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Previous Article | Next Article 
Molecular and Cellular Biology, January 2005, p. 847-853, Vol. 25, No. 2
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.2.847-853.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Marc Lenoir,
Rémy Pujol,
Jean-Luc Puel,
Philippe Brabet, and
Christian P. Hamel*
INSERM, U.583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint-Eloi, Montpellier, France
Received 26 June 2004/ Returned for modification 16 September 2004/ Accepted 10 October 2004
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ABSTRACT |
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INTRODUCTION |
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Progress in the functional characterization of the cochlear fibrocytes has been made with the discovery of proteins expressed in the nonsensory regions. In accordance with their role in ionic and water transport, fibrocytes express various combinations of ion and water channels, carbonic anhydrases II and III (3, 21, 25, 27, 30), as well as gap junction connexins 26, 30, and 31 (7, 12, 37). The findings that fibrocytes also express extracellular matrix proteins (15, 33, 35) and proteins involved in cell-cell signaling, such as bone and cartilage morphogens (2, 18, 20, 31) and the inner ear-specific cochlin, whose function is unknown (19), indicate a broader role and probably a diversity of specific functions that remain to be elucidated. The fact that the alteration of the fibrocyte integrity leads to pathology is an indication of their importance in inner ear physiology. Thus, mutations in some fibrocyte-expressed genes, such as those coding for the chloride-iodide transporter pendrin (6, 23) and cochlin, are responsible for the Pendred syndrome (5) and DFNA9 (19), respectively, and alteration of fibrocytes is observed in first steps of some age-related hearing loss models (9, 36).
We previously showed that, in rats, cochlear fibrocytes express a novel, 6.4-kDa protein that we called otospiralin because of its expression in spiral structures of the cochlea, i.e., the spiral ligament and spiral limbus (4). Otospiralin expression is largely restricted to the inner ear, with only trace amounts of mRNA detected by reverse transcription-PCR (RT-PCR) in brain (24) and the presence of the protein in the inner ear detected only by Western blotting (4). Today, the function of otospiralin remains elusive, although its conservancy from fish to mammals (13) is in accordance with the presence of hair cell organs (i.e., lateral line and cochlea) and thus with vibration detection. Antisense experiments with guinea pigs showed alteration of the cochlear fibrocytes and degeneration of the hair cells, demonstrating the involvement of otospiralin in auditory function (4). These results, along with the lack of known sequence homology, suggested an important and novel role for otospiralin in inner ear biology. We now report that Otos-invalidated mice show hearing impairment. Although the organ of Corti was ultrastructurally normal, some fibrocytes of the spiral ligament and spiral limbus were damaged, suggesting that otospiralin is important for the integrity of the cochlear fibrocytes and for normal performance of the cochlea.
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MATERIALS AND METHODS |
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Embryonic development day 14.1 embryonic stem (ES) cells were transfected with the NotI-linearized plasmid DNA (25 µg) with a Bio-Rad Genepulser (230 V, 500 µF) and were subjected to Geneticin (150 mg/ml active; Life Technologies) and ganciclovir (2 µM; Roche) treatments. Homologous recombination events were identified by PCR using primers specific to the 5' mouse genomic sequence, outside the region used in the targeting vector (5'-AGCCAGGGTAAGTAACAGAGAGTTGCTG-3'), and to the LacZ sequence (5'-GGTACGGGGTCTTCTACCTTTCTCTTCT-3'). Two properly targeted ES clones out of 624 were recovered. The homologous recombination event was confirmed by direct sequencing of the fragment with internal primers. The integrity of the 3' genomic edge was checked with a 1.9-kb HindIII-SphI fragment as a probe, and that of the 5' edge was checked with a 1.3-kb NcoI-DraIII fragment of the LacZ sequence as a probe. Two clones were injected into C57BL/6N blastocysts. Chimeras were mated to C57BL/6N strain for germ line transmission, which was confirmed by PCR using LacZ (5'-GGTACGGGGTCTTCTACCTTTCTCTTCT-3') and mSG6ko5'S2 (5'-CAGAAACAGAAGCACTTATGAA-3') primers.
