Human Molecular Genetics, 2001, Vol. 10, No. 23 2701-2708
© 2001 Oxford University Press
Suppression of the deafness and thyroid dysfunction in Thrb-null mice by an independent mutation in the Thra thyroid hormone receptor 
gene
Department of Human Genetics, Box 1498, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA, 1Physiologisches Institut, Gmelinstrasse 5, and Sektion Sensorische Biophysik, Hals-Nasen-Ohren Klinik, Röntgenweg 11, Universität Tübingen, D-72076 Tübingen, Germany, 2Department of Cell and Molecular Biology, Karolinska Institute, S-17 177 Stockholm, Sweden and 3Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221, USA
Received July 23, 2001; Revised and Accepted September 9, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Deletion of thyroid hormone receptor ß (TRß), a ligand-dependent transcription factor encoded by the Thrb gene, causes deafness and thyroid hyperactivity in Thrb-null (Thrbtm1/tm1) mice and in a recessive form of the human syndrome of resistance to thyroid hormone. Here, we have determined that a targeted mutation (Thratm2) in the related Thra gene, encoding thyroid hormone receptor
suppresses these phenotypes in mice. Thra encodes a TR1 receptor which is non-essential for hearing and a TR2 splice variant of unknown function that neither binds thyroid hormone nor transactivates. The Thratm2 mutation deletes TR2 and concomitantly causes overexpression of TR1 as a consequence of the exon structure of the gene. Thratm2/tm2 mice have normal auditory thresholds indicating that TR2 is dispensable for hearing, and have only marginally reduced thyroid activity. However, a potent function for the Thratm2 allele is revealed upon its introduction into Thrbtm1/tm1 mice, where it suppresses the auditory and thyroid phenotypes caused by loss of TRß. These findings reveal a novel modifying function for a Thra allele and suggest that increased expression of TR1 may substitute for the absence of TRß. The TR isotypes generated by the distinct Thrb and Thra genes represent a small family of receptors that have diverged to mediate different physiological roles; however, the ability of changes in Thra expression to compensate for loss of Thrb indicates that many functions of these genes remain closely related. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Thyroid hormone receptors are ligand-dependent transcription factors and distinct receptor isotypes are encoded by two different genes (1,2). Deletion of thyroid hormone receptor ß (TRß) results in deafness and thyroid hyperactivity in Thrbtm1/tm1 mice (3,4) and in a recessive form of the human syndrome of resistance to thyroid hormone (5). In the typical dominant form of this human resistance syndrome, TRß point mutations result in thyroid hyperactivity and a variable range of additional symptoms (6,7), including mild hearing loss (8). TRß is encoded by the Thrb gene on mouse chromosome 14, and by THRB on human chromosome 3p22 (2). A related TR
1 receptor is encoded by Thra on mouse chromosome 11 and by THRA on human chromosome 17q11.2. THRA mutations have so far not been associated with human disease. However, targeted mutations in mice indicate that Thra and Thrb mediate distinct physiological roles (3,9–13). In contrast to Thrbtm1/tm1 mice, TR1-deficient mice do not have impaired auditory function (14) but exhibit cardiac and thermoregulatory defects (10,12). Thyroid hormone (T3) is essential for cochlear development (15,16). The Thrb and Thra genes are expressed in overlapping patterns in the cochlea, with Thrb being prominent in the organ of Corti, which contains the sensory hair cells, whereas Thra is more widely expressed (17–19). Thrbtm1/tm1 mice, that lack TRß, have a defective auditory-evoked brainstem response (ABR) (4), and although they do not have gross morphological defects in the adult cochlea, they show developmentally retarded expression of a potassium current (IK,f) in the cochlear inner hair cells (IHCs) (14). IK,f is thought normally to transform the immature IHC into a high frequency signal transmitter during cochlear maturation (20). Thrbtm1/tm1 mice also display goiter with excessive levels of thyroid hormones and pituitary thyrotropin (thyroid-stimulating hormone, TSH), indicating another role for TRß in mediating the feedback control of the pituitary–thyroid axis (3,21).
