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First published online September 29, 2004
doi: 10.1242/10.1242/jcs.01393

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Compartmentalization established by claudin-11-based tight junctions in stria vascularis is required for hearing through generation of endocochlear potential

Shin-ichiro Kitajiri^1,2,3,*, Tatsuo Miyamoto^1,*, Akihito Mineharu⁴, Noriyuki Sonoda¹, Kyoko Furuse⁵, Masaki Hata⁵, Hiroyuki Sasaki^5,6, Yoshiaki Mori⁴, Takahiro Kubota⁴, Juichi Ito³, Mikio Furuse¹ and Shoichiro Tsukita^1,2,

¹ Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
² Solution Oriented Research for Science and Technology, Japan Science and Technology Corporation, Sakyo-ku, Kyoto 606-8501, Japan
³ Department of Otolaryngology Head and Neck Surgery, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
⁴ Department of Physiology II, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, 569-8686 Japan
⁵ KAN Research Institute, Kyoto Research Park, Shimogyo-ku, Kyoto 606-8317, Japan
⁶ Department of Molecular Cell Biology, Institute of DNA Medicine, The Jikei University School of Medicine, Nishi-Shinbashi, Minato-ku, Tokyo 105, Japan

View larger version (33K): [in a new window]   Fig. 7. Potassium concentration and endocochlear potential (EP) of endolymph. (A) The method for recording the potassium potential (PP) and EP. A double-barreled K+-selective microelectrode is directly inserted into the scala media of the basal turn of the cochlea of 10-week-old mice under anesthetic. (B) Recordings of PP and EP of endolymph in Cld11+/+ and Cld11-/- cochlea. Cld11+/+ and Cld11-/- cochlea showed similar PPs (70 mV), indicating that endolymph of both Cld11+/+ and Cld11-/- cochlea contained similar K+ concentrations [Cld11+/+, 148±7 mM (n=6); Cld11-/-, 145±6 mM (n=6)]. By contrast, the EP of Cld11-/- cochlea [31±14 mV (n=6)] is significantly lower than that of Cld11+/+ cochlea [95±6 mV (n=6)].

View larger version (75K): [in a new window]   Fig. 1. Stria vascularis and claudin-11 in 10-week-old wild-type mice. (A) Toluidine-blue-stained Epon semi-thin sections of the wild-type cochlea. (left) Stria vascularis (arrowheads) are located on the lateral wall of the cochlear duct. Asterisk, scala media; arrow, the organ of Corti. (See also Fig. 7A.) (right) Stria vascularis is enlarged. Yellow and pink arrowheads represent the marginal and basal cell layers, respectively. (B) Schematic drawing of the cellular architecture of stria vascularis. Three compartments are represented: compartment I (I) is the scala media filled with endolymph; compartment II (II) is the intrastrial space delineated by marginal and basal cell layers; compartment III (III) is connective tissue of the spiral ligament connected to the perilymphatic space. Green cells in compartments II and III are called `intermediate cells' and `fibrocytes', respectively. (C) Double immunofluorescence microscopy of frozen sections of stria vascularis with anti-claudin-11 pAb and anti-occludin mAb (a) or anti-ZO-1 pAb and anti-E-cadherin mAb (b). Occludin is concentrated at TJs of both marginal (yellow arrowhead) and basal (pink arrowhead) cell layers, whereas claudin-11 is detected only in TJs of basal cell layers. In frozen sections, TJs of marginal cells appear as dots, but those of basal cells are observed as discontinuous short lines. ZO-1 and E-cadherin are concentrated at cell-cell borders of both layers but do not colocalize. Bars, 50 µm (A, left), 20 µm (A, right, C).

View larger version (37K): [in a new window]   Fig. 2. Generation of claudin-11-deficient mice. (A) Restriction maps of the wild-type allele, the targeting vector and the targeted allele of the mouse Cld11 gene. The first ATG codon was located in the putative exon 1, which encoded the N-terminal portion (amino acids 1-75) of the claudin-11 molecule containing the first transmembrane domain and the first extracellular loop. The targeting vector contained a pgk neo cassette in its middle portion to delete exon 1 in the targeted allele. The position of the probe for Southern blotting is indicated as a bar. E, EcoRI; B, BglII. (B) Genotype analyses by Southern blotting of EcoRI-digested genomic DNA from wild-type (+/+), heterozygous (+/-) and homozygous (-/-) mice for the mutant Cld11 allele. Southern blotting with the probe indicated in A yielded a 5.8-kb and a 3.8-kb band from the wild type and the targeted allele, respectively. (C) Loss of Cld11 mRNA in the testis of claudin-11-defcient mice examined by reverse-transcription polymerase chain reaction. As a control, the hypoxanthinephosphoribosyl-transferase-encoding gene (HPRT) was equally amplified in all samples.

