Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia

Abstract

WHRN (DFNB31) mutations cause diverse hearing disorders: profound deafness (DFNB31) or variable hearing loss in Usher syndrome type II. The known role of WHRN in stereocilia elongation does not explain these different pathophysiologies. Using spontaneous and targeted Whrn mutants, we show that the major long (WHRN-L) and short (WHRN-S) isoforms of WHRN have distinct localizations within stereocilia and also across hair cell types. Lack of both isoforms causes abnormally short stereocilia and profound deafness and vestibular dysfunction. WHRN-S expression, however, is sufficient to maintain stereocilia bundle morphology and function in a subset of hair cells, resulting in some auditory response and no overt vestibular dysfunction. WHRN-S interacts with EPS8, and both are required at stereocilia tips for normal length regulation. WHRN-L localizes midway along the shorter stereocilia, at the level of inter-stereociliary links. We propose that differential isoform expression underlies the variable auditory and vestibular phenotypes associated with WHRN mutations.

Graphical abstract

Figure 1

Whrn Isoform Expression (A) Diagram of Whrn exons showing deletions in Whrn^wi/wi (orange) and Whrn^tm1b/tm1b (blue), with regions coding for WHRN domains colored (green, PDZ1; blue, PDZ2; red, PDZ3; purple, proline-rich domain). Vertical arrows mark target region of antibodies PB584 and PB595. Horizontal arrows mark locations of primers. (B) Ensembl predictions of Whrn isoforms. Narrow boxes indicate UTRs and wider boxes protein-coding regions. Left: isoform names used in this paper. Right: Ensembl transcript IDs. ENSMUST00000155058 is classified as a retained intron transcript but was recently found in the inner ear (Mathur et al., 2015b) and results in a protein sequence identical to transcript ENSMUST00000119294, one of the WHRN-S isoforms, when translated. (GenBank accession numbers provided in Table S1). Primers used to detect each isoform are shown on the relevant isoform. (C–E) Transcripts detected in wild-type (C), Whrn^tm1b/tm1b, (D) and Whrn^wi/wi, and (E) inner ears. Black lines indicate splice junctions observed by sequencing. Grey lines indicate splice junctions inferred by presence of the isoform, but not observed by sequencing. See also Figure S1.

Figure 2

Auditory Electrophysiology and Whrn Expression in Whrn^wi/wi and Whrn^tm1b/tm1b Mice (A) Mean CAP threshold (±SD) in P20 Whrn^wi mice. (B) Mean ABR threshold (±SD) in P20 Whrn^wi mice. (C) Mean ABR threshold (±SD) in P98 Whrn^wi mice. (D) Mean ABR threshold (±SD) in P98 Whrn^tm1b mice. (E) Mean ABR masked tuning curves (±SD) in P98 Whrn^tm1b mice. Open symbols indicate mean (±SD) 12 kHz probe tone threshold. Filled symbols indicate mean (±SD) masked tuning thresholds. (F) Mean 2F1-F2 DPOAE threshold (±SD) in P98 Whrn^tm1b mice. Red arrows on symbols indicate maximum sound pressure level tested. (G) Waveforms from Whrn^+/wi mouse (P17) showing range of responses. CAP and SP responses are indicated. SP can be either negative (e.g., 12 kHz at 70 dB SPL) or positive (high frequencies, high intensities). (H) Waveforms from a Whrn^wi/wi mutant (P17), showing positive and negative SPs but no CAP. (I) Cochlear microphonic amplitudes plotted as a function of stimulus intensity for a 6 kHz tone. (J) LacZ staining in organ of Corti from Whrn^tm1b/tm1b mice at P5 (left: whole cochlea, right top: magnified) and P28 (bottom right). See also Figure S2.

