Figure 6.
Tprn−/−mice are profoundly deaf at P60 and show abnormalities of IHCs and OHCs. (A–A″) Airyscan confocal images showing TPRN RabmAb immunoreactivity (green) and F-actin (magenta) in P20 WT (Tprn+/+) and TPRN null (Tprn−/−) mouse IHC (A) and OHC stereocilia (A′) and in P10 supporting cells surrounding IHCs and OHCs (A″). TPRN is localized at the base of Tprn+/+ stereocilia and associated with actin cytoskeleton of supporting Deiters’ cells but not detected in Tprn−/− hair cells and supporting cells. (A″) Enlarged side view of hair cell stereocilia showing TPRN localization at the base of OHC stereocilia in all three rows and co-localized with F-actin in ascending processes of Deiters’ cells. (B) Mean ABR thresholds at P60 of Tprn+/+, Tprn+/−, and Tprn−/− littermates at 8, 16, and 32 kHz. Using linear mixed effects regression, we found that Tprn−/− mice exhibited profound deafness at P60 at all frequencies (Tables S4 and S5), progressive deafness from P18 to P30 and P60 (Fig. S5 A; and Tables S2 and S3) and more pronounced early hearing loss at high frequencies (Fig. S5 A and Table S5). The graph displays mean ± SD. **, ***—significant difference in ABR threshold compared with Tprn+/+ animals at P < 0.01 and P < 0.001, respectively. Color of asterisks indicate the group showing the difference. (C–D″) Representative SEM images of IHC and OHC stereocilia bundles of P6 Tprn+/+ (C–C'') and P6 Tprn−/− mouse (D–D''). Note, a Tprn−/− IHC (D) has some stereocilia missing from the first row (arrow) and a less developed third row of stereocilia with more prominent pruning of thin stereocilia/microvilli as compared with a WT IHC stereocilia bundle (C). Tprn−/− OHC stereocilia bundle from apical turn (D′) also has a stereocilium missing from the first row and a shortened stereocilium in the second row (arrows), while all stereocilia in Tprn+/+ apical OHC (C′) are present and are of normal length. Tprn−/− OHC stereocilia bundle from the basal turn of the cochlea (D″) shows multiple missing stereocilia (arrows) from the third row and an altered V shape of the bundle, while the entire third row stereocilia are present in the WT V-shaped OHC from the basal turn (C″). (E and F) Stereocilia abnormalities in P30 Tprn−/− hair cells (E) are similar but less prominent than in P17 TprnN259/N259 hair cells (F). Shortening and disappearance of the third row stereocilia (E, left panel, forked arrow) and shortening of selective stereocilia from longer rows (E and F, left panels, arrows). Middle panels in E and F show OHCs with abnormal hair bundles. Right panels show fusion of IHC stereocilia (arrows). (G and H) TEM images of the synaptic area of a Tprn−/− IHC (H) show abnormal accumulation of endosome-like vesicular structures (arrow) when compared with a Tprn+/+ IHC (G). (I–Q) TEM micrographs of P60 Tprn−/− hair cells. (I) Accumulation of endosome-like vesicles in the cell cytoplasm (arrow) and presence of axosomatic efferent contacts with accumulation of vesicles at the postsynaptic sites (boxed area with an arrow). (J) Swollen and damaged IHC efferent presynaptic terminal. Boxed areas in I and J are enlarged in L–N, correspondingly. (K) Another example of swollen efferent terminal like in M. (O) IHC stereocilia show rootlet fragmentation and breakage at the stereocilia insertion point (top image), splayed rootlets within the cuticular plate, or multiple electron dense spots within stereocilia cores (bottom image). (P and Q) OHC stereocilia show long, prominent rootlets, sometimes penetrating abnormally deep into cytoplasm below the cuticular plate (P) and sometimes showing a hollow center of the rootlet structure (top image) and accumulation of the electron dense spots within the cell body nearby long splayed rootlets (bottom image) (Q). (R–Y) SEM images of P90 Tprn+/+ OC (R), showing three rows of OHC and one row of IHC from the middle turn of the cochlea with representative normal structure OHC hair bundle (S) and IHC hair bundle (T). (U) There are missing OHCs and fused IHC hair bundles in the middle turn of the P90 Tprn−/− OC. (V) Characteristic OHC hair bundle abnormalities with shortened stereocilia of all rows. (W) Common abnormalities of IHC stereocilia: fusion (arrow) and abnormally thin taper with some stereocilia absent likely as a result of breakage at the taper (two adjacent arrows). (X) OHCs from the upper basal turn also show stereocilia fusion (arrow). (Y) Lower basal turn shows complete absence of OHCs and only a few remaining IHCs, some with fused stereocilia bundles (arrow). Scale bars in A, A′, E, F, R, U, and Y are 5 μm, and in A″, G, and N are 2 μm. Scale bars in C–C″, D–D″, H, I, K–M, P, and Q are 1 μm, and in J and O are 500 nm.

