Deafness in Claudin 11-null mice reveals the critical contribution of basal cell tight junctions to stria vascularis function

Abstract

Generation of a strong electrical potential in the cochlea is uniquely mammalian and may reflect recent evolutionary advances in cellular voltage-dependent amplifiers. This endocochlear potential is hypothesized to dramatically improve hearing sensitivity, a concept that is difficult to explore experimentally, because manipulating cochlear function frequently causes rapid degenerative changes early in development. Here, we examine the deafness phenotype in adult Claudin 11-null mice, which lack the basal cell tight junctions that give rise to the intrastrial compartment and find little evidence of cochlear pathology. Potassium ion recycling is normal in these mutants, but endocochlear potentials were below 30 mV and hearing thresholds were elevated 50 dB sound pressure level across the frequency spectrum. Together, these data demonstrate the central importance of basal cell tight junctions in the stria vascularis and directly verify the two-cell hypothesis for generation of endocochlear potential. Furthermore, these data indicate that endocochlear potential is an essential component of the power source for the mammalian cochlear amplifier.

Figure 1.

Localization of TJ proteins in the inner ear of rodents. A, Claudin 11 gene expression is widespread in mouse inner ear. a, X-gal histochemistry reveals expression of lacZ regulated by the endogenous Claudin 11 promoter. 1, Semicircular canals; 2, ampulla; 3, utricle and saccule; 4, suprastrial zone; 5, lateral wall of the cochlear duct; 6, CNS myelin in the eighth cranial nerve. Inset, Strial capillaries (arrow) express Claudin 11. b, Paraffin section of wild-type cochlea labeled with anti-claudin 11 antibodies (37E3) shows expression in the stria vascularis. Inset, Antibodies label the basal cell layer (arrows) but not the marginal cell layer (arrowheads). c, Secondary antibodies do not label a section adjacent to that in b. B, Specificity of anti-claudin 11 antibodies. Antibodies used in Ab label a 24 kDa band on a Western blot of whole brain from wild-type (+/+, lane 1) but not Claudin 11-null (-/-, lane 2) mice. Secondary antibodies do not label the Western blot (+/+, lane 3). M_r, relative mobility (in kilodaltons). C, Claudin 11 expression in rat stria vascularis. The schematic shows intimate relationships between different cells in the stria vascularis. a-h, Confocal micrographs reveal labeling of strial marginal cells (a-d) and basal cells (e-h) using antibodies against TJ proteins claudins 11 and 1, occludin, and ZO-1. D, Immunoelectron micrographs of TJ proteins in basal cells. Colocalization of claudin 11 and occludin (a) ZO-1 and occludin (b) but not claudin 1 (c) at membrane “kiss” sites in basal cell TJs is shown. Cldn 11, claudin 11; Occ, occludin; Cldn 1, claudin 1.

Figure 2.

Freeze fracture showing intercellular junctions in wild-type and Claudin 11-null mice. a, Parallel intramembranous fibrils of claudin 11 TJs (white arrowheads) separate small gap junction domains (black arrowheads) in wild-type P 90 mice. b, Tight junction strands are absent in P90 Claudin 11-null mice, and gap junctions form large domains with defined borders. c, Tight and gap junctions are well developed in P30 wild-type mice and are similar to adults. d, Tight junction domains are absent in null mutant mice but gap junctions are present. Scale bar, 0.15 μm.

Figure 3.

HRP infiltration into the stria vascular is of Claudin 11-null mice. a, HRP is present in the spiral ligament but does not penetrate into the intrastrial space of wild-type cochlea. b, HRP is present in the spiral ligament and stria vascularis from null mutants. Arrowheads, Marginal cell layer; double arrowheads, basal cell layer; black arrows, exogenous HRP tracer activity in the spiral ligament; white arrows, exogenous HRP tracer activity in the stria vascularis; asterisks, endogenous HRP activity in blood vessels; i, intermediate cell nuclei.

Figure 4.

Auditory brainstem responses in Claudin 11-null mice. A, Hearing thresholds for adult wild-type, heterozygous, and null mutant mice using click, 8, 16, and 32 kHz tone pips. B, Auditory brainstem responses from P90 wild-type (+/+) and null (-/-) mice for 32 kHz stimuli at different intensities (99-10 dB SPL). The major ABR Peak I, III, and V for each animal are labeled.

Figure 5.

DPOAE, EP, and ABR latency measurements in Claudin 11-null mice. A, DPOAEs from adult wild-type (+/+; open symbols) and Claudin 11-null mice (-/-; closed symbols). The average noise floor is represented by the gray region along the abscissa. B, Endocochlear potentials from adult wild-type (+/+) and mutant (-/-) mice. C, K⁺ concentrations in endolymph and perilymph from adult wild-type (+/+) and mutant (-/-) mice. D, Latencies of ABR Peak V from adult wild-type (+/+) and mutant (-/-) mice.

Figure 6.

Normal morphologies of hair cells and spiral ganglion neurons in adult Claudin 11-null mice. A, Fluorescence microscopy from phalloidin-labeled hair cell stereocilia in organ of Corti from adult mice. a, Outer and inner hair cells from the basal cochlear turn of a P90 wild-type mouse. b, c, Outer and inner hair cells from the basal cochlear turn (b) and the apical turn (c) of a P90 Claudin 11-null mouse. Asterisks mark sites of individual OHC loss. Scale bar, 10 μm. B, Differential interference contrast microscopy showing cross sections of spiral ganglia in the midcochlear region reveal neuron cell bodies (arrowheads) from P90 wild-type (a) and Claudin 11-null (b) mice. Scale bar, 40 μm.

Figure 7.

Freeze fracture showing normal marginal cell TJs and basolateral infoldings in Claudin 11-null mice. A, Claudin 1 TJ strands (arrowheads) in marginal cells from wild-type (a, c) and null mutant (b, d) mice at P90 (a, b) and P30 (c, d). Scale bar, 100 nm. B, Basolateral infoldings in marginal cells from wild-type (a, c) and null mutant (b, d) mice at P90 (a, b) and P30 (c, d). Characteristic packing of intramembranous particles reflects the presence of Na⁺/K⁺ ATPases and other integral proteins (star). Asterisk in d marks atypical morphology that may be a sign of localized edema in the stria vascularis of an adult mutant. Scale bar, 100 nm.

Figure 8.

Freeze fracture showing normal endothelial cell TJs in Claudin 11-null mice. Intramembranous fibrils (arrowheads) in strial endothelial cell membranes from adult wild-type (a) and Claudin 11-null (b) mice are morphologically similar. Scale bar, 200 nm.

Figure 9.

Northern blotting of cochlear RNA shows temporal expression profiles of intercellular junction, ion channel, and ATPase genes. Total RNA obtained from pooled cochleas of P6.5, P14.5, P30, and P100 wild-type (+/+) and Claudin 11-null mice (-/-) probed with cDNAs indicated on the left. 28S rRNA is included as a loading control.