Heart position and heart tube looping direction in the developing zebrafish. Whole-mount in situ hybridization with BMP4 alone. Embryos, at the h.p.f. indicated, are viewed dorsally, with rostral to the top. Bar 200 μm. (A) Heart primordia are present in the lateral plate mesoderm (thick arrows) at 17 h.p.f. Thin arrows denote olfactory regions. (B) At the onset of heart tube assembly at 19 h.p.f., BMP4-expressing cells form a shallow cylinder (arrow), oriented parallel to the dorsal–ventral axis. (C) By 22 h.p.f., the heart tube cylinder (arrowhead) has become oriented in a nearly left–right direction. (D, E) At 28–30 h.p.f., the rostral end of the heart tube (open arrows) continues to migrate back toward the midline. Closed arrow denotes visceral arch. (F) By 48 h.p.f., the heart tube (outlined by the dots) has clearly looped, with the ventricle (portion between the white arrows and the black arrows) lying to the right of the atrium (portion between arrowheads and white arrows).

(A) Method of measuring heart tube angle at 24 h.p.f., illustrated in three flh homozygotes. The examples shown have heart angles of -20°, 0°, and +40°. a, atrium; e, eye; v, ventricle. Arrowheads denote the left and right first aortic arch. Bar 200 μm. (B) Heart tube angle (in degrees) as a function of h.p.f. in brs homozygotes. The embryonic midline is denoted as 0°. The center (origin) of the polar coordinate plot is the site of the caudal end of the heart tube.

flh homozygote embryos show lack of coupling of initial heart tube position and gut chirality. Whole-mount in situ hybridization with zebrafish GATA6 probe which visualizes both the heart tube position and the liver primordium at 30 h.p.f. All embryos are viewed dorsally, with rostral to the top. Liver primordia in B–F are shown by arrows. (A, B, D, and E) The same magnification; (C and F) slightly larger magnification. Bars 200 μm. (A) Normal pattern with heart on the left and the liver primordium on the left. (B) flh embryo with a wild-type pattern. (C) flh embryo with heart on left and duplicated liver primordia. (D) flh embryo with heart on right and liver on left. (E) flh embryo with heart on right and a midline liver primordium. (F) flh embryo with heart on right and duplicated liver primordia.

Zebrafish claudin7 marks the newly looping distal foregut (open arrows), seen in brs embryos viewed dorsally (A) and from a more lateral perspective (B). (C) An example of a ntl homozygote scored while live at the onset of heart contraction as having a left-sided heart; the foregut is midline. din embryos scored as having a left-sided initial heart position display a left-sided foregut (open arrows) at 36 h.p.f. (D) while those with a right-sided initial heart position have a right-sided gut (open arrows) at 36 h.p.f. (E). Finally, similar findings are observed in cyc^b16 embryos; embryos with left-sided hearts go on to have left-sided foregut (open arrows) seen dorsally (F) and from a posterior oblique view (G), while embryos with right-sided hearts develop a right-sided gut (H). (I–P) Randomization of initial heart tube position is correlated with a failure to correctly express antivin and Pitx2M. antivin is expressed in the left lateral plate mesoderm (open arrow) and left diencephalon (closed arrow) beginning at 19 h.p.f. (I) and continuing at 21 h.p.f. (J) in brs embryos. antivin expression is bilateral in embryos homozygous for ntl (K, L) and absent in embryos homozygous for din (M). Slightly later, Pitx2 is also expressed in left lateral plate mesoderm in brs (N), but not in din (O), or spt (P).