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The Journal of Neuroscience, December 15, 2001, 21(24):9792-9800

Retardation of Cochlear Maturation and Impaired Hair Cell Function Caused by Deletion of All Known Thyroid Hormone Receptors

Alfons Rüsch¹, Lily Ng², Richard Goodyear³, Dominik Oliver¹, Igor Lisoukov², Björn Vennström⁴, Guy Richardson³, Matthew W. Kelley⁵, and Douglas Forrest²

¹ Physiologisches Institut and Sektion Sensorische Biophysik, Hals-Nasen-Ohren Klinik, Röntgenweg 11, Universität Tübingen, D-72076 Tübingen, Germany, ² Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029, ³ School of Biological Sciences, The University of Sussex, Falmer, Brighton, BN19QG, United Kingdom, ⁴ Department of Cell and Molecular Biology, Karolinska Institute, S-17 177, Stockholm, Sweden, and ⁵ National Institute of Deafness and Communication Disorders, National Institutes of Health, Rockville, Maryland 20850

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The deafness caused by early onset hypothyroidism indicates that thyroid hormone is essential for the development of hearing. We investigated the underlying roles of the TR1 and TR thyroid hormone receptors in the auditory system using receptor-deficient mice. TR1 and TR, which act as hormone-activated transcription factors, are encoded by the Thra and Thrb genes, respectively, and both are expressed in the developing cochlea. TR is required for hearing because TR-deficient (Thrb^tm1/tm1) mice have a defective auditory-evoked brainstem response and retarded expression of a potassium current (I_K,f) in the cochlear inner hair cells. Here, we show that although TR1 is individually dispensable, TR1 and TR synergistically control an extended array of functions in postnatal cochlear development. Compared with Thrb^tm1/tm1 mice, the deletion of all TRs in Thra^tm1/tm1Thrb^tm1/tm1 mice produces exacerbated and novel phenotypes, including delayed differentiation of the sensory epithelium, malformation of the tectorial membrane, impairment of electromechanical transduction in outer hair cells, and a low endocochlear potential. The induction of I_K,f in inner hair cells was not markedly more retarded than in Thrb^tm1/tm1 mice, suggesting that this feature of hair cell maturation is primarily TR-dependent. These results indicate that distinct pathways mediated by TR alone or by TR and TR1 together facilitate control over an extended range of functions during the maturation of the cochlea.

Key words: cochlea; development; tectorial membrane; hair cell; thyroid hormone receptor; transcription factor

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Thyroid hormone is essential for the development of hearing. Deafness arises if there is insufficient hormone available during sensitive periods in the fetal and possibly early neonatal period in humans (Trotter, 1960; Morreale de Escobar et al., 1996) and in the neonatal period in rodents (Deol, 1973; Van Middlesworth and Norris, 1980; Uziel, 1986). Despite the well known requirement for thyroid hormone, however, relatively little is known of the receptor pathways underlying the actions of this hormone in the auditory system.

The TR1 and TR thyroid hormone receptors, encoded by the related Thra and Thrb genes, respectively, act as hormone-activated transcription factors (Sap et al., 1986; Weinberger et al., 1986), and both are expressed in the developing cochlea. The Thrb gene is expressed in the organ of Corti, which contains the sensory hair cells, where it is prominently expressed in the greater epithelial ridge. Thra is more widely expressed throughout the cochlea (Bradley et al., 1994; Lauterman and ten Cate, 1997; Knipper et al., 1998). The expression patterns of TR1 and TR suggest that the cochlea is a direct site of action of thyroid hormone, consistent with findings of morphological abnormalities in the organ of Corti in hypothyroid rodents (Deol, 1973, 1976; Uziel et al., 1981; O'Malley et al., 1995). Hypothyroidism retards the development of the greater epithelial ridge and malforms the tectorial membrane (TM), which normally contacts the stereociliary bundles of the mechanosensitive hair cells. An intact TM is necessary for the response of the hair cell to acoustic stimulation and for the tuning of basilar membrane motion mediated by the electromotility of the outer hair cells (Dallos et al., 1996; Legan et al., 2000; Steel and Kros, 2001). Although thyroid hormone is known to be required for the morphological differentiation of the organ of Corti, less is known of the role of this hormone in the development of the physiological functions of the cochlea.

