Figure 8.
Abnormal stereocilia development due to constitutive absence or long-term overexpression of TPRN in vivo. (A and B) Whole mounts of cochlea from P7 WT or Tprn−/− mice were stained with antibodies against TRIOBP-5 (green) and phalloidin (magenta) to reveal stereocilia. (A′–B′) Enlarged images of the OHC hair bundles indicated by arrows in A and B. (A″–B″) Enlarged images of the IHC hair bundles indicated by arrows in A and B. Number of TRIOBP-5 puncta at bases of immature short-row stereocilia was significantly reduced in P7 Tprn-deficient hair cells. Two arrows point to immature stereocilia rows in A′ compared with B′ and in A″ compared with B″. (C) Quantification of TRIOBP-5 puncta at the bases of different rows of stereocilia in P7 Tprn+/+ and in Tprn-deficient mouse model Tprnin103/in103. Fifty-six WT and Tprn-deficient IHCs from at least three mice were analyzed per group. Data are means ± SEM. ***P < 0.001 by unpaired two-sided t test. (D–G) Cochlear whole mounts from P1 (D and E) or P3 (F and G) of Tprn+/+ and Tprnin103/in103 mice immunostained for TRIOBP-5 and counterstained for F-actin using phalloidin. (D′–E′) Enlarged images of TRIOBP-5–stained IHC bundles in D and E. (F–G′) As early as P3, there was a reduction of TRIOBP-5 puncta at the base of short-row stereocilia of Tprn-deficient mice, better appreciated on enlarged images (F′–G′). (H and I) Representative SEM images of IHC stereocilia bundles from the middle turn of the cochlea of P7 WT (H) and Tprnin103/in103 (I) mice. (J) Quantification of the number of stereocilia in different rows. At least 48 IHCs from three mice in each group were analyzed. Data are represented as mean ± SEM. ***P < 0.001 by unpaired two-sided t test. (K and K′) A low-magnification image showing stereocilia of P7 WT hair cells infected at P2 with AAVs expressing FL HA-TPRN. On average, 64.9 ± 6.5% of IHCs exhibited excessive stereocilia elongation. A total of three mice were analyzed, and 27–64 IHCs were assessed in the apical region of the cochlea of each mouse. Arrows indicate cells with apparently normal stereocilia, likely due to the absence of AAV transduction. (L and L′) Degeneration of P7 WT IHC stereocilia infected at P2 with AAVs expressing FL HA-TPRN. (K–L′) Hair cells are counterstained with phalloidin to reveal F-actin and an anti-HA antibody to detect HA-TPRN (green). (M–Q) P7 WT hair cells infected at P2 with AAVs expressing HA-TPRN1–400 (M), HA-TPRN1–300 (N), HA-TPRN1–170 (O), TPRN1–63 (P), or tdTomato (Q). Note, multiple rows of TRIOBP-5 puncta in hair cells expressing TPRN1–400, TPRN1–300, or TPRN1–170. (N) Extra rows of TRIOBP-5 puncta are visualized at the apical surface of infected cells (arrows point to row 5 stereocilia) as compared with a noninfected cell in the middle of the image in N or cells expressing TPRN1–63 or tdTomato (P and Q). (R–S) SEM images of P7 WT noninfected (R) and infected with AAVs expressing HA-TPRN1–170 (R′ and S) hair cells. On average, 5.5 ± 2.1% of IHCs exhibited negligible changes to the hair bundle, 87.4 ± 2.7% exhibited supernumerary rows of short stereocilia with increased thickness (R′), and 7.0 ± 2.9% showed over-elongated stereocilia (S). A total of five mice were analyzed, and 32–41 IHCs were assessed in the apical region of the cochlea of each mouse. Scale bars: 5 µm in A–Q, except 1 µm in H, I, R, and S. (T) Relative intensity of TRIOBP-5 in rows 1–3. Significantly increased intensity of TRIOBP in row 3 stereocilia in hair cells expressing TPRN1–400, TPRN1–300, or TPRN1–170. Data are represented as mean ± SEM. ***P < 0.001 by unpaired two-sided t test. tdTomato: n.s. P = 0.4854. TPRN1–63: n.s. P = 0.0504. WT, TPRN1–400, and TPRN1–300n = 47/3 cells/mice; TPRN1–170 and tdTomato n = 50/3 cells/mice; and TPRN1–63n = 30/3 cells/mice.

