The hepatic vasculature is essential for liver development, homeostasis and regeneration, yet the developmental program of hepatic vessel formation and the embryonic origin of the liver vasculature remain unknown. Here we show in mouse that endocardial cells form a primitive vascular plexus surrounding the liver bud and subsequently contribute to a substantial portion of the liver vasculature. Using intersectional genetics, we demonstrate that the endocardium of the sinus venosus is a source for the hepatic plexus. Inhibition of endocardial angiogenesis results in reduced endocardial contribution to the liver vasculature and defects in liver organogenesis. We conclude that a substantial portion of liver vessels derives from the endocardium and shares a common developmental origin with coronary arteries.
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We thank A. Nagy (Mount Sinai Hospital and Samuel Lunenfeld Research Institute) and J. Rossant (Hospital for Sick Children, University of Toronto) for sharing the Kdrfl mouse line and H. Zeng (Allen Institute) for sharing the Ai66 mouse line. We also thank Shanghai Biomodel Organism Co., Ltd., for mouse generation. This work was supported the Ministry of Science and Technology of China (2012CB945102 and 2013CB945302), the National Science Foundation of China (91339104, 31271552, 31222038, 31301188, 31571503 and 31501172), the Shanghai Basic Research Key Project (14JC1407400), the Major Program of Development Fund for the Shanghai Zhangjiang National Innovation Demonstration Zone (ZJ2014-ZD-002 and 2014-2016), the Shanghai Institutes for Biological Sciences (SIBS) President Fund, the Sanofi-SIBS Fellowship, the SIBS Postdoctoral Fund, AstraZeneca, the China Postdoctoral Science Foundation (2015M570389 and 2015M581669), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2015218), the Shanghai Yangfan Project (15YF1414000) and the Shanghai Rising Star Program (15QA1404300).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 The developing heart is composed of ventricle, atrium and sinus venosus at E8.0 and E8.5.
(a,b) Whole-mount view of E8.0–E8.5 wild-type mouse hearts stained for cardiac troponin I type 3 (TNNI3). Arrows indicate sinus venosus (SV). (c,d) Immunostaining for TNNI3 and PECAM on sections of E8.0 and E8.5 embryos showed that TNNI3+ SV is located on the flanks of foregut diverticulum (f.d.; dotted line). (e) Schematic showing the heart and foregut diverticulum in E8.0–E8.5 embryos. The developing heart at E8.0–E8.5 is composed of bulbus cordis, ventricle, atrium and SV. Scale bars, 100 μm. Each image is representative of three individual heart samples.
(a) Schematic showing the ventricle, atrium and sinus venosus (SV) of the developing heart. (b) Whole-mount view of E8.5 Nfatc1-IRES-CreER embryo stained for TNNI3. Embryos were sectioned and stained for estrogen receptor (ESR; as a surrogate for NFATC1) and TNNI3 (right). (c) Immunostaining for ESR and TNNI3 on consecutive sections of Nfatc1-IRES-CreER embryo. Scale bars, 100 μm.
(a) Whole-mount view of E8.5 Nfatc1-GFP embryo. The schematic (bottom) shows NFATC1 expression in ventricle (V), atrium (A) and sinus venosus (SV). (b) Immunostaining for GFP as a surrogate for NFATC1 and FOXA2 for foregut diverticulum (f.d.) on E8.0 and E8.5 Nfatc1-GFP embryonic sections. (c) Immunostaining for GFP, VE-cadherin (VE-CAD) and HNF4A as a hepatocyte marker in liver bud (LB) on E9.5–E11.5 Nfatc1-GFP embryonic sections. NFATC1 expression was not detectable in E9.5–E11.5 SV. NFATC1 was not expressed in liver vasculature. Scale bars, 100 μm.
