Fig. 5

HDAC inhibitors inhibit neuromast cell proliferation. (a) Confocal images of neuromasts from a 5dpf control and from 5dpf embryos treated with 0.1μM TSA, 100μM VPA or 10μM MS-275 that have been labeled by BrdU to detect proliferating cells. The number of BrdU-labeled cells is much larger in control than in HDAC inhibitor-treated embryos. Red spots represent BrdU signal. HCs are labeled with an antibody against myosin VI (green). Scale bar=10μm. (b) Quantification of replicating cells in the neuromasts for each experimental condition. The BrdU index is higher in the control relative to the HDAC inhibitor-treated fish. The proliferating cells of the first four neuromasts along the body, L1 to L4, were recorded on one side of each fish. Bars are mean±s.e.m. and n=36 neuromasts per condition. **P<0.001. (c) Confocal images of neuromasts from a 5dpf control and from 5dpf embryos treated with 0.1μM TSA, 100μM VPA or 10μM MS-275. HDAC inhibitor treatment decreased the numbers of both BrdU-positive HCs and BrdU-positive SCs in neuromasts. White arrows indicate HCs that were derived from proliferating SCs. Scale bar=10μm. (d, e) Quantification of replicating cells co-labeled with Myosin VI or Sox2 per neuromast (NM) in control and HDAC inhibitor-treated larvae. Bars are mean±s.e.m. and n=36 neuromasts per condition. **P<0.001. (f) Real-time RT–PCR analysis showed that TSA treatment increases the mRNA expression of p21, p27 and p53. The bars represent the means±s.d. of the ratios of p21, p27 and p53 to GAPDH in the real-time RT–PCR data. *P<0.05.