart rate response to injection of T3 into hypothyroid mice (28, 44). The mice heterozygous and homozygous for the TR{alpha}2 ablation both exhibited a 10% increase in basal heart rate as compared with wt controls, despite their lower serum T3 levels. This indicates that the increase in TR{alpha}1, as opposed to loss of or reduction in TR{alpha}2, may be the cause for the elevated heart rate.

Deficiency for TR{alpha}1 but not TRß results in a lowered body temperature (24). Interestingly, both the hetero- and homozygous TR{alpha}2 mutant mice showed a similar increased temperature, indicating that elevated TR{alpha}1 expression causes this physiological response.

Relative Contributions of TR{alpha}1 Overexpression and Loss of TR{alpha}2
Recently, Macchia et al. (44) described pituitary-thyroid axis function in TR{alpha}0/0 mice, a strain that lacks expression of all TR{alpha} isoforms. These mice exhibit an increased sensitivity to T3 in regulation of target genes, attributed by the authors to a silencing effect of TR{alpha}2. Our results from the TR{alpha}2-deficient mouse strains do not refute or support this suggestion since the ablation of TR{alpha}2 expression was accompanied by an inevitable increase in TR{alpha}1, thus making it difficult to discriminate between a role for TR{alpha}2 in modifying a hormonal response and an increased activity of TR{alpha}1.

Because of the reduced serum levels of TH one could expect that the hetero- and homozygous TR{alpha}2 mice would show a hypothyroid phenotype in target tissues. However some of the alterations observed, including decreased body weight, increased heart rate, and increased body temperature, are consistent with a hyperthyroid phenotype. It is possible that the increase in TR{alpha}1 could lead to an increased activity of the receptor, provided intracellular availability to hormone is adequate.

The observation that the heterozygous mice in all aspects studied in this report exhibited a phenotype intermediate or identical to that of homozygotes suggests that the loss of TR{alpha}2 has had a much smaller impact as compared with the increased expression of TR{alpha}1. The phenotypic changes described by us cannot be attributed solely to the loss of TR{alpha}2; in fact, they could with few exceptions be explained as an effect of overexpressed TR{alpha}1. The effects of TR{alpha}2 protein on T3-mediated gene regulation in vitro have previously been described to be weak (18), suggesting that further analyses of the finer details of TH action in our mouse strain is required to assess the role of TR{alpha}2 in physiology.

The discovery of TR{alpha}2 in mammalian, but not amphibian, avian, or fish species despite extensive investigation, suggests that a role for TR{alpha}2 would represent only a refinement of the basic function of the (Thra) TR{alpha} gene. Accordingly, TR{alpha}2 may be viewed as having an active role in control of normal TR signaling, or at another extreme, as having no physiological role but instead representing a consequence of how the mammalian genome arose. The phenotypes led us to consider the explanation that the ability to divert TR{alpha} gene expression toward production of TR{alpha}2, irrespective of if it has an activity, may represent a means for adjusting the activity of the T3 binding TR{alpha}1 protein to physiologically appropriate levels. Our data neither refute nor support these alternative hypotheses but do allow us to conclude that the ratio of expression of TR{alpha}1:TR{alpha}2 plays an important, widespread regulatory role in mammalian physiology.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Targeting Vector
The TR{alpha} gene contains 10 exons (45), which give rise to two different mRNAs. The first nine exons encode the TR{alpha}1 protein, whereas the TR{alpha}2 mRNA is generated from alternative splicing from a donor site 128 bp after the start of exon 9 to the acceptor site for exon 10. Because little is known about the individual role of the TR{alpha}1 and TR{alpha}2 proteins, we decided to delete independently the TR{alpha}1 (24) and the TR{alpha}2 genes. To generate the TR{alpha}2 mutant mice, we modified our TR{alpha}1 targeting vector (24), so that it contains the entire coding sequence of exon 9, followed by a strong SV40 polyadenylation signal and a thymidine kinase (TK) promoter-neo cassette pMCneo/poly(A) (Stratagene, La Jolla, CA) in the opposite transcriptional orientation. The strong SV40 polyadenylation site blocked transcription into exon 10, effectively abrogating expression of the TR{alpha}2 protein. Exon 10 of the TR{alpha} gene and the last exon of the rev-erbA{alpha} gene were left intact, thus avoiding disturbances of transcription of rev-erbA{alpha}.