We showed previously that TR-deficient (Thrb^tm1/tm1) but not TR1-deficient (Thra^tm1/tm1) mice have impaired auditory-evoked brainstem responses (Forrest et al., 1996; Rüsch et al., 1998). The deletion of TR also results in deafness in a human kindred with recessive resistance to thyroid hormone (Refetoff et al., 1967), and mild hearing loss has been reported in 20% of the dominant cases of this syndrome that are associated with TR point mutations (Brucker-Davis et al., 1996). Thrb^tm1/tm1 mice have developmentally retarded expression of a potassium current, I_K,f, in their inner hair cells (Rüsch et al., 1998). I_K,f normally becomes active with the onset of auditory function at approximately postnatal day 13 (P13) and is thought to transform the immature hair cell into a high-frequency signal transmitter (Kros et al., 1998). Thrb^tm1/tm1 adult mice however, do not display major hypothyroid-like cochlear malformations (Forrest et al., 1996). Therefore, to investigate interactions between Thra and Thrb in cochlear development, we generated Thra^tm1/tm1Thrb^tm1/tm1 mice that lack all known TRs (Göthe et al., 1999). The results reveal that TR1 and TR together facilitate control over an extended and novel array of functions in cochlear maturation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mouse strains. The Thrb^tm1Df targeted mutation deletes all known Thrb products (Forrest et al., 1996). The Thra^tm1Ven mutation specifically deletes the TR1 receptor product of Thra while leaving intact the TR2 nonreceptor splice variant product of this gene (Wikström et al., 1998). Some histology and physiological studies were performed on mutant mice with a mixed background of equal parts 129/Sv, C57BL/6J, 129OlaHsd, and BALB/c strains. To remove background variability for auditory-evoked brainstem response (ABR) measurements, Thrb^tm1 and Thra^tm1 mutations were separately backcrossed for 12 and 9 generations, respectively, to create congenic C57BL/6J strains. These strains were intercrossed to generate Thra^tm1/tm1Thrb^tm1/tm1 mice on a C57BL/6J background; wild-type (wt) C57BL/6J mice do not show hearing loss until >6 months of age (Zheng et al., 1999). ABR results were similar on the mixed background except that variation was more marked. Thra^+/tm1Thrb^+/tm1 or Thra^tm1/tm1Thrb^+/tm1 mice were interbred to generate Thra^tm1/tm1Thrb^tm1/tm1 mice; double mutants themselves displayed infertility (Göthe et al., 1999). Genotypes were determined by PCR as described (Forrest et al., 1996; Wikström et al., 1998). Animal experiments followed all applicable guidelines and approved institutional protocols at Mount Sinai School of Medicine, the Karolinska Institute, and the University of Tübingen.

ABR. ABR tests were performed with a SmartEP ABR system, version 2.1, from Intelligent Hearing Systems (Miami, FL) essentially as described (Zheng et al., 1999). Mice were anesthetized with avertin (0.25 mg/gm 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 was presented with a rise-fall time of 1.5 msec, at a rate of 25/sec. ABR thresholds were determined using decreasing intervals of 10 dB sound pressure level (SPL), which were reduced to 5 dB SPL determine the lowest threshold with visually recognizable ABR peaks on a normalized scale. Results were comparable with previous recordings with an older version of the apparatus from Intelligent Hearing Systems (Forrest et al., 1996). Slightly lower thresholds were detected at 32 kHz in the present work, possibly because of the elimination of background strain variability or minor differences in the physical set up of the machines. Both machines were calibrated by the manufacturer. The ABR was analyzed on two series of mice on the congenic background. In addition to the results shown in Figure 1A, a second series of n = 52 mice (males and females at ages of 5-12 weeks), including an additional n = 7 Thra^tm1/tm1Thrb^tm1/tm1 mice, gave comparable results.

View larger version (36K): [in this window] [in a new window]   Figure 1.   ABR thresholds in mice with single or combined deletions of TR1 and TR. A, Mean ABR thresholds ± SEM (in decibels of SPL) for wild-type (wt), Thratm1/tm1 (atm1/tm1), Thrbtm1/tm1 (btm1/tm1), Thratm1/tm1Thrbtm1/tm1 (atm1/tm1btm1/tm1) mice, or other combined mutant strains. All genotypes were on a uniform, congenic C57BL/6J (N > 10) background. Responses to click, 8, 16, and 32 kHz stimuli are shown. Groups shown contained n = 7-8 mice at 5-13 weeks of age. Thresholds were undetectable or were significantly elevated (p < 0.01) in Thratm1/tm1Thrbtm1/tm1 mice compared with wt or Thrbtm1/tm1 mice for all stimulus frequencies tested. B, C, Representative ABR waveforms for wt and Thratm1/tm1Thrbtm1/tm1 mice in response to click (B) and 16 kHz (C) stimuli. Waveforms are shown on a 4 µV fixed scale for comparison (actual thresholds were determined on a normalized scale for sensitivity; see Materials and Methods). Thresholds are underlined. No waveform was detectable for Thratm1/tm1Thrbtm1/tm1 mice for the click stimulus. Only weak, atypical waveforms were detected for the 16 kHz stimulus shown.

Whole-cell recording. The recording technique has been reported previously (Kros et al., 1998; Rüsch et al., 1998). Briefly, hair cells were studied after acute dissection of the most apical half-turn of the organ of Corti from mice at different postnatal ages. The isolated piece of the organ of Corti was mounted in a chamber and perfused at 10 ml/hr with an extracellular solution composed of (in mM): 144 NaCl, 0.7 NaH₂PO₄, 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 (Life Technologies).

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 hair cells (Platzer et al., 2000) (in mM): 135 KCl, 0.1 CaCl₂, 3.5 MgCl₂, 5 K₂EGTA, 2.5 Na₂ATP, and 5 HEPES-KOH, pH 7.3. 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. Voltages were also corrected for the liquid junction potential between the intracellular and extracellular solutions (4 mV; as calculated by computer software by Peter Barry, University of New South Wales, Australia). Fifteen inner hair cells (IHCs) of the most apical half-turn of the cochlea from Thra^tm1/tm1Thrb^tm1/tm1 mice had a mean resting membrane potential of 69 ± 7 mV, which was similar to the mean 70 ± 8 mV measured in 12 Thra^tm1/tm1Thrb^+/tm1 and Thra^tm1/tm1 mice. The IHC fast current I_K,f was measured at 25 mV between 2.4 and 3.6 msec after the onset of the depolarizing voltage step, as shown in Figure 4A. The fits of the developmental expression pattern are according to a sigmoidal logistic growth curve:
where I is current (in nanoamperes), s is a slope factor (d¹), t is time (measured in days), and t_1/2 is the time at which I is halfway between I_max and I_min.