Figure 3

Differential Whrn Isoform Expression Affects Stereocilia Bundle Morphology (A and B) Scanning electron microscopy (SEM) images of OHCs (A) and IHCs (B) from P21 Whrn^+/tm1b mice. Scale bars, 2 μm. (C and D) SEM images of OHCs (C) and IHCs (D) from P21 Whrn^tm1b/tm1b mice. Scale bars, 2 μm. (E and F) SEM images of OHCs (E) and IHCs (F) from P21Whrn^wi/wi mice. Scale bars, 2 μm. (G–I) Confocal fluorescence images of IHC stereocilia (labeled with phalloidin) from P10 Whrn^+/tm1b (G), Whrn^tm1b/tm1b (H), and Whrn^wi/wi (I) mice. Scale bars, 5 μm. (J) Stereocilia length (±SD) from the tallest (row 1) and middle (row 2) row of IHC bundles and the tallest row (row 1) of OHCs of Whrn^+/tm1b, Whrn^tm1b/tm1b, and Whrn^wi/wi mice. (K–P) SEM images of vestibular stereocilia bundles in the extrastriolar and striolar region of the utricle of P21 Whrn^+/tm1b (K and L), Whrn^tm1b/tm1b (M and N), and Whrn^wi/wi (O and P) mice. Scale bars, 2 μm. (Q–V) Confocal fluorescence images of vestibular stereocilia bundles in the extrastriolar and striolar region of the utricle in P10 Whrn^+/tm1b (Q and R), Whrn^tm1b/tm1b (S and T), and Whrn^wi/wi (U and V) mice. Scale bars, 5 μm. (W and X) Length of the tallest stereocilia (±SD) from extrastriolar (W) and striolar (X) vestibular bundles. See also Figure S3.

Figure 4

Whrn Isoforms Show Distinct Localizations within Stereocilia (A–C) WHRN (green) localizes in IHC stereocilia (red) to two distinct regions in P10 Whrn^+/tm1b mice: stereocilia tips of the tallest row (white arrowhead) and proximal to the tips of the shorter row of stereocilia (yellow arrowhead). Boxed region in (A) is shown in high-magnification (B). (D and E) WHRN (green) localizes in OHC stereocilia (red) predominantly to the shorter row of stereocilia in P10 Whrn^+/tm1b mice. (F–H) WHRN (green) localizes in IHC stereocilia (red) to stereocilia tips of the tallest row (F, arrowhead) in Whrn^tm1b/tm1b mice. WHRN localization to shorter stereocilia is not detected. Boxed region magnified in (G). (I and J) WHRN (green) localization is not detected in OHC stereocilia (red) in Whrn^tm1b/tm1b mice (I). (K–N) WHRN label in Whrn^wi/wi mutant stereocilia. WHRN (green) is not detected in Whrn^wi/wi stereocilia (red) from IHCs (K and L) or OHCs (M and N). Scale bars, 2 μm. See also Figure S4.

Figure 5

WHRN-S Localization at Stereocilia Tips Overlaps with EPS8 and Both Proteins Are Required for Normal Stereocilia Elongation (A and B) Localization of WHRN and EPS8 to stereocilia tips. SIM image of IHC stereocilia (green) from P11 Whrn^tm1b/tm1b mice. WHRN-S (blue) and EPS8 (red) colocalize at stereocilia tips (arrowheads). (C) High mag of stereocilium from box in (A) and corresponding fluorescence intensity profile (below). (D) EPS8 (red) in OHC stereocilia (green) from P11 Whrn^tm1b/tm1b mice. (E and F) EPS8 (red) immunofluorescence in stereocilia (green) from P10 Whrn^wi/wi mutants shows stereocilia tip localization (arrowheads) in IHCs (E) and OHCs (F). (G and H) WHRN (red) immunofluorescence in stereocilia (green) from P6 Eps8 null mouse. No stereocilia tip localization observed in IHCs (G) or OHCs (H). WHRN-L labeling is still detected in OHCs (H). (I) Schematic summarizing WHRN isoform and EPS8 localization in stereocilia of cochlear hair cells, and vestibular stereocilia bundle morphology in wild-type, Whrn^tm1b/tm1b, and Whrn^wi/wi mice. Scale bars, 2 μm.