The Thra gene has a complex structure and in addition to TR1, it expresses a C-terminal splice variant, TR2, that neither binds T3 nor transactivates and which has been suggested to act as a weak TR antagonist in vitro (22–24). TR2 is widely co-expressed with TR1 but at 2–6-fold higher levels (25). Unlike TR1 and TRß, which have counterparts in avian, amphibian and fish species, TR2 has been found only in mammals (23). We used gene targeting to generate Thratm2/tm2 mice that lack TR2 and which concomitantly overexpress TR1 (26) as a result of the structure of the 3' exons of the gene (27,28) (Fig. 1). Thratm2/tm2 and Thra+/tm2 mice have mild growth and metabolic phenotypes and slightly low thyroid hormone levels, suggesting that the loss of TR2 and/or increase in TR1 alters the fine control of a range of functions. As TR2 mRNA is expressed in the cochlea, with a similar wide distribution as TR1 (17), we investigated auditory function in Thratm2/tm2 mice. We also introduced the Thratm2/tm2 mutation into Thrbtm1/tm1 mice to assess interactions between Thra and Thrb. The results show that the Thratm2/tm2 mutation is itself innocuous with respect to auditory function, but that it acts as a novel suppressor of the deafness and hormonal phenotypes caused by loss of TRß in Thrbtm1/tm1 mice.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The Thra gene expresses a TR
1 receptor and a TR2 C-terminal splice variant that does not bind thyroid hormone (Fig. 1A). The 2.6 kb TR2 and the 5.2 kb TR1 mRNAs are co-expressed in most tissues, including cochlea, with TR2 typically being more abundant (Fig. 1C; see kidney example in left panel). The Thratm2 mutation introduced a strong SV40 polyadenylation site in exon 9 that precluded TR2 expression by blocking extension to the TR2-specific exon 10 (Fig. 1B). The loss of TR2 expression has been established in brain and liver by northern blot analysis of poly(A)-selected mRNA (26). We further determined the consequence of the mutation in cochlea. Although poly(A)-selected mRNA was unobtainable from the small cochlea, northern blot analysis of total RNA confirmed that the Thratm2 mutation deleted TR2 and terminated all transcripts as a single, novel 2.3 kb mRNA (TR1*) that encoded only TR1 (Fig. 1B and C). Using TR2-specific or TR1/TR2-common probes, the 2.6 kb TR2 band was detected in mice that were wild-type or heterozygous for the Thratm2 mutation (Thra+/tm2Thrbtm1/tm1 genotype, shown as a+/tm2btm1/tm1 in Fig. 1C) but not in Thratm2/tm2 mice (Thratm2/tm2Thrbtm1/tm1 genotype, shown as atm2/tm2btm1/tm1). TR1 mRNA was less abundant and was at the lower limit of detection in cochlear total RNA (Fig. 1C; 1-specific probe). The presence of TR1 mRNA in normal cochlea was confirmed by RT–PCR analysis (Fig. 1D), consistent with previous reports (17–19).
In Thra+/tm2Thrbtm1/tm1 mice, as predicted, both the novel 2.3 kb TR1* mRNA and the 2.6 kb TR2 bands were present, whereas in Thratm2/tm2Thrbtm1/tm1 mice, the novel 2.3 kb TR1* mRNA was the only band detected (Fig. 1C). Since TR2 mRNA is normally more abundant than TR1 mRNA, the Thratm2 mutation unavoidably resulted in higher levels of TR1* mRNA than the natural 5.2 kb TR1 mRNA, which was a consequence of the 3' exon structure of the gene, as has been described by Saltó et al. (26). These results demonstrated deletion of TR2 mRNA in cochlea and indicated a concomitant, relatively high level of expression of TR1-encoding TR1* mRNA.
To determine the results of deletion of TR2 on auditory function, the ABR was analyzed in Thratm2/tm2 mice. Thratm2/tm2 mice displayed no impairment of the ABR, indicating that TR2 was dispensable for auditory function. Thratm2/tm2 and Thra+/tm2 adult mice (indicated as atm2/tm2 and a+/tm2, respectively, in Fig. 2A) responded to normal sound pressure thresholds for click and pure-tone (8, 16 and 32 kHz) stimuli that span the sensitive hearing range of mice. Waveforms with four to five peaks were detected within the first 5 ms of stimulation, typical of the pattern of normal-hearing mice (29) (Fig. 2B). Thrbtm1/tm1 mice (shown as btm1/tm1) also showed waveforms but these were detectable only in response to significantly elevated thresholds [85 dB sound pressure level (SPL) example shown in Fig. 2B], as reported (4). Thus, neither loss of TR2 nor overexpression of TR1 in Thratm2/tm2 mice impaired auditory function.
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3) onto the C57BL/6J strain. (B) Representative ABR waveforms in response to a click stimulus (shown on a fixed 4 µV scale). Waveforms of four to five peaks, typical for mice (29), were detected in the normal range (40–45 dB SPL) for all genotypes except for Thrbtm1/tm1 mice, where much elevated thresholds were required (example shown, 85 dB SPL). Thresholds are underlined.To investigate interactions between the Thra and Thrb genes, the Thratm2 and Thrbtm1 mutations were combined. Remarkably, mice of both Thratm2/tm2Thrbtm1/tm1 and Thra+/tm2Thrbtm1/tm1 genotypes (shown as atm2/tm2btm1/tm1 and a+/tm2btm1/tm1, respectively) had normal ABR thresholds (Fig. 2A and B). Thus, the Thratm2 mutation, which itself does not alter auditory function, dominantly suppressed the auditory defect of Thrbtm1/tm1 mice.
Consistent with the rescued ABR thresholds, the developmental expression of the IK,f potassium current in cochlear IHCs was also largely corrected in Thratm2/tm2Thrbtm1/tm1 mice compared to Thrbtm1/tm1 mice. Using whole cell recordings in short-term cochlear explants, IK,f is normally detected in all IHCs from wild-type mice by postnatal day 14 (P14) and it rises to plateau after P20 (20) (see wild-type curve, Fig. 3B). Figure 3A compares examples of the currents elicited by depolarizing voltage steps in IHCs from normal-hearing control mice (TR1-deficient Thratm1/tm1), Thratm2/tm2Thrbtm1/tm1 double mutant and Thrbtm1/tm1 single mutant mice at P19. IHCs from Thratm2/tm2Thrbtm1/tm1 mice exhibited currents that activated rapidly to steady state levels within 2 ms at potentials between –40 and –14 mV. These currents were not significantly different from those in IHCs from control mice. In contrast, in IHCs of Thrbtm1/tm1 mice, only small outward currents could be activated at potentials between –84 and –14 mV. Moreover, the current kinetics were slower than normal and did not reach steady state during the 10 ms recording interval, indicating the absence of the IK,f current, as reported previously (14).
