1. β-Catenin is temporally regulated during normal liver development
Amanda Micsenyi, Xinping Tan, Tamara Sneddon, Jian-Hua Luo, George K. Michalopoulos, Satdarshan P.S. Monga 
Gastroenterology 
Volume 126, Issue 4, Pages (April 2004)
DOI: /j.gastro

2. Figure 1
Changes in β-catenin during normal liver development. (A) A representative Western blot shows increased levels of β-catenin (top panel) at E12, followed by a decrease at E14 and loss at E16 and E18, followed by significant levels in the adult. A lower-species band, or tβ-catenin (open arrow; top panel), was observed at all stages of development. Actin blot (lower panel) shows comparable loading. (B) Three representative Western blots were scanned, and the normalized integrated optical density was analyzed for statistical significance. There was a significant decrease (P < ) from E14 to E16. (C) A total of 100 or 40 μg of protein from E16, E18, and adult liver and the insoluble pellet from E18 liver (after RIPA extraction) was analyzed by Western blot for β-catenin and other markers of hepatocyte and biliary differentiation as internal controls. There was minimal β-catenin at E16, followed by a complete loss (undetectable even at 100 μg). No β-catenin was detected in the pellet from E18. Significant AFP was present at E16 and E18 (as expected), and none was observed in adult liver. Albumin was present, as expected, at all stages. Cytokeratin (CK)-19 levels were higher in the E16 and E18 than the adult livers, in part because of overloading but also because of the presence of some bipotential stem cells and the formation of bile ducts, plates, and triads at these stages. Ad, adult.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
β-Catenin localization and PCNA staining during normal liver development. (A) β-Catenin localizes (brown staining) to the resident cells at the membrane (arrow), cytoplasm (black arrowhead), and nucleus (white arrowhead) in the E10 liver. Larger cells (stem cells) are more intensely positive and hematopoietic cells in the channels are negative to weakly positive. The inset is a high-power view showing membranous (arrow), cytoplasmic (black arrowhead), and nuclear (white arrowhead) localization of β-catenin. (B) β-Catenin localizes to most cells at E12. It is again seen at the membrane (arrow), in cytoplasm (black arrowhead), and in nucleus (white arrowhead). Hematopoietic cells are negative. (C) At E14, β-catenin stains mainly the membrane of hepatocytes (arrow) and stem cells; a few cells show cytoplasmic localization (black arrowhead). The flattened cells lining the channels are clearly positive for β-catenin as well. (D) At E15, the staining is again similar to that of E14, with membranous (arrow) and cytoplasmic (black arrowhead) β-catenin localization. However, there are more areas of differentiated cells that form clusters along with the negative hematopoietic cells. (E) The staining at E16 is predominantly membranous (arrow), and there are several cells interspersed that are negative for β-catenin. (F) At E18, staining for β-catenin is thin and membranous (arrow) only. (G) At E10, approximately 80%–90% of cells show nuclear positivity to PCNA (arrowhead), and the remaining cells are negative for it (arrow). (H) Approximately 40%–45% of cells are PCNA positive (arrowhead) at E12 that are surrounded by several negatively staining cells (arrow). (I) Less than 25% of cells were PCNA positive (arrowhead) at E14. (J) Approximately 15% of cells maintained PCNA positivity (arrowhead) at E15 while being interspersed among the negative cells (arrow). (K) Less than 10% of PCNA-positive cells were seen at E16 (arrowhead). (L) Approximately 5%–8% of PCNA-positive cells (arrowhead) were observed in this representative section from E18 liver, where most cells were negative (arrow).
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
β-Catenin and PCNA correlation during normal liver development. (A) Mean percentages of PCNA-positive resident liver cells (from 3 different liver sections) at specific stages of development are presented. Also presented are the means from percentages of all resident cells at the same stages of development that show nuclear and cytoplasmic positivity to β-catenin. (B) Graphic representation of these values depicts a decreasing trend in the positivity of both PCNA and nuclear and cytoplasmic β-catenin in the livers with the progression of gestational development. (C) Analysis for correlation of these 2 parameters during liver development showed a correlation coefficient (r) of with an extremely statistically significant P value of , indicating a strong correlation between the nuclear/cytoplasmic β-catenin and proliferation during prenatal hepatic development. CI, confidence interval; N/C, nuclear/cytoplasmic.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
A multifactorial regulation of β-catenin during normal liver development. (A) A representative Western blot demonstrates serine 45/threonine 41-phosphorylated β-catenin during normal liver development (top panel). Even though no β-catenin was detected at E16 and E18 (third panel), there was serine 45/threonine 41-phosphorylated β-catenin at these stages, suggesting its importance in β-catenin degradation. The second panel is a representative blot for serine 33/37/threonine 41-phosphorylated β-catenin and demonstrates levels comparable to the total β-catenin and no reciprocal relationship. The bottom panel shows equal loading, as shown by a β-actin blot. (B) Densitometric analysis depicts a decrease in total β-catenin levels that corresponds to a drastic increase in the serine 45/threonine 41-phosphorylated β-catenin to total β-catenin ratio, indicating the importance of these phosphorylation sites in β-catenin degradation in liver development. (C) Microarray analysis results from pooled liver samples at E11, E14, E17, E18, and adult stages show high expression (signal) of β-catenin at E11, followed by a 2-fold decrease. This change was confirmed by Northern blot analysis, which also showed a noteworthy decrease in ctnnb1 expression after E11 (lower panel). Some other positive internal controls used for validation of the gene array included decreasing expression (signal) of c-kit, an hepatic stem cell marker, and α-fetoprotein, an hepatic stem cell and immature and fetal hepatocyte marker that is not normally expressed in normal adult liver. (D) Representative Western blots show changes in several Wnt pathway components at different stages of liver development. No major changes were observed in the Wnt-1 levels, and Wnt-1 seems to be present throughout development and in the adult stage. Maximum levels of GSK-3β were present at E14 and E16 and coincided with β-catenin degradation. Maximum levels of axin were seen at E14, followed by a gradual decrease. APC, another negative regulator of β-catenin, also peaked at E14, followed by a uniform decrease. Actin (in the lower panel) was a loading control. (E) Increased levels of c-myc were observed at E12 and E14 and coincided with higher β-catenin levels during early liver development (top panel). High cyclin-D1 levels, however, were observed at E14 and E16. Actin blot demonstrated comparable loading. (F) The top panel shows E-cadherin levels during normal liver development; they were low at E12, followed by an increase. An inverse relationship to β-catenin is evident, especially at E12 and E16. The β-actin loading control shows uniform protein loading. The lower 2 panels depict a representative co-precipitation study that shows an association of β-catenin and E-cadherin at all analyzed developmental stages, including E17 and E18, where no normal β-catenin (97-kilodalton species) was present in the immunoprecipitate. This indicates that the lower species (or tβ-catenin) might be associating with E-cadherin especially at these stages or all stages analyzed because the lower species was uniformly present. Ad, adult; IOD, integrated optical density; APC, adenomatous polyposis coli gene product; IP, immunoprecipitation.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Restoration of β-catenin levels in the liver in the perinatal stage. (A) Representative Western blots using whole-cell lysates from pooled livers from newborn pups, their in utero littermates, and 5-day-old pups show increased levels of β-catenin with progressive development. Wnt-1 levels also increased, whereas levels of GSK-3β and axin decreased after birth and at day 5. There was a concomitant decrease in serine 45/threonine 41-phosphorylated β-catenin and an increase in serine 33/37/threonine 41-phosphorylated β-catenin, as seen in the representative blots, again indicating that serine 45/threonine 41 is a crucial indicator of β-catenin degradation in liver. Also, some decrease in E-cadherin was evident. Actin levels in the lowest panel show comparable protein loading. (B) There was more than a 2-fold increase in total β-catenin levels in the livers after birth as compared with in utero littermate livers. Densitometric analysis of the scanned blots from 3 sets of Western blots showed this increase to be statistically significant (P < ). (C) There was more than a 3.5-fold decrease in serine 45/threonine 41-phosphorylated β-catenin (P < ) in the livers after birth. Representative Western blots were subjected to densitometry, and mean integrated optical density (IOD) was calculated, plotted, and compared for statistical significance. (D) Increased expression of ctnnb1 (β-catenin gene) was evident in the postnatal livers as compared with their intrauterine counterparts by Northern blot. Glyceraldehyde phosphate dehydrogenase (GAPDH) expression (in the lower panel) was the loading control. (E) Densitometric analysis of the Northern blots depicts a statistically significant increase (P = ).
Gastroenterology  , DOI: ( /j.gastro )