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Hearing loss caused by progressive degeneration of cochlear hair cells in mice deficient for the Barhl1 homeobox gene

¹ Center for Advanced Biotechnology and Medicine and Department of Pediatrics, UMDNJ-Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA
² Department of Otolaryngology – Head and Neck Surgery, and Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

View larger version (66K): [in a new window]   Fig. 1. Expression of Barhl1 in the developing inner ear and targeted disruption of the Barhl1 locus. (A-C) RNA in situ hybridization analysis of coronal ear sections shows signals in the utricular maculae, cristae of semicircular canals and cochlear hair cells (arrows). (D,E) ß-galactosidase staining (counterstained with Fast Red) of Barhl1+/– inner ear sections labels cochlear outer and inner hair cells, and saccular hair cells. (F,G) ß-galactosidase staining of Barhl1+/– whole-mount organs of Corti and cristae at P5: note the much more intense signals in cochlear outer hair cells compared with inner hair cells and hair cells of the crista. (H) Schematic of the inner ear, indicating the expression levels of Barhl1 in the cochlea and vestibule. (I) Homologous recombination between the wild-type allele and the targeting vector results in the replacement of Barhl1-coding exons (black boxes) with the lacZ marker DNA and a PGK-Neo cassette flanked by two loxp sites (hatched boxes). E, EcoRI; H, HindIII; K, KpnI; S, SalI; ScI, SacI; ScII, SacII; Xb, XbaI; Xh, XhoI. (J) Southern blot analysis of SacI-digested DNA from wild-type, heterozygous and homozygous mutant mice. The 3' probe identifies SacI fragments of 7.8 kb (wild-type allele) and 4.5 kb (targeted allele). (K) PCR analysis of DNA from wild-type, heterozygous and homozygous mutant mice. The wild-type and targeted alleles yield a product of 350 bp and 730 bp (derived from lacZ), respectively. AVC, anterior vertical canal; Co, cochlea; Cr, crista; HC, horizontal canal; IHC, inner hair cell; OHC, outer hair cell; PVC, posterior vertical canal; S, saccule; U, utricule. Scale bars: 50 µm.

View larger version (20K): [in a new window]   Fig. 2. Anomalous auditory brainstem responses (ABRs) in Barhl1-null mice. (A) Representative ABR evoked by 50 dB SPL click stimuli is illustrated for Barhl1+/+ (black line) and Barhl1–/– (red line) mice. The sensitivity, latency and waveform of the ABR response were essentially identical for Barhl1+/+ and Barhl1+/– mice. The auditory stimulus was presented at 0 mseconds. All Barhl1–/– mice either had elevated thresholds or were unresponsive to sound. (B) Mean (±s.d.) click ABR thresholds are shown for Barhl1+/+, Barhl1+/– and responsive Barhl1–/– mice at different ages. (C) Mean (±s.d.) ABR thresholds at different frequencies are shown for Barhl1+/+, Barhl1+/– and responsive Barhl1–/– mice at 3 months of age. Because Barhl1–/– mice did not always respond to even the loudest of the auditory stimuli presented (>70 dB at 4 kHz and >95 dB SPL for all other stimuli; Table 1), thresholds were calculated only for mice with ABR responses (clicks: n=10 out of 10 at one month, seven out 10 at 3 months, and three out of 10 at 10 months; 4 kHz: n=2 out of 10; 8 kHz: n=7 out of 10; 16 kHz: n=5 out of 10; and 32 kHz: n=5 out of 10). Every Barhl1+/+ and Barhl1+/– mouse exhibited responses to acoustic stimulation.

View larger version (101K): [in a new window]   Fig. 3. Generation and progressive degeneration of hair cells in the organ of Corti of Barhl1-null mice. (A,B) RNA in situ hybridization analysis of coronal ear sections shows similar levels of Math1 expression in cochlear hair cells (arrows) between Barhl1+/+ and Barhl1–/– mice at P0. (C,D) Similar levels of myosin VIIa immunoreactivity are seen in cochlear hair cells between Barhl1+/– and Barhl1–/– mice at P6. (E-P) ß-galactosidase staining of Barhl1+/– and Barhl1–/– whole-mount organs of Corti: note the disorganization of outer hair cells (OHC) in the apex of the mutant at P6 (F), followed by the loss of many of these cells from the apical and middle turns by P19 (G,K) and the degeneration of the majority of these cells by P59 (H,L). The outer hair cells in the basal turn, however, show no obvious degeneration by P59 in the mutant (M-P). Similarly, the inner hair cells (IHC) remain normal up to P59 (E-P). Scale bar in P: 100 µm in A,B; 25 µm in C,D; 18.75 µm in E-P.

View larger version (118K): [in a new window]   Fig. 4. Progressive loss of outer hair cells in the organ of Corti of Barhl1-null mice. (A-L) Labeling of Barhl1+/– and Barhl1–/– whole-mount organs of Corti with rhodamine-conjugated phalloidin. Compared to the heterozygote (A-C), many outer hair cells degenerate in the apical and middle turns by P19 (E-G) and only a few residual outer hair cells (arrows) remain by P63 (I-K) in the mutant. By contrast, the basal turn of the mutant is essentially normal except for occasional missing outer hair cells (arrowheads) by P63 (D,H,L). Background staining in mutants presumably results from degenerating and degenerated supporting cells (E-G,I-K). Scale bars: 18.75 µm.

View larger version (101K): [in a new window]   Fig. 5. Cytoarchitectural abnormalities in the organ of Corti of Barhl1-null mice. (A-D) Scanning electron micrographs of the organs of Corti from 3.5-month-old Barhl1+/+ and Barhl1–/– mice. In the apical and middle turns, in contrast to the three rows of organized outer hair cells in the wild type (A), only a few residual outer hair cells remain with disorganized or V-shaped stereocilia in the mutant (B,C, arrows). The outer hair cells in the basal turn and all inner hair cells in the mutant organ of Corti appear to be normal (A-D). (E-J) Light micrographs of semi-thin transverse sections of inner ear sensory epithelia in P66 Barhl1+/– and Barhl1–/– mice. Although the cellular structure of the mutant organ of Corti at the basal turn is similar to the control (E,H), most of the outer hair cells in the apical and middle turns are missing and many Deiters’ cells become lost or irregular in morphology (F,G). The maculae of the utricle are similar between the mutant and control (I,J). DC, Deiters’ cell; IHC, inner hair cell; IP, inner pillar cell; OHC, outer hair cell; OP, outer pillar cell. Scale bars: 10 µm in A-D; 25 µm in E-J.

View larger version (153K): [in a new window]   Fig. 6. Region-specific loss of inner hair cells in the organ of Corti of 10-month-old Barhl1-null mice. Whole-mount organs of Corti from Barhl1+/– (A,C,E) and Barhl1–/– (B,D,F) mice were labeled with rhodamine-conjugated phalloidin. Compared with the heterozygote, no inner hair cells are present within the basal turn of the null mutant (E,F), many of them are missing from the middle turn (C,D), but the large majority of them still remain in the apical turn (A,B). The outer hair cells, however, appear to be entirely missing from the mutant organ of Corti (B,D,F). The arrow points to cell debris presumably resulting from degenerated supporting cells (F). AT, apical turn; BT, basal turn; IHC, inner hair cell; MT, middle turn; OHC, outer hair cell. Scale bar: 18.75 µm.

View larger version (34K): [in a new window]   Fig. 7. Progressive loss of cochlear hair cells along the longitudinal axis of the Barhl1–/– organ of Corti. In the null mice, degeneration of cochlear hair cells follows two reciprocal longitudinal gradients – apical-to-basal for outer hair cells but basal-to-apical for inner hair cells. AVC, anterior vertical canal; Co, cochlea; HC, horizontal canal; IHC, inner hair cell; OHC, outer hair cell; PVC, posterior vertical canal; S, saccule; U, utricule.

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