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Figure 3B shows that the developmental profile of IK,f expression in IHCs of Thratm2/tm2Thrbtm1/tm1 mice (solid line) resembled the normal curve of IK,f expression in wild-type mice (dotted line) rather than the delayed pattern of Thrbtm1/tm1 mice (dashed line), reported previously (14,20). In Thratm2/tm2Thrbtm1/tm1 mice, IK,f expression was detected by P14 and rose steadily, as in wild-type mice. Notably, by P19 all IHCs of Thratm2/tm2Thrbtm1/tm1 mice expressed substantially more IK,f current than IHCs of Thrbtm1/tm1 mice (Fig. 3A). The curve fit of IK,f data points for Thratm2/tm2Thrbtm1/tm1 mice suggested that beyond P30, IK,f was slightly smaller than in wild-type cells (P < 0.001), although it is unlikely that this had major significance as ABR thresholds were normal in Thratm2/tm2Thrbtm1/tm1 mice at this age. Thus, in contrast to the retardation of IK,f expression in Thrbtm1/tm1 mice during postnatal maturation, IK,f was prominently expressed in Thratm2/tm2Thrbtm1/tm1 mice, indicating a substantial rescue of the IK,f defect.
TRß-deficiency in Thrbtm1/tm1 mice (3) and in human resistance to thyroid hormone (5) results in excessive TSH and thyroid hormone levels, reflecting a key role for TRß in the negative feedback regulation of the pituitary–thyroid axis by thyroid hormone. In contrast, Thratm2/tm2 mice have normal serum TSH and marginally low levels of thyroxine (T4) and triiodothyronine (T3) (26). Like the deafness, the hormonal disorder of Thrbtm1/tm1 mice was suppressed by the Thratm2/tm2 mutation. Rather than being elevated as in Thrbtm1/tm1 mice, serum TSH was normal in Thratm2/tm2Thrbtm1/tm1 mice (Fig. 4A). Pituitary levels of TSHß and TSH subunit mRNAs were normal (Fig. 4B). Serum free T4 (FT4) and free T3 (FT3) were slightly but significantly (P < 0.001) below normal and total T3 (TT3) was also slightly low (Fig. 4C). In comparison, Thrbtm1/tm1 mice display 2–3-fold elevated levels of TSH, T4 and T3 (P < 0.001) (3). Histological sections of the thyroid gland in Thratm2/tm2Thrbtm1/tm1 mice appeared like those of Thratm2/tm2 mice, with follicles that had a somewhat thin, flattened epithelium, and lacked the enlargement noted in Thrbtm1/tm1 mice (3) (Fig. 4D). Interestingly, the overall phenotype of Thratm2/tm2Thrbtm1/tm1 mice of normal TSH and slightly low T4 and T3 was similar to that of Thratm2/tm2 mice (26) rather than wild-type mice, indicating that not only is the Thrbtm1/tm1 phenotype suppressed but that the Thratm2/tm2 phenotype dominates. Thus, in the pituitary–thyroid axis, the functional consequences of the increase of TR1 and/or loss of TR2 override the defects due to loss of TRß.
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Thratm2/tm2Thrbtm1/tm1 mice displayed no additional obvious phenotypes. Weight gain in Thra+/tm2Thrbtm1/tm1 and Thratm2/tm2Thrbtm1/tm1 mice was retarded over the first 5 postnatal weeks (Fig. 5), but only to the same degree as in the Thratm2/tm2 strain (26). Thrbtm1/tm1 mice show normal growth rates (3). The lack of additional phenotypes together with the suppression of the auditory and thyroid defects in Thrbtm1/tm1 mice, leads to the conclusion that only beneficial interactions resulted from the combination of Thratm2/tm2 and Thrbtm1/tm1 mutations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hearing and other physiological functions are sensitive to genetic interactions (30,31). Several loci that modify deafness phenotypes have been mapped, including DFNM1 that suppresses human deafness caused by DFNB26 mutations (32), and moth1 and mdfw that modify deafness in tubby (33) and deaf waddler (34,35) mutant mouse strains, respectively. Genetic factors have also been invoked to explain the heterogeneous symptoms in the human syndrome of resistance to thyroid hormone (7,36) which is characterized by thyroid dysfunction and a variety of other symptoms. Profound deafness occurs in a rare recessive form of this syndrome (5) and a 20% incidence of hearing loss is reported in the typical dominant form of the syndrome (6,8). The identification of interacting genes that influence a phenotype would suggest some of the ways in which compensation could occur. The present study indicates that altering the expression levels of the TR
1 and TR2 products of the Thra gene can suppress the deafness and thyroid dysfunction caused by deletion of TRß in Thrbtm1/tm1 mice. Thus, although Thra and Thrb mediate distinct physiological roles as indicated by single gene mutations (3,9,10,12), our results suggest that altering the balance of expression of Thra gene products can compensate for loss of Thrb.
There is little evidence to suggest that the deletion of TR2 per se corrects the phenotypes caused by loss of TRß, although this cannot be entirely excluded. This would presuppose that TR2 somehow causes deafness in Thrbtm1/tm1 mice and in human recessive resistance to thyroid hormone, but such speculation is limited by the lack of a defined function for TR2. The variant structure of TR2 abrogates T3 binding, impairs dimerization and weakens DNA binding, which moreover, is limited to only a subset of TR-binding sites. TR2 is also reported to inhibit transactivation by normal TRs in vitro, albeit weakly (37–39), but receptor interference would be irrelevant in the absence of TRß. Based on the phenotypes of Thratm2/tm2 mice, it has been proposed that the capability of diverting Thra expression into mRNAs encoding either the TR1 receptor or the TR2 non-receptor variant provides a means of modulating TR1 levels (26), regardless of any putative inhibitory activity of TR2. Thratm2/tm2 mice exhibit mild growth retardation, slightly low thyroid hormone levels and an increased heart rate and body temperature that could result from either loss of TR2 or increased TR1 levels. The latter explanation may be supported by the finding that partial phenotypes also occur in Thra+/tm2 mice which overexpress TR1-encoding TR1* mRNA from the mutant allele while still expressing relatively abundant levels of TR2 from the remaining wild-type allele.
Alternatively and more simply, the phenotypic suppression by the Thratm2 allele may be explained by the overexpression of TR1 substituting for loss of TRß. The overexpression of TR1-encoding TR1* mRNA in mice of both Thra+/tm2 and Thratm2/tm2 genotypes could account for the dominance of the phenotypic rescue. Although TR1 and TRß display certain preferences in DNA binding and transactivation (40,41), both can transactivate to a large extent through similar elements in vitro. Also, the exacerbated phenotypes of compound mutant mice lacking both TR1 and TRß, indicate that both receptor types co-regulate several of the same physiological functions and target genes in vivo (21,42). It is noteworthy that a distinct Thra mutation that deletes both TR2 and TR1 was recently reported not to cause deafness nor to rescue ABR thresholds in mice lacking TRß (12), thus strongly supporting the requirement for TR1 to effect the suppressing action of the Thratm2 mutation. Although our findings suggest that TR1 and not TR2 is the critical factor for suppressing the auditory and thyroid phenotypes of Thrbtm1/tm1 mice, these results do not exclude some role for TR2 in other systems (26).
The TRs, like other classes of nuclear receptors such as retinoid and estrogen receptors are encoded by gene families (43). Gene duplication has provided a means of extending the range of specific functions of the small TR family (21,42). However, an interesting implication of the present results is that the divergence of the TR genes has not progressed so far that TR1 is structurally precluded from substituting for TRß since altering the expression levels of Thra can substitute for many of the functions of Thrb. Nonetheless, the possession of two TR genes may be presumed to be beneficial given the presence of both TR1- and TRß-related receptors in mammalian, amphibian, fish and avian species (44). It is not excluded that under normal circumstances a degree of specificity is provided by the structural variations between TR1 and TRß in their N-termini and DNA-binding domains, which might confer receptor-specific interactions with transcription co-factors or target genes (40,41). However, if TR1 overexpression can largely substitute for TRß, such constraints would play a lesser role than relative receptor levels in determining the individual functions of the Thra and Thrb genes.
Another human disorder involving deafness and goiter although with a distinct etiology from the syndrome of resistance to thyroid hormone is Pendred syndrome. Mutations in the PDS gene, which encodes a transporter of chloride and iodide, are associated with both non-syndromic hearing loss and with Pendred syndrome, a recessive disorder of deafness and goiter (45–47). It is unclear why mutations in the same gene result in variable outcomes of deafness or deafness with goiter, but environmental or genetic factors could be involved. Recent studies also suggest that different types of PDS mutation that leave residual transport activity may produce deafness whereas complete loss of activity produces deafness and goiter (48). It remains unclear the extent to which the variable symptoms of resistance to thyroid hormone are due to the THRB mutation type or to independent genetic interactions (6,7). On a speculative note, based on the present study, any change in THRA expression, even if it produces no major phenotype, could potentially ameliorate some of the symptoms.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mouse strains
Generation of Thratm2Ven/tm2Ven mice (nomenclature according to Mouse Genetics Guidelines, Jackson Laboratories) has been described (26). For brevity, homozygous mutants are designated as Thratm2/tm2 (or atm2/tm2 in some figures). Briefly, a polyadenylation site was inserted into exon 10, thus abrogating expression of TR
2. The last exon of the RevErbA gene that overlaps with Thra exon 10 (TR2-specific) on the opposite DNA strand remained intact (27). The mutant mice had a mixed background of 129/OlaHsd x BALB/c. The mutation was backcrossed onto the C57BL/6J strain for two generations and then crossed with Thrbtm1/tm1 mice that lack all known TRß products (3) that were congenic on a C57BL/6J background (N12) (B6.129S1-Thrbtm1Df). The ABR and weight-gain analyses were conducted on mice with these comparable backgrounds that were backcrossed for three or more generations onto C57BL/6J, a background that does not lose hearing until at least 6 months of age (29). An additional study of 119 mice on a mixed background of 129/OlaHsd x BALB/c x 129/Sv x C57BL/6J, showed similar but more variable thresholds, consistent with the presence of endogenous hearing loss genes in 129 substrains (29). Hormones were measured in mice of this mixed background. Genotypes were determined by PCR (3). Animal experiments followed approved institutional protocols at Mount Sinai School of Medicine, the Karolinska Institute and the University of Tübingen.