(a) Immunostaining for NFATC1, HNF4A and VE-CAD on liver sections from E9.5 embryo showed that NFATC1 is expressed in ventricle (V) or atrium (A) endocardium but not in sinus venosus (SV) or liver bud (L). Panels a1 and a2 are magnified images. (b) Immunostaining for NFATC1, HNF4A and ESR or GFP on sections of E9.5 Nfatc1-IRES-CreER or Nfatc1-GFP embryos. NFATC1+ESR+ or NFATC1+GFP+ cells were detected in atrial or ventricular endocardium but not in SV or liver endothelial cells. (c,d) Immunostaining for NFATC1, VE-CAD and HNF4A on sections from E10.5 and E11.5 embryos showed that NFATC1 is expressed in atrial and ventricular endocardium but not in SV or liver endothelial cells. NFATC1+ cells (arrowheads) in liver are close to but not VE-CAD+ endothelial cells. Panels c1, c2, d1 and d2 are magnified images. (e–g) Immunostaining for VE-CAD and NFATC1 on liver sections showed that NFATC1+ cells (arrowheads) are not VE-CAD+ endothelial cells in liver. Scale bars, 500 μm.
(a) Immunostaining for GFP and VE-CAD on liver sections of E12.5, E13.5 and E15.5 Nfatc1-GFP mice. (b) Immunostaining for ESR and VE-CAD on liver sections from E12.5, E13.5 and E15.5 Nfatc1-IRES-CreER mice. Each figure is representative of three individual samples. Scale bars, 0.5 mm.
Supplementary Figure 6 NFATC1+ cells do not generate hepatocytes, stellate cells, smooth muscle cells, cholangiocytes, biliary epithelial cells or fibroblasts in liver.
(a–f) Immunostaining for RFP and HNF4A (a), desmin (Des) (b), αSMA (c), K19 (d), EPCAM (e) and PDGFRA (f) on sections from E15.5–E17.5 Nfatc1-Dre; R26-RSR-RFP embryonic livers. Nuclei were stained with DAPI (blue). Each image is representative of four individual liver samples. Scale bars, 100 μm.
(a) Immunostaining for ESR, VE-CAD and HNF4A on sections from Nfatc1-IRES-CreER embryos. ESR represents NFATC1 expression. ESR is not detected in SV or liver bud (LB) from E9.5–E11.5. Boxed regions are magnified on the right. (b) Schematic showing NFATC1 expression at E9.5–E11.5. (c) Immunostaining for RFP, VE-CAD and HNF4A on sections from Nfatc1-IRES-CreER; Rosa26-RFP embryos. Arrowheads indicate cells derived from NFATC1+ cells (RFP+) in SV or LB. Tamoxifen was injected at E8.0. Scale bars, 100 μm. (d) Schematic showing cells derived from NFATC1+ cells in SV at E9.5–E11.5.
(a) Whole-mount staining of estrogen receptor (ESR) on E8.25, E8.5, E9.0 and E9.5 Npr3-CreER or wild-type embryos. ESR, as a surrogate for NPR3, is expressed in the ventricle (V) and atrium (A) but not in sinus venosus (SV). (b) In situ hybridization for Npr3 on E9.5 embryonic sections. (c,d) Immunostaining for ESR, FOXA2 and PECAM or VE-CAD on sections from E8.5 and E9.0 Npr3-CreER embryos. h.e., hepatic endoderm; LB, liver bud. (e,f) Immunostaining for ESR, HNF4A and VE-CAD on E9.5 and E10.5 Npr3-CreER embryonic sections. NPR3 is expressed in atrium and ventricle endocardium but not in SV. Scale bars, 100 μm.
(a) Schematic showing the tamoxifen induction strategy. (b,c) Immunostaining for RFP, HNF4A and PECAM or VE-CAD on sections from E10.5 and E11.5 Npr3-CreER; Rosa26-RFP embryos. The arrowheads in b indicate RFP+PECAM− cells. (d) Whole-mount bright-field or fluorescence view of E13.5 liver and heart. (e) Immunostaining for RFP and VE-CAD on sections from E13.5 Npr3-CreER; Rosa26-RFP liver shows that cells derived from NPR3+ cells (RFP+; arrowheads) are not VE-CAD+ endothelial cells. (f) Npr3-CreER labels VE-CAD+ endocardial cells in the ventricle (V) of heart from the same embryos. Scale bars: 0.5 mm in d and 100 μm in b, c, e and f.
