Assessment of blood circulation in the developing liver of zebrafish. (A-C) Left lateral 3D confocal sections of liver at the level of sinusoids where blood flow was measured. In the most outer liver layers where the first sinusoids were formed, blood flow was absent at 68 hpf (A). Blood flow was detected in sinusoids of external dorso-lateral part of liver parenchyma at 75 hpf (B) and in all measured sites of liver sinusoids at 120 hpf (C). Red cross, points of measurement at the different focal distance as deep as 70–80 μm from the liver surface; "No" sign, points in sinusoids where no blood flow was detected. The edges of livers are marked by dash lines. Arrows indicate the focused sinusoids for FCS measurement in the picture. (D-F) Confocal projections show three stages of liver vasculogenesis. ECs start to contact the surface layer of hepatocytes in the liver bud at 55 hpf (D), first sinusoids form between external layers of hepatocytes at 75 hpf (E) and have well developed sinusoidal network at 120 hpf (F). Arrows are endothelial cells and sinusoids; arrowheads are clusters of hepatocytes. (G, H) Confocal images demonstrate the sinusoidal network (green) in the liver of 1.5-month-old fish. (H) is a 2x blow-up of the area defined by the white box (G′) to show the sinusoidal network and two rows of hepatocytes between two neighboring sinusoids as indicated by yellow lines. Abbreviations: ec, endothelial cells; in, intestine, L, liver; HPV, hepatic portal vein. In all images anterior is towards the left-hand side. Scale bars are 625 μm in (A-B) and 300 μm in (D-H).
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