1. Volume 124, Issue 1, Pages 202-216 (January 2003)
β-catenin antisense studies in embryonic liver cultures: Role in proliferation, apoptosis, and lineage specification 
Satdarshan P.S. Monga, Hardarshan K. Monga, Xinping Tan, Karen Mulé, Peter Pediaditakis, George K. Michalopoulos 
Gastroenterology 
Volume 124, Issue 1, Pages (January 2003)
DOI: /gast
Copyright © 2003 American Gastroenterological Association Terms and Conditions

2. Fig. 1
β-Catenin in developing liver. (A) β-Catenin at E10 localizes to most of the nonhematopoietic cells in the liver. The cells show membranous (black arrow), cytoplasmic (white arrow), and nuclear (black arrowhead) positivity at this stage. There is moderate to intensive staining for β-catenin at this stage of liver development (original magnification 60×). (B) β-Catenin localizes to the membrane (black arrow) of the cells in an E14 liver. Very few cells show cytoplasmic (white arrows) or nuclear (black arrowhead) localization of β-catenin at this stage. Overall staining appears to be mild to moderate (original magnification 40×). (C) A high-power view emphasizes the membranous (black arrowheads) pattern of β-catenin staining in an E14 liver. Also, the larger cells with larger nuclei show membranous localization of β-catenin, and these cells seem to be present in 4–6-cell-thick clusters or aggregates. The more differentiated hepatocytes show minimal β-catenin staining (white arrows), as do the hematopoietic cells (white arrowheads). Also, flattened cells lining the vascular channels at this stage of liver development show strong β-catenin immunoreactivity (black arrows) (original magnification 60×). (D) c-kit stains liver stem cells (black arrows) at E14, and these cells are larger with larger nuclei than the more mature hepatocytes (original magnification 60×). (E) CK19 stains cells of biliary lineage (black arrow) and bipotential stem cells (black arrowheads) in an E14 liver. The more differentiated hepatocytes (white arrows) are negative for CK19 (original magnification 60×). (F) Immunohistochemical analysis of an E14 liver shows most cells of hepatocytic lineage to be positive for α-FP. Stem cells (black arrows) and immature hepatocytes are positive, whereas differentiated and mature hepatocytes (black arrowheads) and hematopoietic cells (white arrows) are negative for α-FP (original magnification 60×). (G) β-Catenin staining is distinct from other markers of hepatocyte and biliary differentiation, with intense staining around primitive vascular channels and hepatocyte clusters. Overall staining is mild to moderate in an E14 liver (original magnification 10×). (H) c-kit stains stem cells in an E14 liver (original magnification 10×). (I) CK19-positive cells are seen mainly around channels. Some CK19-positive cells are scattered throughout the E14 liver (original magnification 10×). (J) A moderate to intense staining is observed against α-FP in an E14 liver and is scattered throughout (original magnification 10×). (K) An adult mouse liver shows mainly membranous positivity (black arrows) of β-catenin with some subcellular localization (black arrowheads) (original magnification 60×). (L) A section of the portal triad shows cytoplasmic localization of β-catenin in biliary epithelial cells (black arrow) in an adult liver (original magnification 60×). (M) A section around the central vein in an adult liver also shows hepatocyte membrane localization (black arrow) of β-catenin (original magnification 60×).
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Embryonic liver cultures show a significantly reduced size when cultured in the presence of the β-catenin antisense compared with the control antisense. (A) A representative embryonic liver cultured in the presence of Ctrlas shows a healthy and vivid organ that measures about 3 mm in diameter. (B) A representative embryonic liver cultured in the presence of β-Catas for 72 hours shows a clear shrunken organ that measures about 1.4 mm in diameter. (C) Diameters of 5 organ cultures after 72 hours for each condition show a range from 2.8 to 3.1 mm for the controls and 1.25 to 1.5 mm for the antisense-treated group. (D) The bar graph shows an extremely significant decrease in the sizes of organ cultures following antisense treatment after 72 hours in culture (P < ). (E) A representative H & E-stained section of the embryonic liver cultured in the presence of the control PMO displays no change in the cell arrangements, showing the cells in ductular and sheet-like arrangements. (F) General histology remains unaffected in this representative section from the organs cultured in the presence of β-catenin antisense.
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
β-Catenin in embryonic liver cultures and effect of antisense PMO on histology of the embryonic liver cultures. All figures are <40× original magnification unless otherwise noted. (A) A negative control (secondary antibody only) for the immunohistochemistry of the embryonic liver cultures shows no positive brown staining. (B) Membranous localization of β-catenin in the cells in a ductular arrangement (arrow) and a sheet-like arrangement (arrowhead) in a normal embryonic liver culture. (C) This staining pattern is unaffected in the embryonic livers cultured in the presence of the control PMO, with several cells staining for β-catenin in the ductular arrangement (arrow). (D) A substantial decrease in β-catenin protein follows culture in the presence of β-Catas (5 μmol/L) in the ductular area (arrow). (E) Hepatocytes in a sheet-like arrangement show β-catenin–positive cells (arrows) in the embryonic livers cultured in the presence of Ctrlas (5 μmol/L). (F) A significant decrease in β-catenin positivity (arrows) in the hepatocytes is observed in the embryonic livers cultured in the presence of β-catenin antisense at a concentration of 5 μmol/L.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Significant reduction in cell proliferation following the presence of β-catenin antisense in embryonic liver cultures. All figures are <40× original magnification unless otherwise noted. (A) Most of the cells in normal culture are positive for PCNA (arrow). A few cells in the sheet-like arrangement (arrowhead) are negative. (B) The presence of control PMO does not change the PCNA staining in these cultures, with most cells still positive for PCNA (arrow). Again, a minimal number of cells are PCNA negative (arrowhead). (C) Areas of significant reduction in PCNA staining (arrowhead) are observed when these organs are cultured in the presence of the β-catenin antisense PMO. Other areas continue to be positive for PCNA (arrow). (D) A representative section from a normal organ culture shows about 20%–25% of cells staining positive for Ki-67 (arrows), indicating the percentage of cells in S phase of the cell cycle. About 35%–40% of cells were positive in the ductular arrangement and about 20% in the sheet-like arrangement. (E) Ki-67 staining (arrow) of an embryonic liver cultured in the presence of the control PMO also shows about 20% of cells in S phase in the sheet-like arrangement. (F) Another representative section of the control PMO-treated cultures shows about 40% of cells staining positive for Ki-67 (arrow) in the ductular area. (G) A significant decrease in Ki-67–positive cells (arrows) is seen here in the β-Catas–treated cultures, with about 5% of cells in S phase in the sheet-like arrangement of the cells. (H) Another representative section from similarly treated cultures also shows <3% of cells to be Ki-67 positive (arrows). (I) A similar number of Ki-67–positive cells is also observed in the ductular area in another representative section from β-catenin antisense-treated culture with <5% of cells positive for Ki-67 (arrows).
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
β-Catenin loss of function induces apoptosis in embryonic liver cultures and suggests its prosurvival role in developing liver. (A) TUNEL staining of a normal culture shows <5% positive cells (arrows), with more TUNEL-positive cells in the sheet-like arrangement than in the ductular arrangement. (B) A representative section from the control PMO-treated culture shows a similar number of apoptotic nuclei (arrows) in both forms of cellular arrangements with about 5% TUNEL-positive cells in ducts and about 10%–15% positive cells in sheets. (C) Another representative section from Ctrlas-treated culture shows <10% TUNEL-positive cells (arrows) in the sheet-like arrangement. (D) A significant increase in TUNEL-positive cells is evident in this representative section from an embryonic liver cultured in the presence of β-Catas for 72 hours. More than 90% of cells are TUNEL positive (arrows), with an increase to about 80% in the number of TUNEL-positive cells in the ducts and about 90% in sheets. (E) Another representative section shows about 95% apoptotic nuclei (arrows) in the ductular cell arrangement in the antisense-treated culture. (F) A significant increase in the number of TUNEL-positive cells (>90%) (arrows) is clearly evident in another representative culture with β-catenin antisense. Arrow points to increased number of apoptotic nuclei (as in D and E).
