Figure 5

Effects of 1-azakenpaullone are mediated via Wnt/β-catenin pathway. (A) Quantification of fluorescent intensity from an anti-dephosphorylated (active) β-catenin antibody in retinal sections from fish treated with DMSO (vehicle control) or 1-azakenpaullone from 24 to 48 hpf. Average intensity was calculated from approximately 750 μm² area either in central retina or peripheral retina. DMSO-treated controls show significantly more active β-catenin in peripheral retina compared to central retina, while fish treated with 1-azakenpaullone have elevated levels of active β-catenin in central retina, comparable to that in control-treated peripheral retina. (B-E) Dominant-negative TCF3 suppresses the expansion of the CMZ induced by GSK inhibition. Tg(hsp70:ΔTCF3-GFP)^w26 zebrafish embryos, which express a dominant-negative TCF3 (dnTCF) under control of the heat shock promoter, and their wild-type siblings were heat shocked at 36 hpf for 1 h at 39.5°C and then treated with either 0.025% DMSO or 2.5 μM 1-azakenpaullone. Fish were given a pulse of BrdU 2 h prior to fixation at 3 dpf. (B) Wild-type siblings treated with DMSO show a normal CMZ, with BrdU-positive and PCNA-positive cells at the retinal margin. (C) DMSO-treated fish expressing dnTCF showed a decrease in the number of cells at the retinal margin that were BrdU/PCNA immunoreactive. (D) In wild-type sibling fish treated with 1-azakenpaullone, there is a dramatic expansion of the proliferative progenitors marked with PCNA and BrdU. (E) Transgenic fish expressing dnTCF and treated with 2.5 μM 1-azakenpaullone show no expansion of the CMZ, with few dividing cells at the retinal margin, indicating that dnTCF suppresses the 1-azakenpaullone induced expansion of the progenitor pool. Scale bar = 100 μm.