Supplemental Information. Notch Inhibition Induces Cochlear Hair Cell. Regeneration and Recovery of Hearing. after Acoustic Trauma.

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1 Neuron, Volume 77 Supplemental Information Notch Inhibition Induces Cochlear Hair Cell Regeneration and Recovery of Hearing after Acoustic Trauma Kunio Mizutari, Masato Fujioka, Makoto Hosoya, Naomi Bramhall, Hirotaka James Okano, Hideyuki Okano, and Albert S.B. Edge 1

2 Figure S1. Cochlear architecture in a mouse exposed to 8 16 khz octave-band noise at 116 db SPL for 2 h (A) The loss of outer hair cells (myosin VIIa) was apparent throughout the cochlea and loss of inner hair cells in the 22 khz region. (B) At this noise exposure intensity loss of hair cells was accompanied by preservation of supporting cells (Sox2) in most of the cochlea, with the exception of the 22 khz region. (C) Merged images show the base (Piece 1), mid-base (Piece 2), mid-apex (Piece 3), and apex (Piece 4) and the corresponding frequencies. Scale bar is 500 µm. 2

3 Figure S2. Analysis of the effect in vivo on the brainstem response and hair cell morphology of LY administered systemically to young adult noise-damaged mice (A) Treatment with LY by injection intraperitoneally at 50 mg/kg daily for 5 days increased the responsiveness of the ear at low frequency after 2-4 months. Significant improvements in threshold of the ABR were found between 1 and 3 months at 4, 8 and 16 khz. (B) Stereociliary bundles (white arrows) could be detected on hair cells after phalloidin staining. Hair cells (CtBP2-stained) were increased in the apex of the cochlea. Asterisks indicate p < Scale bars are 50 µm. 3

4 Figure S3. Labeling of a mature mouse cochlea with the reporter strain (A) The Sox2-CreER mouse crossed to the reporter line, mt/mg, treated with tamoxifen at P21 was examined as a whole mount after dissection. Any cells that express Sox2 at P21 would be expected to be GFP-positive after removal of the STOP sequence, whereas cells that do not will retain the tomato label. Supporting cells from the 5 to the 45 khz regions were labeled by GFP from the Sox2-Cre reporter (Sox2-lineage; green). In contrast, myosin VIIa-labeled hair cells 4

5 (blue) displayed tdtomato labeling (tdtomato; red), and this pattern was retained from the 5 to the 45 khz region (see bundles in the merged image; Merge). Note that some pillar cells were not labeled by the GFP from the reporter (22.6 khz for example), presumably due to incomplete Cre activity. (B) The reporter mouse was subjected to intense noise (see Fig. 4). xy-projections from the control (contralateral) ear in the 8 khz area. White bracket shows the outer hair cell region. No double-labeled cells were found in the control ear. (C) xz-projections confirm the lack of double-labeled hair cells in the control, contralateral ear. Scale bar is 50 µm. (D, E) No double-labeled cells were found in the LY treated ear without noise exposure at 8 and 22.6 khz cochlear regions. White bracket shows the outer hair cell region. Scale bar is 50 µm. 5

6 Figure S4. Effect of carrier treatment on normal ears (A, B) No significant threshold shifts were observed in DPOAE (A) or ABR (B) before (Pre Surgery: open circles) or 1 wk after (Post Surgery: filled circles) surgery and delivery of PEG-400 to the ear. 6

7 Figure S5. DPOAE measurements after LY treatment of noise-exposed ears (A) No significant changes were observed in DPOAE thresholds after noise exposure up to 3 months after surgery. Thresholds were calculated before noise exposure (Pre Noise: open circles), 1 day after noise exposure (Post Noise: filled circles), 1 week after drug treatment (1 W: open squares), 1 month after treatment (1 Mo: crosses), and 3 months after treatment (3 Mo: filled triangles) (n = 5 in each group). When no response was observed at 80 db (maximum acoustic output of the system) the threshold was designated as 85 db. (B) No significant differences in DPOAE threshold were found 3 months after drug treatment compared to 1 day after noise exposure between control and LY treated ears (n = 5 in each group). Error bars are standard error of the mean. 7

