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© 2001 Oxford University Press

Human Molecular Genetics, 2001, Vol. 10, No. 22 2493–2500

Inner ear localization of mRNA and protein products of COCH, mutated in the sensorineural deafness and vestibular disorder, DFNA9 Nahid G. Robertson1,2, Barbara L. Resendes2,3, Jason S. Lin2, Charles Lee2,3, Jon C. Aster1,3, Joe C. Adams3,4 and Cynthia C. Morton1,2,3,* 1Department

of Pathology and 2Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women’s Hospital, Boston, MA 02115, USA, 3Harvard Medical School, Boston, MA 02115, USA and 4Department of Otology and Laryngology, Massachusetts Eye and Ear Infirmary, Boston, MA 02114, USA Received July 10, 2001; Revised and Accepted August 27, 2001

Missense mutations in the COCH gene, which is expressed preferentially at high levels in the inner ear, cause the autosomal dominant sensorineural deafness and vestibular disorder, DFNA9 (OMIM 601369). By in situ hybridization of mouse and human inner ear sections, we find high-level expression of COCH mRNA in the fibrocytes of the spiral limbus and of the spiral ligament in the cochlea, and in the fibrocytes of the connective tissue stroma underlying the sensory epithelium of the crista ampullaris of the semicircular canals. A polyclonal antibody against the human COCH protein product, cochlin, was raised against the N-terminal 135 amino acid residues of cochlin, corresponding to the Limulus factor C-homology (cochFCH) domain; this domain harbors all five known point mutations in DFNA9. On western blots of human fetal cochlear extracts, anti-cochlin reacts with a cochlin band of the predicted full-length size as well as a smaller isoform. Immunohistochemistry performed with anticochlin shows staining predominantly in the regions of the fibrocytes of the spiral limbus and of the spiral ligament in mouse and in human fetal and adult tissue sections. These sites correspond to those areas that express COCH mRNA as determined by in situ hybridization, and to the regions of the inner ear which show histological abnormalities in DFNA9. The fibrocytes expressing mRNA and protein products of COCH are the very cell types which are either absent or markedly reduced and replaced by eosinophilic acellular material in temporal bone sections of individuals affected with DFNA9. INTRODUCTION Tremendous progress in the discovery and characterization of genes for hereditary hearing loss disorders has occurred in the

past decade (1). Among the growing number of heritable deafness loci that are being identified are those for syndromic, nonsyndromic and mitochondrial disorders (2,3). Non-syndromic deafness loci are designated with the prefix ‘DFNA’ for autosomal dominant, ‘DFNB’ for autosomal recessive and ‘DFN’ for X-linked. Accompanying rapid advances in mapping these loci has been steady progress in identification of genes mutated in deafness. Studies of the roles of these genes in the process of hearing and their dysfunction in deafness can then proceed, employing scientific expertise from various disciplines. The COCH (coagulation factor C homology) gene was isolated initially by organ-specific and subtractive approaches, and found to be expressed abundantly and preferentially in the human cochlear and vestibular labyrinths (4,5). Combining genetic linkage analysis (6) and positional candidate gene strategies, three different missense mutations in COCH were found in three US families with deafness at the DFNA9 locus (7). Subsequently, 15 European kindreds (8,9) and one Australian family (10) were also shown to have different missense mutations in the same domain of COCH. Symptoms of DFNA9 include sensorineural hearing loss starting in the high frequencies, with onset in the second to fourth decades of life and progression to anacusis (11–13). A remarkable feature of this deafness disorder is the prevalence of vestibular malfunction documented both by clinical and histopathological findings. The perturbations of balance are more variable, as opposed to complete penetrance of the deafness phenotype. Other compensatory mechanisms by the visual and proprioceptive systems may attenuate the symptoms of balance dysfunction. Comprehensive vestibular testing in the European DFNA9 families (9,13) and in one of the American families (14), has demonstrated the presence of variable degrees of vestibular malfunction. A rare opportunity in the study of DFNA9 is the availability of postmortem temporal bone sections from affected individuals, which has revealed striking histopathological changes unique to this disorder (11,15,16). In this study, by using complementary techniques for localization of the abundantly expressed COCH transcript, and development and use of an antibody against its protein product, cochlin, valuable insight into the

*To whom correspondence should be addressed at: Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA. Tel: +1 617 732 7980; Fax: +1 617 738 6996; Email: [email protected]

