Inhibition of a transcriptional repressor rescues hearing in a splicing factor–deficient mouse

(A) qRT-PCR data for utricular expression of the indicated genes in Srrm4^bv/bv and Tg(DnRest);Srrm4^bv/bv mice versus WT mice, at E16.5. Bracket indicates REST target genes. Values are mean ± SEM (n = 4–5 mice per genotype, Welch’s unpaired t test, false discovery rate–adjusted *P < 0.05, **P < 0.01). (B) RT-PCR data for alternative splicing of the indicated exons (ex.) in the utricles of E16.5 WT, Srrm4^bv/bv, and Tg(DnRest);Srrm4^bv/bv mice (n = 3 mice per genotype). The primers (arrowheads) were designed from constitutive exons (white boxes) flanking the tested alternative exons (red boxes). Exons included in specific products are depicted next to gel images; reduction in box height indicates that a stop codon is present.

Source data are available for this figure.

(A) In situ hybridization of sections of the apical turn of the cochlea from mice of the indicated genotypes (P0), with probes complementary to Srrm3 (red) and Myo6 (blue) (n = 3 mice per genotype). Upper panels show bright-field images; lower panels show fluorescence of Srrm3 probe. Inner hair cells (IHCs) are encircled by dashed lines. Scale bar, 20 μm. (B) Gene trap mutagenesis of Srrm3. Top: Schematic of the gene trap insertion site in Srrm3. Exons 3 and 4 (gray boxes), introns (horizontal lines), and gene trap (orange box) are indicated. Bottom: Results of qRT-PCR testing of Srrm3 expression in the utricle and organ of Corti of Srrm3^gt/gt versus WT mice at P1. Values are mean ± SEM (n = 3 mice per genotype, one-sample t test, theoretical mean = 0, false discovery rate–adjusted *P < 0.01). (C) F-actin staining of whole-mount preparations of middle-turn region of organs of Corti (left column) and utricles (right column) from E19 mice of the indicated genotypes (n is shown in Fig 3D and E). The rows of outer hair cells (vertical lines) and IHCs (arrowheads) are indicated. Scale bars, 20 μm. (D, E) Numbers of cells with stereocilia (hair) bundles in the IHC row of organ of Corti (D) and in the utricle (E) of E19 mice of the indicated genotypes. IHC data (D) are shown for the apical (ap), middle (m), and basal (b) turn regions of the organ of Corti. Each symbol represents value for a single mouse; blue lines indicate means. (F) qRT-PCR data for utricular expression of the indicated genes in E16.5 Srrm3^gt/gt;Srrm4^bv/bv and Tg(DnRest);Srrm3^gt/gt;Srrm4^bv/bv mice versus WT mice. Values are mean ± SEM (n = 4–5 mice per genotype, Welch’s unpaired t test, false discovery rate–adjusted *P < 0.05, **P < 0.01). (G) RT-PCR analysis of alternative splicing of the indicated exons (ex.) in the utricles of E16.5 WT, Srrm3^gt/gt;Srrm4^bv/bv, and Tg(DnRest);Srrm3^gt/gt;Srrm4^bv/bv mice. Exon composition of RT-PCR products is depicted next to gel images (n = 3 mice per genotype). Red boxes represent the tested alternative exons; white boxes represent constitutive exons complementary to the RT-PCR primers; reduction in box height indicates that a stop codon is present.

Source data are available for this figure.

(A) F-actin–stained DIV9 organ of Corti cultures derived from E19 mice of the indicated genotypes (n is shown in Fig 4B). Positions of inner hair cells (IHCs) (arrowheads) and OHCs (vertical lines) are indicated next to images. Scale bar, 20 μm. (B) Counts of IHCs (top) and OHCs (bottom) with stereocilia (hair) bundles in DIV9 organ of Corti cultures derived from E19 mice of the indicated genotypes. Each symbol represents value for a single culture (one culture per mouse); blue lines indicate means. (C) Expression levels of the indicated REST target genes as determined by qRT-PCR in OHC-containing cell clusters isolated from DIV5 organ of Corti cultures of the indicated genotypes. Values are mean ± SEM (n = 3–4 mice per genotype, one-sample t test, theoretical mean = 0, false discovery rate–adjusted ∗P < 0.05). (D) RT-PCR analysis of alternative splicing of the indicated exons in OHC-containing cell clusters isolated from DIV5 organ cultures of the indicated genotypes (n = 3 mice per genotype). Exon composition of RT-PCR products is depicted next to gel images. Red boxes represent the tested alternative exons; white boxes represent constitutive exons complementary to the RT-PCR primers; reduction in box height indicates that a stop codon is present. (E, F) F-actin–stained (green) and mCherry-immunostained (red) DIV9 organ of Corti cultures derived from E19 mice of the indicated genotypes. (E, F) The cultures were incubated with AAV-mCherry (E) or AAV-DnRest (F) on DIV0 (n = 4–5 cultures for each mouse genotype–AAV genotype combination). Positions of OHCs (vertical lines) and IHCs (arrowheads) are indicated next to images. Scale bars, 20 μm.

Source data are available for this figure.

(A) Schematic of Tg(Rest pro-EGFP). Top: Organization of DNA elements inserted into mouse Rest in a bacterial artificial chromosome to create Tg(Rest pro-EGFP). Rectangles indicate UTR in exon (ex.) 2 of Rest (gray), EGFP-encoding sequence (green), and polyadenylation signal (white). Bottom: Position of modified exon 2 (vertical lines) in Rest (arrow), within the bacterial artificial chromosome (curved gray line) that was used for the production of Tg(Rest pro-EGFP) mice. (B) EGFP (green) and MYO7A (red) immunofluorescence in cochlear sections from Tg(Rest pro-EGFP) mice at E16, E19.5, and P9 (n = 2–3 mice per time point). Inner hair cells (IHCs) (arrowheads) and OHCs (arrows) are indicated. Scale bar, 20 μm. (C) Violin plots of Tg(Rest pro-EGFP) expression in IHCs (left graph) and OHCs (right graph) at the indicated times, as determined by EGFP immunofluorescence. EGFP signal was quantified as the percentage of that in non-hair cells in the same sections. Each symbol represents value for an individual cell (n = 2–3 mice per time point). Blue lines indicate medians. (D) In situ hybridization of a cochlear section from a P0 WT mouse, with probes complementary to Rest (red) and Myo6 (blue) (n = 2 mice). Upper panel shows bright-field image; lower panel shows fluorescence of Rest probe. IHC is encircled by dashed line; OHCs are encircled by dotted lines. Scale bar, 20 μm. (E) Expression of Srrm3 and Srrm4 in OHC-containing regions from AAV-Rest-transduced versus AAV-mCherry–transduced WT organ of Corti cultures as determined by qRT-PCR. The timing of organ culture preparation (E17), AAV infection (DIV0), and isolation of OHC-containing cell clusters from the cultures (DIV5) are shown above the graph. Values are mean ± SEM (n = 4 cultures per AAV genotype, one-sample t test, theoretical mean = 0, false discovery rate–adjusted ∗P = 0.0042). (F) Schematics of identified regulatory interactions among SRRM4, REST, and SRRM3 in perinatal IHCs (left diagram), vestibular hair cells (VHCs, left diagram), and OHCs (right diagram). Arrows represent activating interactions; T-shaped lines represent inhibitory interactions. Red text and line indicate a difference in Rest regulation in OHCs versus IHCs and vestibular hair cells.