Auditory-evoked brain stem response
ABR tests were performed using the SmartEP ABR system, version 2.1, from Intelligent Hearing Systems essentially as described by Zheng et al. (29). Mice were anesthetized with avertin (0.25 mg/g body weight) and active, reference and ground electrode needles were placed subcutaneously at the vertex, ventrolateral to the left ear, and ventrolateral to the right ear, respectively. Binaural stimulation (click, 8, 16 or 32 kHz) was presented to the mice with a rise–fall time of 1.5 ms, at a rate of 25/s. ABR thresholds were identified by visually recognizable ABR peaks on a normalized scale. Responses were bandpass filtered below 100 and above 3000 Hz and amplified 100 000 times. Thresholds were comparable to previous recordings with a previous version of the ABR system from Intelligent Hearing Systems (4), except for slightly lower thresholds at 32 kHz, which may be due to differences in the high frequency transducers or in genetic background.
Whole-cell recording
The recording technique has been reported previously (14,20). Briefly, cochlear IHCs were acutely dissected from the most apical half-turn of the organ of Corti from mice between P12 and P59. The isolated piece of the organ of Corti was mounted in an experimental chamber and perfused at 10 ml/h with an extracellular solution composed of (in mM) 144 NaCl, 0.7 NaH2P04, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 5.6 D-glucose and 10 HEPES–NaOH pH 7.3. Vitamins and amino acids for Eagle’s minimal essential medium were added from concentrate (Gibco/BRL).
Membrane currents and voltages were studied at room temperature (20–25°C) by whole-cell patch-clamp using an Axopatch 200B amplifier. Patch pipettes were filled with an intracellular solution proven to sustain Ca2+ currents in IHCs (49) (in mM): 110 K-gluconate, 20 KCl, 0.1 CaCl2, 4 MgCl2, 5 K2EGTA, 10 Na-phosphocreatine, 4 Na2ATP, 0.3 GTP, 5 HEPES–KOH pH 7.4. No differences in the potassium currents were found compared with KCl-based intracellular solutions as used previously (14). Currents under voltage clamp are presented with capacitive transient and linear leak currents subtracted; all voltages were corrected for the voltage drop across the uncompensated series resistance, Rs (0.5–5 M
; mean ± SD = 1.0 ± 0.2 M; n = 33). Voltages were also corrected for the liquid junction potential between the intra- and extracellular solutions (–10 mV; calculated by computer software by P.Barry, University of New South Wales, Australia). Thirty-one IHCs of the most apical half-turn of the mouse cochlea had a mean resting membrane potential of –66 ± 4 mV and a mean membrane capacitance of 7.2 ± 1.6 pF.
The fast potassium current, IK,f, was measured at –25 mV between 2.4 and 3.6 ms after the onset of the depolarizing voltage step (Fig. 2A). The fits of the developmental expression pattern are according to a sigmoidal logistic growth curve:
I = (Imax – Imin)/(1 + exp(–s(t – t1/2))) + Imin
where I is current (nA), s is a slope factor (day–1), t is time (days) and t1/2 is the time at which I is halfway between Imax and Imin. IK,f was then plotted versus the depolarizing membrane potential to obtain I–V plots. IK,f was measured at –25 mV from these plots by interpolating data points negative and positive to –25 mV and plotted versus the age of the mouse studied. Statistical tests are two-tailed Student’s t-tests.
RNA analysis
Cochleae including the bony capsule from 12–16 mice of each genotype at 3–6 months of age were dissected then pooled for preparation of total RNA using TriReagent (Molecular Research Center). Sequences of oligonucleotides were: exon 9b/TR1-specific antisense northern blot probe, 5'-GACTTCCCGCTTCACCAAGCTGCTGCTCAAGCTGCCGGACCTGCGG-3'; exon 10/TR2-specific antisense northern blot probe, 5'-CTGAGGCTTTAGACTTCCTGATCCTCAAAGACCTCCAGGAAGAGTGGG-3'; exon 9/TR1-specific antisense for PCR (primer A1), 5'-CCTCAAAGACCTCCAGGAAGAGTGGG-3'; exon 10/TR2-specific antisense for PCR (primer A2), 5'-GCTGCTCAAGCTGCCGGACCTGCGG-3'; exon 8/TR common exon (sense) for PCR (primer C), 5'-CCTCCTGAAGGGCTGCTGCATGG-3'.
Samples (15 µg) of cochlear total RNA were analyzed by northern blot and were electrophoresed longer than usual to resolve TR2 and TR1* bands. For northern probes, oligonucleotides were 5' end-labeled using [
-32P]ATP and T4 polynucleotide kinase. A 1.8 kb cDNA of mouse TR1 (50) was 32P-labeled by random-priming for a TR1/TR2common probe. Hybridization was performed in Quikhyb solution (Stratagene) at 60°C for oligonucleotide probes and at 68°C for the cDNA probe. Washes were at 60°C for common and TR1 probes and at 50°C for the TR2 probe. The filter was hybridized successively with TR1-specific, TR2-specific then cDNA common probes. Autoradiographic exposure times were: common probe, 1 day, TR1-specific, 9 days, TR2-specific, 6 days. Northern blot analysis of TSH, TSHß and G3PDH (glyceraldehyde-3P-dehydrogenase) as a control, in pituitary RNA samples was performed as described (42).