Generation of TR{alpha}2 Mutant Mice
Homologous recombination in E14 embryonic stem cells (ES cells) and the screening of positive clones were done as previously described (24). The same probes were used in the Southern blot analyses: 5'-probe (5'p) recognizing a 23-kb fragment from the wt allele and a 13-kb band from the mutated allele; the 3'-probe (3'p) detecting the 23-kb band from the wt allele and a 9.9-kb band from the mutated allele (see Fig. 1BGo). An exon 10 probe (ex10p) was used as an internal probe to exclude the presence of extra bands due to complete or partial integration of the targeting vector into the putative positive clones. Two more independent DNA digestions (XbaI and StuI) (Fig. 1BGo) were carried out to confirm the correct integration of the targeting event. The ex8–9 probe, PCR generated from genomic DNA with a 5'-primer at the end of exon 8 (Ex8.5') and a 3'-primer at the start of exon 9 (Ex9.3') detected the expected bands, a 2.8-kb and a 5.6-kb bands from the wt and the mutated allele, respectively, in the XbaI digestion; and a 1.3-kb band from both alleles after StuI digestion.

Blastocyst injections with the positive clone 165 were performed as described previously (24). From the chimeras generated, one male transmitted the mutation to its offspring when crossed with BALB/c female, yielding mice with a mixed 129/Ola x BALB/c genetic background. Due to poor reproduction and pup rearing by females carrying the targeted allele, +/- and -/- mice were generated by female +/- x male -/- intercrosses, whereas wt animals were generated by +/+ x +/- or +/+ crosses. The wt and mutant lines were intercrossed every two to three generations to avoid genetic drift between them. Some experiments were carried out with wt and mutant mice derived from crossing the 129/Ola+BALB/c line with mice having a mixed 129/sv and C67/B6J background. Mutant and wt mice from this line of mice were generated as described above. This line had an improved, but still impaired, reproduction. They differed little from the original strain in other respects. Pups were genotyped at 2–3 wk of age.

The animals were kept at the facilities in New York and Stockholm as described previously (29). The specifics for breeding the TR{alpha}2 mice can be obtained from B. Vennström upon request.

For readability, we will denote the mouse strains described above as TR{alpha}2-/-. However, the mice will be deposited at The Jackson Laboratories (Bar Harbor, ME) under the name Thratm2Ven/tm2Ven. The mouse strains TR{alpha}1-/- and TRß-/- are available at The Jackson Laboratories under the designations Thratm1Ven and Thrbtm1Df.

Estrous Cycle
Estrous cycling was determined by examination of vaginal smears taken daily from mice that were housed individually. Smears were taken at approximately the same time every day for up to 40 d.

RT-PCR
Heart, liver, and brain were dissected from adult mice (3 months old). Heterozygous and homozygous embryos were taken at E15 and wt embryos at E13. RNA was prepared with the Ultraspec kit (Biotex Laboratories, Inc., Houston, TX) according to the supplier’s instructions. Total embryo or 100 mg of tissue were used for RNA preparation. cDNA was obtained with the SuperScript preamplification system (Life Technologies, Inc., Gaithersburg, MD) using an oligo-dT primer. The amount of RNA used for each cDNA preparation varied from 1.1 to 4 µg. To perform each PCR reaction we used one-tenth of the cDNA or 110–400 ng of total RNA. The primers used in the PCR reaction were: 5'-Sal ex9 hybridizing to the beginning of exon 9 of the TR{alpha} locus; UTex9, 3'-primer recognizing the untranslated region of exon 9, only present in the wt allele; SV40, 3'-primer recognizing a specific sequence of the SV40 polyA+, introduced and present only in the mutated allele; 5'-ex8-1, 5'-primer recognizing exon 8; 3'-ex9-1, 3'-primer hybridizing to exon 9; 5'-ex8-2, 5'-primer recognizing exon 8 and finally 3' ex10-2, 3'-primer hybridizing to exon 10. PCRs were performed according to the SuperScript preamplification system recommendations, with 57 C annealing temperature for 35 cycles.

