fli-1 and Nkx-2.5 expression in heart precursors. Dorsal views of whole-mounted zebrafish embryos after in situ hybridization, anterior to the left in A–C and to the top in D–F. (A–C) fli-1 expression in precardiac cells at 10 somites (10 s), in bilateral condensations lateral to the midbrain. Expression becomes progressively expanded along the AP axis, extending more posteriorly on the left side (compare arrowheads in B) in the forming heart, flanked anteriorly and posteriorly by narrow bands of expression in the head mesoderm. (D–F) Nkx-2.5 is expressed in the heart at the same stage as fli-1 in bilateral heart fields. (D) Nkx-2.5 expression becomes broader on the left side of the midline at 20 s, as does fli-1 (compare arrowheads in B and D). (E, F) Expression of Nkx-2.5 (E), largely confined to the heart tube, and fli-1 (F) asymmetrically in the tube and in surrounding mesoderm, beneath the pharyngeal arches. Scale bars, 100 μm.

Expression of DM-GRASP protein on the left and rtk2 mRNA on the right in endodermal precursors of the visceral organs. Dorsal views of whole-mounted embryos, anterior to the left. (A) zn-5 immunoreactive cells (brown) are found in both the atrium and ventricle of the heart tube, the endodermal lining of the pharyngeal arches, as well as a cluster of 15–20 cells lying to the left of the notochord (arrow). (B) Higher magnification view of zn-5-labeled cells on the left side just posterior to the pharyngeal arches at the level of the second somite (arrow). (C) rtk2 expression (arrow) is found in a cluster of 5–10 cells lying ventrally and to the right of the midline at the level of somite 3. a, atrium; e, eye; fp, floor plate; n, notochord; pa, pharyngeal arch; s1–s3, somites 1–3; v, ventricle. Scale bars, 100 μm.

Ectopic shh reverses or disrupts gene expression in cardiac precursors and visceral precursors. Dorsal views of whole-mounted embryos, anterior to the left. (A) Uninjected wild-type control showing BMP4 expression on the left side of the midline in the heart tube. (B) Embryo injected with shh + GFP mRNA that localized to the right side, as determined by staining with anti-GFP (brown), which is easily visible in the right otic capsule (arrow) and right eye. (C) Uninjected control showing distribution of rtk2 mRNA on the right side of the gut. (D) Reversal in rtk2 expression following injection of shh + GFP on the right side. (E) DM-GRASP in the differentiated heart (arrow) and gut (arrowhead) on the left side. (F) Injected embryo with heart and gut on the right. (G, H) Higher magnification views of reversals in large (arrow) and small (arrowhead) clusters of DM-GRASP expressing cells in the gut. Scale bars, 100 μm.

Ectopic BMP signaling reverses LR asymmetry in heart precursors. Dorsal view of whole-mounted embryos, anterior to the left, except F which is an anterior view with dorsal to the top. (A, B) In situ hybridization showing assymetric expression of fli-1 in the putative endocardium (arrow) in uninjected control and reversed in a BMP4-injected embryo. (C, D) BMP4 expression itself is expressed with reversed asymmetry relative to the midline (white line) following BMP2 injection. (E, F) DM-GRASP expression in an uninjected control (E) and a BMP4-injected embryo (F). Scale bar, 100 μm.

Midline mutants disrupt heart and gut asymmetries. Dorsal views of whole-mounted wild-type (A–C), flh (D–F), and oep (G–I) mutant embryos, anterior to the top, except middle panels which are anterior views with dorsal to the top. (A, D, G) BMP4 expression in the primitive heart tube. (B, E, H) DM-GRASP expression in the ventricle (arrows). (C, F, I) DM-GRASP expression in the hepatic diverticulum (arrows). Scale bars, 100 μm.