Nitrite-exposed embryos were treated with 100 mg/l sodium nitrite from 10 hpf. (A–F) Histological sections showing defective valve development caused by nitrite exposure occurred from 48 hpf. At 36 hpf, the embryos exposed to nitrite exhibited similar histological heart structure (B) to that of control zebrafish, comprising one layer of myocardium and one layer of endocardium (A). At 48 hpf, endocardial cells of control embryos exhibited cuboidal shape in AVC between two chambers (C) but the exposed embryos did not have cuboidal endocardial cells in AVC (D). At 76 hpf, invagination of endocardial cells into cardiac jelly in AVC in control embryos formed superior primitive valve leaflet consisting of multilayer of cells (E); however, no superior primitive valve leaflet (no multilayer cells in the superior part of AVC) was formed in nitrite-exposed embryos (F). (G–N) Cardiac looping in zebrafish embryos shown by the expression of cmlc2 revealing abnormal cardiac looping caused by nitrite exposure occurred as early as 43 hpf. The expression pattern of cmlc2 was not affected by nitrite exposure at 36 hpf (G–H) and 40 hpf (I–J), but slightly abnormal (shown in white dotted curve) at 43 hpf (K–L) and obviously abnormal (shown in white dotted curve) at 48 hpf (M–N). (O–X) Nitrite exposures altered the expressions of molecular makers of valve progenitors at 48 hpf. nppa was not expressed in the AVC (rectangular box) of control embryos (O) but was ectopically expressed in the AVC of nitrite-exposed embryos (P). Compared to control embryos, nitrite exposure significantly decreased or abolished expressions of bmp4 (Q, R), vcana (S, T), notch1b (U, V) and has2 (W, X) in AVC. The number shown in the lower right-hand corner was the number of embryos exhibiting the typical phenotype shown in the panel to the number of embryos totally observed. Black arrow: position of cuboidal endocardial cells; Black arrowhead: position of superior primitive leaflet; White arrow: endocardium; White arrowhead: myocardium.
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