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. 2019 Mar 18;29(6):921-934.e4.
doi: 10.1016/j.cub.2019.01.051. Epub 2019 Feb 28.

GPSM2-GNAI Specifies the Tallest Stereocilia and Defines Hair Bundle Row Identity

Abigail L D Tadenev  1 , Anil Akturk  1 , Nicholas Devanney  1 , Pranav Dinesh Mathur  2 , Anna M Clark  2 , Jun Yang  3 , Basile Tarchini  4
Affiliations

GPSM2-GNAI Specifies the Tallest Stereocilia and Defines Hair Bundle Row Identity

Abigail L D Tadenev et al. Curr Biol. .

Abstract

The transduction compartment of inner ear hair cells, the hair bundle, is composed of stereocilia rows of graded height, a property essential for sensory function that remains poorly understood at the molecular level. We previously showed that GPSM2-GNAI is enriched at stereocilia distal tips and required for their postnatal elongation and bundle morphogenesis-two characteristics shared with MYO15A (short isoform), WHRN, and EPS8 proteins. Here we first performed a comprehensive genetic analysis of the mouse auditory epithelium to show that GPSM2, GNAI, MYO15A, and WHRN operate in series within the same pathway. To understand how these functionally disparate proteins act as an obligate complex, we then systematically analyzed their distribution in normal and mutant bundles over time. We discovered that WHRN-GPSM2-GNAI is an extra module recruited by and added to a pre-existing MYO15A-EPS8 stereocilia tip complex. This extended complex is only present in the first, tallest row, and is required to stabilize larger amounts of MYO15A-EPS8 than in shorter rows, which at tips harbor only MYO15A-EPS8. In the absence of GPSM2 or GNAI function, including in the epistatic Myo15a and Whrn mutants, bundles retain an embryonic-like organization that coincides with generic stereocilia at the molecular level. We propose that GPSM2-GNAI confers on the first row its unique tallest identity and participates in generating differential row identity across the hair bundle.

Keywords: EPS8; GNAI; GPSM2; MYO15A; WHRN; hair bundle; hair cell; staircase-like organization; stereocilia.