Abnormal stereocilia development due to constitutive absence or long-term overexpression of TPRN in vivo. (A and B) Whole mounts of cochlea from P7 WT or Tprn−/− mice were stained with antibodies against TRIOBP-5 (green) and phalloidin (magenta) to reveal stereocilia. (A′–B′) Enlarged images of the OHC hair bundles indicated by arrows in A and B. (A″–B″) Enlarged images of the IHC hair bundles indicated by arrows in A and B. Number of TRIOBP-5 puncta at bases of immature short-row stereocilia was significantly reduced in P7 Tprn-deficient hair cells. Two arrows point to immature stereocilia rows in A′ compared with B′ and in A″ compared with B″. (C) Quantification of TRIOBP-5 puncta at the bases of different rows of stereocilia in P7 Tprn+/+ and in Tprn-deficient mouse model Tprnin103/in103. Fifty-six WT and Tprn-deficient IHCs from at least three mice were analyzed per group. Data are means ± SEM. ***P < 0.001 by unpaired two-sided t test. (D–G) Cochlear whole mounts from P1 (D and E) or P3 (F and G) of Tprn+/+ and Tprnin103/in103 mice immunostained for TRIOBP-5 and counterstained for F-actin using phalloidin. (D′–E′) Enlarged images of TRIOBP-5–stained IHC bundles in D and E. (F–G′) As early as P3, there was a reduction of TRIOBP-5 puncta at the base of short-row stereocilia of Tprn-deficient mice, better appreciated on enlarged images (F′–G′). (H and I) Representative SEM images of IHC stereocilia bundles from the middle turn of the cochlea of P7 WT (H) and Tprnin103/in103 (I) mice. (J) Quantification of the number of stereocilia in different rows. At least 48 IHCs from three mice in each group were analyzed. Data are represented as mean ± SEM. ***P < 0.001 by unpaired two-sided t test. (K and K′) A low-magnification image showing stereocilia of P7 WT hair cells infected at P2 with AAVs expressing FL HA-TPRN. On average, 64.9 ± 6.5% of IHCs exhibited excessive stereocilia elongation. A total of three mice were analyzed, and 27–64 IHCs were assessed in the apical region of the cochlea of each mouse. Arrows indicate cells with apparently normal stereocilia, likely due to the absence of AAV transduction. (L and L′) Degeneration of P7 WT IHC stereocilia infected at P2 with AAVs expressing FL HA-TPRN. (K–L′) Hair cells are counterstained with phalloidin to reveal F-actin and an anti-HA antibody to detect HA-TPRN (green). (M–Q) P7 WT hair cells infected at P2 with AAVs expressing HA-TPRN1–400 (M), HA-TPRN1–300 (N), HA-TPRN1–170 (O), TPRN1–63 (P), or tdTomato (Q). Note, multiple rows of TRIOBP-5 puncta in hair cells expressing TPRN1–400, TPRN1–300, or TPRN1–170. (N) Extra rows of TRIOBP-5 puncta are visualized at the apical surface of infected cells (arrows point to row 5 stereocilia) as compared with a noninfected cell in the middle of the image in N or cells expressing TPRN1–63 or tdTomato (P and Q). (R–S) SEM images of P7 WT noninfected (R) and infected with AAVs expressing HA-TPRN1–170 (R′ and S) hair cells. On average, 5.5 ± 2.1% of IHCs exhibited negligible changes to the hair bundle, 87.4 ± 2.7% exhibited supernumerary rows of short stereocilia with increased thickness (R′), and 7.0 ± 2.9% showed over-elongated stereocilia (S). A total of five mice were analyzed, and 32–41 IHCs were assessed in the apical region of the cochlea of each mouse. Scale bars: 5 µm in A–Q, except 1 µm in H, I, R, and S. (T) Relative intensity of TRIOBP-5 in rows 1–3. Significantly increased intensity of TRIOBP in row 3 stereocilia in hair cells expressing TPRN1–400, TPRN1–300, or TPRN1–170. Data are represented as mean ± SEM. ***P < 0.001 by unpaired two-sided t test. tdTomato: n.s. P = 0.4854. TPRN1–63: n.s. P = 0.0504. WT, TPRN1–400, and TPRN1–300n = 47/3 cells/mice; TPRN1–170 and tdTomato n = 50/3 cells/mice; and TPRN1–63n = 30/3 cells/mice.

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