Tprn −/− mice are profoundly deaf at P60 and show abnormalities of IHCs and OHCs. (A–A″) Airyscan confocal images showing TPRN RabmAb immunoreactivity (green) and F-actin (magenta) in P20 WT (Tprn+/+) and TPRN null (Tprn−/−) mouse IHC (A) and OHC stereocilia (A′) and in P10 supporting cells surrounding IHCs and OHCs (A″). TPRN is localized at the base of Tprn+/+ stereocilia and associated with actin cytoskeleton of supporting Deiters’ cells but not detected in Tprn−/− hair cells and supporting cells. (A″) Enlarged side view of hair cell stereocilia showing TPRN localization at the base of OHC stereocilia in all three rows and co-localized with F-actin in ascending processes of Deiters’ cells. (B) Mean ABR thresholds at P60 of Tprn+/+, Tprn+/−, and Tprn−/− littermates at 8, 16, and 32 kHz. Using linear mixed effects regression, we found that Tprn−/− mice exhibited profound deafness at P60 at all frequencies (Tables S4 and S5), progressive deafness from P18 to P30 and P60 (Fig. S5 A; and Tables S2 and S3) and more pronounced early hearing loss at high frequencies (Fig. S5 A and Table S5). The graph displays mean ± SD. **, ***—significant difference in ABR threshold compared with Tprn+/+ animals at P < 0.01 and P < 0.001, respectively. Color of asterisks indicate the group showing the difference. (C–D″) Representative SEM images of IHC and OHC stereocilia bundles of P6 Tprn+/+ (C–C'') and P6 Tprn−/− mouse (D–D''). Note, a Tprn−/− IHC (D) has some stereocilia missing from the first row (arrow) and a less developed third row of stereocilia with more prominent pruning of thin stereocilia/microvilli as compared with a WT IHC stereocilia bundle (C). Tprn−/− OHC stereocilia bundle from apical turn (D′) also has a stereocilium missing from the first row and a shortened stereocilium in the second row (arrows), while all stereocilia in Tprn+/+ apical OHC (C′) are present and are of normal length. Tprn−/− OHC stereocilia bundle from the basal turn of the cochlea (D″) shows multiple missing stereocilia (arrows) from the third row and an altered V shape of the bundle, while the entire third row stereocilia are present in the WT V-shaped OHC from the basal turn (C″). (E and F) Stereocilia abnormalities in P30 Tprn−/− hair cells (E) are similar but less prominent than in P17 TprnN259/N259 hair cells (F). Shortening and disappearance of the third row stereocilia (E, left panel, forked arrow) and shortening of selective stereocilia from longer rows (E and F, left panels, arrows). Middle panels in E and F show OHCs with abnormal hair bundles. Right panels show fusion of IHC stereocilia (arrows). (G and H) TEM images of the synaptic area of a Tprn−/− IHC (H) show abnormal accumulation of endosome-like vesicular structures (arrow) when compared with a Tprn+/+ IHC (G). (I–Q) TEM micrographs of P60 Tprn−/− hair cells. (I) Accumulation of endosome-like vesicles in the cell cytoplasm (arrow) and presence of axosomatic efferent contacts with accumulation of vesicles at the postsynaptic sites (boxed area with an arrow). (J) Swollen and damaged IHC efferent presynaptic terminal. Boxed areas in I and J are enlarged in L–N, correspondingly. (K) Another example of swollen efferent terminal like in M. (O) IHC stereocilia show rootlet fragmentation and breakage at the stereocilia insertion point (top image), splayed rootlets within the cuticular plate, or multiple electron dense spots within stereocilia cores (bottom image). (P and Q) OHC stereocilia show long, prominent rootlets, sometimes penetrating abnormally deep into cytoplasm below the cuticular plate (P) and sometimes showing a hollow center of the rootlet structure (top image) and accumulation of the electron dense spots within the cell body nearby long splayed rootlets (bottom image) (Q). (R–Y) SEM images of P90 Tprn+/+ OC (R), showing three rows of OHC and one row of IHC from the middle turn of the cochlea with representative normal structure OHC hair bundle (S) and IHC hair bundle (T). (U) There are missing OHCs and fused IHC hair bundles in the middle turn of the P90 Tprn−/− OC. (V) Characteristic OHC hair bundle abnormalities with shortened stereocilia of all rows. (W) Common abnormalities of IHC stereocilia: fusion (arrow) and abnormally thin taper with some stereocilia absent likely as a result of breakage at the taper (two adjacent arrows). (X) OHCs from the upper basal turn also show stereocilia fusion (arrow). (Y) Lower basal turn shows complete absence of OHCs and only a few remaining IHCs, some with fused stereocilia bundles (arrow). Scale bars in A, A′, E, F, R, U, and Y are 5 μm, and in A″, G, and N are 2 μm. Scale bars in C–C″, D–D″, H, I, K–M, P, and Q are 1 μm, and in J and O are 500 nm.

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