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DVDY_23714_sm_SuppFigS1.tif10776KSupp. Fig. S1. DiI was injected into the left side of the secondary heart field (SHF) (A1–3) and the right second pharyngeal arch at stage 14 (B1–4). A: The embryo was exteriorized from the nascent amniotic cavity and rotated to allow DiI to be injected into the left SHF (arrow in A1, 2), which was located in the visceral mesoderm caudal to the second pharyngeal arch (Ph2) (A3). B: DiI was injected into the right second pharyngeal arch (arrowhead in B1, 2), and histological sections were made. Mesenchymal cells that were contiguous with the OFT myocardium were labeled with injected DiI (arrowhead in B3, 4). OFT, outflow tract; OV, otic vesicle; Ph2, second pharyngeal arch.
DVDY_23714_sm_SuppFigS2.tif5450KSupp. Fig. S2. Schematic drawing of the distribution of DiI in all of the examined samples. DiI was injected into the SHF or AHF at stage 14, and then the embryos were reincubated and sacrificed at stage 31. Serial cross-sections were produced and observed using a fluorescent microscope, and DiI-positive regions were plotted on a scheme of the intracardiac structure viewed from the apex. Blue dots, myocardium; Red dots, cushion mesenchyme; LV, left ventricle; LVOT, left ventricular outflow tract; RV, right ventricle; RVOT, right ventricular outflow tract; ph1, first pharyngeal arch; ph2, second pharyngeal arch; SHF, secondary heart field.
DVDY_23714_sm_SuppFigS3.tif4119KSupp. Fig. S3. Mesenchymal cells in the first and second pharyngeal arches expressed Islet1 and Nkx2.5. Stage-14 embryos were fixed with 4% paraformaldehyde in PBS for 1 hr at room temperature and doubly stained with anti-Nkx2.5 (SC8697, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-Islet1 (clone 40.2D6, Developmental Studies Hybridoma Bank, Indianapolis, IN) antibodies followed by secondary antibody mixture (FITC-conjugated donkey anti-mouse IgG and RITC-conjugated donkey anti-goat IgG, Chemicon, Temecula, CA). Mesenchymal cells that were contiguous with the OFT myocardium in the first and second pharyngeal arches expressed Nkx2.5 and Islet1 (arrowheads in A2–4 and B2–4). The visceral mesoderm of the pharyngeal region expressed Nkx2.5 and Islet1 (arrowheads in C2–4). The myocardium of the distal OFT expressed both Nkx2.5 and Islet1 (OFT in A2–4 and B2–4), but the expression of Islet1 was weak in the myocardium of the proximal OFT (OFT in C2). OFT, outflow tract. Scale bar = 50 μm.
DVDY_23714_sm_SuppFigS4.tif12021KSupp. Fig. S4. Heart progenitor cells in the first pharyngeal arches that had been labeled at stage 12 migrated to the ventral wall of the right ventricular OFT (RVOT). DiI was injected into the right first pharyngeal arch (r-ph1) and DiO was injected into the left first pharyngeal arch (l-ph1) at stage 12, and the embryos were sacrificed at stage 31. DiI-labeled cells were found on the right ventral side of the OFT (arrow in A2), and DiO-labeled cells were detected as a sharp band in the ventral wall of the OFT (arrowheads in A3). Intracardiac and histological observations showed that DiI-positive cells were distributed in the ventral wall of the right ventricular OFT (RVOT) close to the aortic valve (arrow in B2 and C2), and DiO-positive cells were seen in the ventral wall of the RVOT close to the pulmonary valve (arrowheads in B3 and C3). A1–4: Ventral view of the stage 31 heart. B1–4: Intracardiac observation viewed from the apex. C1–4: Histological section indicated by line C1 in A1. RA, right atrium; RV, right ventricle; RVOT, right ventricular OFT; LA, left atrium; LV, left ventricle; LVOT, left ventricular OFT.
DVDY_23714_sm_SuppFigS5.tif6549KSupp. Fig. S5. The core mesenchyme of the pharyngeal arch was successfully labeled with DiI. DiI was injected into the core region of the right second pharyngeal arch (Ph2) at stage 14, and then the embryos were sacrificed. Histological sections were produced and then stained with anti-HNK1 antibody. B: The core mesenchyme of the Ph2 was successfully labeled with DiI. HNK1-positive mesenchymal cells, which presumably originated from the neural crest, were distributed in the anterolateral region of the Ph2 (C).

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