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First published online 4 October 2006
doi: 10.1242/dev.02591

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Suppression of C/EBP expression in periportal hepatoblasts may stimulate biliary cell differentiation through increased Hnf6 and Hnf1b expression

¹ Department of Biology, Faculty of Science, Shizuoka University, 836 Oya, Surugaku, Shizuoka City, Shizuoka 422-8529, Japan.
² Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba 260-8670, Japan.
³ Anadys Pharmaceuticals, 3115 Merryfield Row, San Diego, CA 92121, USA.

View larger version (77K): [in a new window]   Fig. 1. C/EBP and its mRNA expression in mouse liver development. Immunohistochemistry reveals that hepatoblasts and hepatocytes express C/EBP, localized in their nuclei, in 12.5-(A,B), 14.5-day (D) and neonatal (F) liver sections. (B) Double staining of C/EBP and DAPI. (C,E) Cytokeratin immunostaining of D and F, respectively. Pearl-like structures and biliary epithelial cells are negative for C/EBP (arrows in C-F). In situ hybridization using Cebpa antisense probes shows the absence of Cebpa mRNA in biliary epithelial cells (arrows) of 16.5-day (G) and 17.5-day (I) mouse liver sections. Hepatocytes are positive for Cebpa mRNA. Sense probes give no significant signal on a 16.5-day liver section (H). PV, portal vein. Scale bars: 50 µm.

View larger version (97K): [in a new window]   Fig. 2. Pseudoglandular development in Cebpa knockout mouse livers. Hematoxylin and Eosin staining indicates that periportal hepatoblasts form pearl-like structures in 14.5-day and 17.5-day wild-type livers (arrowheads) (A-C), while pseudoglandular or pearl-like structures are abundant in the whole liver parenchyma of knockout livers (small arrows) (E-G). Biliary epithelial cells differentiate only around the portal vein and a typical intrahepatic bile duct is formed in a neonatal wild-type liver (D). In a neonatal knockout liver, pseudoglandular structures with large lumina still abundantly develop and no normal bile duct is seen even around the portal vein (H). HV, hepatic vein; PV, portal vein. Scale bars: 50 µm.

View larger version (79K): [in a new window]   Fig. 3. Periportal cells of pseudoglandular structures in the 17.5-day knockout liver express biliary cell markers. PAS staining indicates that wild-type hepatocytes store abundant glycogen (A). Immunohistochemistry for cell type-specific markers and lectin histochemistry also show that wild-type hepatocytes express HNF4 in their nuclei (B), and that periportal biliary epithelial cells are negative for HNF4 (arrowhead; B), but are positive for bile duct-specific cytokeratin, laminin, DBA-binding sites and nidogen (C-F). The knockout liver develops many pseudoglandular structures, which are composed of hepatocyte-like cells poorly accumulating glycogen (G). Although most pseudoglandular structures are positive for bile duct-type cytokeratin (I), other bile duct markers are reactive only with periportal pseudoglandular structures (J-L). Staining pattern of HNF4 in the knockout liver resembles that of the wild-type liver; periportal biliary cells are negatively immunostained (arrows; H). HV, hepatic vein; PV, portal vein. Scale bars: 50 µm.

View larger version (51K): [in a new window]   Fig. 4. Pseudoglandular structures under the hepatic capsule express bile duct markers in the 17.5-day knockout liver. Pseudoglandular structures under the hepatic capsule (arrows) are reactive with fluorescent DBA (B) and an anti-nidogen antibody (D) in the knockout liver. Hepatocytes under the capsule react with neither DBA (A) nor the anti-nidogen antibody (C) in the wild-type liver. Scale bars: 50 µm.

View larger version (49K): [in a new window]   Fig. 5. Expression of cell-adhesion molecules in pseudoglandular structures of the 17.5-day knockout liver. Intrahepatic biliary epithelial cells (arrows) are negatively or weakly immunostained for 3 and ß4 integrin subunits in the wild-type liver (A,B) although extrahepatic bile duct cells are moderately immunostained for ß4 integrin subunits (B). E-cadherin expression is strong in biliary epithelial cells, but they have weak N-cadherin expression in the wild-type liver (arrows) (C,D). In the knockout liver, cells of periportal pseudoglandular structures moderately express both integrin subunits (arrowheads) (E,F). In addition, almost all cells of pseudoglandular structures strongly or moderately express E-cadherin and N-cadherin (G,H) but those around the portal vein are negative for N-cadherin (arrowhead, H). PV, portal vein. Scale bars: 50 µm.

View larger version (111K): [in a new window]   Fig. 6. Higher proliferation of pseudoglandular cells in the knockout mouse liver. (A-D) PCNA immunostaining shows an increase of proliferation in pseudoglandular cells in 17.5-day (C) and neonatal (D) knockout mouse livers (arrowheads), compared with wild-type mice (arrows, A,B). PV, portal vein. Scale bars: 50 µm. Proliferation indexes of hepatic cells, given as number of PCNA-positive cells per portal biliary epithelial cell (light brown) or nonperiportal hepatocyte/pseudoglandular cell (purple), show a decrease and increase of proliferation in periportal biliary epithelial cells and nonperiportal pseudoglandular cells of the knockout mouse liver, respectively, compared with wild-type mice (E; wild type, n=3; knockout, n=3). Bars indicate the standard deviations of the means.