2494 Human Molecular Genetics, 2001, Vol. 10, No. 22

Figure 1. Schematic representation of the deduced amino acid sequence of human COCH, encoding the protein cochlin, shows a predicted signal peptide (SP), followed by a region homologous to a domain in factor C of Limulus (FCH), an ivd1, and two vWFA-like domains (vWFA1 and vWFA2) separated by an intervening domain (ivd2). Five missense mutations in the FCH domain, causing the DFNA9 deafness and vestibular disorder, are indicated by arrows. The positions of all cysteine residues are shown. (A) The region of the COCH cDNA (a 660 bp BamHI–PstI fragment) corresponding to amino acid residues 266–487, spanning ∼50% of the vWFA1 and vWFA2 regions and ivd2, was used to derive riboprobes for tissue in situ hybridization. (B) The peptide sequence corresponding to this region of the COCH cDNA (amino acid residues 27–161) containing the FCH domain and ivd1 was expressed as a his-tagged fusion protein in bacteria, purified, and used as immunogen to develop a polyclonal antibody against human cochlin. Comparison of cochlin and Vit1 shows very similar structures of the two proteins, with utilization of the same domains in the same order (with a longer ivd1 domain in Vit1). All cysteine residues are conserved between the two proteins except for one cysteine in the signal peptide of cochlin which would be cleaved out in the mature protein.

role of this gene in analogous structures and functions in the cochlear and vestibular labyrinths can be gained. Correlation of these findings to the characteristic histopathological and clinical phenotypes in DFNA9 can provide further understanding of the pathogenesis of this disorder. RESULTS AND DISCUSSION Tissue expression of COCH mRNA We previously showed very high levels of COCH expression predominantly in human fetal cochlea and vestibule using northern blots with RNAs from a variety of tissues (4). Very faint message levels were also detected in human fetal cerebrum and eye. In the mouse, a wider range of expression was seen, including the spleen, cerebrum, thymus, cerebellum and medulla, and very faint levels in eye and lung (5). In view of distinctive histopathological findings from temporal bone sections of individuals with DFNA9 (11,15,16), localization of COCH expression within the cochlea and vestibule is warranted to study the role of COCH in inner ear function and pathogenesis of DFNA9. By previous in situ hybridization in the chicken inner ear (7) we detected intense hybridization of Coch mRNA in the neural and abneural cartilaginous plates (corresponding to the mammalian spiral limbus, osseous spiral lamina and spiral ligament) adjacent to the basilar papilla (sensory epithelium, homologous to the mammalian organ of Corti) and in the stroma of the crista ampullaris of the semicircular canals. However, there are marked differences between mammalian and avian inner ears in both structure and function, with

different and more numerous cell types and a more complex morphology in the mammalian system. We have performed in situ hybridization and immunohistochemistry in the human and mouse cochlear and vestibular labyrinths for further localization of COCH mRNA and to allow for better comparison with the DFNA9 histopathology. Using a riboprobe from the two von Willebrand factor type A-like (vWFA) domains of human COCH (Fig. 1A) on human fetal inner ear sections (Fig. 2A and B), strong hybridization was detected in fibrocytes located in the spiral limbus and throughout the spiral ligament (fibrocyte types I, III and IV), including the suprastriatal zone. The area of the external sulcus and root cells also showed a particularly high intensity of hybridization, presumably from the external sulcus cells, as root cells are not well developed at this stage. Because the spiral ligament constitutes a large percentage of the total mass of the membranous cochlea, the abundance of COCH mRNA in this area reflects its overall high-level expression in this organ. This localization was also confirmed by in situ hybridization on adult mouse temporal bone sections (Fig. 2C and D). In the vestibular labyrinth, high levels of COCH mRNA were found in the stromal cells underlying the sensory epithelium in the crista ampullaris of the semicircular canals (Fig. 2B). These fibrocytic cells surround the Schwann cell bodies and nerve fibers, which do not show COCH expression. Cells in the wall of the semicircular canal also showed the presence of COCH mRNA. Hybridization of cochlear and vestibular sections with a negative control sense COCH riboprobe did not show a localized signal in these structures. The cochlear and vestibular labyrinths have apparent similarities in their structure and function. For example, proteins associated with hair cells, such as the unconventional myosins,

Human Molecular Genetics, 2001, Vol. 10, No. 22 2495

Figure 2. In situ hybridization of the COCH gene. Silver grains, reflecting hybridization of 33P-labeled COCH probe, were captured with bright field microscopy and pseudocolored red. For tissue morphology, Hoescht 33258 counterstaining of nuclei was visualized by fluorescence microscopy with a DAPI filter and pseudocolored blue. (A) Cross section of a cochlear duct in human fetal cochlea (19 week developmental age) shows intense COCH hybridization in the fibrocytes of the spiral limbus (large arrow) and throughout the spiral ligament (fibrocyte types I, III and IV) (small arrows). (B) In the human fetal vestibular system (crista ampullaris of a semicircular canal), a strong COCH signal is detected in the stromal cells (fibrocytes) underlying the sensory epithelium (large arrow) and in the surrounding wall of the semicircular canal (small arrows). (C) Cross section of several cochlear ducts of the adult mouse and (D) a higher magnification of one of the cochlear ducts, showing COCH hybridization in the same areas as seen in the human cochlea: fibrocytes of the spiral limbus (large arrow) and of the spiral ligament (small arrows). Size bars are indicated in microns.

have been shown to be important in both systems. However, many of the genes expressed in the cochlea and responsible for deafness have not been studied in the vestibular system. Localization of COCH in mesodermally derived tissues in the support structures underlying and adjacent to the sensory epithelium and surrounding neural fibers of both organs may point toward other common proteins responsible for maintenance of the precise architecture and ionic balance necessary for proper mechanosensory transduction in these analogous systems. Generation of antibody against cochlin A polyclonal antibody was raised against a 135 amino acid peptide (∼14 kDa) that included the factor C-homology region of human cochlin (cochFCH domain) and the short intervening domain (ivd1) N-terminal to the first vWFA domain (Fig. 1B), fused to a bacterial trpLE sequence (14 kDa). All five residues, encoded in exons 4 and 5, found to be mutated in DFNA9 to date are included in this peptide, which also contains four conserved cysteine residues (7–9). Therefore, this domain is

Figure 3. Immunoblots using anti-cochlin total IgYs (isolated from yolks of immunized chickens). Reactivity is seen to the his-tagged cochFCH–TrpLE fusion peptide (∼28 kDa) (lane 1) expressed in bacteria, which was used to raise antibody against cochlin. To demonstrate reactivity against the cochFCH portion, cochFCH –GST ( ∼40 kDa) was tested (lane 2); it is also recognized by the anti-cochlin antibody. A smaller degradation product is also present in this lane. As a negative control, an unrelated his-tagged fusion peptide (otoraplin, ∼16 kDa) (lane 3) expressed in the same bacterial strain was tested and did not react with the anti-cochlin antibody. Immunoblot of total protein extracted from human fetal membranous cochlea (lane 4) shows two predominant bands of ∼60 kDa (corresponding to the full-length predicted coding region of COCH) and ∼40 kDa. In parallel immunoblots, preimmune IgY did not react with any of the bacterially expressed cochlin fusion peptides or protein in cochlear extracts (lanes 1– to 4–).

likely to be important for the proper structure and function of cochlin. Chicken antisera and total yolk immunoglobulin (IgY) were obtained and tested on western blots for reactivity to cochlin protein. Immune IgY recognized the bacterially expressed 28 kDa cochFCH–trpLE fusion peptide that was used as the immunogen (Fig. 3) as well as a cochlin–glutathione S-transferase (GST) fusion protein (∼40 kDa) that lacked the (his)9 or the bacterial trpLE moieties, indicating the presence of antibody specific for the cochFCH domain. The antibody did not react with a bacterially expressed control protein (otoraplin, ∼16 kDa) in lysates prepared from the same bacterial strain, indicating that extraneous antibodies were not detectable against bacterial proteins. Preimmune sera and IgYs were also used as negative controls and did not react with cochlin. Cochlin expression in the mammalian inner ear Western blot analysis was performed to assess expression of cochlin in the membranous cochlea (Fig. 3). Two predominant bands of ∼60 and 40 kDa were detected in human fetal cochlea using the anti-cochlin antibody. The larger protein band corresponds to the expected size of full-length cochlin deduced from the cDNA. Based on the findings of Ikezono et al. (17), the 40 kDa band likely represents a cochlin isoform. These investigators showed that cochlin was the most abundant protein in the bovine inner ear, where it exists as three distinct isoforms. Sequencing of these isoforms revealed that their N-terminal amino acids correspond to residues 25, 133 and 152 in full-length, unprocessed cochlin. The largest cochlin isoform starts directly after the signal peptide, while the midsized protein starts in ivd1 (29 residues upstream of the start of