PCR Primer Sequences
5'-Sal ex9: 5'-GGA GTC GAC CGA GAA GAG TCA GGA UTex9: 5'-CAG GGG AAA TCT AGG CCA AGG AAC SV40: 5'-CAC TGC ATT CTA GTT GTG GT 5'-ex8-1: 5'-TCC GCT ACG ACC CTG AGA GTG AC 5'-ex8-2: 5'-GAA TGG TGG CTT GGG TGT GGT CT 3'-ex9-1: 5'-TGG GAG GAA GGA GAG AAG AGA T 3'-ex10-2: 5'-GAC CTG CGG ACC CTG AAC AAC Ex8.5': 5'-GGC TGT GCT GCT AAT GTC AAC Ex9.3': 5'-GCG TCG ACA GCA AGT TCATTT ATG GCC

Northern Blot Analysis
Polyadenylated RNAs were prepared from tissues as previously described (46). Northern blots were performed as before (47), and the probes used for the analyses were the complete cDNA of TR{alpha}1 recognizing both TR{alpha}1 (5.8 kb) and TR{alpha}2 (2.6 kb) variants, specific probes for the two isoforms TR{alpha}1 and TR{alpha}2, and cDNA probes for TSH{alpha} and TSHß. Levels of expression were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. Quantification was done with PhosphorImagers from Fuji Photo Film Co., Ltd. (Stamford, CT) or Molecular Dynamics, Inc. (Sunnyvale, CA).

Gel Mobility Shift Assays and Nuclear T3 Binding Determinations
Nuclear extracts from brain and liver of wt and homozygous mice were prepared and analyzed using an F2 TRE as earlier described (29). Ligand binding experiments were performed as previously described (29) to determine the extent to which T3 binding was increased because of TR{alpha}1 overexpression.

Hormone Assays
FT3 and FT4 were obtained from 1.5 to 5-month-old mice using a competitive RIA kit (Amerlex-MAB FT3 and FT4 kit) from Amersham Pharmacia Biotech (Piscataway, NJ) as described before (24). TSH and total T3 and total T4 levels in serum were determined as previously described (48). Recombinant bovine TSH was purchased from Sigma (St. Louis, MO). GH levels were measured as described earlier (49). The computer program StatView 4.5 (SAS Institute Inc., Cary, NC) was used in ANOVA analyses to compare all the groups in our hormone assays and also to compare weight and growth curves.

Histology and Immunostaining
Thyroid glands of wt and TR{alpha}2-/- mice were embedded in paraffin, sectioned (5 µm thick), and stained with hemotoxylin and eosin according to standard protocols. TSH immunostaining was performed as previously described (29).

Serum Parameters
Serum IGF-I levels were measured by double-antibody IGF binding protein-blocked RIA (49).

Serum osteocalcin levels were measured using a monoclonal antibody raised against human osteocalcin (Rat-MID osteocalcin ELISA, Osteometer Biotech, Copenhagen, Denmark). The sensitivity of the osteocalcin assay was 21.1 ng/ml, and intra- and interassay coefficients of variation (CVs) were less than 10%. Serum leptin levels were measured by a RIA (Crystal Chem, Inc., Chicago, IL) with intra- and interassay CVs of 5.4 and 6.9%, respectively. Serum corticosterone levels were measured by a RIA (ImmunoChem, ICN Biomedicals, Inc., Costa Mesa, CA) with intra- and interassay CVs of 6.5 and 4.4%, respectively.

Measurement of TRAP activity was performed with the TRAP 5b immunoassay (50). TRAP was purified from human osteoclasts as described, and the purified enzyme was used as antigen to develop a polyclonal TRAP-antiserum in rabbits. A 1:1,000 dilution of the antiserum was used. In the immunoassay, the antiserum was incubated on antirabbit IgG-coated microtiter plates (EG & G Wallac, Inc., Turku, Finland) for 1 h. Diluted mouse serum samples (200 µl) were incubated in the wells for 1 h, and bound enzyme activity was detected using 8 mmol/liter 4-nitrophenyl phosphate as substrate in 0.1 mol/liter sodium acetate buffer, pH 6.1, for 2 h at 37 C. The enzyme reactions were terminated by adding 25 µl of 0.32 mol/liter NaOH to the wells, and A405 was measured using model 2 Victor equipment (EG & G Wallac, Inc.).