RT–PCR
RT–PCR reactions were performed using cDNA templates made with oligo(dT) primers and 10 µg of cochlear total RNA or 1 µg of brain poly(A)-selected RNA. PCR reactions used primer pairs A1 and C (1-specific) or A2 and C (2-specific) (see above). The TR2 primers encompass both variant I and variant II forms of TR2; TR2 variant II results from an alternative splice acceptor within exon 10 (25,38). PCR conditions: annealing at 60°C for 30 s, extension at 72°C for 45 s and denaturation at 95°C for 30 s.
Hormone determinations
Levels of serum thyrotropin (TSH) and thyroid hormones (free thyroxine, FT4; total and free triiodothyronine, TT3, FT3, respectively) were determined using radioimmunoassays, as described (42). FT4 and FT3 values were similar in females or males, whereas female TSH levels were consistently 40–50% lower than in males.
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ACKNOWLEDGEMENTS |
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We thank W.Wood, D.Gordon and E.C.Ridgway for cDNA clones, A.Parlow for reagents for TSH assays, Professor J.P.Ruppersberg, Department of Physiology, and Professor H.P.Zenner, Ear, Nose and Throat Hospital, University of Tübingen, for providing laboratory space and equipment, and I.Jones for comments on the manuscript. This work was supported in part by the German Research Council (DFG, to A.R.), the Swedish Cancer Fund (to B.V.) and by NIH (DC 03441), March of Dimes Birth Defects Foundation and an Irma T.Hirschl Award (to D.F.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 212 659 6735; Fax: +1 212 849 2508; Email: douglas.forrest@mssm.edu Correspondence may also be addressed to Björn Vennström. Email: bjorn.vennstrom@cmb.ki.se
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REFERENCES |
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1 Sap, J., Muñoz, A., Damm, K., Goldberg, Y., Ghysdael, J., Leutz, A., Beug, H. and Vennström, B. (1986) The c-erbA protein is a high affinity receptor for thyroid hormone. Nature, 324, 635–640.[Medline]
2 Weinberger, C., Thompson, C.C., Ong, E.S., Lebo, R., Gruol, D.J. and Evans, R.M. (1986) The c-erb-A gene encodes a thyroid hormone receptor. Nature, 324, 641–646.[Medline]
3 Forrest, D., Hanebuth, E., Smeyne, R.J., Everds, N., Stewart, C.L., Wehner, J.M. and Curran, T. (1996) Recessive resistance to thyroid hormone in mice lacking thyroid hormone receptor ß: evidence for tissue-specific modulation of receptor function. EMBO J., 15, 3006–3015.[ISI][Medline]
4 Forrest, D., Erway, L.C., Ng, L., Altschuler, R. and Curran, T. (1996) Thyroid hormone receptor ß is essential for development of auditory function. Nat. Genet., 13, 354–357.[ISI][Medline]
5 Refetoff, S., DeWind, L.T. and DeGroot, L.J. (1967) Familial syndrome combining deaf-mutism, stippled epiphyses, goiter, and abnormally high PBI: possible target organ refractoriness to thyroid hormone. J. Clin. Endocrinol. Metab., 27, 279–294.[ISI][Medline]
6 Refetoff, S., Weiss, R.E. and Usala, S.J. (1993) The syndromes of resistance to thyroid hormone. Endocr. Rev., 14, 348–399.[ISI][Medline]
7 Beck-Peccoz, P. and Chatterjee, V.K. (1994) The variable clinical phenotype in thyroid hormone resistance syndrome. Thyroid, 4, 225–232.[ISI][Medline]
8 Brucker-Davis, F., Skarulis, M.C., Pikus, A., Ishizawar, D., Mastroianni, M.-A., Koby, M. and Weintraub, B.D. (1996) Prevalence and mechanisms of hearing loss in patients with resistance to thyroid hormone (RTH). J. Clin. Endocrinol. Metab., 81, 2768–2772.[Abstract]
9 Fraichard, A., Chassande, O., Plateroti, M., Roux, J., Trouillas, J., Dehay, C., Legrand, C., Gauthier, K., Kedinger, M., Malaval, L. et al. (1997) The T3R gene encoding a thyroid hormone receptor is essential for post-natal development and thyroid hormone production. EMBO J., 16, 4412–4420.[ISI][Medline]
10 Wikström, L., Johansson, C., Saltó, C., Barlow, C., Campos Barros, A., Baas, F., Forrest, D., Thorén, P. and Vennström, B. (1998) Abnormal heart rate and body temperature in mice lacking thyroid hormone receptor 1. EMBO J., 17, 455–461.[ISI][Medline]
11 Kaneshige, M., Kaneshige, K., Zhu, X., Dace, A., Garrett, L., Carter, T.A., Kazlauskaite, R., Pankratz, D.G., Wynshaw-Boris, A., Refetoff, S. et al. (2000) Mice with a targeted mutation in the thyroid hormone ß receptor gene exhibit impaired growth and resistance to thyroid hormone. Proc. Natl Acad. Sci. USA, 97, 13209–13214.
12 Gauthier, K., Plateroti, M., Harvey, C.B., Williams, G.R., Weiss, R.E., Refetoff, S., Willott, J.F., Sundin, V., Roux, J.P., Malaval, L. et al. (2001) Genetic analysis reveals different functions for the products of the thyroid hormone receptor locus. Mol. Cell Biol., 21, 4748–4760.
