Figure 1.
Activation of Notch in differentiating hair cells causes profound deafness.
A. Schematic diagram demonstrating constructs for NICD expression in hair cells. (1) Cre is expressed in early developing hair cells under the Gfi1 promoter, causing recombination of a floxed-stop cassette and expression of NICD (2). The cross also contains a ROSA26-lacZ allele, which is similarly recombined upon expression of Cre and marks by lineage the cells expressing NICD (3). B–C. Hearing assessments of Gfi1-NICD mutants and their littermate Cre-negative controls at 5weeks of age. B. Auditory brainstem recordings (ABRs) show that Gfi1-NICD mutants have no responses to the highest sounds levels (80 dB SPL). C. Measurements of distortion product otoacoustic emissions (DPOAEs) also show raised thresholds, but have some responses to sound. To determine significance, a 2-way ANOVA was performed, with pairwise comparisons to determine significance for each frequency, * = p<0.001.
Figure 2.
At P20, NICD-expressing hair cells in the cochlea have downregulated hair cell markers.
A–I Paraffin sections through the P20 cochlea stained for hair cell markers, SOX2, and ß-galactosidase. A, D, G. Calretinin, parvalbumin, and myosin VI all show expression in the inner and outer hair cells at P20 (although calretinin expression is weak in outer hair cells at P20). B, E, H. Both calretinin and parvalbumin are shut off in the Gfi1-NICD inner and outer hair cells, whereas myosin VI is generally still expressed in the outer hair cells in the mutant (H). Gfi1-NICD inner and outer hair cells express SOX2 and the inner hair cells have a more basally-located nucleus than in controls (B,E,H, arrows and insets in C,F,I showing higher power images of inner hair cells expressing ß-galactosidase and SOX2). J. Quantification of relative expression levels of indicated markers in the Gfi1-NICD (NICD) inner hair cells and littermate controls [Cre(-)]. Scale bar in I = 100 microns ( = 50 microns for inset panels).
Figure 3.
At P6, Some hair cell markers are downregulated in the inner hair cells.
A–I. Frozen sections through the P6 cochlea stained for hair cell markers, SOX2, and ß-galactosidase. A, D, G. Calretinin, parvalbumin, and myosin VI all show expression in the inner and outer hair cells at P6 (although parvalbumin expression is weaker in outer hair cells at P6). B, E, H. Both calretinin and parvalbumin are downregulated in the Gfi-NICD inner hair cells, although outer hair cell expression is largely maintained. Interestingly, myosin VI (G–I) is generally indistinguishable from the controls at this time point. Scale bar in I = 100microns. J–O. Wholemount cochlea stained for hair cell (parvalbumin) and supporting cell (SOX2) markers. Many of the inner hair cells have shut off parvalbumin and upregulated SOX2 by this time point (M–O, arrows). A few outer hair cells have downregulated parvalbumin (M–O, arrowhead), but most have upregulated SOX2.
Figure 4.
NICD-expressing inner hair cells show morphology changes and abnormal P27KIP1 expression at P11.
A–D. Paraffin sections from Gfi1-NICD cochlea and littermate controls at P11. A′-D′ show higher power views of the inner hair cells, which demonstrate morphological and molecular changes due to Notch activation. The changes in the inner hair cells in the Gfi1-NICD mutants (B,C,D and B′C′,D,) include weaker MYO6 expression, lack of the normal flask-like shape, more basally positioned nulcei, and nuclear P27KIP1 expression normally only observed in supporting cells at this time. Scale bar in D = 100 microns for A–D, and in D′ = 25 microns for A′–D′.
Figure 5.
The mature supporting cell marker NKAα1 is upregulated in NICD-expressing inner and outer hair cells.
A–F. Paraffin sections from Gfi1-NICD and Cre-negative control cochleae at P20. A–C. Control sections showing the normal expression of NKAα1 in the phalangeal cells (phc) surrounding the inner hair cells (ihc), which are also coexpressed with SOX2. Deiter's cells also show expression of SOX2, although not NKAα1. D–F. Gfi1-NICD expressing inner hair cell has upregulated NKAα1 (arrowhead). Surprisingly, the NICD-expressing outer hair cells (ohc) have also upregulated NKAα1, although much more weakly than in the inner hair cells. Both inner and outer hair cells also show upregulation of SOX2. Scale bar = 100 microns.
Figure 6.
NICD-expressing hair cells exhibit expected markers in the vestibular regions. A–F
. Frozen sections through the vestibular regions of P6 inner ears. Hair cell markers (calretinin and Myosin VI; B and E) are expressed similarly to controls (A and D) in the indicated regions of the vestibular system. Sections were co-labeled with ß-galactosidase expression, which is shown in a different panel (C, F) to better show the expression. Scale bar in F = 100 microns.
Figure 7.
Auditory testing of Gfi1-SOX2 mutants demonstrates that SOX2 expression causes hearing impairment.
A. Auditory brainstem recordings (ABRs, left) of Gfi1-SOX2 mutants and littermate controls at 5 weeks of age show that SOX2-expressing mice have significantly raised thresholds compared to controls. B. Measurements of distortion product otoacoustic emissions (DPOAEs) of the same mice tested for ABR, also show raised thresholds at 5 weeks. To determine significance, a 2-way ANOVA was performed, with pairwise comparisons to determine significance for each frequency: ** = p<0.001, * = p<0.05.
Figure 8.
Histological assessment of SOX2-expressing hair cells shows no morphological changes or downregulation of hair cell markers over time.
A–D. Both calretinin and myosin VI are expressed normally in Gfi1-SOX2 mutants (B,D) at P6 when compared to controls (A,C). A′–D′. Expression of SOX2 alone (shown also in A–D) demonstrates the upregulation of SOX2 in the inner and outer hair cells in Gfi1-SOX2 mutants (asterisks indicate hair cell nuclei expressing SOX2 in B′–D′). Scale bar in D = 100 microns for A–D. E–H. Expression of indicated hair cells markers in Gfi1-SOX2 mutants (F,H) is maintained normally at P20 when compared to controls (E,G). Scale bar in H = 100 microns for E–H.