Capacitance. Motility-related nonlinear capacitance was measured as described previously (Oliver and Fakler, 1999). Briefly, outer hair cells (OHCs) were whole-cell voltage clamped, and their membrane capacitance was monitored using a software lock-in technique while the voltage was ramped from 120 to +50 mV. Capacitance was plotted as a function of membrane potential and fitted with the derivative of a Boltzmann function (Santos-Sacchi, 1991). Values of nonlinear capacitance are given relative to the linear membrane capacitance of the cell (in femtofarads per picofarad) as determined from current transients induced by 10 mV voltage steps.

Endocochlear potentials. Procedures followed described methods (Steel and Barkway, 1989; Rüsch et al., 1998). Mice were anesthetized with 20% urethane at 0.01 ml/g body weight. For Thra^tm1/tm1Thrb^tm1/tm1 mice, the surgical procedure had to be modified because of their 15-fold enlarged thyroid gland (Göthe et al., 1999). A tracheotomy was not performed, and the head was held at the dorsal skull using dental cement. The cochlea was exposed as described previously (Rüsch et al., 1998). Potential measurements used a custom-made amplifier as a high impedance voltmeter. Statistical tests were two-tailed Student's t tests.

Histology, cell counts, and electron microscopy. Mice were killed with CO₂, then the temporal bones were isolated rapidly and fixed in 3% glutaraldehyde/2% paraformaldehyde in PBS by overnight immersion. Thra^tm1/tm1Thrb^tm1/tm1 cochlear samples were decalcified in 0.2 M EDTA in PBS for 7-21 d, then embedded in methacrylate (Immunobed; Polysciences, Warrington, PA) and sectioned at 3-5 µm on a rotary microtome for histology; sections were stained with thionin. Cochleas from n  3 mice per genotype at a given age were studied. Other cochleas were embedded in OCT glue and cryosectioned at 7-10 µm for immunostaining. Antibodies against - and -tectorins were used at 1:1000 dilution, and specific fluorescent staining was detected using FITC-conjugated secondary antibodies. Staining against otogelin was performed as described (Legan et al., 2000).

For cell counts, after fixation, the bony labyrinth, scala vestibuli, and Reissner's membrane were dissected to expose the apical surface of the sensory epithelium. The sensory epithelium was cut into thirds and placed on glass slides as whole mounts. Total numbers of hair cells were counted for 3 cochleas per genotype for P8 pups and for adults using differential interference contrast microscopy.

For transmission electron microscopy, cochleas were rapidly removed, placed in PBS, the oval and round windows were removed, and a small hole was made in the apex of the bony capsule. Fixative (2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.2, containing 1% tannic acid) was gently perfused through the opened windows, and the apical hole and cochleas were then immersed in the same fixative for a further 2 hr. Tissue pieces were washed three times in 0.1 M sodium cacodylate buffer, pH 7.2, post-fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate, pH 7.2, washed with cacodylate buffer, and decalcified in 0.5 M EDTA for 10-14 d at 4°C. After decalcification, tissues were dehydrated through a series of ascending concentrations of ethanol, equilibrated with propylene oxide, and imbedded in TAAB 812 resin (TAAB Laboratories Equipment Ltd., Reading, UK). Blocks were cured for 2 d at 60°C. For light microscopy of Thrb^tm1/tm1 mice, 1-µm-thick sections were cut with glass knives and stained with Toluidine blue.

Ultrathin sections were cut with a diamond knife, double stained with uranyl acetate and lead citrate, and viewed with a Hitachi 7100 electron microscope operating at 75 kV. Cochleas from a total of 10 1- to 7-month-old wild-type mice, seven 4- to 6-month-old Thra^tm1/tm1 Thrb^tm1/tm1 mice, and six 1- to 4-month-old Thrb^tm1/tm1 mice were examined.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Combined roles of TR and TR1 in auditory function

The auditory-evoked brainstem response, an overall measure of auditory function, is impaired in TR-deficient Thrb^tm1/tm1 mice but is normal in TR1-deficient Thra^tm1/tm1 mice (Forrest et al., 1996; Rüsch et al., 1998). To investigate interactions between the Thra and Thrb genes, we analyzed the ABR in Thra^tm1/tm1 Thrb^tm1/tm1 mice lacking all known TRs (Fig. 1). Thra^tm1/tm1 Thrb^tm1/tm1 mice were 30% smaller than wt mice but were viable, and despite fertility problems, thrived reasonably well (Göthe et al., 1999), thus allowing study of auditory function. ABR thresholds were assessed on a uniform, congenic C57BL/6J background to preclude hearing loss because of background strain variations.