DXA
BMC and areal BMD (BMC/cm2) were measured as described earlier (29) using the pDEXA Sabre (Norland Medical Systems, Inc., Fort Atkinson, WI) with Sabre Research 3.6 software. In vivo measurements were performed with three mice in the same scan. To avoid interscan variations a wt mouse was included as an internal control in each scan. Ex vivo measurements of the left femur and tibia and vertebrae L6 were performed on excised bones placed on a 1-cm-thick Plexiglas table. High-resolution scans were performed (line spacing: tibia and femur, 0.02 cm; vertebrae, 0.01 cm). In vivo measurements of animals were performed to measure total body BMC. Medium resolution scans were performed (line spacing, 0.05 cm).

Peripheral Quantitative Computerized Tomography
Tomographic measurements were performed using the STRATEC pQCT XCT (software version 5.4B; Norland Medical Systems, Inc.) operating at a resolution of 70 µm as previously described (51).

Histological Staining and Growth Plate Measurements
Right femurs were excised and fixed in 4% buffered paraformaldehyde, and were subsequently decalcified, embedded in paraffin, and sectioned. Sections were stained with Alcian Blue/van Gieson stain. The width of growth plates was measured as previously described (30). For measurements of total growth plate and the hypertrophic layer, the average of 30 measurements was calculated. The width of the proliferative layer was calculated by subtracting the width of the hypertrophic layer from the width of the total growth plate.

Fat Measurements
We have previously developed a combined DXA image analysis procedure for the in vivo prediction of fat content in mice (52). The interassay CV for the measurements of percent fat area was less than 3%.

Telemetry
Telemetry assays were performed in adult mice (3–5 months old): seven wt, seven heterozygous, and six homozygous mice. Electrocardiogram records, body temperature, and locomotor activity were analyzed as described previously (53). The mice were allowed to recover at least 7 d before the recordings of each individual mouse kept in its own cage were begun. After 48 h of baseline registration, the animals were injected daily with T3 (Sigma, St. Louis, MO; 0.1 mg/kg sc) for 4 d at 1300 h.


    ACKNOWLEDGMENTS
 
Dr. Ernest Arenas provided constructive comments on the manuscript. We thank Dr. Patrick Chomez, Dr. Lilian Wikström, Dr. Carrolee Barlow, Dr. Monika Andersson, Dr. Angel Campos Barros, Dr. Igor Lisoukov, Mrs. Lilian Sundberg, Miss SuSan Oh, and Miss Hanna Rahtu for their contributions to this project. We are grateful to Dr. James Gurr for the gift of the TSH clones and Dr. W. Wood for providing mouse TR clones. Dr. A. F. Parlow, National Institutes of Health, and the US Department of Agriculture provided hormone assay reagents. Drs. Sari Alatolo and Jussi Halleen, Turku University, performed the much appreciated TRAP assays. We also thank the MouseCamp Transgene Facility at Karolinska Institute for blastocyst injections.


    FOOTNOTES
 
This work was supported by the Swedish Medical Research Council, the Swedish Cancer Foundation, the Swedish Foundation for Strategic Research, the Lundberg Foundation, the Swedish Association for Rheumatic Diseases, the Novonordisk Foundation, the Human Frontiers Science Program, Astrazeneca R&D, Torsten and Ragnar Söderbergs Stiftelser, the Göteborg Medical Society, the March of Dimes Birth Defects Foundation, by a Hirschl Award, and National Institutes of Health. C.S. was supported in part by the Ministerio de Educación y Cultura of Spain.

1 Present address: Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Solna, Sweden. Back

Abbreviations: BMC, Bone mineral content; BMD, bone mineral density; CV, coefficient of variation; DXA, dual x-ray absorptiometry; G3PDH, glyceraldehydes-3-phosphate dehydrogenase; pQCT, peripheral quantitative computed tomography; TH, thyroid hormone; TRAP, tartrate-resistant acid phosphatase; TRE, thyroid hormone response element; wt, wild type.

Received for publication June 21, 2001. Accepted for publication August 27, 2001.


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 DISCUSSION
 MATERIALS AND METHODS
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