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. 2008 Sep 16:8:84.
doi: 10.1186/1471-213X-8-84.

Requirement of vasculogenesis and blood circulation in late stages of liver growth in zebrafish

Svetlana Korzh  1 , Xiufang Pan, Marta Garcia-Lecea, Cecilia Lanny Winata, Xiaotao Pan, Thorsten Wohland, Vladimir Korzh, Zhiyuan Gong
Affiliations

Requirement of vasculogenesis and blood circulation in late stages of liver growth in zebrafish

Svetlana Korzh et al. BMC Dev Biol. .

Abstract

Background: Early events in vertebrate liver development have been the major focus in previous studies, however, late events of liver organogenesis remain poorly understood. Liver vasculogenesis in vertebrates occurs through the interaction of endoderm-derived liver epithelium and mesoderm-derived endothelial cells (ECs). In zebrafish, although it has been found that ECs are not required for liver budding, how and when the spatio-temporal pattern of liver growth is coordinated with ECs remains to be elucidated.

Results: To study the process of liver development and vasculogenesis in vivo, a two-color transgenic zebrafish line Tg(lfabf:dsRed; elaA:EGFP) was generated and named LiPan for liver-specific expression of DsRed RFP and exocrine pancreas-specific expression of GFP. Using the LiPan line, we first followed the dynamic development of liver from live embryos to adult and showed the formation of three distinct yet connected liver lobes during development. The LiPan line was then crossed with Tg(fli1:EGFP)y1 and vascular development in the liver was traced in vivo. Liver vasculogenesis started at 55-58 hpf when ECs first surrounded hepatocytes from the liver bud surface and then invaded the liver to form sinusoids and later the vascular network. Using a novel non-invasive and label-free fluorescence correction spectroscopy, we detected blood circulation in the liver starting at approximately 72 hpf. To analyze the roles of ECs and blood circulation in liver development, both cloche mutants (lacking ECs) and Tnnt2 morphants (no blood circulation) were employed. We found that until 70 hpf liver growth and morphogenesis depended on ECs and nascent sinusoids. After 72 hpf, a functional sinusoidal network was essential for continued liver growth. An absence of blood circulation in Tnnt2 morphants caused defects in liver vasculature and small liver.

Conclusion: There are two phases of liver development in zebrafish, budding and growth. In the growth phase, there are three distinct stages: avascular growth between 50-55 hpf, where ECs are not required; endothelium-dependent growth, where ECs or sinusoids are required for liver growth between 55-72 hpf before blood circulation in liver sinusoids; and circulation-dependent growth, where the circulation is essential to maintain vascular network and to support continued liver growth after 72 hpf.