To assess otospiralin mRNA expression in mice, RT-PCR was performed on cochlear RNA (Rneasy kit; QIAGEN) from 5-day-old animals with exon 2 forward (5'-ATGCAGCCCTGTCTGCTGTGGTGG-3') and exon 4 reverse (5'-GTCCTCCTGGTAGGGAACATGGAA-3') primers (270-bp amplicon). The inner ear-specific otoconin-95 (Ocn-95) transcript was amplified with 5'-GTGTGACAAGGCTGCTGTGGAGTGC-3' and 5'-CTCCGTCTGGTGACTTGAGGCTTTG-3' primers as a positive control (788-bp amplicon). The absence of the protein was tested by Western blotting using the polyclonal antibody SE3950.
X-Gal staining and histological analysis. Dissected inner ears were fixed for 40 min in 4% paraformaldehyde (PFA), stained with X-Gal buffer (0.4-mg/ml 5-bromo-4-chloro-3-indolyl-D-galactoside, 2 mM potassium ferricyanide, 2 mM potassium ferrocyanide, 4 mM MgCl2 in phosphate-buffered saline [PBS]) overnight at 37°C. They were decalcified for 3 days in a 16.8% EDTA solution after the staining step. The stained inner ears were embedded in paraffin as described previously (4), sectioned (14 µm), and counterstained with eosin for histological analysis.
Immunohistological analysis. For cochleas, 21-day-old mice were decapitated under deep anesthesia (pentobarbital [Nembutal], 50 mg/kg), their cochleas were quickly removed, and they were pierced at their apex and fixed overnight in 4% PFA. They were then decalcified 4 days in 10% EDTA, dehydrated in ethanol, cleared, and embedded in paraffin. Sections (7 µm) were deparaffinized, blocked in 30% goat serum, and incubated with SE3950 antiotospiralin antiserum diluted at 1/400. Bound primary antibodies were revealed by fluorescence using fluorescein isothiocyanate-conjugated secondary antibody (1/100).
Functional assessment: surgical procedure. Otos+/+ (n = 10) and Otos–/– (n = 10) mice were anesthetized with an intraperitoneal injection of a 6% sodium pentobarbital solution (Sanofi, Montpellier, France) at 0.3 ml/kg of body weight and artificially breathed with a respirator (NBS NarcoBio-Systems). Rectal temperature was measured with a thermistor probe to control and maintain the body temperature at 38.5 ± 1°C, using a heated underblanket. Heart rate (normal range 260 to 330 per min) was monitored with an oscilloscope via electrocardiogram electrodes.
The pinna and the external meatus were resected bilaterally to the level of the tympanic annulus, and the animal was mounted in the head-holder of a stereotaxic apparatus to expose the bulla. A ventral approach was used to visualize the cochlea. Then a silver recording electrode was placed on the round window membrane of the cochlea. A reference electrode was inserted into the neck musculature.
Electrophysiology: gross cochlear and auditory nerve potentials. Cochlear potentials were elicited with tone bursts of a 1-ms rise/fall time and a 9-ms total duration generated by an arbitrary function generator (type 9100R; LeCroy Corporation, Chestnut Ridge, N.Y.). The signals were passed through a programmable attenuator and presented to the ear in a free field via a JBL 075 earphone (JBL, Northridge, Calif.). Ten frequencies were tested (2, 4, 6, 8, 10, 12, 16, 20, 26, and 32 kHz), with increasing levels of 5 dB from a 0- to 100-dB sound pressure level (SPL). The rate of presentation was 10 bursts per s. Cochlear responses were amplified (gain, 2,000) by a differential amplifier (Grass P511K), averaged (256 samples), and saved on a Pentium PC computer (100 Mhz; Dell Dimension). The stored potentials could then be digitally filtered with a low-pass setting at 2.5 kHz to measure the compound action potential (CAP) of the auditory nerve, and the summating potential (SP) reflecting the summed intracellular dc receptor potential, mainly generated by the inner hair cells (IHCs) (1). Filtering cochlear potentials with a band-pass filter centered on the frequency of tone burst stimulation allows extraction of the cochlear microphonic (CM) reflecting the summed intracellular ac receptor potential mainly generated by outer hair cells (OHCs) (17).
Statistical analysis of the data. To evaluate the significance of the functional difference between Otos+/+ and Otos–/– mice, statistical tests were performed on Sigmaplot 2000 for Windows (version 6.1). All comparisons between means were performed with Student's paired two-tail t tests. The data values are expressed as means ± standard error.