View larger version (20K): [in a new window]   Fig. 3. Deafness in Cld11-/- mice. (A) ABR to stimuli of 0-80 dB sound pressure level (16 kHz) in 10-week-old Cld11+/+ and Cld11-/- mice. (B) Hearing thresholds of 10-week-old Cld11+/+, Cld11+/- and Cld11-/- mice at various sound frequencies. Cld11+/+ and Cld11+/- mice show normal hearing thresholds (10-20 dB SPL), whereas Cld11-/- mice show abnormally increased hearing thresholds (40-60 dB SPL).

View larger version (74K): [in a new window]   Fig. 4. Stria vascularis in 10-week-old Cld11-/- mice. (A) Toluidine-blue-stained Epon semi-thin sections of the cochlea of Cld11-/- mice. (left) Stria vascularis (arrowheads) are located on the lateral wall of the cochlear duct and there are no obvious gross morphological malformations compared with the Cld11+/+ cochlea (Fig. 1A). Asterisk, scala media; arrow, the organ of Corti. (See also Fig. 7A.) (right) The stria vascularis is enlarged. Yellow and pink arrowheads represent the marginal and basal cell layers, respectively. (B) Double immunofluorescence microscopy of frozen sections of Cld11-/- stria vascularis with anti-claudin-11 pAb and anti-occludin mAb (a) or anti-ZO-1 mAb and anti-E-cadherin pAb (b). Claudin-11 completely disappears from TJs of basal cell layers (pink arrowhead) and signals of other TJ markers such as occludin and ZO-1 also appear to be decreased in basal cells. Occludin- and ZO-1-positive TJs are normally detected from marginal cell layers (yellow arrowhead). The E-cadherin staining pattern suggests that the cellular architecture is not extensively disorganized. Bars, 50 µm (A, left), 20 µm (A, right, B).

View larger version (168K): [in a new window]   Fig. 5. Freeze-fracture replica electron microscopy. As previously reported (Janke, 1975a; Janke, 1975b; Gulley and Reese, 1976), TJs of basal cell layers of Cld11+/+ stria vascularis are characterized by many parallel strands (arrows) that do not extensively anastomose (a). In a low-power replica image of Cld11-/- stria vascularis, marginal (M) and basal (B) cell layers can be identified (b). Arrows and arrowheads represent the basal cellular processes of marginal cells and cell-cell contact sites of basal cells, respectively. Asterisk, scala media. At the most apical region of the lateral membrane of marginal cells of Cld11-/- stria vascularis, TJ strands (arrows) are well developed continuously (c) but, at the cell-cell contact planes between adjacent basal cells on the same replica, no TJ strands are observed (d). Bars, 200 nm (a), 2 µm (b), 200 nm (c,d).

View larger version (59K): [in a new window]   Fig. 6. Tracer-permeability assay of the stria vascularis of 10-week-old Cld11+/+ and Cld11-/- mice. An isotonic solution containing freshly made biotinylation reagent was injected into the perilymph space from the round window of the cochlea and, after a 5 minute incubation followed by washing with PBS, the cochlea was dissected out, fixed and frozen. Frozen sections were double stained with anti-E-cadherin pAb (red) and streptavidin (green) to detect cell layers and bound biotin, respectively. In the Cld11+/+ stria vascularis, the biotinylation reagent diffused freely through the connective tissue, represented as `compartment III' in Fig. 1B, but did not get into the intrastrial space (`compartment II' in Fig. 1B). In sharp contrast to this, in the Cld11-/- cochlea, diffusion of the biotinylation reagent was not stopped at the basal cell layers but the reagent appeared to pass through them to reach the intrastrial space. Yellow and pink arrowheads represent the marginal and basal cell layers, respectively. Bar, 20 µm.

View larger version (40K): [in a new window]   Fig. 8. Two models for the mechanism behind generation of EP. The `single-cell model' hypothesizes that the Na+ conductance of the basolateral membranes of marginal cells (blue lines) generates a large positive membrane voltage, which is the source for the positive EP (90 mV; red zone). In this model, TJs in marginal cells (green) are thought to be essential for EP generation. In the `two-cell model', the K+ conductance in the inner membranes (blue lines) of basal cells and of intermediate cells, which are connected to basal cells through gap junctions (GJ), are assumed to generate the source of EP (90 mV; red zone). In this model, the involvement of marginal cells in the generation of EP was limited to the maintenance of the low K+ concentration in the intrastrial space and TJs in basal cells (green) play a crucial role.

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