Expression of Myc and Fgf pathway genes during HC regeneration in zebrafish neuromasts by in situ hybridization.

mycb (A-D), fgf10a (E-H), fgf3 (I-L), fgfr1a (M-O), fgfr2 (P-R) expression were shown in the lateral line neuromast L1 from 5-dpf zebrafish at different time points following neomycin treatment (e.g. Neomycin-6hr, 6 hrs after neomycin treatment). Ctr, untreated fish larvae. Dotted blue lines marked the boundary of neuromasts. Bold dotted lines marked the expression areas (M,O,P,R). (S) An illustration of a neuromast demarcated by differential fgfr expression patterns, including HC region, fgfr1a(+) only region, fgfr1a/r2(+) region and fgfr1a(-) region. A-P, anterior-posterior; D-V, dorsal-ventral. Scale bars: 10 μm.

Myc inhibition suppresses HC regeneration in zebrafish neuromasts.

(A) HCS1-labeled HC number was significantly reduced after treatment by c-MYC inhibitor 10058F4 that was dose-dependent. ***p<0.001. n = 20 larvae for each group. (B) A significant reduction in HC number was observed after the treatment by Int-H1-S6A, F8A, a peptide inhibitor of c-MYC, which was dose-dependent. ***p<0.001. n = 15 larvae for each group. Three independent experiments were performed for each comparison study with similar results. (C-H) Inhibitor Int-H1-S6A, F8A blocked the expression of c-myc target gene odc1. In situ hybridization showed up-regulation of odc1 expression in the neuromast 3 or 6 hrs after neomycin treatment (D, G); whereas Int-H1-S6A, F8A markedly suppressed odc1 expression (E, H). Scale bars: 10 μm.

Inhibition of c-Myc blocked proliferation and down-regulated vimentin during HC regeneration.

(A) A significant reduction in the number of proliferating neuromast cells (BrdU⁺) was seen 18 or 24 hrs after c-MYC inhibitor Int-H1-S6A, F8A (Myc-pep, 100 nM) treatment. Ctr, time-matched control fish without neomycin treatment. (B) c-MYC peptide inhibitor mainly blocked proliferation-derived HCs (HC-BrdU⁺) at 72 hrs. ***p<0.001; **p<0.01. A and B, n = 15 for each group. Two independent experiments were performed with similar results. (C-H) In situ hybridization of atoh1a on 5-dpf zebrafish neuromasts 18 (C-E) or 24 hrs (F-H) after neomycin treatment, with either Myc-pep or DMSO in the media. Ctr, time-matched control fish without neomycin treatment. atoh1a up-regulation by neomycin treatment was blocked in the Myc-pep treatment group (D-E, G-H). (I-Q) Vimentin was down-regulated by c-Myc during HC regeneration. 5-dpf neuromasts were labeled with vimentin and DAPI 18 hrs after neomycin treatment, with either Myc-pep (O-Q) or DMSO (L-N) in the media. No Trt, time-matched control fish without neomycin treatment (I-K). Scale bars: 10 μm.

Inhibition of Fgf signaling suppresses HC regeneration.

(A) A significant reduction in the number of HCs (HCS1⁺) regenerated after SU5402 treatment that was dose-dependent. Ctr, time-matched fish without neomycin treatment. (B) A significant reduction in the number of HCs regenerated in hsp70l:dn-fgfr1:GFP (Hsp) zebrafish neuromasts at 32°C or 37°C. AB: wild type AB fish. Ctr, time-matched transgenic fish without neomycin treatment. (C) A significant reduction in proliferation and transdifferentiation derived HCs after 20 μM SU5402 treatment for 72 hrs. ***p<0.001; **p<0.01. For all statistical analysis, n = 15 for each group. Three (for A) and two (for B,C) independent experiments were performed with similar results. (D) Fgf targets etv4 and spry4 were down-regulated by SU5402 (20 μM) in L1 neuromasts during HC regeneration. Scale bars: 10 μm.

Neuromast SCs with different capacities in HC regeneration and proliferation.

Hybrid larvae of pou4f3:GFP and ET20 fish ablated in HCs only (A,D), HC/fgfr1a(+) SCs (B,E), or HC/fgfr1a(-) SCs (C,F) were stained with HCS1 (A-C) or BrdU (D-F) antibody, to illustrate proliferating (BrdU⁺) and regenerated HCs (HCS1⁺). (G) Quantification showed that only HC/fgfr1a(+) SC ablation significantly reduced the regenerated HCs. (H) Quantification showed that HC/fgfr1a(+) SC ablation increased the number of proliferating cells; whereas HC/fgfr1a(-) SC ablation reduced the proliferating cells. (G,H) ***p<0.001; *p<0.05. n = 12 in each group. Scale bars: 10 μm.

c-Myc and Fgf pathway inhibitors block HC regeneration.

5-dpf zebrafish larvae were treated with different inhibitors or DMSO after neomycin-induced HC death. 72 hrs later, the HCs were labeled with Yo-Pro-1. The pictures of whole fish (left) and enlarged neuromast L1 (right) showed the reduction of HC number in the inhibitor-treated neuromasts. Scale bars: left panel, 50 μm; right panel, 10 μm.

The Myc inhibitor does not induce apoptosis.

5-dpf zebrafish larvae with neomycin treatment were then treated with or without 100 nM c-MYC inhibitor Int-H1-S6A, F8A for 72 hrs (G-I, J-L). Larvae without neomycin and inhibitor treatment (No Trt) and larvae collected 1 hr after neomycin treatment were used as controls. The fish were stained with HCS1 antibody (A,D,G,J) to label HCs and TUNEL assay (B,E,H,K) to measure apoptosis. No significant difference in apoptosis signal was observed between inhibitor-treated and non-treated fish (TUNEL⁺ cells per neuromast: 0.4 ± 0.2 for No Trt, n = 14; 0.4 ± 0.1 for Neo 72hr, n = 15; 0.6 ± 0.2 for Neo/Myc-pep 72hr, n = 15). All TUNEL signals were from outside of the neuromast (I,L). In the positive control (D-F), a significant increase in the TUNEL⁺ cells were seen inside the neuromast. Scale bars: 10 μm

Blockade of Fgf signaling in heat shocked transgenic fish.

In situ hybridization showed Fgf targets etv4 (A,B) and spry4 (C,D) were relatively down-regulated in hsp70l:dn-fgfr1:GFP (Hsp) zebrafish neuromasts at 37°C compared to control. Scale bars: 10 μm

Laser ablation of HCs and SCs in zebrafish neuromasts.

Hybrid larvae of pou4f3:GFP and ET20 fish were used to ablate HCs alone (A,B), and HC/fgfr1a(+) SCs (C-D), or HC/fgfr1a(-) SCs (E-F). Neuromasts before (A,C,E) and after ablation (B,D,F) were shown. The red circles are the target areas for ablation. A-P, anterior-posterior; D-V, dorsal-ventral. Scale bar, 10 μm. (G) In situ hybridization of fgfr1a confirmed the ablation of fgfr1a(+) cells in HC/fgfr1a(+) ablation group, where fgfr1a is undetectable; whereas in HCs or HC/fgfr1a(-) ablation group, fgfr1a signal is still present. Similar ablation did not change fgfr2 signal. Ctr, unablated neuromasts labeled with fgfr1a. Scale bars: 10 μm.