TEM. For transmission electron microscopy (TEM), following the last electrophysiological test, animals were heavily anesthetized and the cochleas were quickly removed and perfused with a freshly prepared fixative containing 3.5% glutaraldehyde in sodium cacodylate buffer (0.1 M, pH 7.2). They were rinsed in cacodylate buffer, postfixed in 2% osmic acid for 2 h, rinsed twice again before dehydration, and embedded in Spurr resin at 70°C. Blocks were trimmed to separate the different cochlear coils and remounted for transverse sections. Semithin sections were examined in Nomarski optics before cutting of thin sections (80 to 100 nm) with a Leica-Reichert ultra-microtome. Grids, counterstained with uranyl acetate and lead citrate, were observed with a transmission electron microscope (Hitachi 7100). Three series of sections were taken from each block (two blocks per coil) so as to thoroughly examine at least six different levels of the organ of Corti per cochlea.
SEM. For scanning electron microscopy (SEM), after decapitation of the animal under deep anesthesia (pentobarbital, 50 mg/kg), cochleas were removed from the temporal bone and perfused with a fixative solution of 2.5% glutaraldehyde in 0.1 M pH 7.3 phosphate buffer and then immersed in the same fixative solution for 2 h. Then, the otic capsule was dissected out. The stria vascularis and the tectorial and Reissner membranes were removed. After being rinsed in the phosphate buffer, the samples were dehydrated in a graded series of ethanol (30 to 100%), critical point dried in CO2, coated with gold palladium, and observed with a Hitachi S4000 microscope.
Antibody production. The peptide corresponding to amino acids 22 to 33 at the N terminus of the peptide signal-cleaved mouse otospiralin (NH2-KPMPEEADPHTQC-COOH) was coupled to keyhole limpet hemocyanin and injected into rabbits for antiserum production (Eurogentec, Seraing, Belgium). Antiserum SE3950 was obtained, and its specificity was checked by immunoblot analysis using the preimmune serum as a negative control.
Western blotting. Tissues were harvested in cold PBS and homogenized in sample buffer (11), and the lysate was centrifuged to remove detergent-insoluble material and separated by 16.5% polyacrylamide sodium dodecyl sulfate-polyacrylamide gel electrophoresis in Tris-Tricine (22). After gel electrophoresis, proteins were transferred electrophoretically to nitrocellulose membranes. Blots were incubated with SE3950 antiotospiralin antibody diluted at 1/1,000 according to Towbin et al. (32) and revealed by chemiluminescence using a peroxidase-conjugated secondary antibody (Boehringer Mannheim, Mannheim, Germany). A polypeptide range marker was used for calibration (Bio-Rad, Richmond, Calif.).
Animal handling. The care and use of animals followed the animal welfare guidelines of the Institut National de la Santé et de la Recherche Medicale, under the approval of the French Ministère de l'Agriculture et de la Forêt. All efforts were made to minimize the number of animals used and their suffering.
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RESULTS |
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phage containing the entire mouse Otos gene was isolated by screening a FIX II genomic DNA library derived from a 129/X1SvJ mouse strain. The restriction enzyme map was established (Fig. 1a). We constructed a vector by replacing most of the Otos coding sequence, including part of exon 2 and exons 3 and 4, with a neomycin-ß-galactosidase (ß-Gal) cassette. After electroporation into ES cells, two homologous recombinants were identified by PCR and confirmed by Southern hybridization using two probes, including one outside the vector (Fig. 1b). Both ES clones were used to generate chimeric mice by injection into mouse blastocysts. The chimeras were mated to C57BL/6N mice, and germ line transmission was confirmed by PCR analysis of tail DNA from F1 progeny. Mating of heterozygous male and female mice generated F2 progeny with the three genotypes in normal proportions (23% homozygous mutant, 51% heterozygous, and 26% wild-type mice) indicating that the absence of otospiralin is not embryonic lethal. The absence of otospiralin expression in the F2 homozygous mutant mice was confirmed by RT-PCR (Fig. 1c) and Western blotting (Fig. 1d). Otos–/– mice exhibited neither growth development nor gross anatomy differences compared with their Otos+/+ and Otos+/– counterparts.
