Volume 141, Issue 4, Pages 1432-1438.e4 (October 2011) Embryonic Ductal Plate Cells Give Rise to Cholangiocytes, Periportal Hepatocytes, and Adult Liver Progenitor Cells  Rodolphe Carpentier, Regina Español Suñer, Noémi van Hul, Janel L. Kopp, Jean–Bernard Beaudry, Sabine Cordi, Aline Antoniou, Peggy Raynaud, Sébastien Lepreux, Patrick Jacquemin, Isabelle A. Leclercq, Maike Sander, Frédéric P. Lemaigre  Gastroenterology  Volume 141, Issue 4, Pages 1432-1438.e4 (October 2011) DOI: 10.1053/j.gastro.2011.06.049 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Lack of apoptosis and very low proliferation in the ductal plate. (A and B) Mouse and human ductal plates were analyzed at the stages indicated. No evidence for apoptosis (activated caspase 3 and TUNEL) was found. (C) Only a few SOX9+ ductal plate cells were proliferating (Ki67; arrowhead). dp, ductal plate; pv, portal vein; asterisk, lumen of developing duct. Bar = 20 μm. Gastroenterology 2011 141, 1432-1438.e4DOI: (10.1053/j.gastro.2011.06.049) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Expression of CreERT2 in developing liver is restricted to SOX9-expressing ductal plate cells. Pregnant female mice were injected with tamoxifen 18 hours before collection of the liver at the stages indicated. The livers were stained to detect CreERT2 and biliary (SOX9) or hepatoblast (HNF4) markers. dp, ductal plate; pv, portal vein. Bar = 20 μm. Gastroenterology 2011 141, 1432-1438.e4DOI: (10.1053/j.gastro.2011.06.049) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 The ductal plate gives rise to cholangiocytes lining interlobular bile ducts and periportal hepatocytes. Pregnant female mice were injected with tamoxifen at E15.5, and the SOX9-CreERT2;ROSA26RYFP offspring were analyzed at the time points indicated. (A) The YFP+ progeny of the ductal plate consisted of interlobular ducts and of hepatocytes, which persisted at least until 7 months of age. The biliary markers (SOX9, OPN, CK19) were expressed in the YFP+ ducts but not in YFP+ hepatocytes; the latter expressed the hepatocyte marker HNF4. (B) The YFP+ hepatocytes were restricted to the periportal zone and expressed periportal (CPS1) but not perivenous (GS) markers. cv, central vein; hc, hepatocyte; id, interlobular bile duct; pv, portal vein. Bar = 20 μm. Gastroenterology 2011 141, 1432-1438.e4DOI: (10.1053/j.gastro.2011.06.049) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 The YFP+ progeny of the ductal plate includes cholangiocytes lining the ductules and the canals of Hering. (A) Laminin (Lam) fully surrounds ductules (yellow dashed line) and interlobular ducts (white dashed line). (B) (Upper panels) Canals of Hering (yellow dashed line) were delineated by hepatocytes with a large nucleus and cholangiocytes expressing cytokeratin (pan-cytokeratin antibody); the white dashed line separates the cholangiocyte from the hepatocyte. Laminin is only found along the basal pole of the cholangiocytes. (Middle and lower panels) Canals of Hering are also identified by carcinoembryonic antigen–related cell adhesion molecule (CEACAM) staining, which marks the bile canaliculi at the apical pole of hepatocytes. The middle pictures show a canal of Hering lined by a YFP+ hepatocyte and a YFP+ cholangiocyte. In A and B, the right 3 pictures correspond to magnified areas delineated in the left picture by a white box. CoH, canal of Hering; d, ductule; id, interlobular bile duct; pv, portal vein. Bar = 10 μm. Gastroenterology 2011 141, 1432-1438.e4DOI: (10.1053/j.gastro.2011.06.049) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 CDE diet–induced oval cells derive from the ductal plate. Pregnant female mice were injected with tamoxifen at E15.5, and 4-week-old SOX9-CreERT2;ROSA26RYFP offspring were fed a CDE diet. YFP+ oval cells express typical markers (CK19, SOX9, OPN, TROP2). The percentage of YFP+ oval cells ([YFP+SOX9+/SOX9+] × 100) per periportal area can reach close to 100%. A total of 13,419 SOX9+ oval cells were counted over 146 periportal areas. OVc, oval cell; pv, portal vein; W, week after birth. Bar = 10 μm. Gastroenterology 2011 141, 1432-1438.e4DOI: (10.1053/j.gastro.2011.06.049) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 DDC diet–induced oval cells derive from the ductal plate. Pregnant female mice were injected with tamoxifen at E15.5, and 4-week-old SOX9-CreERT2;ROSA26RYFP offspring were fed a DDC diet for 2 weeks. Atypical ductular structures were induced. They coexpress YFP and biliary/oval cells markers SOX9, OPN, and CK (pan-cytokeratin antibody). The pictures on the right correspond to magnified areas delineated by a white box in the left panels. ads, atypical ductular struture; pv, portal vein; W, week after birth. Bar = 10 μm. Gastroenterology 2011 141, 1432-1438.e4DOI: (10.1053/j.gastro.2011.06.049) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Expression and activity of SOX9-CreERT2. (A) Distribution of CreERT2expression in SOX9+ ductal plate cells. Pregnant female mice were injected with tamoxifen 18 hours before collection of the liver at E14.5, E15.5, and E16.5. Countings at all stages were pooled. (B) Activity of SOX9-CreERT2 is specific to tamoxifen-treated liver. The livers of noninjected SOX9-CreERT2;ROSA26RYFP embryos at E15.5 and E18.5 and of SOX9-CreERT2;ROSA26RYFP mice at postnatal day 2 were immunostained for YFP. In the absence of tamoxifen injection, no YFP is detectable. pv, portal vein; dp, ductal plate; *, lumen of developing duct; id, interlobular duct. Gastroenterology 2011 141, 1432-1438.e4DOI: (10.1053/j.gastro.2011.06.049) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Proliferation of duct cells and of ductal plate–derived hepatocytes, as well as tamoxifen-induced ectopic expression of SOX9. (A) Percentage of Ki67+ hepatocytes showing a decreasing proliferation rate of hepatocytes from birth to postnatal day (P) 55. No significant difference was found between YFP+ and YFP− hepatocytes regarding their proliferation rate (2-way analysis of variance, P > .2; number of hepatocytes counted: 5724 [P5], 3138 [P15], 4104 [P40], 5540 [P55]). (B) The percentage of Ki67+SOX9+ cholangiocytes showed a decreasing proliferation rate from birth to P55 (number of SOX9+ cholangiocytes counted: 685 [P5], 189 [P15], 240 [P40], 345 [P55]). (C) Tamoxifen induces ectopic expression of SOX9 in adult hepatocytes. Eight-week-old mice were injected with vehicle (0.5 mg corn oil per kg body wt or 0.3 mg ethanol per kg body wt) or vehicle and tamoxifen (10 mg tamoxifen per kg body wt), and livers were harvested 18 hours later. Vehicles did not affect SOX9 expression. Tamoxifen induced SOX9 expression in the hepatocytes independently of the vehicle. W, week; pv, portal vein. Bar = 20 μm. Gastroenterology 2011 141, 1432-1438.e4DOI: (10.1053/j.gastro.2011.06.049) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Origin of oval cells. The distribution of YFP+ oval cells per periportal area does not correlate with that of the YFP+ interlobular cholangiocytes. Gastroenterology 2011 141, 1432-1438.e4DOI: (10.1053/j.gastro.2011.06.049) Copyright © 2011 AGA Institute Terms and Conditions