(a) Schematic showing the two strategies employed, Dre-rox and Cre-loxP lineage tracing. Strategy 1 labels NFATC1+ endocardium in the atrium, ventricle and SV. Strategy 2 labels NFATC1+ atrium and ventricle endocardium but subtracts SV labeling from strategy 1. (b–e) Whole-mount images of E10.5 embryos and immunostaining for RFP and PECAM on embryonic sections. RFP+ cells are detected in NFATC1+NPR3+ atrium and ventricle endocardium. Tamoxifen was injected at E8.0. Scale bars: white, 1 mm; yellow, 100 μm.
(a) Images of embryonic sections stained by two Vegfa in situ hybridization probes. The red dotted line circles liver bud. (b) Immunostaining for VEGFR2 and PECAM on embryonic sections shows expression of VEGFR2 in SV endocardium (arrowheads). (c) Immunostaining for VEGFR2, PECAM and HNF4A on embryonic sections shows expression of VEGFR2 in vasculature surrounding the liver bud. Scale bars, 100 μm.
Supplementary Figure 12 Endocardial cells contribute to sinusoidal, central vein, portal vein and arterial endothelial cells.
(a) Immunostaining for RFP, CK19 and VE-CAD on liver sections from E17.5 Nfatc1-IRES-CreER; Rosa26-RFP embryo. Panels 1 and 2 are magnified regions showing that Nfatc1-IRES-CreER labeled cells (RFP+) contribute to endothelial cells in portal vein (PV; green arrowheads) and central vein (CV; white arrowheads) and also the sinusoidal endothelial cells in between the portal vein and central vein. BD denotes bile duct in the portal region. Tamoxifen was administered at E8.0. (b) Immunostaining for RFP, CK19 and VE-CAD on liver sections from adult Nfatc1-IRES-CreER; Rosa26-RFP mice. P8w, postnatal 8 weeks. Tamoxifen was administered at E8.0. A, artery. Panels 1 and 2 are magnified regions showing that RFP+ cells contribute to a subset of endothelial cells in portal artery (yellow arrowheads), portal vein (green arrowheads) and central vein (white arrowheads). (c) Immunostaining for RFP, αSMA and EPHB4 on liver sections shows that NFATC1+ cells contribute to endothelial cells in both EPHB4− portal artery (PA) and EPHB4+ portal vein (PV). (d) Immunostaining for RFP, PROX1 and LYVE1 shows that NFATC1+ cells contribute to PROX1+LYVE1+ lymphatic vessels (arrowheads). PROX1 is also expressed in hepatocytes (H). Scale bars, 100 μm.
Supplementary Figure 13 Endocardium-derived liver endothelial cells respond to injury and proliferate during regeneration.
(a) Schematic showing the experimental strategy. PH, partial hepatectomy as a model for liver injury and regeneration. (b) Quantification of the percentage of proliferating endothelial cells among RFP+ cells from Nfatc1-IRES-CreER; Rosa26-RFP livers. *P < 0.05; n = 4. (c) Immunostaining for RFP, Ki-67 and VE-CAD on sections from sham or partial hepatectomy livers. Arrowheads indicate proliferating Ki-67+RFP+ endothelial cells. (d) Immunostaining for RFP, PHH3 and VE-CAD on sections from sham or partial hepatectomy livers. Arrowheads indicate proliferating PHH3+RFP+ endothelial cells. (e) Immunostaining for RFP, PCNA and VE-CAD on sections from sham or partial hepatectomy livers. Arrowheads indicate proliferating PCNA+RFP+ endothelial cells. Each image is representative of four individual samples. Scale bars, 100 μm.
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Zhang, H., Pu, W., Tian, X. et al. Genetic lineage tracing identifies endocardial origin of liver vasculature. Nat Genet 48, 537–543 (2016). https://doi.org/10.1038/ng.3536
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