Fig. 4

bach2a is necessary for thoracic duct (TD) development in zebrafish larvae.

(A) Confocal images of the TD in 4-dpf control MO- (10 ng), bach2a MO-(3.75 ng), vegfc MO- (10 ng), bach2b MO- (10 ng), or bach2a gRNA-injected Tg(fli1:EGFP)y1 larvae. A white arrow indicates the presence of a TD and a white asterisk, its absence. Scale bar, 20 μm. (B) Percentage of 4-dpf Tg(fli1:EGFP)y1 larvae with an intact TD after injection with control MO (10 ng) or bach2a MO (3.75 ng, nControl MO = 46; nbach2a MO = 52). Error bars, mean ± SEM; *P < 0.0001. (C) Percentage of larvae with a TD after injection with vegfc MO (10 ng, nControl MO = 46; nvegfc MO = 52). Error bars, mean ± SEM; *P < 0.0001. (D) Quantification of bach2b morphants with an intact TD after injection with indicated bach2b MO concentrations (3.75, 5, or 10 ng, nControl MO = 18; nbach2b MO-3.75ng = 30; nbach2b MO-5ng = 30; and nbach2b MO-10ng = 10; P ≥ 0.1356). Error bars, mean ± SEM. (E) Percentage of TD-containing larvae injected with bach2b gRNA (125 ng, nControl MO = 35 nbach2b gRNA = 42; P = 0.5461). Error bars, mean ± SEM. (F) Confocal images of a TD (white arrow) in 4-dpf Tg(fli1:EGFP)y1: homozygous bach2a mutants (bach2amut−/−) derived from bach2amut+/− F2 incross. An asterisk indicates absence of a TD and a white arrow, its presence. Scale bar, 20 μm. (G) Analysis of Tg(fli1:EGFP)y1 4-dpf progeny obtained from F2 bach2a heterozygous intercross. Random selection from the pool of siblings injected at the one-cell stage with control MO (10 ng, nbachmut + Control MO = 50), bach2a MO (3.75 ng, nbach2amut + bach2a MO = 75), bach2b MO (3.75 ng, nbach2amut + bach2b MO = 55), or bach2bgRNA (125 ng, nbach2amut + bach2b gRNA = 137) was found to maintain, after genotyping, the expected Mendelian ratios of inheritance. Error bars, mean ± SEM; *P < 0.0003. (B, C, D, E, G) Wilcoxon rank sum test in panels (B, C, E) and Kruskal–Wallis test in panels (D, G).