the vWFA1). The smallest-sized isoform contains only 10 amino acids in ivd1, with the remainder consisting entirely of vWFA domains; this likely accounts for the failure of our antibody to recognize this species. The different isoforms of cochlin may arise from mRNA splice variants, or could result from post-translational proteolytic processing. Immunohistochemistry was performed using the anticochlin antibody on mouse, human adult and human fetal cochlear sections (Fig. 4). Immunostaining for cochlin was observed in the area of the fibrocytes of the spiral limbus and spiral ligament (mainly fibrocytes types I and III), in addition to extracellular spaces in these structures. Staining in the extracellular space at the attachment of the basilar membrane in the spiral ligament has also been seen with some other antibodies, so the specificity of staining at this site is interpreted with caution. The predominant immunostaining pattern obtained with the anti-cochlin antibody in the spiral limbus and spiral ligament corresponds to COCH mRNA expression detected by in situ hybridization.

Areas of the cochlea which express cochlin also contain a network of gap junctions (18) thought to play an important role in fluid homeostasis by recycling K + ions through the spiral limbus and spiral ligament, back into the endolymph of scala media, which bathes the hair cells. Loss of normal fibrocytes and accumulation of the homogeneous acidophilic material in the spiral ligament and spiral limbus caused by COCH mutations could interfere with adequate intercellular ion flow through these gap junctions and disrupt the integrity of this system critical for proper cochlear function. The late-onset and progressive nature of DFNA9 as a result of COCH missense mutations may suggest gain of a novel deleterious property of cochlin, affecting the structure and function of this protein, or its interaction with other extracellular components of the inner ear. This in turn could lead to accumulation of the acidophilic deposits and cell death, disrupting the integrity of the support structures, neural elements and ionic balance necessary for normal functioning of the cochlear and vestibular organs.

Correlation to DFNA9 histopathologic findings

Different domains of cochlin

We have used two complementary approaches to localize expression of COCH in the human and mouse cochlear and vestibular organs to allow for a close comparison to inner ear histopathology of individuals with COCH mutations causing deafness and vestibular dysfunction at the DFNA9 locus. A striking feature of the DFNA9 disease phenotype is the presence of abundant deposits of a homogeneous acidophilic ground substance that almost completely fills the lateral wall of the cochlear duct in the area of the spiral ligament, the spiral limbus and osseous spiral lamina, as well as in the stroma of the crista ampullaris and the macula in the vestibular system (11,15,16). These areas closely parallel the sites of the distribution of mRNA and protein products of COCH in the cochlea and vestibule. DFNA9 histopathological findings also include marked loss of cellularity in both organs, including loss of the fibrocytes in the spiral limbus, the spiral ligament and the stromal cells of the crista and macula; these are the very cell types that express COCH abundantly in the normal human fetal and mouse inner ears. Neuroepithelial degeneration of the organ of Corti, crista and macula, and the dendrites and ganglia of the cochlear and vestibular nerves are also seen in some DFNA9-affected inner ear sections (11,15,16). The sequence of events leading to deafness and balance dysfunction in DFNA9, the exact composition of the acidophilic material and the relationship to cellular and neuronal atrophy remain to be elucidated. Previous staining of sections has shown these deposits to contain a mucopolysaccharidetype substance (11). Recent electron microscopic examination of these deposits shows dense, highly branched and disarrayed, non-parallel microfibrils with a granular substance (possibly glycosaminoglycans) scattered among the fibrils (14). There is an apparent degradation and loss of the normal fibrillar type II collagen in the same areas. The spatial correlation of cochlin expression and the abnormal acidophilic deposits in DFNA9 suggests either a direct effect of the mutated protein in these locations, or possible altered binding of cochlin with other proteins such as the fibrillar collagens.

