Analysis of Pbx expression during zebrafish cardiac development. (A–G) Pbx immunostaining at (A–B) 10 s (14 hpf), (C–F) 21 s (19.5 hpf), and (G–H) 48 hpf. (A–B) Pbx expression (magenta) in wild type 10 s embryo co-stained with Hoechst to label nuclei (green). (A) Dorsal view, anterior to the left. The line in (A), approximately at the level of hindbrain rhombomere 3, shows where we took an optical cross-section view for (B). Arrows in (B) show Pbx expression in ALPM cells next to the neural tube (nt in A,B). (C–F) Pbx expression (magenta) in Tg(myl7:EGFP)twu34 (green) 21 s (C–D) control embryo and (E–F) pbx4-/- embryo, at low (C,E) and high (D,F) magnification. Arrow in (D) points to cells co-expressing myl7:EGFP and Pbx. Dorsal view, anterior to the left. (G) Pbx expression (magenta) in Tg(myl7:EGFP)twu34 (green) 48 hpf embryo. Ventral view, anterior to the top. V, ventricle. A, atrium. (H–I) Pbx expression (magenta) in wild-type 48 hpf embryo co-stained for proepicardial pan-cytokeratin expression (green) and myocardial cells (MF20, red), at low (H) and high (I) magnification. V, ventricle. Arrows in (H,I) point to two pan-cytokeratin-expressing cells that do not express Pbx, compared to neighboring cardiomyocytes and pan-cytokeratin/Pbx co-expressing cells (arrowheads, I). Scale bar for (A): 100 µm. Scale bar for (B–D): 50 µm. Scale bar for (H): 10 µm.