A second rotation in the heart at 28–30 hpf converts the D/V axis back to the L/R axis. Cells are labeled with cmlc2::Dendra (green) and cmlc2::dsRED (red). A--H are dorsal views at 18–20 hpf; A2–H2 are ventral views at 48 hpf. White lines indicate the embryonic midline. WT embryos with left (A) or right (B) GFP at 20 hpf display a left-directed rotation of the heart at 28–30 hpf (A1 and B1). This rotation positions the original left cells from the dorsal side of the heart tube to the left of the looped heart (A2 and A3) where they contribute to the outer atrial (A2, arrowhead) chamber curvature. The original right cells are repositioned from the ventral side of the linear heart to the right of the looped heart at 48 hpf (B2 and B3) where they contribute to the outer curvature of the ventricle (B2, arrow) and the inner curvature of the atrium (B2, arrowhead). swt morphants with left jog and right loop display the same leftward rotation of the heart and contribution of original left cells to the inner ventricular (C2, arrow) and outer atrial (C2, arrowhead) chamber curvatures (C–C3). The asterisk in C marks GFP-expressing cells at 18 hpf that are to the right of midline. The asterisk in C2 marks the likely localization of these cells at 48 hpf on the right side of the looped heart. swt morphants with left GFP at 20 hpf (D) and exhibiting right jog and left loop display a right-directed rotation of the heart at 28–30 hpf (D1). This rotation positions the left, GFP-expressing cells to the left of the 48 hpf heart (D2 and D3) where they consistently contribute to the outer curvature of the ventricle (D2, arrow) and the inner curvature of the atrium (D2, arrowhead). spaw morphants with left jog and right loop display the same direction of heart-tube rotation and positioning of original left and right cardiac cells at 48 hpf as both WT and swt embryos with left jog and right loop (E–E3). The asterisk in E marks GFP-expressing cells at 18 hpf that are to the right of midline. The asterisk in E2 marks the likely localization of these cells at 48 hpf on the right side of the looped heart. spaw morphants with right jog and left loop show the same reversed direction of heart-tube rotation as swt morphants with this phenotype (F1). In this embryo, the original right cells (F) are repositioned to the right of the heart at 48 hpf (F2 and F3) and contribute to the inner curvature of the ventricle (F2, arrow) and the outer curvature of the atrium (F2, arrowhead). The same reversed direction of heart-tube rotation is observed in spaw morphants with right jog and right loop (G1). In this embryo, the original left cardiac cells (G) are positioned to the left of the heart at 48 hpf (G2 and G3). These cells are localized to the outer curvature of the ventricle (G2, arrow) and the inner curvature of the atrium (G2, arrowhead). spaw morphants lacking directional jog also lack a directed rotation of the heart tube at 28–30 hpf (H1). spaw morphants with original right expression of GFP at 20 hpf (H) display a maintenance of these cells on the right of the heart at 24–28 hpf and at 48 hpf (H2 and H3). In this embryo, the right GFP-expressing cells are localized to the inner curvature of the ventricle (H2, arrow) and the outer curvature of the atrium (H2, arrowhead). L, left; R, right; N, no; D, dorsal; V, ventral.