Figure 4

Figure 4

Direct differentiation of SHF progenitors establishes the zebrafish arterial valve. (A–B″) Representative midline section of immunohistochemistry on Tg(kdrl:GFP) (green) embryo at 54 hpf (A–A″) and 70 hpf (B–B″) for cardiac troponin (white, membrane), DAPI (white, nuclear) (A and B), Islet1/2 (Isl1/2, magenta) (A′ and B′), and Sox9 (cyan) (A″ and B″). (A–A″) At 54 hpf, cells of the distal OFT, including within the arterial valve primordia (yellow) co-express Isl1/2 and Sox9 (n = 19). (B–B″) By 70 hpf, cells within the arterial valve primordia (yellow) have down-regulated Isl1/2 (B′) but maintain expression of Sox9 (B″) defining them as VICs (n = 12). (C–D″) Representative midline sections of lineage tracing of endothelial cells in the OFT of Tg(kdrl:GFP); Tg(kdrl:Cre); Tg(-3.5ubb:loxP-EGFP-loxP-mCherry) embryos (magenta) at 70 hpf (C–C″) and 118 hpf (D–D″). (C–C″) Cells of the arterial valve primordia at 70 hpf (yellow), identified between distal-most myocardium (cyan) and endocardium (green), are not of endocardial origin (n = 16). (D–D″) At 118 hpf following primordia remodelling, there is no recombination observed in the arterial valve leaflets (yellow, arrowheads denote tips of leaflets) (n = 14). (E–F″) Representative midline sections of lineage tracing of neural crest cells in the OFT of Tg(kdrl:GFP); Tg(sox10:iCre, cryaa:DsRed2); Tg(-3.5ubb:loxP-EGFP-loxP-mCherry) embryos (magenta) at 70 hpf (E–E″) and 118 hpf (F–F″). (E–E″) Cells of the arterial valve primordia at 70 hpf (yellow), identified between distal-most myocardium (cyan) and endocardium (green), are not of neural crest origin (n = 12/14). (F–F″) At 118 hpf following primordia remodelling, there is no recombination observed in the arterial valve leaflets (yellow) (n = 8/12, arrowheads denote tips of leaflets). At both stages, recombination is present in the distal tip of the bulbus arteriosus (E′ and F′, asterisk). (G) Summary of expression data from (A–F). V, ventricle; BA, bulbus arteriosus. Scale bars: 10 μm.