Figure 1.
Gentamicin exposure results in rapid inner and outer hair cell loss.
A: Acutely isolated cochlear explant (stage P2). Atoh1/nGFP expression (green) labels inner and outer hair cell nuclei. Red lines subdivide auditory sensory epithelium into apex, mid apex, mid base and base. Scale bar 100 μm. B–M: Hair cell phenotype 24 hours after exposure to various concentrations of gentamicin. Confocal images are taken from the central part of mid apical segment (A, white box). Brackets mark outer hair cell domain (ohc), arrowheads point to inner hair cell domain (ihc). Myosin VI antibody staining (Myo6, red) labels cytoplasmic domain of hair cells. Atoh1/nGFP transgene (green) labels hair cell nuclei. Scale bar 50 μm. B–D: control (0 μg/ml gentamicin). E–G: 10 μg/ml gentamicin. H–J: 50 μg/ml gentamicin. White arrow marks Myo6 positive hair cell debris. K–M: 100 μg/ml gentamicin. N: Quantification of B-M. Hair cell numbers were analyzed in the mid apical segment. Data expressed as mean ±SEM (n = 3 cochlear explants from 2 independent experiments; ** p≤0.0001, Student's t-test; p>0.05 was considered not significant (n.s.)).
Figure 2.
Supporting cell phenotype in the hair cell damaged cochlea.
A–B': P2 Lfng/GFP transgenic control (A, A') and gentamicin treated (B, B') cochlear cultures after 1 DIV. Deiters cell (DC), outer pillar cell (oPC) and inner phalangeal cell nuclei (iPh) are visualized by native Lfng/GFP expression (green); hair cells are immuno-stained with myosin VI antibody (Myo6, red). A, B: Confocal images of supporting cell layer. A' B': orthogonal section of z-stack projections of supporting cell and hair cell layer. 24 hours after initial gentamicin insult, Lfng/GFP positive supporting cell layer is disorganized (B) and myosin VI positive hair cell debris (red) is engulfed by Lfng/GFP positive supporting cells (green) (B'). DAPI (blue) labels cell nuclei. Scale bar 50 μm. C–F: Confocal images of P2 Atoh1/nGFP transgenic control (C, E) and gentamicin treated (D, F) cultures after 2 DIV. Prox1 immuno-staining (red) marks Deiters cells (DC), outer pillar cells (oPC) and inner pillar cell (iPC) nuclei and Atoh1/nGFP expression (green) marks hair cell nuclei. Distinct oval nuclear morphology of inner pillar cells seen in control cultures (C) is largely lost in gentamicin treated cultures (D). Scale bar: 50 μm. G: Relative mRNA levels of Notch effector genes (bracket) and Notch target genes (Jag1, Sox2) were analyzed in undamaged (control, white bar), hair cell damaged (gentamicin +DMSO, grey bar) and hair cell damaged and Notch inhibited (gentamicin +DAPT, purple bar) cochlear epithelia after 38 hours in culture using q-PCR. Gentamicin was added at plating and washed out after 14 hours of culture. DAPT or vehicle control DMSO was added following gentamicin treatment for 24 hours. Hair cell specific genes (Atoh1, Pou4f3, and Fgf8) and supporting cell specific genes (Prox1, S100a1) were analyzed as experimental controls. Data expressed as mean ±SEM (n = 5, independent experiments analyzed * p≤0.05, Student's t-test, p>0.05 was considered not significant (n.s)).
Figure 3.
Inhibition of Notch signaling allows for hair cell regeneration in gentamicin damaged cochlea.
A–C: Hair cell phenotype in control (A, A'), gentamicin +DMSO (B, B') and gentamicin +DAPT (C, C') treated cultures after a total of 3 DIV. Atoh1/nGFP expression (green) labels hair cell nuclei and myosin VI immuno-staining marks hair cells (Myo6, red). All images shown are from the cochlear mid apical region. A'–C': higher magnification of A–C. Asterisk in B' mark faint nGFP expression in inner phalangeal cells. Scale bar 50 μm. D: Quantification of hair cell density in mid apex, mid base and base of gentamicin + DMSO (grey) and gentamicin +DAPT (red) treated cochlea cultures after 3 DIV. Data expressed as mean ±SEM (n = 3, cochlea explants from 2 independent experiments; ** p-value <0.005; * p-value ≤0.05). E–J: EdU (green) incorporation in control (E, F), gentamicin +DMSO (G, H) and gentamicin +DAPT (I, J) treated cultures. All images shown are from the cochlear mid apical region. E, G, I: Confocal images of hair cell layer. Myosin VI immuno-staining marks hair cells (Myo6, red). F, H, J: Projections of confocal z-stacks, imaging supporting cell layer. Sox2 immuno-staining (blue) marks supporting cells. No EdU positive hair cell (Myo6, red) or EdU positive supporting cell nuclei (Sox2, green) are observed in control (E, F), gentamicin (G, H), or gentamicin +DAPT (I, J) treated cochlea cultures. White arrows in J mark EdU positive cells at the lateral edge of sensory epithelium. Scale bar 50 μm.