The deduced amino acid sequence of COCH (Fig. 1) shows a mosaic molecule consisting of a secretion signal peptide followed by two different types of domains, which are also found in combination with other motifs in proteins with diverse functions. It is thought that modules may serve similar functions in different contexts, but they have been shuffled and rearranged in the course of evolution, and in juxtaposition with a unique set of other domains may create novel associations. vWFA-like domains are found in a large number of proteins, the majority of which are components of the extracellular matrix (such as matrilins and non-fibrillar collagens VI, VII, XII and XIV). However, they also occur in proteins involved in hemostasis, cell adhesion and the complement and immune systems (19–21). A common function of the vWFA domain seems to be the capacity to bind proteins such as the fibrillar collagens, glycoprotein GpIb and complement C3b. A conserved motif within vWFA domains for protein binding has been identified and designated as a metal ion-dependent adhesion site (MIDAS motif) (22). The two vWFA modules in cochlin are likely to interact with other connective tissue elements of the cochlea and vestibule, such as type II collagen fibers that are expressed in the same areas of the inner ear as cochlin. All mutations found to date in cochlin, responsible for the pathology manifested in DFNA9, occur in the N-terminal region, homologous to a domain in factor C of the ancient invertebrate, Limulus (horseshoe crab). Factor C is a serine protease consisting of a number of different domains of known function, which are also conserved in other mammalian proteins. In response to binding lipopolysaccharide endotoxin on gram-negative bacteria cell surface, factor C initiates a coagulation cascade for host defense (23,24). The function of the factor C domain homologous to that in COCH is not known, but it is contained within a region of factor C shown to bind lipopolysaccharides (25). Alterations in this cysteinecontaining region of cochlin may alter its overall structure and solubility or disrupt the function of this or the downstream vWFA domains.


Figure 4. Immunohistochemistry using anti-cochlin antibody on (A) 6-week mouse cochlea, (B) human adult cochlea, (C) 18-week and (D) 22-week developmental age human fetal cochlea. In all panels, cross sections of cochlear ducts are shown. Immunostaining is seen as a reddish brown DAB reaction product. In (A) and (B), no tissue counterstain was applied; in (C) and (D), tissue was counterstained with H&E. (A) In the basal turn of the mouse cochlea, type I fibrocytes (I) of the spiral ligament, the spiral limbus (vertical arrow) and the petrous bone adjacent to Rosenthal’s canal (open arrowhead) are stained. In the second turn, most of the staining is in the spiral limbus (vertical arrow) and the extracellular matrix adjacent to the insertion of the basilar membrane (horizontal arrow). (B) In the basal turn of a human adult cochlea, type I and type III fibrocytes within the spiral ligament are immunostained, as is the extracellular matrix of the limbus (vertical arrow) and the spiral ligament at the attachment of the basilar membrane (horizontal arrow), similar to that seen in the second turn of the mouse. In the human fetal cochlear sections (C and D), immunostaining for cochlin is seen in the fibrocytes and in the extracellular matrix of the spiral limbus (vertical arrow) underlying the interdental cells, and in the spiral ligament (horizontal arrow) in the area of the collagen fibrils. The staining in the 18-week human fetal cochlea (C) is weak in the area of the spiral ligament, but by 22 weeks (D), staining in this region is more prominent. Size bars are indicated in microns.

The ‘factor C-homologous’ (FCH) motif appears to be quite rare among known proteins. An extensive database homology search (26) has revealed the presence of this domain in three other mammalian proteins: Lgl1 (late gestation lung protein) (27), a partial cDNA related to a CUB domain (an extracellular

module in functionally diverse proteins) (28) and a predicted protein from a BAC on human chromosome 2 (GenBank accession no. AC007363). The FCH domain was referred to as the LCCL module (26) for its presence in Limulus factor C, COCH and Lgl1. Lgl1, which contains two tandem FCH/LCCL