8 SUPPLEMENTAL EXPERIMENTAL PROCEDURES Distortion product otoacoustic emission (DPOAE) measurements For measurement of DPOAEs at 2f 1 f 2, the primary tones were set so that the frequency ratio (f 2 /f 1 ) was 1.2 and so that f 2 level was 10 db below f 1 level. For each f 2 /f 1 primary pair, levels were swept in 5 db steps from 20 to 80 db SPL (for f 2 ). At each level, both waveform and spectral averaging were used to increase the signal-to-noise ratio of the recorded ear-canal sound pressure, and the amplitude of the DPOAE at 2f 1 f 2 was extracted from the averaged spectra, along with the noise floor at nearby points in the spectrum. Iso-response curves were interpolated from plots of DPOAE amplitude versus sound level. Threshold was defined as the f 1 level required to produce a DPOAE at 0 db SPL. Noise-induced hearing loss in the mature cochlea (Description of Figure S1) We used a noise-induced hearing loss model in young adult mice. At an exposure intensity of 116 db SPL (8-16 khz), which leads to permanent hearing loss and major hair cell death especially in the outer hair cells region (Wang et al., 2002), almost all inner hair cells were preserved (from the apical tip to the 22 khz area), while the outer hair cells showed severe loss. Moreover, almost all supporting cell were preserved (Figure S1). Thus 116 db noise exposure was selected to produce a hair cell loss model for the investigation of hair cell regeneration. Systemic LY treatment ameliorates hearing loss in the mature noise-damaged cochlea (Description of Figure S2) Preliminary range finding experiments for drug treatment were carried out by systemic injection and were limited by toxicity. A minimal dosing regimen for an effect on the thymus weight was chosen (Hyde et al., 2006; Wong et al., 2004). Of 12 mice administered 50 mg/kg for 5 days, 6 8

9 could be tested for ABR at 3 months, the final time point of the LY treatment. The rest died within the first week due to severe diarrhea and weight loss. Mice that survived also suffered from weight loss (approximately 15% loss in 3 days), with a loss of epithelial cells of the stomach and increase in secreting cells in the gastrointestinal tract from esophagus to colon and severe atrophy of the spleen; atrophy of the thymus (total number of the cells was decreased to 1/40 and double-positive fraction of CD4 and CD8 was decreased from 78.6% to 1.23%), and changes in skin color. These changes resulted from Notch inhibition as reported in previous papers (Hadland et al., 2001; Wong et al., 2004). A small threshold shift (Figure S2A) that achieved statistical significance by comparison of the control and treated animals after 1 month and persisted to 3 months was observed at 4, 8 and 16 khz. The number of outer hair cells was increased, and the increase was most apparent at the regions where the damage was most severe (low frequency, Figure S2A). The hair cells appeared to have stereociliary bundles (phalloidin) and synapses (neurofilament and CtBP2) (Figure S2B). Model for lineage tracing of supporting cells (Description of Figure S3) To visualize supporting cell transdifferentiation into hair cells, we used a reporter line: mt/mg mice (Muzumdar et al., 2007) crossed with Sox2-CreER mice. In the mt/mg mice, cells that have undergone Cre recombination are labeled by expression of membrane-bound GFP (GFP; green fluorescence), and non-recombined cells express tdtomato (red fluorescence) after tamoxifen treatment. The result is a Cre-reporter line that can be used for lineage tracing of Sox2-positive cells. In the double-transgenic mouse cochlea, after estrogen receptor activation by tamoxifen in Sox2-positive cells, supporting cells expressed green fluorescence from GFP and hair cells retained red fluorescence from tdtomato (Figure S3). 9

10 Effect of carrier injection on ABR and DPOAE (Description of Figure S4) To rule out the influence of the injection of carrier and exposure of the inner ear, we tested whether carrier-treated ears showed changes in ABR or DPOAE, a measure of outer hair cell function. No significant differences were found after injection of carrier alone (Figure S4). DPOAE evaluation in noise-exposed ears treated with LY (Description of Figure S5) We also tested whether DPOAE threshold shifts could be seen in noise-exposed ears treated with LY Because the thresholds were above 70 db SPL at all frequencies after noise exposure and DPOAEs are difficult to distinguish from noise floor at intense input signals, our expectation was that for the small changes induced by alterations in apical hair cell number documented above, the DPOAE measurement would not be distinguishable above the noise floor, and, indeed, the alterations were not significant (Figure S5). 10