13 Hashimoto, K., Curty, F.H., Borges, P.P., Lee, C.E., Abel, E.D., Elmquist, J.K., Cohen, R.N. and Wondisford, F.E. (2001) An unliganded thyroid hormone receptor causes severe neurological dysfunction. Proc. Natl Acad. Sci. USA, 98, 3998–4003.
14 Rüsch, A., Erway, L., Oliver, D., Vennström, B. and Forrest, D. (1998) Thyroid hormone receptor ß-dependent expression of a potassium conductance in inner hair cells at the onset of hearing. Proc. Natl Acad. Sci. USA, 95, 15758–15762.
15 Deol, M.S. (1973) An experimental approach to the understanding and treatment of hereditary syndromes with congenital deafness and hypothyroidism. J. Med. Genet., 10, 235–242.[ISI][Medline]
16 Uziel, A., Gabrion, J., Ohresser, M. and Legrand, C. (1981) Effects of hypothyroidism on the structural development of the organ of Corti in the rat. Acta Otolaryngol., 92, 469–480.[Medline]
17 Bradley, D.J., Towle, H.C. and Young, W.S.,III (1994) and ß thyroid hormone receptor (TR) gene expression during auditory neurogenesis: evidence for TR isoform-specific transcriptional regulation in vivo. Proc. Natl Acad. Sci. USA, 91, 439–443.
18 Lauterman, J. and ten Cate, W.-J.F. (1997) Postnatal expression of the -thyroid hormone receptor in the rat cochlea. Hear. Res., 107, 23–28.[ISI][Medline]
19 Knipper, M., Bandtlow, C., Gestwa, L., Kopschall, I., Rohbock, K., Wiechers, B., Zenner, H.-P. and Zimmermann, U. (1998) Thyroid hormone affects Schwann cell and oligodendrocyte gene expression at the glial transition zone of the VIIIth nerve prior to cochlea function. Development, 125, 3709–3718.[Abstract]
20 Kros, C., Ruppersberg, J. and Rüsch, A. (1998) Expression of a potassium conductance in inner hair cells at the onset of hearing in mice. Nature, 394, 281–284.[Medline]
21 Gauthier, K., Chassande, O., Plateroti, M., Roux, J.-P., Legrand, C., Pain, B., Rousset, B., Weiss, R., Trouillas, J. and Samarut, J. (1999) Different functions for the thyroid hormone receptors TR and TRß in the control of thyroid hormone production and post-natal survival. EMBO J., 18, 623–631.[ISI][Medline]
22 Izumo, S. and Mahdavi, V. (1988) Thyroid hormone receptor isoforms generated by alternative splicing differentially activate myosin HC gene transcription. Nature, 334, 539–542.[Medline]
23 Lazar, M., Hodin, R., Darling, D. and Chin, W. (1988) Identification of a rat c-erbA -related protein which binds deoxyribonucleic acid but does not bind thyroid hormone. Mol. Endocrinol., 2, 893–901.[Abstract]
24 Koenig, R.J., Lazar, M.A., Hodin, R.A., Brent, G.A., Larsen, P.R., Chin, W.W. and Moore, D.D. (1989) Inhibition of thyroid hormone action by a non-hormone binding c-erbA protein generated by alternative mRNA splicing. Nature, 337, 659–661.[Medline]
25 Mitsuhashi, T. and Nikodem, V. (1989) Regulation of expression of the alternative mRNAs of the rat -thyroid hormone receptor gene. J. Biol. Chem., 264, 8900–8904.
26 Saltó, C., Kindblom, J., Johansson, C., Wang, Z., Gullberg, J., Nordström, K., Mansén, A., Ohlsson, C., Thorén, P., Forrest, D. et al. Ablation of thyroid hormone receptor 2 and a concomitant overexpression of 1 yields a mixed hypo- and hyperthyroid phenotype in mice. Mol. Endocrinol., in press.
27 Lazar, M., Hodin, R., Darling, D. and Chin, W. (1989) A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbA- transcriptional unit. Mol. Cell. Biol., 9, 1128–1136.
28 Laudet, V., Begue, A., Henry-Duthoit, C., Joubel, A., Martin, P., Stehelin, D. and Saule, S. (1991) Genomic organization of the human thyroid hormone receptor (c-erbA-1) gene. Nucleic Acids Res., 19, 1105–1112.
29 Zheng, Q.Y., Johnson, K.R. and Erway, L.C. (1999) Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses. Hear. Res., 130, 94–107.[ISI][Medline]
30 Friedman, T., Battey, J., Kachar, B., Riazuddin, S., Noben-Trauth, K., Griffith, A. and Wilcox, E. (2000) Modifier genes of hereditary hearing loss. Curr. Opin. Neurobiol., 10, 487–493.[ISI][Medline]
31 Nadeau, J.H. (2001) Modifier genes in mice and humans. Nat. Rev. Genet., 2, 165–174.[ISI][Medline]
32 Riazuddin, S., Castelein, C.M., Ahmed, Z.M., Lalwani, A.K., Mastroianni, M.A., Naz, S., Smith, T.N., Liburd, N.A., Friedman, T.B., Griffith, A.J. et al. (2000) Dominant modifier DFNM1 suppresses recessive deafness DFNB26. Nat. Genet., 26, 431–434.[ISI][Medline]
33 Ikeda, A., Zheng, Q.Y., Rosenstiel, P., Maddatu, T., Zuberi, A.R., Roopenian, D.C., North, M.A., Naggert, J.K., Johnson, K.R. and Nishina, P.M. (1999) Genetic modification of hearing in tubby mice: evidence for the existence of a major gene (moth1) which protects tubby mice from hearing loss. Hum. Mol. Genet., 8, 1761–1767.