motifs, is a developmentally regulated gene expressed in lung mesenchyme and involved in lung maturation, possibly through regulation of extracellular matrix degradation (27). The specific function of the FCH/LCCL motif in these newly identified proteins is not known. Multiple alignment comparison and secondary structure prediction of the FCH/LCCL domains (26) suggest the presence of six β-strands and two α-helices in this module, with positions of all five of the COCH mutations occurring at or near critical conserved residues predicted to be necessary for proper folding. An analysis of circular dichroism spectra of a bacterially expressed recombinant FCH/LCCL module of human COCH has been performed (26), predicting 54% β-sheet and 9% α-helix content of this domain. New family of ‘cochlin-related’ proteins The predicted peptide from the human chromosome 2 BAC containing a factor C-homologous domain (26) appears to be the human homolog of a recently isolated protein, designated Vit1, from the vitreous of the bovine eye (29). Human and mouse VIT1 cDNAs were cloned using degenerate primers to the bovine peptide sequence (29). The protein sequence of Vit1 (Fig. 1) reveals a modular structure very similar to that of cochlin: a secretion signal peptide followed by a FCH domain, an intervening domain (ivd1) and two tandem vWFA-like modules, also separated by a short intervening domain (ivd2). The only apparent structural difference between the two proteins is the length of the ivd1 (∼125 amino acids in Vit1, ∼35 amino acids in cochlin). The longer ivd1 in Vit1 may represent an additional domain, but it does not contain any cysteine residues. All cysteines, including the ones in ivd2, are conserved between Vit1 and cochlin. Furthermore, COCH and VIT1 possess the same exon/intron boundaries of their vWFA domains, which differ from some boundaries of other vWFA-containing genes (29). COCH and VIT1 are the only genes reported to date with evolutionary utilization of the same combination of modules in the same order, indicating that they may be closely related members of the same family of proteins with similar functions. The vitreous gel of the eye is a network of connective tissue elements, including thin collagen fibrils (predominantly type II, some types IX, V/XI), hyaluronan and Vit1, speculated to have a bridging role between these components for stabilization of the vitreous gel (29). Cochlin and Vit1 are similar in their colocalization with collagen fibrils, suggesting possible interactions with these proteins. The occurrence of these two closely related proteins in the inner ear and the eye may be a reflection of their common role in the matrices of these two sensory organs and parallel studies of these proteins may provide valuable insight into their roles in the respective organs. MATERIALS AND METHODS Tissue in situ hybridization Riboprobe construct. A 660 bp BamHI–PstI fragment from human COCH cDNA (GenBank accession no. AF006740) spanning nucleotides 850–1515 (corresponding to amino acid residues 266–487), was subcloned into the Bluescript

KS plasmid vector (Stratagene, La Jolla, CA). This region (Fig. 1A) contains ∼50% of the first vWFA-like domain (vWFA1), 50% of the second vWFA domain (vWFA2) and the intervening domain (ivd2). The plasmid was linearized with BamHI or EcoRI for generating antisense and sense riboprobes with T3 or T7 RNA polymerase (Promega, Madison, WI) and radiolabeled with 33P-UTP (New England Nuclear, Boston, MA). Tissues. Human inner ear tissues were obtained in accordance with guidelines established by the Human Research Committees at Brigham and Women’s Hospital and the Massachusetts Eye and Ear Infirmary. Adult mouse temporal bones were obtained following approval by the Harvard Medical School Standing Committee on Animals. Tissues were fixed in 4% paraformaldehyde, decalcified at the same time in 1% acetic acid or after fixation in 0.1 M EDTA, and then embedded in paraffin by standard histologic procedures. Some mouse ears were fixed in a mixture of 10% formalin and 1% acetic acid. In situ hybridization. Tissue sections were deparaffinized, fixed in 4% paraformaldehyde in PBS, and treated with proteinase K. After washing in 0.5× SSC, sections were covered with hybridization solution [50% deionized formamide, 0.3 M NaCl, 20 mM Tris (pH 8.0), 5 mM EDTA, 1× Denhardt’s solution, 10% dextran sulfate and 10 mM dithiothreitol] and prehybridized for 2 h at 55°C. 33P-labeled antisense and sense RNA probes (3 × 106 cpm/slide) were then added to the hybridization solution and incubated for 12–18 h at 55°C. After hybridization, sections were washed for 20 min in 2× SSC, 10 mM β-mercaptoethanol and 1 mM EDTA, treated with RNase A (20 µg/ml) for 30 min at room temperature, and washed at high stringency (0.1× SSC, 10 mM β-mercaptoethanol and 1 mM EDTA) for 2 h at 60°C. Sections were dehydrated, dipped in photographic emulsion NTB-3 (Kodak, Rochester, NY) and stored at 4°C. After 14 days of exposure, sections were developed and counterstained with 2 µg/ml Hoechst 33258 (Sigma, St. Louis, MO) and slides were mounted with 50% Canada balsam in methyl salicylate (Sigma). Generation of antibody against cochlin The portion of the human COCH cDNA encoding the Limulus factor C-homology domain (cochFCH domain) mutated in DFNA9, and a short intervening domain (ivd1) N-terminal to the first vWFA-like domain (Fig. 1B), was subcloned into the pMM vector (30). This construct drives the expression of cochFCH (134 amino acid residues, ∼14 kDa) fused to the trpLE peptide and an N-terminal (his)9 tag (31). As a result of the hydrophobic trpLE domain (∼14 kDa), the his-tagged cochFCH–trpLE fusion peptide (28 kDa) localizes to inclusion bodies (30). BL21(DE3)pLysS E.coli (Invitrogen, Carlsbad, CA) transformed with the cochFCH fusion peptide construct were grown to an OD 600 of 0.8 and then induced with 1 mM IPTG for 3 h at 37°C. Inclusion bodies were isolated and solubilized from the cell pellets in 8 M urea and purified over a column of Ni2+-NTA agarose (Qiagen, Valencia, CA) to isolate the cochFCH fusion peptide. This peptide was then used as an immunogen in chickens (Cocalico Biologicals, Reamstown, PA). Both polyclonal antisera and total immunoglobulins (IgYs) from egg yolks were collected and used in subsequent experiments.


