Arterial valve primordia cells are distinct from smooth muscle cells. (A–D) Representative mRNA in situ hybridization analysis of elastin b (elnb) expression during arterial valve primordia formation. elnb is not expressed at 46 hpf (A) (n = 17) and initiates at 54 hpf (B, arrowhead) (n = 17), and the domain expands between 62 and 70 hpf (C–D) (62 hpf, n = 17; 70 hpf, n = 17). (E–H) Midline resin sections of embryos stained for elastin fibres by Miller’s elastin between 46 and 70 hpf. No elastin fibres are present at 46–54 hpf (E and F) (46 hpf, n = 13; 54 hpf, n = 12). Fibres are present at 62 hpf (G) (n = 11), and by 70 hpf (H, n = 12), the majority of the bulbus arteriosus has elastin fibres, but these are not present in the primordia (arrowheads in F–H). (I–J″) Representative midline resin section of immunohistochemistry on Tg(kdrl:HsHRAS-mCherry) (magenta) embryos incubated with the NO sensor DAF-FM (green) and stained with cardiac troponin (cyan) and DAPI (white) at 54 hpf (I–I″) and 70 hpf (J–J″). At 54 hpf, there is weak, sparse DAF-FM staining of the putative SHF-derived smooth muscle (n = 13/18), and there is no staining in the arterial primordia (yellow region). At 70 hpf (J–J″), DAF-FM staining is present throughout the majority of the bulbus arteriosus, with some cells negative at the distal-most point. There is no staining of the arterial valve primordia (yellow region) (n = 13). (K–L″) Representative midline resin section of immunohistochemistry on embryos incubated with the NO sensor DAF-FM (green) and stained with cardiac troponin (cyan), MLCK (magenta), and DAPI (white) at 54 hpf (K–K″) and 70 hpf (L–L″). As in (I′), at 54 hpf, there is weak, sparse DAF-FM staining of the putative SHF-derived smooth muscle and no staining in the arterial primordia (yellow region) (n = 11/14). In all embryos, MLCK signal is present in the valve primordia and undifferentiated SHF. At 70 hpf (L–L″), MLCK signal is membrane restricted, absent from the arterial primordia and marks the entirety of the bulbus arteriosus (arrowhead), overlapping with DAF-FM staining (n = 14). V, ventricle; BA, bulbus arteriosus. Scale bars: 10 μm.
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