The Tg(myo6b:hDTR) zebrafish. (A) Schematic representation of the human diphtheria toxin receptor (hDTR) construct driven by the hair cell-specific myo6b promoter and representation of the hair cell-specific ablation approach. Diphtheria toxin (DT) is internalized in cells specifically expressing the human version of the DT receptor, triggering cell death. (B) qRT-PCR analysis on untreated Tg(myo6b:hDTR) zebrafish saccule and utricle showing expression of hDTR and myo6b relative to wild-type (WT) animals. The values are represented as the mean ± SEM from three independent samples. (C) Schematic depicts a lateral view of a whole zebrafish larva with neuromasts (gray circles). The P1 neuromast examined for imaging is indicated by magenta fill. In situ HCR using probes targeting hDTR and myo6b in hair cells of a single P1 neuromast of 3 dpf WT and Tg(myo6b:hDTR) embryos is shown. Brightness and contrast adjusted 50 and 25%, respectively. Scale bar 20 μm. (D) Schematic depicts the left ear of a larval zebrafish with anterior macula indicated by magenta fill. Lateral views of inner ears are shown following HCR in situ with probes targeting hDTR and myo6b in 3 dpf WT and Tg(myo6b:hDTR) embryos. Brightness and contrast adjusted 65 and 35%, respectively. Scale bar 20 μm.

Tg(myo6b:hDTR) larval zebrafish show hair cell loss and regeneration in lateral line neuromasts after in vivo DT treatment. (A) Schematic representation of the larval DT exposure treatment. (B) Wild-type (WT) and Tg(myo6b:hDTR) (hDTR) larvae were exposed to three concentrations of DT (0.5, 1, and 1.5 μg/mL) for various durations (3, 6, 8, 10, and 12 h). Neuromast viability was monitored daily by YO-PRO-1 labeling 0 to 72 h post-incubation. Shown is the quantification of YO-PRO-1 labeled hair cells. The values are represented as the mean ± SEM from eight fish. (C) Regeneration time-course showing P1–P4 over three days of recovery. Larvae were exposed to 1 μg/mL of DT for 6 h. Scale bar 100 μm.

Adult Tg(myo6b:hDTR) injected fish exhibit spatial disorientation and impaired balance. (A) Injected WT fish (WT), (top) and Tg(myo6b:hDTR) (hDTR), (bottom) with 10 ng of DT 3 days-post injection. (B) Saccule and utricle from untreated (–DT) or treated (+DT) WT and Tg(myo6b:hDTR) fish. Saccule and utricle from treated (+DT) WT and Tg(myo6b:hDTR) fish were isolated 3 days post-DT. Scale bar 100 μm.

Tg(myo6b:hDTR) adult zebrafish hair cell loss and regeneration in the inner ear after DT treatment. Saccule and utricle were isolated at specified timepoints following DT administration in Tg(myo6b:hDTR) fish. Close examination of hair cells with phalloidin (green channel) and hair cell bodies with anti-myosin VI/VIIa (red channel). Scale bar 100 μm. For qualitative purposes, brightness was increased 10% and contrast by 20% across all images.

Tg(myo6b:hDTR) adult zebrafish hair cell loss and regeneration in the inner ear after DT treatment. (A) Tg(myo6b:hDTR) saccule and utricle used for quantification were isolated at specified timepoints following DT administration and hair cells were labeled with phalloidin (green channel). Scale bar 100 μm. (B) Quantification of phalloidin positive hair cell number after DT injection. Error bars demonstrate the mean ± SEM.

Figure 6. Diphtheria toxin induced hair cell death is primarily due to apoptosis. (A) Saccule and utricle from WT and Tg(myo6b:hDTR) (hDTR) inner ear tissues 2 days post-DT. Hair cells are labeled with phalloidin (green channel) and cleaved caspase-3 (red channel) positive cells were detected 2 days post-DT. (B) Quantification of cleaved caspase-3 positive cells in saccule and utricle dissected on days 1–5 following DT injection. Scale bar 100 μm. For qualitative purposes, brightness and contrast increased by 20%.