2-PCPA treatment inhibits neuromast cell proliferation. (A-B) Effect of 2-PCPA on neuromast cell proliferation. Confocal projections of neuromasts of 5 dpf control (A) and 75 µM 2-PCPA treated larvae (B) that have been labeled by BrdU for proliferating cells. The number of BrdU-labeled cells was much larger in control than in 2-PCPA treated larvae, and BrdU-labeled cells often located in the periphery of the neuromast. Green spots represented BrdU signal. HCs were labeled with FM1-43FX (red) and nuclei were stained with DAPI (blue). (C-D) 2-PCPA treatment decreased the numbers of both newly formed HCs and SCs within neuromasts. Confocal projections of neuromasts of control (C) and 2-PCPA treated larvae (D) at 5 dpf. HCs were labeled with Myosin VIIa (blue) and SCs were stained with Sox2 (red). Green spots represented BrdU signal. White arrows indicated hair cells that were derived from cell proliferation. Scale bar =10 µm. (E) Quantification of replicating cells in neuromasts in the experimental conditions. BrdU index was higher in the control relative to the 2-PCPA treated larvae (n: total number of fish per 2-PCPA dose). (F) Mean Myosin VIIa-expressing cells colabeled with BrdU per neuromast (NM) in control and 2-PCPA treatment larvae. (G) Quantification of replicating cells colabeled with Sox2 per neuromast (NM) under each condition. The labeled cells of the first four neuromasts along the body, L1 to L4, were recorded on one side of each fish. Bars are mean ± SEM (one-way ANOVA *P<0.05, **P<0.001). (H,I) Real-time RT-PCR analysis showed that 2-PCPA treatment increased the expressions of p21 and p27 at mRNA levels. The bars represent the mean ± SEM of ratio of p21 and p27/GAPDH in the real-time RT-PCR data.
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