34 Noben-Trauth, K., Zheng, Q.Y., Johnson, K.R. and Nishina, P.M. (1997) mdfw: a deafness susceptibility locus that interacts with deaf waddler (dfw). Genomics, 44, 266–272.[ISI][Medline]
35 Zheng, Q.Y. and Johnson, K.R. (2001) Hearing loss associated with the modifier of deaf waddler (mdfw) locus corresponds with age-related hearing loss in 12 inbred strains of mice. Hear. Res., 154, 45–53.[ISI][Medline]
36 Weiss, R.E., Marcocci, C., Bruno-Bossio, G. and Refetoff, S. (1993) Multiple genetic factors in the heterogeneity of thyroid hormone resistance. J. Clin. Endocrinol. Metab., 76, 257–259.[Abstract]
37 Liu, R.T., Suzuki, S., Miyamoto, T., Takeda, T., Ozata, M. and DeGroot, L.J. (1995) The dominant negative effect of thyroid hormone receptor splicing variant 2 does not require binding to a thyroid response element. Mol. Endocrinol., 9, 86–95.[Abstract]
38 Reginato, M., Zhang, J. and Lazar, M. (1996) DNA-dependent and DNA-independent mechanisms regulate the differential heterodimerization of the isoforms of the thyroid hormone receptor with retinoid X receptor. J. Biol. Chem., 271, 28199–28205.
39 Tagami, T., Kopp, P., Johnson, W., Arseven, O. and Jameson, J. (1998) The thyroid hormone receptor variant 2 is a weak antagonist because it is deficient in interactions with nuclear receptor corepressors. Endocrinology, 139, 2535–2544.
40 Lezoualc’h, F., Hassan, A.H.S., Giraud, P., Loeffler, J.-P., Lee, S.L. and Demeneix, B.A. (1992) Assignment of the ß-thyroid hormone receptor to 3, 5, 3'-triiodothyronine-dependent inhibition of transcription from the thyrotropin-releasing hormone promoter in chick hypothalamic neurons. Mol. Endocrinol., 6, 1797–1804.[Abstract]
41 Strait, K.A., Zou, L. and Oppenheimer, J.H. (1992) ß1 isoform-specific regulation of a triiodothyronine-induced gene during cerebellar development. Mol. Endocrinol., 6, 1874–1880.[Abstract]
42 Göthe, S., Wang, Z., Ng, L., Nilsson, J., Campos-Barros, A., Ohlsson, C., Vennström, B. and Forrest, D. (1999) Mice devoid of all known thyroid hormone receptors are viable but exhibit disorders of the pituitary–thyroid axis, growth and bone maturation. Genes Dev., 13, 1329–1341.
43 Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, P., Schütz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P. et al. (1995) The nuclear receptor superfamily: the second decade. Cell, 83, 835–839.[ISI][Medline]
44 Laudet, V. (1997) Evolution of the nuclear receptor superfamily: early diversification from an ancestral orphan receptor. J. Mol. Endocrinol., 19, 207–226.[Abstract]
45 Everett, L.A., Glaser, B., Beck, J.C., Idol, J.R., Buchs, A., Heyman, M., Adawi, F., Hazani, E., Nassir, E., Baxevanis, A.D. et al. (1997) Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nat. Genet., 17, 411–422.[ISI][Medline]
46 Coyle, B., Reardon, W., Herbrick, J.A., Tsui, L.C., Gausden, E., Lee, J., Coffey, R., Grueters, A., Grossman, A., Phelps, P.D. et al. (1998) Molecular analysis of the PDS gene in Pendred syndrome. Hum. Mol. Genet., 7, 1105–1112.
47 Li, X.C., Everett, L.A., Lalwani, A.K., Desmukh, D., Friedman, T.B., Green, E.D. and Wilcox, E.R. (1998) A mutation in PDS causes non-syndromic recessive deafness. Nat. Genet., 18, 215–217.[ISI][Medline]
48 Scott, D.A., Wang, R., Kreman, T.M., Andrews, M., McDonald, J.M., Bishop, J.R., Smith, R.J., Karniski, L.P. and Sheffield, V.C. (2000) Functional differences of the PDS gene product are associated with phenotypic variation in patients with Pendred syndrome and non-syndromic hearing loss (DFNB4). Hum. Mol. Genet., 9, 1709–1715.
49 Platzer, J., Engel, J., Schrott-Fischer, A., Stephan, K., Bova, S., Chen, H., Zheng, H. and Striessnig, J. (2000) Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2+ channels. Cell, 102, 89–97.[ISI][Medline]
50 Wood, W.M., Ocran, K.W., Gordon, D.F. and Ridgway, E.C. (1991) Isolation and characterization of mouse complementary DNAs encoding and ß thyroid hormone receptors from thyrotrope cells: the mouse pituitary-specific ß2 isoform differs at the amino terminus from the corresponding species from rat pituitary tumor cells. Mol. Endocrinol., 5, 1049–1061.[Abstract]
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