Fig 1.
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.
Fig 2.
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.
Fig 3.
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.
Fig 4.
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.
Fig 5.
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.