Altered Cochlear Fibrocytes in a Mouse Model of DFN3 Nonsyndromic Deafness

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Science  27 Aug 1999:
Vol. 285, Issue 5432, pp. 1408-1411
DOI: 10.1126/science.285.5432.1408


DFN3, an X chromosome-linked nonsyndromic mixed deafness, is caused by mutations in the BRN-4 gene, which encodes a POU transcription factor. Brn-4-deficient mice were created and found to exhibit profound deafness. No gross morphological changes were observed in the conductive ossicles or cochlea, although there was a dramatic reduction in endocochlear potential. Electron microscopy revealed severe ultrastructural alterations in cochlear spiral ligament fibrocytes. The findings suggest that these fibrocytes, which are mesenchymal in origin and for which a role in potassium ion homeostasis has been postulated, may play a critical role in auditory function.

Hereditary deafness affects about 1 in 2000 children and 70% of the cases occur nonsyndromically (in the absence of other associated clinical features). DFN3, an X chromosome-linked nonsyndromic deafness, is clinically characterized by a conductive hearing loss, a flow of perilymph during the opening of the stapes footplate, and progressive sensorineural deafness (1). Genetic studies have shown that DFN3 is caused by mutations in BRN-4/RHS2/POU3F4, which encodes a POU transcription factor (2). The role of Brn-4 in the development of auditory function, however, remains unclear. Mutations in BRN-3.1/BRN-3c, which encodes another POU factor, are responsible for hereditary nonsyndromic deafness, DFNA15, and targeted mutagenesis in mice has suggested that the protein plays a critical role in differentiation of hair cells in the inner ear (3). During development, Brn-4 is expressed throughout the inner ear in the mesenchyme of both the cochlear and vestibular aspects but not in tissues derived from neuroepithelial or neuronal cells (4).

To analyze Brn-4 function in vivo and to elucidate possible mechanisms underlying DFN3, we generated Brn-4-deficient mice by targeted mutagenesis. A targeting vector was constructed that enabled us to replace a 2-kb genomic sequence containing the entire coding region ofBrn-4 with a pgk-neo (neomycin-resistance gene driven by phosphoglycerate kinase gene promoter) cassette (Fig. 1A) (5). Homologous recombination was confirmed in 1 of 330 G418-resistant embryonic stem (ES) cell clones (Fig. 1B). This clone was injected into blastocysts from C57BL/6 mice and the resulting chimeras were mated to C57BL/6 mice. Germ line transmission of the mutant Brn-4 allele was confirmed by Southern blotting. Mating of heterozygous F1females with male chimeras yielded homozygous males and females. Gel mobility-shift assays (6) indicated that the homozygous mutants had no DNA binding activity attributable to Brn-4 (Fig. 1C). Mice deficient for Brn-4 appeared to be normal and were fertile.

Figure 1

Targeted inactivation of the Brn-4gene. (A) Diagram of the targeting vector and wild-type and mutant alleles. Brn-4 open reading frame is indicated by the black box; exon is indicated by the open box. neo, neomycin-resistance gene driven by phosphoglycerate kinase gene promoter (pgk-neo); DTA, diphtheria toxin A-chain gene; A, Acc I; E, Eco RI; H, Hind III; B, Bam HI; Xb, Xba I. (B) Southern blot analysis of recombinant (#9), randomly integrated (#18), and wild-type (W) ES cell clones by Xba I digestion. Map locations of probes a and b are shown in (A). The mutant allele was detected as a 4.4-kb fragment. Homologous recombinant ES clone (#9) shows only the mutant allele band because mouse Brn-4 is located on the X chromosome. (C) DNA binding activity of Brn-4 protein in brain extracts at postnatal day 0 from wild-type male (W), heterozygous female (E), and homozygous mutant female (O) mice, analyzed by gel-shift assay. An extract of Brn-4-transfected NIH 3T3 cells and an assay mix without cell extract (probe) were used as controls. Arrowhead indicates band shifted by Brn-4 binding. Arrows indicate bandshifts caused by Brn-1 (upper) and Brn-2 (lower).

To assess the auditory function of Brn-4-deficient mice, we measured the auditory brainstem response (ABR) (7). Wild-type male mice showed a typical ABR waveform and waves I to V were clearly identified above the 30-decibel (dB) sound pressure level (SPL). In contrast, Brn-4 mutant male mice showed an ABR response only with stimuli above a 90-dB SPL (Fig. 2A). At 11 weeks of age, the average ABR threshold in wild-type mice was 23-dB SPL for males and 25-dB SPL for females, whereas the average in Brn-4-deficient mice was 89-dB SPL for males and 92-dB SPL for homozygous females, which is indicative of profound deafness (Fig. 2B). The ABR consisted of multiple waves, corresponding to successive activation of nuclei transmitting neural signals from the periphery to the central region. Thus, the lack of wave I at 30- to 70-dB SPL in mutant mice suggested that the cause of deafness resided in neurons of the spiral ganglion or more peripheral regions, including the cochlea and conductive apparatus. Brn-4-deficient mice could swim normally, which indicates that they had a normal vestibular function.

