Additional Supporting Information may be found in the online version of this article.

Sup.Fig.1.tif3689KSupporting Fig. 1. Immunostaining of Alcam, desmin, SMA, and Wt1 in E18.5 and adult livers. E18.5 embryonic (A) and adult livers (B) were immunostained with antibodies against Alcam, desmin, SMA, and Wt1. Nuclei were counterstained with DAPI. Wt1 is faintly expressed in MCs, but not in HSCs in E18.5 liver. Expression of Alcam is evident in immature hepatocytes around the portal veins (pv) at E18.5 and hepatocytes and biliary epithelial cells in the portal area in the adult liver. bd, bile ducts; cv, central veins; ha, hepatic artery; hsc, hepatic stellate cells; mc, mesothelial cells. Bar, 10 μm.
Sup.Fig.2.tif7404KSupporting Fig. 2. Supporting Fig. 2. No contribution of Wt1+ MC/SubMCs to SECs, Kupffer cells, hepatoblasts, and blood cells in developing livers. Immunostaining of lacZ and markers for SECs (CD31, Flk1), Kupffer cells (F4/80), hepatoblasts (E-cadherin), leukocytes (CD45), and erythrocytes (Ter119) in E12.5 livers after tamoxifen injection at E10.5. No lacZ expression in SECs, Kupffer cells, hepatoblasts, leukocytes, and erythrocytes. hb, hepatoblasts; hsc, hepatic stellate cells; kc, Kupffer cells; sec, sinusoidal endothelial cells. Bar, 10 μm.
HEP_24119_sm_suppinfofig3.tif8294KSupporting Fig. 3. Wt1+ MC/SubMCs give rise to HSCs and PMCs during liver morphogenesis. (A-C) Conditional labeling of Wt1+ MC/SubMCs using the Wt1CreERT2 and Rosa26mTmGflox mice. Upon tamoxifen treatment, the membrane-tagged GFP is expressed in Wt1+ MC/SubMCs instead of red fluorescence protein (tomato) (A). After tamoxifen injection at either E10.5 (B) or E11.5 (C), embryos were immunostained with antibodies against Alcam, CD31, desmin, GFP, and SMA at E13.5 (B) or E14.5 (C). Note that expression of GFP is seen in desmin+ HSCs and PMCs in E13.5 or E14.5 livers. No GFP expression in CD31+ endothelial cells in the veins. Alcam+ MC/SubMCs express GFP. No GFP expression in the wild type littermate (control, Wt1+/+). (D) Quantification of Wt1+ MC/SubMC-derived HSC/PMCs in the Wt1CreERT2/+; Rosa26mTmGflox/+ developing livers. The percentages of the GFP+ HSCs and PMCs in E13.5 (B) and E14.5 livers (C). These percentages were obtained from total 9,782 (E13.5, 4,873; E14.5, 4,909) and 11,330 (E13.5, 3,332; E14.5, 7,998) desmin+ cells in the ML and LL, respectively. Results are means ± SD of 5 independent sections. **P < .01. ec, endothelial cells; hsc, hepatic stellate cells; mc, mesothelial cells; pmc, perivascular mesenchymal cells; submc, submesothelial cells. Bar, 50 μm (B, left), 10 μm (B, right, C).
SupportingTable1.doc59KSupporting Table 1. List of antibodies for immunohistochemistry

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