Thra^tm1/tm1Thrb^tm1/tm1 mice (shown as a^tm1/tm1b^tm1/tm1 in Fig. 1A) had significantly exacerbated defects compared with Thrb^tm1/tm1 mice (shown as b^tm1/tm1) (p < 0.01) for click and pure tone stimuli (8, 16, 32 kHz) that span the sensitive hearing range of mice. Thra^tm1/tm1Thrb^tm1/tm1 mice lacked any detectable click response at the upper limit of stimulation of the testing apparatus (100 dB SPL) and showed only weak, atypical responses for high-frequency stimuli (16 and 32 kHz). The waveforms of the residual responses to high frequencies could only be evoked with much elevated stimulus intensities (85 dB SPL threshold for 16 kHz shown in Fig. 1C) and were abnormal because the initial peaks within the first 2-3 msec of stimulation that are usually the most prominent in mice (Zheng et al., 1999) were absent or delayed. Thra^+/tm1Thrb^tm1/tm1 mice had intermediate thresholds between those of Thrb^tm1/tm1 and Thra^tm1/tm1Thrb^tm1/tm1 mice, indicating a dosage function for TR1. Thra^tm1/tm1Thrb^+/tm1 mice, however, had normal thresholds, indicating that a single Thrb⁺ wild-type allele was sufficient to sustain auditory function. The results demonstrate major interactions between Thra and Thrb in the auditory system.

Cochlear morphology in TR-deficient mice

Morphological abnormalities were detected in the cochlea in Thra^tm1/tm1Thrb^tm1/tm1 mice that indicated a role for TRs during the postnatal differentiation of the greater epithelial ridge of the organ of Corti (Fig. 2). During the first postnatal week, the TM, an extracellular matrix (Richardson et al., 1987), is secreted and extends from the spiral limbus across the inner sulcus to contact the hair cells in the sensory epithelium (Fig. 2A,C and see schematic diagram in Fig. 2M). The inner sulcus opens in association with the retraction of the epithelial cell layer underlying the TM. In Thra^tm1/tm1Thrb^tm1/tm1 pups at P8, the TM was enlarged and deformed and remained attached to the underlying epithelium; moreover, no inner sulcus was formed (Fig. 2B,D). The tunnel of Corti between the inner and outer pillar cells had not opened, and the pillar cells formed a pyramidal rise above the surface of the sensory epithelium (Fig. 2D).

View larger version (78K): [in this window] [in a new window]   Figure 2.   Cochlear malformations in Thratm1/tm1Thrbtm1/tm1 (A-H) and Thrbtm1/tm1 (I-L) mouse strains. A-H, Thratm1/tm1Thrbtm1/tm1 and wt control mice at postnatal day 8 (A-D) and as adults (7- to 8-week-old) (E-H). A, C, Low (A) and higher (C) magnification view of a mid-modiolar, midbasal turn of the cochlea from a wt pup at P8 showing the tectorial membrane (TM) extending over the inner sulcus (IS) to the hair cells (the TM is slightly retracted from the OHCs because of shrinkage during fixation). The tunnel of Corti (TC) has opened between the inner and outer pillar cells. The filled arrowhead indicates an IHC, and the three arrows indicate OHCs. Abbreviations and symbols are the same in other panels; see M, for full description. B, D, Low (B) and higher (D) magnification view of a midbasal turn from a Thratm1/tm1Thrbtm1/tm1 pup. The TM is enlarged, the IS has not formed, the tunnel of Corti has not opened, and the pillar cells protrude (open arrowhead) above the epithelium between the IHC and OHCs. The greater epithelial ridge below the TM is markedly thicker than in the wt pup. Scale bars: A (same for B), C (same for D), 100 µm. E, Apical turn of the cochlea from an adult wild-type mouse. F, G, H, Apical (F), mid (G), and basal (H) turns of the cochlea of a Thratm1/tm1Thrbtm1/tm1 adult. The tunnel of Corti and IS have opened. An occasional but minimal loss of hair cells was evident in mid- and basal turns. The TC is present but appears somewhat misshapen. The TM is enlarged and deformed, and in basal turns is often shriveled and retracted into the IS. Scale bar: E (same in F-H), 50 µm. I-L, Cochlear basal turn from Thrbtm1/tm1 and wt control mice at P9 (I, J) and at P20 (K, L). J, In Thrbtm1/tm1 pups at P9, the formation of the IS is retarded, the TM is slightly enlarged, the underlying epithelial cell layer is thicker than in wt controls, and the tunnel of Corti (TC) is unopened. K, In wt mice at P20, the organ of Corti has matured (the TM in this example is slightly lifted above the OHCs because of shrinkage during fixation). L, In Thrbtm1/tm1 mice at P20, the IS has opened, but the TM is slightly enlarged; the epithelium lining the IS is slightly thicker than in wt controls. The Thrbtm1/tm1 phenotype is milder than in Thratm1/tm1Thrbtm1/tm1 mice. I-L represent 1 µm sections stained with toluidine blue (and thus differ from the 3 µm, thionin-stained sections in A-H). Cochleas from n  3 mice per genotype per age were examined. M, Schematic diagram of major structures of the cochlea. The hair cells reside on the basilar membrane and lie below the tectorial membrane. IHC, Inner hair cell; OHC, outer hair cell; PC, pillar cell; TC, tunnel of Corti; GER, greater epithelial ridge; LER, lesser epithelial ridge of the organ of Corti.

In adult Thra^tm1/tm1Thrb^tm1/tm1 mice, the inner sulcus had opened below the TM and the sensory epithelium was differentiated (Fig. 2E-H). These results suggested that TRs are not required for the subsequent development of the greater epithelial ridge but rather that they determine the correct timing of the developmental progression. The TM however, remained permanently malformed in adults, and it was enlarged in apical and mid-turns of the cochlea (Fig. 2F,G), whereas it was often retracted in basal turns (Fig. 2H). An occasional but minimal loss of hair cells was observed in some adult but not P8 cochleas (Fig. 2G,H). Systematic counts, however, showed that the hair cell loss was not statistically significant (Table 1). No obvious defects were observed in other regions of the cochlea.

View this table: [in this window] [in a new window]   Table 1.   Hair cell densitiesa in adult wild-type and Thratm1/tm1Thrbtm1/tm1 mice

Although TR-deficient mice lack gross malformations in the cochlea as adults (Forrest et al., 1996), these mice showed some delay in the earlier postnatal development of the organ of Corti that represents a milder form of the phenotype present in Thra^tm1/tm1Thrb^tm1/tm1 mice (Fig. 2I-L). The formation of the inner sulcus was delayed, and the tunnel of Corti remained unopened at P9 in Thrb^tm1/tm1 mice (Fig. 2J). The TM also showed some enlargement, although not to the extent occurring in Thra^tm1/tm1Thrb^tm1/tm1 mice. In wt weanlings at P20, the organ of Corti had matured (Fig. 2K), and normal ABR thresholds could be recorded (Forrest et al., 1996). In Thrb^tm1/tm1 mice at P20, however, the TM was slightly enlarged, although it did extend to the hair cells and was not grossly mis-shapen as in Thra^tm1/tm1Thrb^tm1/tm1 mice. Thus, TR has an individual role in the timely differentiation of the organ of Corti and the correct formation of the TM. However, major control of these processes is conferred by TR1 and TR acting together.

The TM also exhibited ultrastructural disarray in Thra^tm1/tm1 Thrb^tm1/tm1 mice (Fig. 3A,B). Transmission electron micrographs showed that the major collagenous fibrils were present but that the striated sheet matrix (Hasko and Richardson, 1987) was disorganized. Thus, TR and TR1 jointly exert major control over both the ultrastructure and the overall form of the TM. The loss of TR alone resulted in a subtle form of this phenotype. Although in adult Thrb^tm1/tm1 mice, the TM was not grossly malformed (Forrest et al., 1996), the organization of the striated sheet matrix was disrupted in some areas. The disorganization was limited to upper regions of the TM (Fig. 3C), whereas lower regions appeared normal (Fig. 3D). Staining with antibodies against - and -tectorins (Legan et al., 2000) and otogelin (Simmler et al., 2000) in cochlear sections from Thra^tm1/tm1 Thrb^tm1/tm1 mice yielded positive signals that confirmed the TM deformities but did not show any gross absence of these major components (Fig. 3E,F).

View larger version (164K): [in this window] [in a new window]   Figure 3.   Tectorial membrane malformation in Thratm1/tm1Thrbtm1/tm1 and Thrbtm1/tm1 mice. A, B, Transmission electron micrographs showing the ultrastructure of the matrix in the region of the TM that overlies the sensory hair cells in the organ of Corti in adult wt (A) and Thratm1/tm1Thrbtm1/tm1 (B) mice. In wt mice, the major 20 nm diameter collagen fibrils (two arrowheads) are seen embedded in a striated sheet matrix formed from alternating light and dark staining, fine diameter filaments (five small arrows). In the Thratm1/tm1Thrbtm1/tm1 mouse, collagen fibrils are present, but the striated sheet matrix is disorganized throughout the TM. C, D, Partial disarray of TM ultrastructure in Thrbtm1/tm1 mice. The TM displays a similar disarray as Thratm1/tm1 Thrbtm1/tm1 mice in the upper region (C) (i.e., those regions that are located furthest from the apical surface of the organ of Corti). The lower region (D) appears normal. Micrographs shown are from a 7-month-old wt mouse (A), a 6-month-old Thratm1/tm1Thrbtm1/tm1 mouse (B), and a 4-month-old Thrbtm1/tm1 mouse (C, D). Scale bar: C (same in A, B, D), 200 nm. E, F, Representative immunostaining for -tectorin (E) and -tectorin (F) in cochlear sections of adult wt and Thratm1/tm1 Thrbtm1/tm1 mice. In Thratm1/tm1Thrbtm1/tm1 mice, the malformation of the TM is evident, but it is still immunoreactive for the tectorins. Similar results were found for otogelin (data not shown).

Cochlear physiology in TR-deficient mice

We investigated a role for TRs in the physiological differentiation of hair cells because mouse hair cells mature during the early postnatal period when thyroid hormone is required for the development of hearing. With the onset of auditory function at approximately P13, IHCs express the fast-activating potassium current I_K,f that is associated with maturation of the IHC (Kros et al., 1998). Normally, I_K,f expression begins by P13 and plateaus after P20 (Fig. 4B, wild-type curve), whereas we have shown that I_K,f induction is retarded in Thrb^tm1/tm1 mice (Fig. 4B, Thrb^tm1/tm1 curve) (Rüsch et al., 1998). The induction of I_K,f was found to be similarly retarded in Thra^tm1/tm1Thrb^tm1/tm1 mice. Figure 4A shows examples of the voltage-activated currents of a control IHC from a Thra^tm1/tm1 mouse at P21, which activated rapidly to steady state levels within 2 msec at potentials between 43 and +1 mV, whereas the fast current was absent in a Thra^tm1/tm1 Thrb^tm1/tm1 mouse at P25. Analysis of IHCs (n = 13) from Thra^tm1/tm1Thrb^tm1/tm1 mice over a range of postnatal ages showed that I_K,f was eventually expressed and followed a logistic growth function that reached half-maximal expression at a time point (t_1/2) of 33.6 d with a slope factor s = 0.22/d (Fig. 4B, Thra^tm1/tm1Thrb^tm1/tm1 curve fit), compared with the wt curve, where t_1/2 = 17.5 d and s = 0.42/d (see Materials and Methods) (Rüsch et al., 1998). I_K,f values of IHCs (n = 15) from normal-hearing Thra^tm1/tm1 or Thra^tm1/tm1Thrb^+/tm1 control mice closely followed the previously described developmental profile of wt mice (Kros et al., 1998; Rüsch et al., 1998) (Fig. 4B).