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DISCUSSION |
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The auditory impairment observed in the Otos–/– mice is clearly different from the severe deafness seen in the guinea pig cochleas treated with otospiralin antisense oligonucleotides (4). In the latter experiment, hair cells were lost while they remained intact in the Otos–/– mice. Such a difference may rely upon long-term adaptation of the cochlea to the congenital absence of otospiralin in the Otos–/– mice or, alternatively, be linked to the fact that oligonucleotides in antisense experiments may have targeted, in addition to otospiralin, another factor important for hair cell survival. Nonetheless, damage to the mesenchymal fibrocytes of the spiral ligament and spiral limbus, which was the only histological abnormality observed in the Otos–/– mice, was strictly identical to that observed in guinea pig cochleas treated with otospiralin antisense oligonucleotides (4), indicating that this is a characteristic feature of the lack of otospiralin.
The fact that cytoplasmic alterations predominate in those fibrocytes which produce otospiralin (i.e., type II in spiral ligament and some fibrocytes of spiral limbus, as seen from the otospiralin promoter activity) suggests that the lack of otospiralin exerts a direct effect on fibrocytes themselves. There are examples of animal models in which fibrocyte degeneration is due to a molecular defect involving primarily fibrocytes with relatively few structural alterations in the neurosensory epithelium (14, 16, 38). These fibrocyte alterations may lead to severe auditory impairment, which has been attributed to important modifications of the production and composition of the endolymph. In the Slc12a2–/– mice (14), which are deficient for the Na+-K+-2Cl– cotransporter that is normally expressed in type II and IV fibrocytes and stria vascularis, there is a collapse of the Reissner membrane, indicating that the volume of the endolymph is considerably decreased, presumably because of insufficient K+ secretion. In mice deficient for the Pou transcription factor Pou3f4, which in wild-type animals is only expressed in fibrocytes of the spiral ligament and spiral limbus (16, 38), there is remarkable damage to type II and IV fibrocytes, while the organ of Corti remains normal. As in Otos–/– mice, fibrocytes show larger extracellular spaces. In addition, in the Pou3f4–/– model, scarcity of mitochondria and of membrane infoldings in fibrocytes suggests a diminished metabolism. These mice are severely deaf, as are human DFN3 patients with mutation in the BRN4 gene. Although it is not known what function of the fibrocytes is impaired, it is likely that the production of some component of the endolymph is also abnormal, since there is a dramatic reduction in endocochlear potential. In Otos–/– fibrocytes, we observed changes that more or less resemble those of both Slc12a2–/– and Pou3f4–/– mouse models, but it is clear that the moderate auditory dysfunction found in Otos–/– mice implies a more subtle pathogenic mechanism than that in play in Slc12a2–/– and Pou3f4–/– mice.
Pathological processes with moderate functional impairment are age-related auditory loss (presbyacusis). Mouse models of presbyacusis have shown IHC and OHC loss with neuronal loss and alterations of the stria vascularis and fibrocytes. In these models, there are indications that damage to the lateral wall of the cochlea (i.e., stria vascularis and fibrocytes) could be an early event preceding the loss of hair cells and therefore be a primary cause of the pathology (9, 29). In the quiet-aged gerbil model (29), many vacuoles are observed in type II fibrocytes along with an increase in their surrounding extracellular spaces, presumably a sign of edema. These features of fibrocyte alteration along with moderate auditory impairment resemble the pathology occurring in Otos–/– mice. Thus, it is reasonable to address the question of whether the alteration of fibrocytes seen in Otos–/– mice leads to more profound changes in cochlear function and ultrastructure with time, such as hair cell and neuronal loss. This awaits backcrossing of Otos–/– mice on another genetic background to avoid the age-related hearing loss that occurs in C57BL/6N and 129 Olahsd-related strains.
In conclusion, we have shown that the lack of otospiralin leads to fibrocyte alterations and hearing impairment, enhancing the importance of the nonsensory regions of the cochlea in auditory function.
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ACKNOWLEDGMENTS |
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This study was supported by grants from the Institut de la Santé et de la Recherche Médicale (INSERM). B.D. has a fellowship from Caisse d'Epargne, Montpellier, and Région Languedoc-Roussillon, France.
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FOOTNOTES |
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Present address: INSERM U.567, Institut Cochin, 75014 Paris, France.
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