Precancerous Lesions Upon Sporadic Activation of β-Catenin in Mice Isabelle Coste, Jean–Noël Freund, Simone Spaderna, Thomas Brabletz, Toufic Renno  Gastroenterology  Volume 132, Issue 4, Pages 1299-1308 (April 2007) DOI: 10.1053/j.gastro.2007.01.029 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Generation of S37F mice. (A) Partial amino acid sequence of wt and mutated exon 3 of β-catenin. Note that in addition to the Ser to Phe substitution at position 37, an EcoRI restriction site was generated without modification of the Ile at position 35. (B) Status of the β-catenin locus on homologous recombination of the targeting vector in ES cells. Several latent allele configurations are possible, depending on the occurrence of branch migration and/or gene conversion. These configurations are tested by digestion of ES cell genomic DNA with EcoRI and BglI followed by Southern blotting with an internal probe (thick arrows). (C) Example of such a Southern blot analysis of allele configuration. Asterisks indicate ES clones corresponding to a Latent 2 configuration. (D) Example of mouse genotyping. Exon 3 was amplified from tail DNA, and the amplification product was treated with EcoRI. DNA from mice containing the S37F mutation carries the new EcoRI restriction site described above and generates 2 additional bands upon digestion (samples 2 and 3). Gastroenterology 2007 132, 1299-1308DOI: (10.1053/j.gastro.2007.01.029) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Gastric lesions in S37F mice (delineated by dashed lines). (A) Normal gastric tissue organization (Harris staining). (B) In the dysplastic lesions, the surface epithelium showed brush borders (arrows, H&E staining). (C) The lesions developed as ramified glands in the mucosal height (Harris staining). (D) In vivo recombination of the latent β-catenin allele in the gastric lesions but not in adjacent normal tissue. Genomic DNA was isolated by laser-capture microdissection from the lesions or from the adjacent normal mucosa, amplified with a pair of primers spanning exon 2 to intron 8, and digested with EcoRI. (E) β-catenin staining of the normal mucosa-labeled cell membranes with a faint labeling at the surface (arrow). (F) β-catenin was also membranous at the bottom of the ramified lesions, although cytoplasmic accumulation occurred in the surface epithelium (arrows). (G) Proliferating cell nuclear antigen and (H) Ki67 stainings point to the proliferating cells located at the bottom of the ramified lesions (white arrowheads), whereas postmitotic cells are located at a higher position (black arrowheads). Arrows indicate proliferative cells located in the isthmus region of normal adjacent gastric mucosa. (I) β-catenin labeling of the same lesion as in G and H showed membrane staining in the proliferative cell compartment (white arrowhead) and increased accumulation in the postmitotic cells (black arrowhead). (J) H/K–adenosine triphosphatase and (K) histidine decarboxylase staining showed loss of these gastric markers in the lesions, whereas (L) sialomucins were expressed in the epithelial cells of the lesions but not in the adjacent normal gastric mucosa. (M) Cdx1 and (O) Cdx2 were not expressed in the lesions but in the duodenum (N and P). Antibodies are listed in Materials and Methods. Bar = 50 μm except for B, where bar= 20 μm. Gastroenterology 2007 132, 1299-1308DOI: (10.1053/j.gastro.2007.01.029) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Dedifferentiated gastric cells in S37F mice do not acquire intestinal phenotype. Shown is immunohistochemical staining for the indicated intestinal cell markers on sections of gastric lesions and duodenum of the same animal. Antibodies used are listed in Materials and Methods. Gastroenterology 2007 132, 1299-1308DOI: (10.1053/j.gastro.2007.01.029) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Activation of β-catenin decreases Shh. (A) Shh staining in gastric and duodenal tissue of S37F mouse. Gastric lesion is delineated by dashed lines. (B) Human gastric cell line 23132 was incubated in presence of the Wnt pathway inducer LiCl or control (NaCl). Shh and β-catenin immunostaining was performed using specific antibodies (see Materials and Methods). (C) Quantification of Shh and β-catenin immunofluorescence staining using the Lucia software (Nikon). (D) Real-time reverse-transcription PCR quantification of actin, lam5c2, or Shh messenger RNA from 23132 cells treated with NaCl or LiCl. For absolute copy number of the target transcripts, purified PCR fragments were used to produce a standard curve. Gastroenterology 2007 132, 1299-1308DOI: (10.1053/j.gastro.2007.01.029) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Characterization of papillomas in S37F mice. (A) Normal mammary tissue in a wild-type female mouse. (B) Perimammary papilloma with nipple hypertrophy in a female S37F mouse. (C) Histologic section of a papilloma from an S37F mouse showing acanthosis and hyperkeratosis. (D) In vivo homologous recombination of the β-catenin latent allele in the papilloma but not in adjacent normal tissue. Genomic DNA was isolated from the indicated tissues, amplified with a pair of primers spanning exon 2 to intron 8, and digested with EcoRI. (E and F) β-catenin immunohistochemical staining at low and high magnification of a papilloma from an S37F mouse reveals strong membranous accumulation in the membranes of keratinocytes but not in nuclei. (G and H) β-catenin staining at low and high magnification of normal adjacent skin. Sections were counterstained with hematoxylin to visualize the nuclei in blue. (I and J) Ki67 staining at low and high magnification of an S37F papilloma and (K and L) of the normal adjacent skin shows few labeled nuclei (arrows). (M) Alcian blue staining and (N) Muc2 immunohistochemical staining show absence of sialomucins and Muc2 expression in the papilloma. (O) Shh immunofluorescent detection in an S37F papilloma and (P) in normal adjacent skin shows absence of expression in the papilloma and strong fluorescent staining in hair follicles of the normal skin. (C) Bar= 200 μm. (E, G, I, K, M, and N) Bar= 100 μm. (O and P) Bar = 50 μm. (F, H, J, and L) Bar= 25 μm. Gastroenterology 2007 132, 1299-1308DOI: (10.1053/j.gastro.2007.01.029) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Blue nevi in skin of S37F mice after exposure to suboptimal DMBA/TPA carcinogenic treatment. (A) Skin biopsy specimens from an S37F mouse showing areas of hyperpigmentation. (B) Quantification of hyperpigmented areas in wild-type and S37F mice (experiment 1, 5 wild-type and 6 S37F male mice; experiment 2, 6 wild-type and 6 S37F female mice). (C) Histologic analysis of a hyperpigmented area revealing a blue nevus consisting of melanocyte aggregates with melanin overproduction. Gastroenterology 2007 132, 1299-1308DOI: (10.1053/j.gastro.2007.01.029) Copyright © 2007 AGA Institute Terms and Conditions