1. Volume 131, Issue 5, Pages 1561-1572 (November 2006)
Conditional Deletion of β-Catenin Reveals Its Role in Liver Growth and Regeneration 
Xinping Tan, Jaideep Behari, Benjamin Cieply, George K. Michalopoulos, Satdarshan P.S. Monga 
Gastroenterology 
Volume 131, Issue 5, Pages (November 2006)
DOI: /j.gastro
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2. Figure 1
Successful deletion of β-catenin in hepatocytes in the conditional Ko mice. (A) Targeting strategy for conditional inactivation of the gene for β-catenin. Cre-mediated excision of the floxed β-catenin allele (top) leads to the generation of a floxdel allele (bottom) lacking exons 2–6. (B) Representative genotyping of mice and livers for identifying the Ctnnb1 loxP/loxP; Cre+/- (conditional knockouts) showing simultaneously floxed allele and cre in tail DNA. Polymerase chain reaction also confirmed the presence of the recombined β-catenin allele (floxdel allele) in the liver. (C) Representative Western blot analysis showing a dramatic decrease in total β-catenin protein in the whole-liver lysates from the conditional β-catenin null mice and their age- and sex-matched littermate controls. (D) Representative immunohistochemistry for β-catenin in 2-month-old control littermate livers show its localization in hepatocytes (D1) (100×), which is predominantly membranous at higher magnification (D2) (400×). A uniform lack of β-catenin is observed in Ko livers (D3) (100×) and can be appreciated at higher magnification (D4) (400×). Some β-catenin continued to stain nonparenchymal cells (arrowheads).
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Appreciable decrease in LW/BW ratio is evident in the β-catenin conditional null mice. This deficit was around 20% before 2 months of age and in older mice, and ranged anywhere from 15% to 35%. The difference between the Ko and age-matched control littermates was statistically significant.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
The phenotype in β-catenin Ko livers was a result of the combination of an increase in apoptosis and a decrease in basal hepatocyte proliferation. (A) Normal liver section (50×) from 3-month-old control littermate shows a normal frequency of apoptotic nuclei (arrows). (B) A higher magnification (200×) of A shows TUNEL-positive apoptotic nuclei (arrowheads). (C) Liver section (50×) from 3-month-old β-catenin conditional knockout shows an aberrant frequency of apoptotic hepatocytes (arrows). (D) A higher magnification (200×) of C shows several hepatocytes with TUNEL-positive apoptotic nuclei (arrowheads). (E) PCNA-positive hepatocytes (arrowheads) are seen in 1-month-old age- and sex-matched Con liver at lower magnification (100×). (F) A higher magnification (200×) of E shows PCNA-positive hepatocytes (arrowheads) in Con liver. (G) Fewer PCNA-positive hepatocytes (arrowheads) were seen in the 1-month-old Ko liver. (H) Higher magnification of G (200×) shows a smaller number of PCNA-positive hepatocytes (arrowheads). (I) PCNA-positive hepatocytes (arrowheads) in 3-month-old Con liver from age- and sex-matched littermate (200×). (J) PCNA-positive hepatocytes (arrowheads) in 3-month-old Ko liver (200×). (K) Ki-67–positive hepatocytes (arrowheads) in 3-month-old Con liver from age- and sex-matched littermate (200×). (L) Ki-67–positive hepatocytes (arrowheads) in 3-month-old Ko liver (200×).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Increased apoptosis and decreased basal proliferation of hepatocytes seen in livers lacking β-catenin. (A) Significant increase in apoptosis in β-catenin conditional null livers in 1- and 3-month-old mice as compared with their control littermates (*P < .05). (B) A significant decrease in hepatocyte proliferation in the absence of β-catenin (**P < .001) is evident in Ko livers in 1-month-old mice only.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Specific changes in Wnt pathway components and targets of β-catenin in the absence of β-catenin in conditional null mice. (A) Lack of β-catenin is confirmed at 2, 3, 6, and 12 months in the whole-cell lysates from the Ko livers as compared with their controls by Western blot analysis. Although low levels of dephosphorylated or active form (β-catenin-A), are observed in control livers, this was nondetectable in the Ko livers. Total GSK3β levels appeared to be increased in Ko livers. However, its ser9-phosphorylated form (inactive) was increased simultaneously. E-cadherin levels remained unaltered except in 12-month-old Ko livers. β-actin confirmed equal loading. (B) Densitometry shows higher levels of total GSK3β, especially at 2 and 3 months in the Ko livers. (C) Densitometry shows increased levels of ser9-GSK3β in 3-month-old and older livers in the Ko as compared with the Con livers. (D) Decreased levels of EGFR in the Ko livers at 1, 2, 6, and 12 months as