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Inhibition of β-catenin retards hepatocyte maturation and induces loss of biliary differentiation. All figures are <40× original magnification unless otherwise noted. (A) A representative section from Ctrlas-treated embryonic liver culture shows Hep-Par–positive cells (arrows) in all layers in the ductular arrangement of cells, as well as in other cells of hepatocytic lineage in the sheet-like arrangement. This was similar to the Hep-Par localization in the normal embryonic liver cultures. (B) Hep-Par staining remains unaffected following β-catenin inhibition, with cells in ducts and sheets continuing to stain for this marker of hepatocyte lineage (arrows). (C) Albumin-positive cells (arrows) are observed in all layers of ductular arrangement as well as in the sheet-like arrangement in the Ctrlas-treated cultures (original magnification 60×). (D) Albumin staining remains unaffected (arrows) in the organs cultured in the presence of β-Catas (original magnification 60×). (E) c-kit–positive cells (arrows) were only observed in the innermost or luminal layer in the ductular cellular arrangement in this representative control PMO-treated culture (original magnification 60×). (F) A substantial increase in c-kit–positive cells in the ductular arrangement is evident in this representative section from the β-Catas–treated cultures, indicating a deficit in maturation of hepatocytes. There is increased c-kit immunoreactivity in the inner (arrows) as well as outer (arrowhead) layers in ducts. An overall increase in c-kit–positive cells is also seen in the sheet-like cellular arrangement (original magnification 60×). (G) CK19 immunostaining of control PMO-treated cultures shows the innermost layer of the ductal array to be CK19 positive (arrows). This layer is also positive for Hep-Par and albumin and some cells are also c-kit positive in this layer, indicating its bipotential capacity (as shown earlier). Some cells in the sheet-like arrangement are also CK19 positive. (H) A complete loss of CK19 immunoreactivity is observed in the innermost layer of the ducts (arrowheads) in this representative section from embryonic livers cultured with β-catenin antisense for 72 hours, indicating loss of biliary differentiation potential in these cells following β-catenin inhibition. (I) Another representative section from the β-Catas–treated cultures shows loss of CK19 positivity (arrowheads) in the innermost layer of the ducts. Arrowheads point to the loss of CK19 positivity. (J) The innermost layer in the ductular layer shows CK19-positive cells (arrows) in the embryonic liver cultured in the presence of the Ctrlas for 24 hours. (K) A significant loss of CK19-positive cells (arrowheads) in 24-hour β-Catas–treated cultures. Very few CK19-positive cells (arrow) were seen in the innermost ductular layer of such cultures, suggesting a role for β-catenin in early biliary specification of the bipotential stem cells. (L) A significant loss of CK19 positivity (arrowheads) is observed after 48 hours of antisense treatment in the ductular arrangements, suggesting a role in survival of the biliary epithelial cells.
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

8. Fig. 7
c-Myc and cyclin D1 remain unaffected following β-catenin inhibition in embryonic liver cultures. (A–C) Representative sections from normal culture, control PMO-treated cultures, and β-catenin antisense-treated cultures showing comparable c-Myc staining (arrows) (original magnification 60×). (D–F) Representative immunohistochemical stains showing no change in cyclin D1 staining (arrows) in the embryonic livers cultured without and with control PMO and antisense PMO, respectively (original magnification 40×).
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

9. Fig. 8
A schematic representation of differentiation of hepatic progenitors shows a multistep role for β-catenin in the cascade of hepatocyte maturation and bile duct differentiation by affecting cell proliferation and apoptosis in liver development.
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions