Ush1c gene expression levels in the ear and eye suggest different roles for Ush1c in neurosensory organs in a new Ush1c knockout mouse

Abstract

Usher syndrome (USH) is the most common form of deaf-blindness in humans. Molecular characterization revealed that the USH gene products form a macromolecular protein network in hair cells of the inner ear and in photoreceptor cells of the retina via binding to PDZ domains in the scaffold protein harmonin encoded by the Ush1c gene in mice and humans. Although several mouse mutants for the Ush1c gene have been described, we generated a targeted null mutation Ush1c mouse model in which the first four exons of the Ush1c gene were replaced with a reporter gene. Here, we assessed the expression pattern of the reporter gene under control of Ush1c regulatory elements and characterized the phenotype of mice defective for Ush1c. These Ush1 knockout mice are deaf but do not recapitulate vision defects before 10 months of age. Our data show LacZ expression in multiple layers of the retina but in neither outer nor inner segments of the photoreceptor layers in mice bearing the knockout construct at 1-5 months of age. The fact that Ush1c expression is much higher in the ear than in the eye suggests a different role for Ush1c in ear function than in the eye and may explain why Ush1c mutant mice do not recapitulate vision defects.

Figure 1

Targeted disruption of Ush1c. The native Ush1c gene (A), the properly targeted deletion locus (A) and the targeting vector (B) are shown. The first four exons of the native gene were replaced with a β-gal/neo cassette downstream of the putative promoter region to drive β-gal reporter expression and positive selection by neomycin resistance. A herpes simplex virus thymidine kinase gene (TK) cassette was added downstream of the 3′ sequence to permit negative selection of ES cells. (C) Restriction maps of the wild-type and the properly targeted deletion locus for Southern-blot analysis of embryonic stem (ES) cells and mutant mice. (D) Southern-blot analysis was used for screening of embryonic stem (ES) cells. A probe to the 3’ end of the targeting vector yielded a 13.1 Kb fragment for the wild type allele and a 7.6 Kb fragment for the allele targeted for disruption. Likewise, a probe to the 5’ end yielded fragments of 9.3 Kb and 7.2 Kb for the wild type allele and the targeted allele respectively. Arrows indicate targeted clones on each blot. (E) The presence of the neomycin gene is detected in heterozygous KO mice (lane 1) as a 280-bp band in PCR from tail DNA. Wild-type mice (lane 2) lack the neo gene, but give a 200-bp band for the Tcrd gene. (F) Ush1c PCR yields a 512-bp band for the endogenous Ush1c gene and a 150-bp band for the KO allele from tail DNA. Lane 2, heterozygous knockout; lane 3, wild-type, lane 4, homozygous Ush1c knockout.

Figure 1

Targeted disruption of Ush1c. The native Ush1c gene (A), the properly targeted deletion locus (A) and the targeting vector (B) are shown. The first four exons of the native gene were replaced with a β-gal/neo cassette downstream of the putative promoter region to drive β-gal reporter expression and positive selection by neomycin resistance. A herpes simplex virus thymidine kinase gene (TK) cassette was added downstream of the 3′ sequence to permit negative selection of ES cells. (C) Restriction maps of the wild-type and the properly targeted deletion locus for Southern-blot analysis of embryonic stem (ES) cells and mutant mice. (D) Southern-blot analysis was used for screening of embryonic stem (ES) cells. A probe to the 3’ end of the targeting vector yielded a 13.1 Kb fragment for the wild type allele and a 7.6 Kb fragment for the allele targeted for disruption. Likewise, a probe to the 5’ end yielded fragments of 9.3 Kb and 7.2 Kb for the wild type allele and the targeted allele respectively. Arrows indicate targeted clones on each blot. (E) The presence of the neomycin gene is detected in heterozygous KO mice (lane 1) as a 280-bp band in PCR from tail DNA. Wild-type mice (lane 2) lack the neo gene, but give a 200-bp band for the Tcrd gene. (F) Ush1c PCR yields a 512-bp band for the endogenous Ush1c gene and a 150-bp band for the KO allele from tail DNA. Lane 2, heterozygous knockout; lane 3, wild-type, lane 4, homozygous Ush1c knockout.

Figure 2

RT-PCR analysis of gene expression in KO mice. (A) 2% agarose gel electrophoresis of RT-PCR products to detect the Ush1c a1 isoform (296 bp) in mice at P25. Lane M: 100-bp DNA ladders; lanes 1, 2 and 3, RT-PCR products from inner ears of Ush1c^−/−, Ush1c^+/− and Ush1c^+/+ mice, respectively; lanes 4, 5 and 6, RT-PCR products from eye tissue of Ush1c^−/−, Ush1c^+/− and Ush1c^+/+mice, respectively. Lower panel, expression level of GAPDH as the control; Upper panel, expression level of Ush1c a1. (B) Gene expression in the inner ears of the of Ush1c^−/− and Ush1c^+/− mice. The gene being assayed (by real-time RT-PCR, N=5) is indicated on the X axis and RQ is relative quantitation. (C) Gene expression in the eye tissue of Ush1c^−/− (white bars) and Ush1c^+/−(black bars) mice. The error bars in (B) and (C) refer to one standard deviation (SD). The conditions for the real time RT-PCR experiment (N=5) performed for gene expression in the eye were identical to the assay conditions in the inner ears, but the expression level of Ush1c was too high to be presented in one histogram panel. N=5 for both (B) and (C).

Figure 3

Western blot analysis with an anti-harmonin antibody to show that there is no protein production in the inner ears of Ush1c^−/− (knockout) mice. Ears from two mice of each genotype were assayed for harmonin protein. Me1: Ush1c^−/− ear 1, Ce1: Ush1c^+/− ear 1. Bands for two isoforms of harmonin were detected and quantitated against an actin control band using ImageQuant software.

Figure 4

Ush1c reporter gene expression in the inner ear. The lacZ reporter gene for Ush1c expression was visualized by histochemical reaction of its product, β-gal. Whole mount preparations of inner ears from P0 mice A show Ush1c expression is limited to the neurosensory epithelia: apical turn (AT), middle (MT), basal (BT) and hook (H) of the organ of Corti (OC) in the cochlea, the maculae of the saccule (S) and utricle (U), cristae ampullares (CA) of the semicircular canals. B Surface preparations of the dissected organ of Corti of P24 mice show expression in the three rows of outer hair cells (OHC) and one row of inner hair cells (IHC) on the apical turn of the cochlear duct. A uniform expression pattern appears along the entire length of the cochlear duct in Ush1c^−/− mice. C Enlarged views of corresponding middle turn of (−/−, heavier stained) and (+/− lighter stained). D Midmodiolar cross section through the cochlea of a P24 mouse shows β-gal expression in the inner and outer hair cells (IHC and OHC) and supporting cells (pillar cells and Deiters cells) of the basal and middle turns. Upper panel is an enlargement of the rectangle box. ST: Scala tympani, SM: Scala media, SV: Scala vestibule, SG: spiral ganglion, E–H Cross section through saccule (S) and utricle (U), cristae ampullares (CA) show only neurosensory epithelia are stained. Wildtype (+/+) is not stained (data not shown). Scale bars D, F–H are 50 µm unless otherwise labeled.

Figure 5

ABR thresholds in Ush1c^−/− and Ush1c^+/− littermate mice. Mean ABR thresholds are plotted in response to click or tone-burst stimuli at frequencies shown on the X axis at P22 (n=5) in Ush1c^−/− mice (black bars). In Ush1c^−/− mice, no ABR was detectable at any frequency up to 100 dB SPL (decibels sound pressure level) at all timepoints tested. Mean ABR thresholds at the same frequencies are shown for Ush1c^+/− mice (light bars) at P22. Standard deviations from the mean are shown by vertical error bars. The dark columns without bars indicate all mice have the same 100 value. All the STDEVs were 0 and could not be shown on the chart.

Figure 6

Scanning Electron Microscopy of organ of Corti surface preparations to examine hair cell morphology. At birth (P0), Ush1c^−/− mice (lower panels) exhibit damage predominantly in the OHCs, with kinociliar rotation deviating roughly 20° from normal. Severe damage at P21, P100, and P120 exhibits progressive degeneration of the hair cells and stereocilia of the cochlea in Ush1c^−/− mice compared to the rigid organization of the age-matched wild type controls (upper panels). Some of the stereocilia of the outer hair cell bundles are missing in the KO mice, and the inner hair cells are all severely splayed. Bar = 10 µm for all panels.

Figure 7

Retinal histology from KO and control mice shows normal phenotype and lacZ expression in eyes of Ush1c knockout. (A) Eyes from Ush1c^−/− mice (left panel, n=3) compared with Ush1c+/+ littermate controls (right panel, n=3) at 12 months of age. Each layer is indicated as follows: ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), inner segments (IS) and outer segments (OS). (B) LacZ expression in the multiple layers of retina (but not the IS nor OS layers) in the littermate mice bearing the KO construct and higher lacZ expression for −/− (n=3) than +/− (n=4) at P17, No expression of lacZ was observed in the wild-type littermate +/+ (n=2). Scale bar = 50 µm.