Figure 2

ABRs of wild-type andBrn-4-deficient mice. (A) ABR waveforms of wild-type (+/Y) and Brn-4 mutant (−/Y) males at 11 weeks of age, measured at 20- to 50-dB SPL and 70- to 100-dB SPL, respectively. (B) Scatterplots of ABR thresholds for 11-week-old male and female mice. Horizontal bars denote averages for each group. +/Y and +/+, wild type; −/+, heterozygote; −/Y and −/−, Brn-4-deficient mice.

To determine whether the mutant mice had an impaired conductive apparatus, we examined the middle ear structure of 11-week-old males under a dissecting microscope. The structures of the tympanic membrane and an ossicular chain—consisting of malleus, incus, and stapes—appeared to be normal in mutant mice. Intact structures were confirmed by histological analysis (8). Although human DFN3 patients showed stapes fixation (1), the flexibility of auditory ossicle junctions and the mobility of stapes footplates of the mutant mice were indistinguishable from those of wild-type mice.

The absence of gross defects in the middle ear organs suggested that the primary lesion resided in the inner ear cochlear system or in the auditory nerves. Analysis of the inner ear structures of 11-week-old Brn-4-deficient male mice by light microscopy (9) revealed the normal appearance of the cochlear duct, including the organ of Corti, the spiral ganglion, and the stria vascularis (Fig. 3, A and B). The organ of Corti had a well-differentiated structure consisting of inner and outer hair cells and several types of supporting cells, including pillar cells and Deiters' cells resting on the basilar membrane (Fig. 3, D and E). The tectorial membrane was also normal in the mutant mice. Thus, although Brn-4 is highly expressed in the developing inner ear (4), we detected no gross abnormality of the inner ear in the Brn-4-deficient mice.

Figure 3

Histological analysis of the cochlear duct of Brn-4 mutants and expression of Brn-4 in the cochlear duct of mice. (A) Morphology of the basal turn of the cochlea of an 11-week-old wild-type male. (B) Basal turn of the cochlea of an 11-week-old Brn-4 mutant male. Scale bars = 100 μm. (C) In situ hybridization analysis of Brn-4 mRNA expression. Cochlear section of a wild-type female at postnatal day 0 is shown. Brn-4 mRNA expression is evident as a blue precipitate. The following structures are indicated: spiral ligament (SLg), suprastrial zone (ssz), Reissner's membrane (RM), stria vascularis (filled arrowheads), organ of Corti (open arrowheads), spiral limbus (SLm), spiral ganglion (SG). (D andE) Images at higher magnification showing the organ of Corti of a wild-type male (D) and a Brn-4 mutant male (E). IH, inner hair cells; OH, outer hair cells; TM, tectorial membrane. Scale bars = 10 μm.

We next analyzed Brn-4 mRNA expression in the inner ears of wild-type neonatal mice by in situ hybridization (10) (Fig. 3C). The highest level of expression was detected in the spiral ligament, a connective tissue structure containing three types of fibrocytes (types 1, 2, and 3) (11) as predominant cellular components. Type 1 fibrocytes occupy the region beneath the stria vascularis. Type 2 fibrocytes are found in the suprastrial zone and the central area of the spiral ligament beneath the spiral prominence. Type 3 fibrocytes form a cell layer attached to the otic capsule. Our results suggested that all three fibrocyte types expressedBrn-4. Reissner's membrane, the edge of the spiral limbus, and the region between the spiral limbus and the spiral ganglion also contained Brn-4 mRNA but in lower amounts than the spiral ligament. The organ of Corti (including the inner and outer hair cells), the stria vascularis, the spiral limbus, and the spiral ganglion were negative for Brn-4, which suggests thatBrn-4 expression is limited to cells of mesodermal origin.