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Figures

Figure 1.
Figure 1.. Similar hair bundle defects in Gpsm2−/−, Myo15ash2/sh2 and (Gpsm2−/−; Myo15ash2/sh2) double-mutant and in Gpsm2−/−, Whrnwi/wi and (Gpsm2−/−; Whrnwi/wi) double mutants at P6/P7.
(A) Medial views of IHC and OHC hair bundles from Gpsm2−/−, Myo15ash2/sh2, and Gpsm2−/−; Myo15ash2/sh2 double mutant and unaffected littermate control using SEM. (B) Quantification of IHC stereocilia height in row 1, height differential between row 1 and row 2, number of stereocilia in row 1, total number of rows and stereocilia diameter in row 1 (solid-colored bars) and row 3 (black-edged bars) for the genotypes indicated. (C) Medial views of IHC and OHC hair bundles from Gpsm2−/−, Whrnwi/wi, and Gpsm2−/−; Whrnwi/wi double mutant and unaffected littermate control using SEM. (D) Same set of IHC quantification as in (B) for the genotypes indicated. See Figure S1 for OHC quantifications and lateral views of hair bundles in IHCs and OHCs. For all images and quantifications, IHCs were selected at a half-turn position up from the cochlear base. At least 10 IHCs were analyzed for 3 to 5 animals per genotype, and 3 stereocilia were measured per cell for height, height differential and diameter (see Tables S1, S2 for complete dataset). Each point on the graphs represents one animal (average +/− SEM). Statistics were calculated using one-way ANOVA with Tukey's multiple comparisons test, with a single pooled variance (ns, p>0.05; *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001). Green asterisks reflect significance compared to control, whereas orange asterisks reflect significance compared to Gpsm2−/−. Asterisks in the diameter graphs refer to row 3. In all panels and figures, the lateral (abneural) side of the auditory epithelium is up and the medial (neural) side is down. Scale bars are 1 μm. See also Figure S1, S2.
Figure 2.
Figure 2.. Distinct stereocilia distribution for WHRN-GPSM2-GNAI3 and MYO15A-EPS8 in postnatal and embryonic hair cells.
(A-C) Coimmunostainings for GPSM2+WHRN (A), GNAI3 or GPSM2+MYO15A (B) and GPSM2 or GNAI3+EPS8 (C) in P0 IHCs at the cochlear base (left) and P7 IHCs at the apical turn (right). All proteins colocalized at row 1 tips (solid arrowheads; see also Figure S3A-C for P7 OHCs). In addition, GPSM2 and GNAI3 localized at the bare zone (solid arrows), WHRN localized at the base of stereocilia (hollow arrows), and MYO15A and EPS8 could be weakly detected at row 2 stereocilia tips (hollow arrowheads). Note that younger bundles tended to fall medially (away from the BZ) during mounting, whereas more mature bundles often fell laterally. (D-E) E16.5 OHCs (D) and IHCs (E) coimmunostainings at the cochlear base. MYO15A and EPS8 were already abundantly enriched at stereocilia tips, whereas WHRN and GPSM2-GNAI3 were low or undetectable there in the same cells (arrowheads). GPSM2-GNAI3 and WHRN already localized to the bare zone (arrow) and the base of stereocilia (hollow arrow), respectively. (F) E18.5 OHCs coimmunostainings at the cochlear base for the proteins indicated. Low amounts of GNAI3 and GPSM2 colocalized with more abundant EPS8 and MYO15A only in central stereocilia, and EPS8 and MYO15A showed a wider distribution encompassing peripheral stereocilia (left and center panels). By contrast, WHRN enrichment matched GPSM2 in central stereocilia (right panels). (G) E18.5 IHCs MYO15A immunostaining. MYO15A was enriched in roughly comparable amounts at stereocilia tips across rows. Two to three rows can be distinguished. In all figures, solid arrows indicate the bare zone, hollow arrows indicate the stereocilia base, solid arrowheads indicate row 1 stereocilia tips, hollow arrowheads indicate row 2 tips and F-act indicates phalloidin staining. (A-C and G) were acquired in Airy mode (single z) (see Methods). Scale bars are 5μm. See also Figure S3.
Figure 3.
Figure 3.. MYO15A and WHRN are required for GPSM2-GNAI3 enrichment at row 1 stereocilia tips.