View larger version (21K): [in this window] [in a new window]   Figure 4.   Delayed expression of the IK,f fast current in inner hair cells of Thratm1/tm1Thrbtm1/tm1 mice. A, Fast-activating currents (IK,f) in IHCs of a Thratm1/tm1 mouse at P21 and a Thratm1/tm1Thrbtm1/tm1 mouse at P25. The fast component IK,f was largely missing in the Thratm1/tm1Thrbtm1/tm1 IHC. Arrows and bars indicate the time window during which the amplitude of IK,f was measured at 25 mV. The voltage step protocol used is indicated above the traces of the Thratm1/tm1Thrbtm1/tm1 cell. B, Developmental profile of IK,f at 25 mV in IHCs of Thratm1/tm1Thrbtm1/tm1 mice. Currents were measured as indicated by the arrow and bar in A, and they were plotted versus the depolarizing membrane potential to obtain I-V plots. IK,f was then measured at 25 mV from these plots by interpolating data points negative and positive to 25 mV and plotted versus the postnatal age of the mouse. The solid line is the previously determined curve for IK,f in IHCs of wt mice and the broken (dashed) line the curve for Thrbtm1/tm1 mice as reported (Rüsch et al., 1998). The dotted line is the logistic growth function fitted to the data points plotted for IHCs of Thratm1/tm1Thrbtm1/tm1 mice (circular points), where the current maximum and minimum were fixed to the values of the wt fit [4.28 nA and 208 pA (0.208 pA)]; t1/2 = 33.6 d and slope = 0.22/d. Data points for IK,f from IHCs of normal-hearing Thratm1/tm1Thrb+/tm1 (filled circles), and Thratm1/tm1 littermates (inverted triangles) followed the normal wt developmental profile.

The similar pattern of retardation in Thra^tm1/tm1Thrb^tm1/tm1 and in Thrb^tm1/tm1 mice indicated that I_K,f induction is primarily dependent on TR. The eventual rise of I_K,f in IHCs of Thra^tm1/tm1Thrb^tm1/tm1 mice however, shows that none of the known TRs are ultimately necessary for I_K,f induction and indicates rather that TRs confer correct timing over I_K,f expression during IHC maturation. IHCs of Thra^tm1/tm1Thrb^tm1/tm1 mice at stages when I_K,f was absent did develop small currents with slow kinetics that did not reach steady state during the 15 msec time interval shown in Figure 4A. This type of current resembled the I_K,s slow current component described previously for IHCs of wt mice (Kros et al., 1998), suggesting that despite the lack of I_K,f, IHCs in Thra^tm1/tm1Thrb^tm1/tm1 mice retained other functional properties of IHCs.

We also investigated whether TRs control the acquisition of the physiological properties of the OHCs, the second cochlear hair cell type. Most prominently, OHCs display a unique electromotility that is believed to facilitate the active amplification process of the cochlea (Holley, 1996). The underlying electromechanical transduction mechanism is based on conformational changes of a voltage-sensitive membrane motor protein, recently identified as prestin (Zheng et al., 2000). We assessed electromechanical transduction by measuring the voltage-dependent capacitance prestin imposes on the OHC membrane (Santos-Sacchi, 1991; Zheng et al., 2000) (Fig. 5). OHCs from Thra^tm1/tm1Thrb^tm1/tm1 pups at P8 had a significantly reduced (p < 0.001) nonlinear capacitance (mean ± SD; 87 ± 32 fF/pF) compared with the normal values of Thra^tm1/tm1Thrb^+/tm1 littermates (379 ± 228 fF/pF) or the values described previously for wt pups at P8 (290 ± 47 fF/pF) (Rüsch et al., 1998). OHCs of Thrb^tm1/tm1 pups at P8 were previously reported to have slightly reduced nonlinear capacitance (150 ± 46 fF/pF) (Rüsch et al., 1998). Thus, TRs are important for the acquisition of OHC electromotile properties. The slight impairment in Thrb^tm1/tm1 pups suggests that TR has a specific role. However, the more marked defect in Thra^tm1/tm1Thrb^tm1/tm1 pups suggests that this function is largely coregulated by both TR1 and TR.

View larger version (17K): [in this window] [in a new window]   Figure 5.   Reduced nonlinear capacitance in outer hair cells in Thratm1/tm1 Thrbtm1/tm1 mice. A, Measurements of nonlinear capacitance in a Thratm1/tm1Thrbtm1/tm1 mouse and a control (Thratm1/tm1Thrb+/tm1) mouse. B, Plot of values of the nonlinear capacitance in five OHCs of three Thratm1/tm1Thrb+/tm1 control mice and in 10 OHCs of three Thratm1/tm1 Thrbtm1/tm1 mice at P8. The capacitance was significantly smaller in OHCs from Thratm1/tm1Thrbtm1/tm1 mice (p < 0.001).