Analysis of fibrocyte ultrastructure by transmission electron microscopy (TEM) (12) revealed remarkable pathological changes in the spiral ligament fibrocytes in all turns of the cochlea of 11-week-old Brn-4-deficient mice. In wild-type mice, type 2 fibrocytes in the suprastrial zone had a highly convoluted shape with numerous cytoplasmic extensions (Fig. 4, A and B) and showed an abundance of mitochondria (Fig. 4B). In mutant mice, these fibrocytes had markedly fewer cytoplasmic extensions and both the volume of cytoplasm and the number of mitochondria were dramatically reduced (Fig. 4, E and F). Type 1 fibrocytes filling the area beneath the stria vascularis also had few mitochondria (Fig. 4G) and the surrounding extracellular matrix was extremely sparse compared with wild-type mice (Fig. 4C). Further analysis of Brn-4 mutants by transmission and scanning electron microscopy revealed no pathological features in other structures of the inner ear, including inner and outer hair cells (Fig. 4, D and H), the stria vascularis, the spiral ganglia, and the auditory nerve (13).

Figure 4

Electron microscopic features of the cochlear duct of Brn-4 mutants (−/Y) (E–H) and wild-type mice (+/Y) (A–D) at 11 weeks of age. (A andE) TEM analysis of the suprastrial zone of the spiral ligament. T2, type 2 fibrocyte. Scale bars = 2 μm. (Band F) Magnification of rectangular regions in (A) and (E), respectively. Arrowheads indicate plasma membrane projections. Note the dramatic difference in cell shape and the reduced number of mitochondria (Mt) in the mutant. (C and G) TEM analysis of fibrocytes behind the stria vascularis. T1, type 1 fibrocyte; EF, extracellular filament. Scale bars = 2 μm. (D and H) Scanning electron microscopy of hair cell stereocilia, showing normal shape and arrangement in both Brn-4 mutant and wild-type mice. IH, inner hair cell; OH, outer hair cell. Scale bars = 10 μm.

According to a recent hypothesis (11), type 2 fibrocytes may take up perilymphatic K+ ions by means of their Na+,K+-adenosine triphosphatase (ATPase) activity. These K+ ions may then be transported to the stria vascularis by type 1 fibrocytes through gap junctions and ultimately may be secreted into the endolymph; this would contribute to generation of the endocochlear potential (EP), a resting potential maintained in the extracellular fluid that bathes the upper surface of mechanosensory hair cells. Because the pathological findings in the spiral ligament fibrocytes described above strongly suggested fibrocyte dysfunction in Brn-4-deficient mice, we measured EP values in the mutant mice (14). In 11-week-old wild-type mice the average value was 85 mV for males and 91 mV for females, whereas in the mutants it was only 38 mV for males and 39 mV for females (Fig. 5). This mutant phenotype suggests that Brn-4 plays a critical role in the generation or maintenance of the EP by controlling development of fibrocytes along the cochlear duct. Any abnormality of fibrocytes in the spiral ligament would be expected to disrupt K+ transport, leading to depression of the EP. A reduced EP would explain the elevation of ABR thresholds in mutants as the receptor potential of hair cells depends on the magnitude of the EP (15). The idea that fibrocytes contribute to the generation or maintenance of the EP is thus strongly supported by our present finding.

Figure 5

Decreased EP in Brn-4-deficient males and females at 11 weeks of age. −/Y, male mutant; +/Y, male wild-type control; −/−, female mutant; −/+, female heterozygote control. Horizontal bars denote averages for each group of mice.

In the past 5 years, 13 human genes have been identified that are responsible for hereditary nonsyndromic deafness (16). Mouse models harboring mutations in the homologous genes are available for Brn 3.1/Brn-3cand for the myosin VIIA (shaker 1) and myosin XV (shaker 2) genes. In all three of these models, the mice suffer sensorineural deafness because of defects in sensory hair cells of the inner ear. Our analysis of Brn-4-deficient mice has indicated that cochlear fibrocytes, which are nonsensory mesenchymal cells specific to the cochlear duct, may also play an important role in auditory function. Given the high level of Brn-4 expression in fibrocytes, the pathological changes may be a cell autonomous consequence of Brn-4 deficiency, as is the case for other members of the POU transcription factor family (17). Because the number of fibrocytes in Brn-4-deficient mice is similar to that in wild-type mice (9), Brn-4 may be essential for the differentiation or function of fibrocytes but not for their survival. The fibrocytes are rich in Na+,K+-ATPase and the gap junction protein connexin 26 (11), which are thought to be essential for K+ transport, and mutations in the GJB2 gene encoding connexin 26 have been shown to be responsible for DFNB1, another nonsyndromic deafness (18). Neither GJB2nor the Na+,K+-ATPase gene, however, appears to be a target of Brn-4-mediated regulation, because Brn-4-deficient fibrocytes showed no dramatic changes in the expression of either gene. Identification of Brn-4 target genes in cochlear fibrocytes may help to elucidate the role of these cells in auditory function.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: tnoda{at}


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