View larger version (92K): [in a new window]   Fig. 7. In situ hybridization analysis of Hnf6 and Hnf1b mRNAs in fetal and neonatal livers. In the wild-type liver, hepatoblasts weakly or moderately express Hnf6 and Hnf1b mRNAs at 12.5 days (A,D), but strong signals of both mRNAs are confined to periportal biliary structures in later development (arrowheads; B,C,E,G). Epithelial cells of the extrahepatic bile duct are strongly positive for both mRNAs (A,D,F). In the knockout liver, Hnf6 and Hnf1b mRNAs are upregulated in hepatoblasts at 12.5 days (arrows; H,M), and in pseudoglandular structures at 15.5 days (star; I,N). Cells of pseudoglandular structures under the hepatic capsule (arrowheads; J,O) and extrahepatic bile duct cells (K,P) strongly express both mRNAs. Also in the neonatal the knockout liver, Hnf6 and Hnf1b mRNAs are upregulated in hepatocytes (L,Q,R) compared with those in the wild-type liver. (R) The nonperiportal region. Periportal epithelial cells and hepatocytes are strongly positive (arrowheads; L,Q). ED, extrahepatic bile duct; PV, portal vein; SI, small intestine. Scale bars: 50 µm. (S) Positive signal profile of Hnf6 mRNA localization from the portal vein to the parenchyma in 15.5-day wild-type (red tracing) and knockout (blue tracing) livers. The level of Hnf6 mRNA expression is upregulated in the parenchymal region of the knockout liver. The use of `weak', `moderate' or `strong' expression of Hnf6 mRNA in the text is based on the profile analyses of positive signals.

View larger version (145K): [in a new window]   Fig. 8. In situ hybridization analysis of Jag1 and Notch2 mRNAs in fetal and neonatal livers. In the wild-type liver, hepatoblasts very weakly express Jag1 and Notch2 mRNAs at 12.5 days (arrows; A,B,G,H), but epithelial cells of periportal biliary structures become positive for both mRNAs in later development (arrows; C-F,I,K,L). Epithelial cells of the extrahepatic bile duct are moderately positive for both mRNAs (A,G,J). Endothelial cells of the portal vein and hepatic artery (*; E), and their connective tissue cells express Jag1 mRNA (A-F). Arrowheads indicate Jag1 mRNA-positive hemopoietic cells in the neonatal wild-type liver (F). In the 12.5-day knockout liver, although Jag1 mRNA expression in hepatoblasts is at a normal level (arrow; M), Notch2 mRNA is upregulated in hepatoblasts (arrow; Q). Hepatoblasts under the capsule (large arrowheads) are positive for Notch2 mRNA (R). In 15.5-day and neonatal knockout livers, Jag1 and Notch2 mRNAs are upregulated in pseudoglandular structures (star), but periportal epithelial cells are more strongly stained (small arrowheads; N-P,S,T). Jag1 mRNA expression is downregulated in endothelial cells of the portal vein (N), compared with that in the 17.5-day wild-type liver (C,D,E). ED, extrahepatic bile duct; PV, portal vein. Scale bars: 50 µm.

View larger version (84K): [in a new window]   Fig. 9. Liver fragments of knockout fetuses develop cystic structures in the testes of Scid mice. Liver fragments of 12.5-day wild-type and knockout fetuses were transplanted and maintained for 2 months in the testes of Scid mice, and histologically and histochemically analyzed for expression of cell type-specific markers. The wild-type liver developed normal hepatic tissues, in which the portal vein and hepatic vein differentiated (A,B). Hepatocytes store PAS-positive glycogen. Periportal biliary cells (arrows) are immunostained for bile duct-specific cytokeratin (C), type IV collagen (E) and nidogen (F) but are negative for DBA-binding sites (D). By contrast, the knockout liver developed abundant cystic structures strongly expressing bile duct markers in the testis (G-L). Normal hepatic tissues are also observed with cystic structures in a few knockout transplants (G; Hematoxylin and Eosin staining). HV, hepatic vein; PV, portal vein. Scale bars: 50 µm.

View larger version (13K): [in a new window]   Fig. 10. Bile duct development and C/EBP. The portal environment induces suppression of C/EBP expression, which leads to downregulation of hepatocyte-specific genes and upregulation of Hnf6 and Hnf1b genes. The deposition of basal laminar components, expression of DBA- and SBA-binding sites, and suppression of HNF4 expression are not downstream of the action of C/EBP. Jag1 and Notch2 mRNA expression might be downstream of C/EBP. CK, bile duct-specific cytokeratin.