BIX01294 treatment decreases H3K9me2 levels in zebrafish neuromasts (NMs). (A) Both the BIX01294 (BIX)-treated group and DMSO control group were morphologically normal following 1 h neomycin treatment. (B1,D1) H3K9me2 immunofluorescence (red) is of relatively high intensity in the control zebrafish NMs, and the H3K9me2 level increased at 48 h after neomycin damage for 1 h. (C1,E1) BIX01294 treatment reduced the H3K9me2 level, and the intensity of H3K9me2 was significantly decreased with the addition of 20 µM BIX01294 for 48 h following 1 h neomycin damage. (B2-E3) Some degree of decreased hair cell (HC; green) regeneration after neomycin treatment for 1 h was also observed after exposure to BIX01294 for 48 h compared to the neomycin treatment controls, while a reduction in HCs was not obvious in the non-neomycin control groups. HCs were labeled with anti-GFP antibody (B2-C2) or green fluorescence (D2-E2), and nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bar = 10 µm. (F) The reduced H3K9me2 level by 20 µM BIX01294 was reconfirmed with western blotting analysis at 48 h after neomycin damage. β-actin was included as the control. Mean ± SEM for three experimental replicates. **p < 0.01.

BIX01294 treatment reduces HC regeneration in the zebrafish lateral line. (A) We treated 5 days post-fertilization (dpf) Tg (brn3c:mGFP) zebrafish with neomycin for 1 h and then treated them for 24 h or 48 h with BIX01294. HCs in these fish are GFP⁺ (green), and nuclei are stained with DAPI (blue). Scale bars = 10 µm. (B) The average number of GFP⁺ cells per NM in larvae treated with or without 20 µM BIX01294 for 24 h or 48 h after neomycin damage for 1 h. Bars are mean ± SEM, and n = total number of embryos. **p < 0.001. (C) We treated 5 dpf wild-type zebrafish with neomycin for 1 h and then treated them for 24 h or 48 h with BIX01294, and HC-specific Myosin-VI immunostaining (red) was used to label HCs. Nuclei are stained with DAPI (blue). Scale bars = 10 µm. (D) The average number of Myosin-VI⁺ cells per NM in larvae treated with or without 20 µM BIX01294 for 24 h or 48 h after neomycin damage for 1 h. Bars are mean ± SEM, and n = total number of embryos. **p < 0.001.

BIX01294 treatment reduces FM1-43FX⁺ cells in the course of regeneration. (A) The functional HCs of 5 dpf wild-type larvae are stained with FM1-43FX (red) with or without exposure to BIX01294 for 24 h or 48 h after neomycin damage for 1 h. Nuclei are stained with DAPI (blue). Scale bars = 10 µm. (B) The average number of FM1-43FX⁺ cells per NM in larvae treated with or without 20 µM BIX01294 for 24 h or 48 h after neomycin damage. Bars are mean ± SEM, and n = total number of embryos. **p < 0.001.

Inhibition of G9a/GLP with UNC0638 also decreases the regeneration of HCs in lateral line NMs. (A) We treated 5 dpf Tg (brn3c:mGFP) zebrafish with neomycin for 1 h and then treated them for 24 h or 48 h with 10 µM UNC0638. HCs in these fish are labeled with anti-GFP antibody (green), and nuclei are stained with DAPI (blue). Scale bars = 10 µm. (B) The average number of GFP⁺ cells per NM in larvae treated with or without 10 µM UNC0638 for 24 h or 48 h after neomycin damage for 1 h. Bars are mean ± SEM, and n = total number of embryos. **p < 0.001. (C) The functional HCs are stained with FM1-43FX (red). Nuclei are stained with DAPI (blue). Scale bars = 10 µm. (D) The average number of FM1-43FX⁺ cells per NM in larvae treated with or without 10 µM UNC0638 for 24 h or 48 h after neomycin damage. Bars are mean ± SEM, and n = total number of embryos. **p < 0.001. (E) HCs in the lateral line NMs were stained with Myosin-VI (red). Nuclei are stained with DAPI. Scale bars = 10 µm. (F) The average number of Myosin-VI⁺ cells per NM in larvae treated with or without 10 µM UNC0638 for 24 h or 48 h after neomycin damage for 1 h. Bars are mean ± SEM, and n = total number of embryos. **p < 0.001.

BIX01294 significantly suppresses cell proliferation and induces reduced supporting cell (SC) production. We treated larvae at 5 dpf with neomycin and monitored cell proliferation with or without BIX01294 over the next 2 days. (A1-D2) The BrdU antibody shows dividing cells (green) in the NMs of the zebrafish lateral line, and HCs are stained with Myosin-VI (red). Scale bars = 10 µm. (A3-D4) Lateral line SCs are stained with Sox2 antibody (red), and nuclei are stained with DAPI (blue). The BrdU antibody shows dividing cells (green) in the NMs of zebrafish. Scale bars = 10 µm. (E) BrdU⁺ cells were counted in control and BIX01294-treated larvae at 24 h and 48 h after neomycin damage for 1 h. (F) Quantification of the ratio of BrdU⁺ HCs in control and inhibitor-treated larvae at 24 h and 48 h after neomycin incubation for 1 h. (G,H) The number of SCs and quantification of the ratio of BrdU⁺ SCs in control and BIX01294-treated larvae at 24 h and 48 h after neomycin incubation for 1 h. Bars are mean ± SD, and n = total number of embryos. **p < 0.001.

BIX01294 significantly down-regulates the Wnt and Fgf signaling pathways. Localization of markers of Wnt (wnt4a, wnt10a, and axin2) and Fgf signaling (fgf3, fgf10a, fgfr1, pea3, and atoh1a) with whole mount in situ hybridization in 6 dpf control embryos and BIX01294-treated embryos with or without neomycin treatment for 1 h. Expression of wnt4a, wnt10a, axin2, fgf3, fgf10a, fgfr1, pea3, and atoh1a was significantly down-regulated during the neomycin-induced regeneration period after BIX01294 treatment for 24 h. The ratios of representative images to total images were labeled, respectively.

The decreased HC regeneration after BIX01294 treatment following neomycin damage can be recued with the addition of bFGF and OF 6-bromoindirubin-3-oxime (BIO). (A) Co-treatment of bFGF and BIO with BIX01294 after neomycin damage increases the number of HCs (Myosin-VI⁺, red) compared to the BIX01294-only treatment. Nuclei are stained with DAPI. Scale bars = 10 µm. (B) The average number of Myosin-VI⁺ cells per NM in larvae treated with or without BIX01294 for 48 h after neomycin damage for 1 h and the average number of Myosin-VI⁺ cells per NM after the addition of bFGF and BIO to controls or BIX01294-treated larvae. Bars are mean ± SEM, and n = total number of embryos. **p < 0.001.

Impact of BIX01294 treatment on apoptosis. Cleaved caspase-3 staining (red) is used to label cell death. HCs are labeled with anti-GFP antibody (green), and nuclei are stained with DAPI (blue). Scale bars = 10 µm.

The representative images for fgf3, wnt4a, wnt10a, atoh1 and pea3.