Figure 4.
Newly generated hair cell-like cells mature in vitro.
Hair cell phenotype of untreated (control A, D, G, K) gentamicin +DMSO (B, E, H, L) and gentamicin +DAPT (C, F, I, M) treated cochlear explants. A–C: Parvalbumin immuno-staining (red) and native Atoh1/nGFP (green) expression mark hair cells. After 3 DIV, Atoh1/nGFP positive cells (green nuclei) in gentamicin + DAPT treated (C) cultures express hair cell marker parvalbumin (red). Scale bar 50 μm. D–F: Fluorescently labeled phalloidin (green) visualizes actin. After a total of 3 DIV, gentamicin + DAPT (F) treated cultures have actin-rich (phalloidin, green) membrane protrusions (white arrows) reminiscent of hair cell stereocilia. Scale bar 25 μm. G–M: Prestin immuno-staining (red) selectively marks outer hair cells. After 6 DIV, the majority of Atoh1/nGFP positive cells co-express prestin in gentamicin + DAPT treated cultures (I, M). Note white asterisk labels Atoh1/nGFP negative epithelial cells at the lateral edge of cochlear epithelium, which express prestin at low levels (H, I, L, M). Scale bar 50 μm. N–O: Q-PCR based quantification of mRNA expression levels of hair cell specific genes in hair cell damaged (gentamicin +DMSO) and hair cell damaged and Notch inhibited (gentamicin +DAPT) cochlear epithelia after 3 DIV (N) and 6 DIV (O). Data expressed as mean ± SEM, (n = 3 independent experiments, *p≤0.05, Student's t-test).
Figure 5.
Newly generated hair cell-like cells originate from Prox1 positive supporting cells.
P2 Prox1-CreER; mTomato/mEGFP transgenic control (A–C), gentamicin+ DMSO treated (D–F) and gentamicin +DAPT treated (G–I) cochlea explant cultures after 3 DIV. Note prior to plating tamoxifen injection at stage P0 and P1 permanently converted ∼ 10% of Prox1-CreER expressing supporting cells into membrane EGFP (mEGFP, green) positive cells. Myosin VI immuno-staining marks hair cells (Myo6, red). A–C: In control cultures, mEGFP positive supporting cells (B, C, green) surround myosin VI positive hair cells (A, C, red). D–F: Gentamicin treatment results in severe loss of myosin VI positive hair cells (D, F, red) and result in spread of mGFP positive supporting cells (E, F, green). G–I: In gentamicin and DAPT treated cultures mGFP positive cells (H, I, green) co-express hair cell specific marker myosin VI and acquire hair cell characteristic cell shape (G, I, red). Scale bar 50 μm.
Figure 6.
Newly formed hair cell-like cells have electrophysiological properties reminiscent of hair cells rather than supporting cells.
A: Voltage step protocol as used in (B). From a holding potential of −70 mV, 200 ms voltage steps to values between −100 and 50 mV, in 10 mV increments were applied. B: Supporting cell recording, here from an untreated preparation after 6 DIV. Due to network coupling, supporting cells could not be voltage clamped and exhibited a typical ‘leaky’ response to a voltage step protocol. C: Supporting cells typically show resting membrane potentials that oscillate over a wide voltage range, here over ∼20 mV. D: Voltage step protocol as used in (E) and (F). From a holding potential of −70 mV, a 50 ms prestep to −130 mV was applied, followed by 200 ms voltage steps to values between −100 and 50 mV, in 10 mV increments. E: Recording from an Atoh1/nGFP positive, hair bundle bearing cell in a gentamicin +DAPT treated culture after 6 DIV shows delayed rectifier potassium currents qualitatively similar to control hair cells. F: Outer hair cell recording in an untreated preparation after 6 DIV shows delayed rectifier potassium currents.