The potassium-rich endolymph of the scala media (see Fig. 2M) is normally maintained at a high positive resting potential that is necessary for auditory function. This endocochlear potential (EP) contributes to the driving force for mechanoelectrical transduction by the hair cells (Rübsamen and Lippe, 1997). The EP was reduced in adult Thra^tm1/tm1Thrb^tm1/tm1 mice (52.3 ± 13.5 mV; p < 0.001) compared with the normal values in Thra^tm1/tm1 Thrb^+/tm1 mice (100.3 ± 9.0 mV) or the values shown previously in wt and Thrb^tm1/tm1 mice (Table 2) (Steel and Barkway, 1989; Rüsch et al., 1998). The occurrence of the low EP only in Thra^tm1/tm1Thrb^tm1/tm1 mice lacking all TRs suggests that the EP is normally coregulated by both TR1 and TR and that these receptors are functionally interchangeable in developing the ability to generate the EP.

View this table: [in this window] [in a new window]   Table 2.   Endocochlear potentials of TR-deficient mouse strains

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The phenotype of Thra^tm1/tm1Thrb^tm1/tm1 mice unmasks a role for TR1 that was not evident in mice lacking only TR1 and also indicates that TR1 and TR together control novel cochlear functions. These common TR functions include a major role in the formation of the TM and in the development of the endocochlear potential and the electromechanical transduction properties of outer hair cells (Table 3). The masking of the full extent of these phenotypes in the single receptor gene deletions suggests a functional overlap between TR1 and TR consistent with their related transactivation properties on several different DNA response elements in vitro (Jeannin et al., 1998; Wahlstrom et al., 1999). These results therefore suggest that the variety of actions provided by two related receptor genes extends the range of functions that may be controlled by thyroid hormone in cochlear development.

View this table: [in this window] [in a new window]   Table 3.   Summary of auditory system phenotypes in mice lacking TR1 (Thratm1/tm1), TR (Thrbtm1/tm1), or both TR1 and TR (Thratm1/tm1Thrbtm1/tm1)

The unique role of TR, evident in the somewhat less severe phenotype in Thrb^tm1/tm1 mice, may reflect differences in receptor expression levels in specific cochlear cell types (Bradley et al., 1994) such that inadequate levels of TR1 fail to substitute for the loss of TR. It is also possible that structural distinctions between TR1 and TR partly constrain the ability of TR1 to regulate a critical subset of TR target genes in the cochlea. TR1 and TR diverge completely in the N terminus, which plays a role in DNA binding stability and in the transactivation properties of the receptor. They also have certain differences in their central DNA binding domains which contribute to functional differences on some response elements in vitro (Lezoualc'h et al., 1992; Sjöberg and Vennström, 1995; Zhu et al., 1997). The identification of the direct, downstream target genes that mediate the physiological actions of TRs in the cochlea may allow the elucidation of the basis of this TR isotype-specificity.

The cochlear abnormalities in Thra^tm1/tm1Thrb^tm1/tm1 mice that lack all known TRs resemble the defects described in hypothyroid mice or rats, which suggests that TR1 and TR together account for the known functions of thyroid hormone in the cochlea. The induction of hypothyroidism in mice and rats during a critical, early window beginning at or before the time of birth (Deol, 1973; Uziel et al., 1981; Uziel, 1986) causes a similar retardation in the formation of the inner sulcus and deformity of the TM as is found in Thra^tm1/tm1Thrb^tm1/tm1 mice. Our findings thus argue against the hypothetical existence of any other unknown TRs or non-TR-mediated mechanism of action of thyroid hormone in cochlear development. A range of cloning and functional studies suggest that TR1 and TR represent the full complement of nuclear TRs (Gauthier et al., 1999; Göthe et al., 1999). However, the growth retardation and other phenotypes of Thra^tm1/tm1Thrb^tm1/tm1 mice are somewhat milder than the phenotypes of severe hypothyroidism, a distinction that has raised the possibility of non-TR pathways of action of thyroid hormone or of hormone-independent actions of TRs in some systems (Göthe et al., 1999). The similar cochlear phenotypes of hormone- or TR-deficient mice, however, make it unlikely that such mechanisms need be invoked in the cochlea. Although current evidence allows us to draw a conclusion regarding cochlear morphology, these comparisons cannot be extended to the physiological defects we report for Thra^tm1/tm1Thrb^tm1/tm1 mice, because cochlear physiology has been little studied in hypothyroid rodents.

The malformation of the TM in Thra^tm1/tm1Thrb^tm1/tm1 mice would impair hair cell mechanosensitive transduction and the tuning of basilar membrane motion, as indicated by other mutations in TM structural components. The complete detachment of the TM in Tecta^ENT/ENT mice with a large deletion in the entactin-like domain of -tectorin reduces the sensitivity of basilar membrane motion by 35 dB (Legan et al., 2000). Also, human -tectorin mutations cause deafness (Verhoeven et al., 1998) and deletions of type XI collagen 2 (McGuirt et al., 1999), and otogelin (Simmler et al., 2000) cause TM abnormalities and impair the ABR. The integrity of the compartments formed by the TM, the inner sulcus, and adjacent interdental cells of the spiral limbus may also be critical for auditory function and may contribute to the control of the ionic microenvironement and the potassium recycling that are necessary for hair cell function (Spicer and Schulte, 1998; Steel and Kros, 2001; Ulfendahl et al., 2001). Because the roles of the inner sulcus and its functional relationship to the TM and hair cells are incompletely understood at present, it is possible that other, as yet unknown functions are disrupted by the loss of TRs.