compared with the age- and sex-matched controls. Glutamine synthatase (arrow) levels consistently were lower in the absence of β-catenin at all stages. (E) Densitometry confirms lower EGFR levels at 1, 2, 6, and 12 months in the Ko livers. (F) Changes in the gene expression of various Cyps in the Ko livers. Although Cyps such as 2E1, 7B1, 51, 1A2, 2d9, 8B1, 2A5, and 4F14 were down-regulated substantially, other Cyps such as 2B10, 2B9, 4A14, and 1A1 showed up-regulation. (G) Down-regulation of Cyp1A2 and Cyp2E1 protein levels in β-catenin knockout mice was confirmed by Western blot analysis using liver microsomal preparations from age- and sex-matched control (Con) or knockout (Ko) mice. Bottom panel confirms equal protein loading as seen by Ponceau Red staining of the membranes.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Lack of β-catenin in hepatocytes affects their proliferation during liver regeneration after partial hepatectomy. (A) β-catenin staining in Con livers at 40 hours after partial hepatectomy (200×). (B) Higher magnification of A reveals chiefly membranous localization of β-catenin at 40 hours after hepatectomy. In addition, some hepatocytes also showed its nuclear localization (arrowheads) (400×). (C) β-catenin staining in Ko livers at 40 hours after partial hepatectomy (200×). Some nonparenchymal cells (arrowheads) showed β-catenin immunoreactivity in the Ko livers. In addition, the appearance of fat droplets was evident in the hepatocytes (arrow). (D) Higher magnification of C reveals the continued lack of β-catenin in hepatocytes at 40 hours after hepatectomy, with some positively staining nonparenchymal cells (arrowheads). Microvesicular steatosis (arrows) is evident in hepatocytes (400×). (E) Ki-67 immunohistochemistry shows several positive hepatocytes (arrowheads) in Con livers at 40 hours after partial hepatectomy (200×). (F) Ki-67 immunohistochemistry shows very few positive hepatocytes (arrowheads) in Ko livers at 40 hours after partial hepatectomy (200×). Several fat droplets (arrows) in hepatocytes can be appreciated in the Ko liver. (G) Several Ki-67–positive hepatocytes (arrowheads) also are observed in Con livers at 3 days (200×). (H) Several Ki-67–positive hepatocytes (arrowheads) also are observed in the Ko livers on the third day of liver regeneration (200×). (I) Few hepatocytes continue to be Ki-67–positive (arrowhead) after 7 days of hepatectomy in the Con livers (200×). (J) Very few isolated Ki-67–positive hepatocytes (arrowhead) are evident in the Ko liver at 7 days after hepatectomy (200×). (K) No Ki-67–positive hepatocytes can be observed in Con liver at 14 days after hepatectomy (200×). (L) A few Ki-67–positive hepatocytes (arrowheads) are observed in Ko livers at 14 days after hepatectomy (200×).
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Suboptimal liver regeneration in the absence of β-catenin is reflected by changes in cell proliferation, apoptosis, and the LW/BW ratio. (A) Less than 50% of hepatocytes are in the S-phase of the cell cycle at 40 hours after hepatectomy in the Ko mice as compared with their littermate controls (**P < .0001). However, high and comparable numbers of cells are Ki-67 positive at 3 days. A significant discrepancy in the Ko livers also is evident at 7 days after hepatectomy (P < .0001), whereas higher numbers of hepatocytes maintain Ki-67 positivity in the Ko livers as compared with the Con livers at 14 days after hepatectomy (P < .05). (B) Significantly higher number of hepatocytes showed apoptotic nuclei at 40 hours and at 14 days after hepatectomy in the Ko livers as compared with their controls. (C) Significantly lower LW/BW ratios are evident in the Ko mice at all stages during liver regeneration. However, the discrepancy is more pronounced at earlier stages (before 3 days), again supporting a delay in onset of liver regeneration in the absence of β-catenin.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Decreased G1 to S-phase transition during liver regeneration in the absence of β-catenin. (A) Differences in gene expression as determined by microarray analysis reveals various cyclins such as A, B, D, E, K, and T to be down-regulated in the Ko livers as compared with their controls at 40 hours after hepatectomy, suggesting a compromise in G1 to S transition. (B) A representative Western blot analysis shows an increase in total protein levels of cyclin-D1 in the Con livers at 40 hours after partial hepatectomy. Normal β-catenin levels are confirmed in this group of animals (top left panel). The Ko livers show very low levels of β-catenin (top right panel) and failure of increase in total cyclin-D1 protein to the same extent as their controls at 40 hours after partial hepatectomy. β-actin in bottom panel confirms equal loading.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions