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Fig. 3 Liver vasculogenesis. (A-C) Left-lateral confocal live images of liver of LiPan/Tg(fli1:EGFP)y1 larva at 50 hpf. Discrete ECs frame outer layers of the liver bud (A-C) while no ECs are present in the inner layer (D-F). Panels A and D are images under a GFP filter, panels B and E under a RFP filter, and panels C and F are combinations of GFP and RFP images. (G, H) Left-lateral confocal live images of LiPan/Tg(fli1:EGFP)y1 liver at 55 hpf: external liver layers (G) and all liver layers (H). ECs establish contact and enter superficial hepatocytes layer. (I) Hematoxylin and eosin (H&E) staining of a cross section of an embryo at 55 hpf to show tightly packed hepatocytes inside the liver. (J, K, M, N) Confocal sections of internal liver layers at 58 hpf (J), 70 hpf (K), 84 hpf (M) and 120 hpf (N). At 58 hpf, RFP negative areas in the innermost liver layers (arrowhead) and a daisy pattern of hepatocytes are formed around this areas (J). At 70 hpf, the daisy pattern of hepatocytes around RFP negative areas extended to the superficial layers and ECs surround the daisy clusters of hepatocytes from outside (K). In some cases, ECs are inside of daisy clusters of hepatocytes (M). By 120 hpf, the size of sinusoids significantly increases (N). (L, O) H&E staining of cross sections of embryos at 70 hpf (L) and 120 hpf (O). Note a daisy pattern of hepatocytes in (L) and erythrocytes with pink cytoplasm in liver sinusoids (O). Abbreviations: dph, daisy pattern of hepatocytes; ec, endothelial cells; e, erythrocyte; h, hepatocyte; in, intestine; nt, notochord; L, liver; s, sinusoid; y, yolk. In all whole mount images anterior is towards the left. Scale bars represent 625 μm except for Panels (I, L, O), where the scale bars are 125 μm.