The TM in Thra^tm1/tm1Thrb^tm1/tm1 mice possesses collagen fibrils and is immunoreactive for tectorins and otogelin, suggesting that TRs are not required for expression of these major TM components. A more subtle role for TRs could be in the control of the correct amount and timing of expression of TM components by the greater epithelial ridge (Rau et al., 1999), which could explain the enlargement of the TM in Thra^tm1/tm1 Thrb^tm1/tm1 mice. The dysregulated secretion of the TM could also be secondary to the more general delay in the reshaping of the greater epithelial ridge during the delayed differentiation of the inner sulcus. The ultrastructural disarray of the TM in both Thrb^tm1/tm1 and Thra^tm1/tm1Thrb^tm1/tm1 mice (Fig. 3) suggests another subtle role, which is at least partly TR-specific, in the formation of the striated sheet matrix of the TM. This could involve, for example, glycosylation or other forms of processing of TM components (Richardson et al., 1987).

This study identifies roles for TRs in the physiological differentiation of both IHCs and OHCs. Immature IHCs and OHCs resemble each other morphologically (Pujol et al., 1997) and functionally (Kros, 1996) before they differentiate into mature hair cell types with distinct properties. In rodent postnatal development, OHCs have been suggested to enter a second phase of differentiation during which they acquire their unique properties including electromotility (Pujol et al., 1997). Although TRs are not required for the commitment to form either IHCs or OHCs, they are required subsequently for the proper maturation of both hair cell types. The defect in OHC nonlinear capacitance in Thra^tm1/tm1Thrb^tm1/tm1 mice is in accord with the altered distortion product otoacoustic emissions, a measure of OHC activity, reported in the hyt/hyt hypothyroid mouse strain (Li et al., 1999). It remains to be determined whether the defects in hair cell maturation are because of the absence of TRs within the hair cells or are indirect, perhaps because of abnormalities in maturation factors or in cell-cell interactions that are normally directed by other regions of the organ of Corti.

The stria vascularis has a major role in generating the endocochlear potential in the scala media of the cochlea (Fig. 2M), and defects in its function could contribute to the reduced endocochlear potential in Thra^tm1/tm1Thrb^tm1/tm1 mice. This may be consistent with the suggested regulation of NaK-ATPases in the stria vascularis by thyroid hormone (Zuo and Rarey, 1996). It is also possible that this defect originates elsewhere in the cochlea, for example in cells of the spiral limbus that may be involved in potassium recycling (Spicer and Schulte, 1998; Steel and Kros, 2001). The IHC and OHC defects together with the abnormal TM and low endocochlear potential could explain the profound abrogation of auditory function found in Thra^tm1/tm1Thrb^tm1/tm1 mice, as indicated by the severely defective ABR. This need not exclude additional roles for TRs in more central auditory pathways, as is suggested in hypothyroid rodents that show changes in innervation and myelination of the cochlear nerve (Uziel, 1986; Knipper et al., 1998), in expression of type 2 deiodinase in brainstem cochlear nuclei (Guadaño-Ferraz et al., 1999) and in pyramidal cell morphology in the auditory cortex (Ruiz-Marcos et al., 1983).

Several features of the cochlear phenotype, including the retarded development of the inner sulcus and IHC I_K,f current reflect delays rather than permanent defects. Thus, other signals or transcriptional pathways (Corey and Breakefield, 1994) must set the ultimate developmental course for these events, whereas TRs confer correct timing. Conceivably, the maturation of auditory function may require activity and sensory inflow during critical periods (Rübsamen and Lippe, 1997; Rüsch et al., 1998), perhaps resembling other sensory systems, such as vision (Katz and Shatz, 1996). As ligand-dependent transcription factors, TRs are well adapted to such a role because they can alter the cochlear transcriptional program in response to temporal signals provided by rising thyroid hormone levels in development (Campos-Barros et al., 2000). Thyroid hormone also has a timing role in amphibian metamorphosis (Huang et al., 2001), suggesting that an interplay between rising hormone levels and specific TRs in target tissues provides a timing control that can be adapted to very different processes in vertebrate development.

	FOOTNOTES

Received July 12, 2001; revised Sept. 18, 2001; accepted Oct. 3, 2001.

This work was supported in part by the German Research Council (Deutsche Forschungsgemeinschaft; A.R.), the Swedish Cancer Fund (B.V.), the Wellcome Trust (Grant 057410/Z/99/Z; R.G. and G.R.), and March of Dimes, the Deafness Research Foundation, National Institutes of Health Grant DC 03441, and a Hirschl Award (D. F.).We thank Prof. J. P. Ruppersberg and Prof. B. Fakler (Department of Physiology, University of Tübingen, Tübingen, Germany) and Prof. H. P. Zenner (Ear, Nose, and Throat Hospital, University of Tübingen, Tübingen, Germany) for providing lab space and equipment and Dr. Christine Petit for otogelin antibody.

A.R. and L.N. contributed equally to this work.

Correspondence should be addressed to Douglas Forrest, Department of Human Genetics, Mount Sinai School of Medicine, Box 1498, 1425 Madison Avenue, New York, NY 10029. E-mail: douglas.forrest{at}mssm.edu.

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