Coimmunostaining for GPSM2 and GNAI3 in Myo15ash2/sh2 and Whrnwi/wi mutant and a control in E18.5 OHCs at the cochlear base (A), P4 OHCs in the apical turn (B), and P4 IHCs in the basal half-turn (C). GPSM2 and GNAI3 were not detected at stereocilia tips (arrowheads) in either mutant. Note that bare zone localization of GPSM2 and GNAI3 (solid arrow) was unchanged. Control at E18.5 is a Myo15ash2/+ littermate of Myo15ash2/sh2, control for P4 OHCs is a Whrnwi/+ littermate of Whrnwi/wi, and control for P4 IHCs is a Gpsm2+/−; Whrnwi/+ littermate of Whrnwi/wi. Note that kinocilium signal for GPSM2 is not specific, as it is also seen in Gpsm2−/− (not shown). Scale bars are 5μm. See also Figure S4.
Figure 4.
Figure 4.. GPSM2 is not required for early GNAI3 trafficking to stereocilia tips, but is required to stabilize GNAI3 and WHRN at tips.
(A-B) GNAI3 and EPS8 coimmunostaining in E18.5 OHCs (A) and GNAI3 immunostaining in P4 IHCs (B) in Gpsm2−/−. In absence of GPSM2, GNAI3 was found in excess at tips, closely matching EPS8 signal at E18.5, but was missing at tips at P4 (arrowheads). Enrichment at the bare zone was by contrast reduced at both stages (arrows). (C) Immunoprecipitation of GNAI3:EGFP with WHRN in HEK293 cells. GNAI3 constructs were wild-type (WT) or carried a N150I mutation preventing binding to GPSM2, and MYC:GPSM2 was cotransfected in lane 2. Preventing direct binding to GPSM2 (N150I) or adding GPSM2 neither decreased nor increased the amount of GNAI3:EGFP immunoprecipitated, respectively. EGFP alone was not immunoprecipitated by WHRN and GNAI3:EGFP was not immunoprecipitated when WHRN was not transfected (see Figure S5A for extra control blots). (D) MYO15A and EPS8 coimmunostaining in E17.5 Whrnwi/wi OHCs showing normal enrichment at tips (arrowheads). (E-F) WHRN immunostaining in E18.5 Gpsm2−/− OHCs. WHRN signal intensity is reduced at stereocilia tips but increased at stereocilia base compared to littermates (E), as illustrated in (F). Arbitrary unit (AU); n= 4 animals, 6 OHCs per animal; Mann-Whitney U test, ***p=0.0002 for tips and base signal. An example of regions of interest (ROIs) for WHRN signal quantification is shown in (F). Control image in (B) shows the same IHC pair as in Figure 3C because these experiments were performed as GPSM2-GNAI3 coimmunostainings in a Gpsm2− × Whrnwi breeding scheme, and the same Gpsm2+/−; Whrnwi/+ sample was used as littermate control. Solid arrows indicate the bare zone, hollow arrows indicate the stereocilia base, and solid arrowheads indicate row 1 stereocilia tips. Scale bars are 5μm. See also Figure S5.
Figure 5.
Figure 5.. GPSM2-GNAI is required for graded MYO15A-EPS8 amounts across stereocilia rows and to limit WHRN to row 1 at tips.
(A) WHRN, MYO15A and EPS8 immunostainings in P4 IHCs of Gpsm2−/−, Atoh1-Cre; PTXa and Myo15ash2/sh2 or Whrnwi/wi mutants. WHRN was ectopically located at the tips of row 2 and further stereocilia rows in Gpsm2−/− and Atoh1-Cre; PTXa, but absent at tips in Myo15ash2/sh2. WHRN-MYO15A-EPS8 appeared uniformly enriched at tips across all rows (including supernumerary rows) in lower amounts than row 1 enrichment in control littermates. (B) Example of a P4 IHC pair used to quantify MYO15A accumulation at stereocilia tips in (C) and (D). The left IHC in this Atoh1-Cre; Gpsm2Flox/− sample retained apparently normal GPSM2 distribution and morphology, and was used as an internal control (see text). ROIs for MYO15A signal quantification are indicated as yellow circles at the tips of rows 1, 2, and 3 (control row 3 represents background signal level). (C-D) MYO15A signal per row for Atoh1-Cre; Gpsm2Flox/− (C) and Atoh1-Cre; PTXa (D). MYO15A signal intensity was normalized for each IHC pair to the average signal at row 1 tips in the control IHC. Two IHC pairs were selected for 3 animals and 5 stereocilia quantified per row for each IHC (n=30; two-way ANOVA with Sidak’s multiple comparisons test; ns, p>0.05; ****p≤0.0001). (E) MYO15A immunostaining in P21 Gpsm2−/− and Atoh1-Cre; PTXa IHCs. As at P4, all mutant stereocilia show an approximately equal amount of MYO15A, which is less than in control row 1. Internal control and mutant IHCs are indicated for each Atoh1-Cre; PTXa image. The same cells are shown in (A) for MYO15A and EPS8 (Control, Gpsm2−/− and Whrnwi/wi), and for WHRN and EPS8 (Atoh1-Cre; PTXa) because these experiments were performed as coimmunostainings. Hollow arrows indicate the stereocilia base, solid arrowheads indicate row 1 stereocilia tips, and hollow arrowheads indicate row 2 tips. Scale bars are 5μm. See also Figure S6, S7.
