Volume 41, Issue 5, Pages 790-798 (November 2004) Suppression of C/EBP α expression in biliary cell differentiation from hepatoblasts during mouse liver development  Nobuyoshi Shiojiri, Kentaro Takeshita, Harufumi Yamasaki, Takeyuki Iwata  Journal of Hepatology  Volume 41, Issue 5, Pages 790-798 (November 2004) DOI: 10.1016/j.jhep.2004.07.011 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Reactivity of anti-C/EBP α (A) and β (B) antibodies in extracts of 12.5-day, 17.5-day and 18.5-day fetal livers, and adult livers. Anti-C/EBP α and β antibodies react with 42- and 30-kDa bands, and 34-kDa bands in adult liver extracts, respectively (lanes e of A and B). In control lanes (lanes f of A and B), the primary antibodies were preabsorbed with respective competitive peptides. C/EBP α bands became strong with development (lanes a, c and e of A). Although expression of 34-kDa C/EBP β is weak in fetal livers, that of 21-kDa C/EBP β is obvious. Lanes b and d of A and B are controls for a and c, respectively. The cell lysates of liver samples were separated by SDS-PAGE using a 12% gel, and transferred electrophoretically to nitrocellulose membranes. After transfer, proteins on the blots were immunostained with antibodies against C/EBP α or C/EBP β. CRM, cross-reactive material. Journal of Hepatology 2004 41, 790-798DOI: (10.1016/j.jhep.2004.07.011) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Expression of C/EBP α during mouse liver development. (A) Sagittal section of a 9.5-day liver primordium area. The endodermal cells of the liver primordium (arrow) were weakly positive for C/EBP α. (B) Magnified view of liver primordium of A. (C) A 10.5-day liver primordium. Nuclei of the hepatic cords are positively immunostained for C/EBP α. (D) Section of a 10.5-day liver primordium preabsorbed with competitive peptides. (E) 12.5-day fetal liver. The liver is positively immunostained. (F) The yolk sac endoderm of 10.5-day embryo is immunoreactive in its cell nuclei. FG, foregut; H, heart; ST, septum transversum mesenchyme. Bar indicates 50μm. Journal of Hepatology 2004 41, 790-798DOI: (10.1016/j.jhep.2004.07.011) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Expression of C/EBP α in extrahepatic and intrahepatic bile ducts. (A, B, C) Cytokeratin (8 and 18) and C/EBP α immunostaining, and their double immunostaining in 13.5-day mouse liver, respectively. Epithelial cells of the extrahepatic bile duct are negative. (D, E, F) Cytokeratin (8 and 18) and C/EBP α immunostaining, and their double immunostaining in 14.5-day mouse liver, respectively. Epithelial cells of the extrahepatic bile duct are negative. (G, H) Double immunostaining with nidogen (red) and C/EBP α (green) in 13.5- and 14.5-day liver parenchyma, respectively. Progenitors of periportal biliary epithelial cells are moderately or weakly positive for C/EBP α in 13.5 and 14.5-day liver (arrows). (I, J) Expression of cytokeratin (8 and 18) and C/EBP α in 17.5-day liver. Periportal, biliary epithelial cells are negative for C/EBP α (arrows). ED, extrahepatic bile duct; PV, portal vein. Bar indicates 50μm. Journal of Hepatology 2004 41, 790-798DOI: (10.1016/j.jhep.2004.07.011) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Expression of C/EBP α (B, D) in neonatal and adult liver. (A, C) Cytokeratin (8 and 18) immunostaining of B and D, respectively. C/EBP α is localized in nuclei of hepatocytes. Arrows indicate epithelial cells of intrahepatic bile ducts, which do not express C/EBP α. IBD, intrahepatic bile duct; PV, portal vein. Bar indicates 50μm. Journal of Hepatology 2004 41, 790-798DOI: (10.1016/j.jhep.2004.07.011) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Expression of C/EBP β in 10.5-day yolk sac (A), and 14.5- (C), 17.5- (E), 7-day (F) and adult (G) livers. (B, D) Cytokeratin (8 and 18) immunostaining of C and E. While nuclear C/EBP β staining of hepatoblasts and hepatocytes is very weak in 14.5-day and 17.5-day fetal mouse livers (C, E), it is clearly seen in 7-day and adult livers (F, G). Nuclear C/EBP β staining in hepatocytes is obvious. PV, portal vein. Bar indicates 50μm. Journal of Hepatology 2004 41, 790-798DOI: (10.1016/j.jhep.2004.07.011) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Expression of mRNAs for AFP (A, C, E) and albumin (B, D, F) during liver primordium formation. (A, B) The liver diverticulum at 9.5 days of gestation. (C, D) The 10.5-day liver primordium. (E, F) The 12.5-day liver. In the 9.5-day liver diverticulum, mRNA for AFP is strongly expressed (A, arrow), but that for albumin is absent (B). In the 10.5-day liver primordium, AFP mRNA is weakly or moderately seen in epithelial cells of extrahepatic bile ducts in addition to hepatoblasts, whose signals are very strong (C). Albumin mRNA is obvious in hepatoblasts and some extrahepatic bile duct cells, especially extrahepatic bile duct cells in the hilus (D). In 12.5-day liver, mRNAs for AFP (E) and albumin (F) are expressed in hepatoblasts. The AFP mRNA staining in extrahepatic bile duct cells is stronger than that of albumin mRNA. ED, extrahepatic bile duct; FG, foregut; PV, portal vein; ST, septum transversum mesenchyme. Bar indicates 50μm. Journal of Hepatology 2004 41, 790-798DOI: (10.1016/j.jhep.2004.07.011) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Suppression of AFP (A, C, E) and albumin (B, D, F) mRNA expression during biliary cell differentiation. (A, B, C, D) 14.5-day liver. (E, F) 17.5-day liver. In 14.5-day liver, although most extrahepatic bile duct cells do not express mRNAs for AFP and albumin, those located in the hilus, presumably hepatic duct cells, are weakly stained for both mRNAs (A, B, arrows). Progenitors of intrahepatic bile duct cells are weakly positive or negative for both mRNAs (C, D, arrows). In 17.5-day liver, most epithelial cells of intrahepatic bile ducts are negative for both mRNAs (E, F, arrows). ED, extrahepatic bile duct; PV, portal vein. Bar indicates 50μm. Journal of Hepatology 2004 41, 790-798DOI: (10.1016/j.jhep.2004.07.011) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 8 OTC expression (B, D) in 15.5-day (A, B) and 17.5-day (C, D) mouse livers. (A, C) Laminin immunostaining in B and D, respectively. While laminin-positive cells of bile duct progenitors express OTC in 15.5-day liver, differentiated biliary epithelial cells are negative for it in 17.5-day liver. Arrows indicate epithelial cells of bile duct structures or their progenitors. PV, portal vein. Bar indicates 50μm. Journal of Hepatology 2004 41, 790-798DOI: (10.1016/j.jhep.2004.07.011) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions