1. Volume 137, Issue 1, Pages 145-155.e3 (July 2009)
Notch Signaling Promotes the Generation of EphrinB1-Positive Intestinal Epithelial Cells 
Bon–Kyoung Koo, Hyoung–Soo Lim, Hee Jin Chang, Mi–Jeong Yoon, Yongwook Choi, Myung–Phil Kong, Cheol–Hee Kim, Jin–Man Kim, Jae–Gahb Park, Young–Yun Kong 
Gastroenterology 
Volume 137, Issue 1, Pages e3 (July 2009)
DOI: /j.gastro
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2. Figure 1
Increased numbers of secretory lineage cells in the intestines of Vil–Cre;Mib1f/f mice. Histochemical (A, B) and immunohistochemical (C–F) analyses of intestinal tissues from 4-week-old Vil–Cre;Mib1+/f mice (A, C, E) and Vil-Cre;Mib1f/f mice (B, D, F). Tissues were stained for periodic acid-Schiff/Alcian blue (goblet cells; A, B), chromogranin A (enteroendocrine cells; C, D), and lysozyme (Paneth cells; E, F). The total number of secretory lineage cells was increased in Vil–Cre;Mib1f/f mice (B, D, F) compared with Vil–Cre;Mib1+/f mice (A, C, E). (A, B) An enlarged image, the boxed area in the left, is shown in the right. Arrows indicate lysozyme-positive cells in the villus. Scale bar, 50 μm.
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3. Figure 2
Loss of proliferating progenitors and induction of p27kip1 in intestinal tissues of Vil–Cre;Mib1f/f mice. Immunohistochemical analysis of 4-week-old Vil–Cre;Mib1+/f (A, C) and Vil–Cre;Mib1f/f (B, D) mice. Tissues were analyzed for levels of proliferating cell nuclear antigen (PCNA), which indicates proliferating cells, and Alcian blue, which indicates goblet cells (A, B). Levels of the cyclin-dependent kinase inhibitor p27kip1 were also assessed (C, D). Scale bar, 30 μm.
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4. Figure 3
Vil–Cre;Mib1f/f mice have defects in generation of EphrinB1-positive IECs from proliferating progenitors. Immunohistochemical analysis of the small intestine from 4-week-old Vil–Cre;Mib1+/f (A, D, left panels), Vil–Cre;Mib1f/f (B, E, middle panels) and Vil–Cre;Mib1f/f;RN1+/RN1 (C, F, right panels) mice. Paraffin sections were analyzed for levels of EphrinB1 (A–C, upper panels, in red) and EphB2 (D–F, lower panels, in red), with double immunostaining for levels of proliferating cell nuclear antigen (PCNA; A–F, in green). Sections from the Vil–Cre;Mib1f/f mice show the loss of EphrinB1 with the accumulation of EphB2. The phenotypes of Vil–Cre;Mib1f/f mice are reverted by the activation of the Notch1 transgene in Vil–Cre;Mib1f/f;RN1+/RN1 mice. Scale bar, 50 μm (A–F).
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5. Figure 4
Notch signaling promotes the generation of EphrinB1-positive IECs. (A) Statistical analysis of BrdU pulse-labeled, 5- to 7-week-old Vil–Cre;Mib1+/f (wt), Vil–Cre;Mib1f/f (Mib1cKO), and Vil–Cre;Mib1f/f;RN1+/RN1 (Mib1cKO;RosaN1) mice. Sections were analyzed by double staining for EphrinB1 and BrdU. The Vil–Cre;Mib1f/f mice are defective in the generation of EphrinB1-positive IECs compared with Vil–Cre;Mib1+/f and Vil–Cre;Mib1f/f;RN1+/RN1 mice. The black and white columns indicate the percentages of EphrinB1-positive and EphrinB1-negative cells, respectively, in the BrdU-positive cells of each genotype. (B) Real-time quantitative PCR analysis of mock- and ΔEN1-retrovirus–infected IEC-6 cells. The IEC-6 cells that expressed activated Notch up-regulated EphrinB1 and Hes1 and down-regulated EphB2. (C–E) Real-time, quantitative PCR analysis of IEC-6 cells. The γ-secretase inhibitor DAPT inhibits the expression of EphrinB1 in cells that express activated Notch (ΔEN1; C). The dominant-negative form of Mastermind (DN) also inhibits the expression of EphrinB1 in these cells (D). Hes1 expression had no effects on the expression of EphrinB1 in these cells (E).
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6. Figure 5
Activation of the β-catenin signaling pathway in Vil–Cre;Mib1f/f intestinal epithelium. Analysis of colons tissue from 4-week-old Vil–Cre;Mib1+/f (A, D), Vil–Cre;Mib1f/f (B, E), and Vil–Cre;Mib1f/f;RN1+/RN1 (C, F) mice with Alcian blue/hematoxylin staining (A–C) and anti–β-catenin antibody immunostaining (E, F). Vil–Cre;Mib1f/f crypts show high levels of cytoplasmic and nuclear β-catenin expression (E, arrows), whereas the others show normal membranous β-catenin expression. Note that Vil–Cre;Mib1f/f;RN1+/RN1 mice contain no Alcian blue-positive cells because of Notch1 activation. Scale bar, 20 μm.