The cochFCH cDNA was also subcloned into pGEX-2TK (Amersham Pharmacia, Piscataway, NJ), which permits expression of a GST (26 kDa)–cochFCH fusion peptide (∼40 kDa total). CochFCH–GST expressed in BL21(DE3)pLysS cells was used as a positive control in immunoblotting with anti-cochlin antibody. For a negative control, a construct from an unrelated protein, otorpalin, was cloned into the (his)6-tagged vector, pRSET (Invitrogen), and expressed in the same bacterial strain for use in immunoblots. Immunoblot Human fetal cochlear tissues were homogenized in a solution of 7 M urea, 2 M thiourea, 2% Triton X-100, 100 mM dithiothreitol and antiproteases, and incubated at 37°C for 1 h. The total cochlear protein lysate, the cochFCH–trpLE fusion peptide and total protein cell lysates from bacteria expressing the cochFCH–GST fusion peptide or the negative control his-tagged peptide were solubilized in 1× sample buffer, boiled for 5 min, separated by 10–15% SDS–PAGE and transferred (32) onto nitrocellulose or Immobilon-P (Millipore, Bedford, MA) membranes using either a wet transfer apparatus (Idea Scientific, Minneapolis, MN) or the Trans-Blot SD semi-dry transfer apparatus (Bio-Rad, Hercules, CA) following each manufacturer’s protocol. Membranes were incubated in blocking buffer (5% Carnation non-fat dry milk, 0.075% Tween 20 in PBS) for 1 h at room temperature, rinsed 4× for 10 min in PBS and 0.075% Tween (PBS-T), incubated for 1 h with preimmune serum or IgYs as negative control or with anti-cochlin polyclonal antisera or total IgYs. Membranes were rinsed 4× for 10 min in PBS-T, incubated with the secondary antibody, horseradish peroxidase (HRP)-conjugated rabbit anti-chicken IgG (Sigma), rinsed 4× for 10 min in PBS-T and incubated for 1–5 min in an equal volume mixture of Super Signal Chemiluminescent Substrate Stable Peroxidase and Luminol Enhancer (Pierce, Rockford, IL) before autoradiography. Immunohistochemistry Human fetal cochlear tissues were prepared in the same manner as for in situ hybridization and embedded in paraffin. Immunostaining was performed by use of a microwave antigen-retrieval protocol as previously described (33). Sections were incubated with anti-cochlin polyclonal antibody (either antisera or total IgYs) or with preimmune serum or IgYs as negative control, for 1 h at room temperature, or over ight at 4°C, washed, and incubated with a secondary biotinylated anti-chicken IgG (Vector Labs, Burlingame, CA). Immunostaining was visualized by incubation with the Vectastain ABC reagent (Vector Labs) followed by 3,3′diaminobenzidine (DAB). Sections were counterstained with hematoxylin and eosin. Adult human and mouse tissues were processed without antigen retrieval procedures, but with a biotinylated tyramine signal amplification technique, as previously described (34). ACKNOWLEDGEMENTS We would like to thank Dr Massimo Loda and Carmen Tam for technical assistance with in situ hybridization provided

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