Figure 6.
Figure 6.. GPSM2 and GNAI are required to establish molecularly distinct stereocilia row identities.
(A-B) TWF2 immunostainings in P33 IHCs. GPSM2 and TWF2 normally adopted complementary localization at row 1 (solid arrowheads) and row 2 (hollow arrowheads) tips, respectively (A), but TWF2 was ectopically found at row 1 in both Gpsm2−/− (A) and Atoh1-Cre; PTXa (B) (arrowheads). Note that the control bundles fell medially upon mounting, whereas the shorter mutant bundles fell laterally. TWF2 levels appeared substantially lower at the stereocilia tips of mutant IHCs compared to control IHCs, particularly in Atoh1-Cre; PTXa samples. (C-D) MYO15A long isoform (MYO15A-long) immunostainings in P8 IHCs. EPS8 and MYO15A-long normally adopted complementary localization at row 1 (solid arrowheads) and row 2 (hollow arrowheads) tips, respectively (C), but MYO15A-long was ectopically found at row 1 in Gpsm2−/− (C), Atoh1-Cre; PTXa and Whrnwi/wi IHCs (D). (E) One representative P4 Isl1-Cre; PTXa IHC pair coimmunostained for GPSM2 and GNAI3 where the apparently normal right IHC served as an internal control (see text and Methods). Top: GNAI3 and GPSM2 were planar polarized at the bare zone (arrow) in the control IHC, but delocalized to the whole apex in the mutant IHC. Bottom: remarkably, GNAI3 and GPSM2 were concomitantly delocalized to all stereocilia rows at tips in the same mutant IHC (arrowheads). Low GPSM2 signal at tips in the mutant IHC was enhanced in the magnified inset. The XZ view shows GNAI3 at tips (black arrowhead) and at the flat IHC surface (black arrow). (F) GNAI3 and GPSM2 immunostainings in P21 IHCs. Delocalization from row 1 to all rows is still clearly observed at maturity in Atoh1-Cre; PTXa IHCs, whereas an internal control IHC only showed GPSM2 at row 1 tips (bottom right IHC). Airy mode was used to acquire images in (A), (B), (D), (F) (single z) and (E) (maximum projection of z-series). In (E), top and bottom panels are maximum intensity projection of flat apical surface and tips level GPSM2-GNAI3 signal, respectively, overlaid with full-depth projection of phalloidin signal. Solid arrows indicate the bare zone, solid arrowheads indicate row 1 stereocilia tips, and hollow arrowheads indicate row 2 tips. Scale bars are 2μm (A-E), 5μm (F).
Figure 7.
Figure 7.. Model describing the contribution of GPSM2-GNAI to IHC hair bundle morphogenesis.
At E18.5 (left), GPSM2-GNAI (blue) is mostly enriched outside the hair bundle at the bare zone, the flat apical sub-region that directly abuts the first stereocilia row (apical view). At this stage, MYO15A-EPS8 (red) broadly localizes at tips throughout the IHC bundle in relatively comparable amounts. 1) Trafficking phase: MYO15A uses WHRN as an adapter to bring low amounts of GPSM2-GNAI (purple) to tips in central stereocilia of the first row only (green arrow). 2) Stabilization phase: by P4 and until maturity (P21) (center), WHRN-GPSM2-GNAI remains restricted to row 1, where it builds up high amounts of MYO15A-EPS8 (green arrow) and specifies the tallest identity of the first row. MYO15A-EPS8 remains enriched at tips in shorter stereocilia but in much lower amounts, as reported previously. In stark contrast, little row-specific differentiation occurs in Gpsm2−/− or PTXa mutant IHCs (right). Stereocilia are uniformly short and maintain an excessive number of rows where MYO15A-EPS8 is enriched in comparable amounts. WHRN is maintained at row 1 tips, but is also ectopically found in all further rows. Although MYO15A-EPS8-WHRN colocalize at tips (brown), they cannot rescue normal elongation in row 1. As a consequence, mature mutant IHC bundles (right) are reminiscent of immature E18.5 embryonic bundles (left).
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