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7. Figure 6
Activation of Notch promotes the GSK3β kinase activity. Immunoblot analysis of Vil–Cre;Mib1+/f (wt) and Vil–Cre;Mib1f/f (mt) intestinal epithelia (A, B) and IEC-6 cells infected with mock and ΔEN1-expressing retrovirus (C). An antibody against unphosphorylated β-catenin was used to detect active β-catenin. Antibodies against β-catenin protein and β-actin were used for normalization. (D, E) Immunoblot analysis of IEC-6 cells that express activated Notch (ΔEN1); mock-transfected cells were used as controls. Cells were incubated with the proteosome inhibitor (MG132) or the GSK3 inhibitor LiCl. An antibody against unphosphorylated β-catenin was used to detect active β-catenin. Antibodies against β-actin protein and β-actin were used for normalization. The IEC-6 cells that expressed activated Notch had higher levels of β-catenin that was phosphorylated at S33/37/T41 (E). (F) Immunocytochemical analysis of IEC-6 cells that express activated Notch (ΔEN1); mock-transfected cells were used as controls. The IEC-6 cells that expressed activated Notch had higher levels of cytoplasmic β-catenin that was phosphorylated at S33/37/T41 (upper right panel). Staining for total β-catenin protein was used as a positive control (lower panels).
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8. Figure 7
Activation of Notch suppresses expression of EphB2 through GSK3β-mediated β-catenin phosphorylation and degradation. (A, B) Immunoblot analysis of IEC-6 cells that express activated Notch (ΔEN1); mock-transfected cells were used as controls. Cells were incubated with GSK3 inhibitors (LiCl, BIO). Levels of β-actin were used for normalization. The cells that expressed ΔEN1 had higher levels of β-catenin that was phosphorylated on S33/37/T41 (A, lane 2); this phosphorylation was inhibited by GSK3 inhibitors (A, lane 2 [LiCl] and lane 3 [BIO]). The phosphorylated β-catenin was degraded in an ubiquitin-dependent manner (Ub, ubiquitin; B).35 (C) Immunoblot analysis of IEC-6 cells that were mock transfected or transfected with a retrovirus that expressed ΔEN1 or Hes1. The over-expression of Hes1 had no effect on the GSK3β-mediated phosphorylation of β-catenin. (D, E) Real-time quantitative PCR analysis of IEC-6 cells. Incubation of cells with BIO restored the expression of EphB2 (D); Hes1 overexpression did not repress the expression of EphB2 (E).
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9. Figure 8
A schematic view of the intestinal Notch signaling pathway in IECs. Notch is cleaved by γ-secretase (which is inhibited by DAPT); the Notch-soluble intracellular domain Nicd then moves into the nucleus where it interacts with RBPj to up-regulate expression of EphrinB1 and Hes1. Hes1 inhibits Math1 and expression of p27kip1 and p57kip2, to maintain proliferative progenitors. The Nicd also activates GSK3β (inhibited by BIO), which leads to phosphorylation, ubiquitination (Ub), and degradation of β-catenin. Because unphosphorylated β-catenin interacts with TCF-4 to up-regulate EphB2 expression, Notch-mediated phosphorylation of β-catenin results in EphB2 down-regulation.
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10. Supplementary Figure 1
Generation of Vil–Cre;Mib1f/f and Vil–Cre;RN1+/RN1 mice. (A) Histologic analysis of the small intestine (upper panels) and colon (lower panels) from 1-week-old Vil–Cre;Mib+/f (left panels) and Vil–Cre;Mib1f/f (right panels) mice. Paraffin sections were analyzed by AB. Sections from the Vil–Cre;Mib1f/f mice show goblet cell metaplasia. Scale bar, 100 μm. (B) Histologic analysis of the small intestine (upper panels) and colon (lower panels) from 5-week-old Vil–Cre;RN1+/+ (left panels) and Vil–Cre;RN1+/RN1 (right panels) mice. Paraffin sections were analyzed by AB. Sections from the Vil–Cre; RN1+/RN1 mice show loss of goblet cells. Scale bar, 50 μm. (C) X-gal staining of small intestine (upper panel) and colon (lower panel) from a 4-week-old Vil–Cre;Rosa-Reporter mouse. These data show the effective recombination activity of Vil–Cre in the intestinal and colonic epithelia. (D) Western blot analysis shows the loss of Mib1 protein expression upon Cre expression driven by the rat Villin-promoter. β-Actin was used for normalization. Note that the Villin–Cre (Vil–Cre) effectively deletes Mib1 in the ileum and colon epithelia. Col, colon; Duo, duodenum; Jej, jejunum; Ile, ileum; mt, mutant (Vil–Cre;Mib1f/f); wt, wild type (Vil–Cre;Mib1+/f).
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