(A) 3D reconstruction of the heart tube at E8.5i, aligned with the notochord vertical (green), showing the axis of the myocardial tube (red) and the last tube section before the bifurcation (dotted black line) used for the measurement of the position of the venous pole. The distance to the notochord (double black arrow) was measured after projection onto the frontal plane. (B) Displacement of the venous pole during the looping process. Positive values indicate a leftward deviation, which we take as significant when the null value (0) lies outside the 95% confidence interval (CI) at E8.5g-h and E8.5i, and outside the 99% confidence interval at E8.5i. Means and standard deviations are shown, with n = 3 at E8.5e-h and 4 at E8.5i-j. (C1–D1) Brightfield images (ventral view) of embryos at the indicated stage, showing the symmetrical DiI and DiO labelling in the right (white arrow) and left (yellow arrow) posterior heart field, respectively. The contour of the heart tube is outlined (dotted white line). (C2–D2) Fluorescent signal on brightfield images (dorsal view) of the isolated heart after 24 hr of embryo culture, in which the labelled regions deriving from the right (white arrows) and left (yellow arrows) injections are visible. The boundary of the heart tube is indicated (dashed white line), showing that both labelled regions are within the heart tube in C2, whereas this is the case only for the right label in D2. (E) Quantification of left-right differences in the ingression of cells from the posterior heart field. At each stage of labelling, the number of embryos, in which the right (white) or left (yellow) label had progressed further into the heart tube after 24 hr of culture, is indicated. Embryos in which no obvious asymmetry between the right and left was observed were equally distributed in the two other categories. A significant difference is observed at E8.5g only (chi-square test with Yates’ correction, n = 11(E8.5d), 10(E8.5e), 17(E8.5f), 24(E8.5g), 9(E8.5h), p-value=1(E8.5d), 0.21(E8.5e), 0.81(E8.5f), <0.01(**, E8.5g), 1(E8.5h). Raw counts are presented in the table below. dRA: dorsal right atrium; dLA: dorsal left atrium; OFT: outflow tract. (F) Whole mount immunostaining of an embryo at E8.5g, showing the second heart field domains, marked by Isl1 (green), used for the quantification of mitotic cells (red), labelled with phosphorylated histone H3 (P–H3). The limit between the anterior (AHF) and posterior (PHF) heart fields is taken as the middle between the heart poles (see Figures 3D–E and 4C). The midline is highlighted by a vertical dotted line. The left headfold (hf) was cut and used as a landmark to orient samples. Examples of optic tissue slices (1, 2) are shown on the right, at the level indicated by the horizontal lines, with arrows (white, right cells, yellow, left cells) pointing to mitotic second heart field cells. Asterisks indicate unspecific bright staining (red asterisk), in yolk sac edges and the foregut cavity, which was removed in the left panel (white asterisk) for better visualisation of the second heart field. (G) Quantification of the number of mitotic cells in the second heart field, shown for individual embryos (n = 2, upper and lower lanes), indicating a significant increase on the right side (chi-square test, p-value=0.02 for the upper embryo and p=0.002 for the lower embryo), with no difference between the anterior and posterior domains. Scale bars: 100 µm.