The expression of Bach1 during embryonic development in zebrafish. Bach1 expression was evaluated in zebrafish embryos that had been stained with (a–c) or without (d–f) a Bach1 antibody at 4 h (a, d), 8 h (b, e), and 24 h (c, f) after fertilization. Scale bar: 100 μm.

Bach1 overexpression induces vascular defects in zebrafish embryos. (a) Zebrafish vasculature was evaluated via fluorescent images (C-D) or confocal images (E and F) in normal 32-hpf Tg(fli1:EGFP) zebrafish embryos (A, C and E) and in embryos that had been injected with 200 pg of Bach1 mRNA (B, D and F) at the single-cell stage. Bach1 mRNA injections disrupted formation of the intersegmental vessels (ISV) (arrow) and the dorsal longitudinal anastomotic vessels (DLAV) (asterisks). (A–D) Scale bar: 100 μm. (E and F) Scale bar: 50 μm. (b) Bach1 mRNA levels were evaluated in normal and Bach1-mRNA–injected embryos via quantitative real-time RT-PCR (n = 8; **P < 0.01 versus Control). (c) Embryos with defects in ISV and DLAV were counted and expressed as a percentage of the total number of embryos in each experimental group (n = 50; *P < 0.05 versus Control).

Bach1 suppresses endogenous Wnt/β-catenin signaling and exogenous Wnt8a stimulated VEGF and IL-8 gene expression in zebrafish. (a) Representative fluorescent images of 36-hpf Tg(TOP:GFP) transgenic zebrafish embryos that had been injected with or without Bach1 mRNA. (b) The fluorescence intensity of GFP was quantified and expressed as the as the ratio of measurements Bach1 mRNA and Control group (n = 12; **P < 0.01 versus Control). (c) IL-8, VEGF₁₆₅, and Bach1 mRNA levels were evaluated in 32-hpf Tg(hsp:wnt8a) embryos following Bach1 mRNA injections at the one cell stage and Wnt pathway activation (heat-shock) at 24 hpf (n = 10; *P < 0.05, **P < 0.01 versus Control; ^##P < 0.01 versus Wnt8a).