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Figures

Figure 1
Figure 1
Liver development in LiPan transgenic zebrafish. (A, B) Initial RFP expression in the liver starts at 48–53 hpf (A) and GFP expression in the exocrine pancreas starts at 67–72 hpf (B). (C) Expression of RFP and GFP at 96 hpf. (D) Cross section of a 96 hpf larvae shows RFP-positive liver. GFP-expressing exocrine pancreas is faintly visible (arrow). The dotted line represents the midline of the larva and left (L) and right (R) sides are indicated. (E, F) Expression of RFP and GFP in the larva at 120 hpf: lateral (E) and dorsal view (F). The dotted horizontal line in (F) represents the midline with the right side at the top. The solid vertical line represents the plan of the cross section in (G). (G) A cross section to illustrate morphology of internal organs and expression of transgenes right (R) sides are indicated. (H, I) Lateral view of RFP-expressing liver at 10 dpf (H) and in a 120-hpf larva. The dotted line represents the midline of the larva and left (L) and 15dpf (I). Abbreviations: e, eye; gb, gall bladder; in, intestine; L, liver; LL, left lobe; RL, right lobe; nt, notochord; p, pancreas; pi, principal islet; pg, pigment; s, somite, VL, ventral lobe. In all whole mount images anterior is towards the left. Scale bars, 125 μm.
Figure 2
Figure 2
Liver and pancreas in adult LiPan fish. (A, B) Ventral view of the three liver lobes (red) in the dissected LiPan zebrafish at 1.5 mpf (A) and 4 mpf (B). The three lobes of liver are indicated by LL, left lobe; RL, right lobe; and VL, ventral lobe. The exocrine pancreas (p) is in green. (C, D) lateral view of LiPan adult: the left side (C) and the right side (D). Other abbreviation: in, intestine. Scale bars, 1500 μm.
Figure 3
Figure 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.
Figure 4
Figure 4
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).
Figure 5
Figure 5
Role of endothelia in liver development. (A-F) Left-lateral views: liver morphogenesis in live LiPan/Tg(fli1:EGFP)y1 larvae; wild type (A, D), LiPan/clo-/- mutants (B, E) and LiPan/Tnnt2 morphants (C, F) at 60 hpf (A-C) and 70 hpf (D-F). Dorsal views of the liver region of the same embryos are shown as inserts in each panel and dash lines indicate the midline with the right side at the top. Note that the liver in LiPan/clo-/- mutants is significantly reduced compared to that in controls and Tnnt2 morphants. The pericardial edemas in both clo-/- mutant and tnnt2 morphant are indicated by arrows (B, C, E, F). (G-I) Left-lateral confocal in vivo projections of liver of LiPan/Tg(fli1:EGFP)y1 larvae in wild type (G), clo-/- mutant (H) and Tnnt2 morphant (I) backgrounds. Abbreviations: bd, bile duct; dph, daisy pattern of hepatocytes; ec, endothelial cells; e, ear; h, hepatocytes; l, liver; s, sinusoid. In all images, anterior is towards the left. Scale bars, 125 μm in (A-F) and 625 μm in (G-I).
Figure 6
Figure 6
Role of circulation in liver development. (A-F) Left-lateral views: liver morphogenesis in live LiPan wild type (A, D), LiPan/clo-/- mutants (B, E) and LiPan/Tnnt2 morphants (C, F) at 80 hpf (A-C) and 120 hpf (D-F). Dorsal views of the liver region of the same embryos are shown as inserts in each panel and dash lines indicate the midline with the right side at the top. Livers in clo-/- mutants and Tnnt2 morphants are located more medial, lack the anterio-ventral and posterior expansion, and are significantly reduced in size compared to the livers in wild type sibling. The cardiac edema in both mutants and morphants is indicated by arrow. (G-I) Left-lateral confocal in vivo projections of liver of LiPan/Tg(fli1:EGFP)y1 larvae at 120 hpf in wild type (G), clo-/- mutant (H) and Tnnt2 morphant (I) backgrounds. In clo-/ mutants ECs are absent (H), whereas in tnnt2 morphants they are present only between hepatocytes of the outer layer (I). (J-L) High-resolution light micrographs of hepatic parenchyma of zebrafish larvae stained with H&E. In 120-hpf wild type sibling, hepatocyte tubules are separated by sinusoids containing erythrocytes (J); in contrast, in 120-hpf clo-/- mutant (K) and Tnnt2 morphant (L), hepatocytes are tightly connected to each other. Note two sinusoids separated by two rows of neighboring hepatocytes as defined by yellow lines. Abbreviations: ec, endothelial cells; e, ear; h, hepatocytes; l, liver; s, sinusoid. In all images, anterior is towards the left. Scale bars, 125 μm in (A-F) and 625 μm in (G-L).
Figure 7
Figure 7
Summary of developmental events during liver vasculogenesis and growth. (A) Timing of liver morphogenesis in zebrafish. According to Field et al. [5], the liver morphogenesis in zebrafish consists of two phases, budding and growth. Our current study showed the formation of three liver lobs during the growth phase. (B) Timing of liver vasculogenesis. Detailed analysis of liver vasculogenesis and precise determination of blood flow initiation in liver sinusoids of live embryonic zebrafish have led us to define three phases of liver vasculogenesis: contact of ECs and hepatocytes, formation of nascent sinusoids, initiation of blood circulation and formation of the vascular network in the liver. (C) Stages of liver growth. Based on our analyses of liver vasculogenesis and growth in wild type as well as in cloche mutant and Tnnt2 morphant larvae, we propose three stages of liver growth: avascular growth as a transition to the growth phase, where ECs are not required; endothelium-dependent, and circulation-dependent growth stage. Approximate periods of the stages of liver vasculogenesis and growth are represented along the time line in hpf or dpf. The green time lines represent the period of liver vasculogenesis.
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