Volume 126, Issue 5, Pages 1236-1246 (May 2004) Inducible cre-mediated control of gene expression in the murine gastrointestinal tract: effect of loss of β-catenin  Heather Ireland, Richard Kemp, Carol Houghton, Louise Howard, Alan R. Clarke, Owen J. Sansom, Douglas J. Winton  Gastroenterology  Volume 126, Issue 5, Pages 1236-1246 (May 2004) DOI: 10.1053/j.gastro.2004.03.020

Figure 1 Inducibility of Cre recombinase in Ahcre mice. (A) Reverse-transcription PCR for cre and hprt (positive controls) in βNF-induced (+) and uninduced (−) Ahcre mice. Shown are liver (li), SI, esophagus (oe), forestomach (fs), glandular stomach (gs), gallbladder (gb), and colon (co). (B–E) Immunohistochemistry for Cre showing nuclear localized reactivity in (B) liver, (C) SI, (D) esophagus, and (E) pancreas. Bars are 25 μm. Gastroenterology 2004 126, 1236-1246DOI: (10.1053/j.gastro.2004.03.020)

Figure 2 Patterns of epithelial Cre-mediated recombination in AhcreR26R mice as shown by tissue staining for β-gal. (A, C, E–I) Animals killed 2–4 weeks after 5 daily IP doses of βNF. (A, B) Liver, (C, D) SI, (B, D) untreated, (E) pancreas, (F) esophagus, (G) glandular stomach, (H) gallbladder, (I) Brunner’s glands. (J–M) Intestinal whole-mounts of SI and colon stained for β-gal 2–6 weeks after treatment. (N) Close-up view of colonic whole-mount. (J) Corn oil only, (K–N) βNF 80 mg/kg treated; (K) single IP dose showing a pronounced proximal to distal expression gradient, (L, N) × 3 IP doses, (M) × 5 IP doses. The whole-mounts are prepared from the duodenum (top left) to the end of the ileum and from below the cecum to the anus (bottom right). Bars are (A–I) 25 μm, (J–M) 1 cm, (N) 200 μm. Gastroenterology 2004 126, 1236-1246DOI: (10.1053/j.gastro.2004.03.020)

Figure 3 Dose responsiveness in the extent of SI recombination in AhcreR26R mice. (A) Southern blot of purified intestinal epithelium 4 weeks after βNF treatment analyzed by phosphorimaging. Bands corresponding to the intact (2.1 kb) and recombined (3.6 kb) ROSA26 locus are shown after IP injection with (track 1) corn oil only, (track 2) single dose of βNF (8 mg/kg), (track 3) single dose of βNF (80 mg/kg), (track 4) 3 doses of βNF (80 mg/kg), (tracks 5, 6) 5 doses of βNF (80 mg/kg). (B) Bar chart showing percentage recombination as determined by direct counting of X-gal-stained crypts in whole-mounts at 25% (open bar), 50% (striped bar), and 75% (black bar) of the length of the SI distal from the stomach and the midregion of the colon (stippled bar). n = 3 or 4, error bars = SE. Gastroenterology 2004 126, 1236-1246DOI: (10.1053/j.gastro.2004.03.020)

Figure 4 Epithelial selectivity in intestinal recombination in βNF (× 5 IP doses) induced AhcreR26R mice. (A) H&E-stained denuded SI showing tissue remaining after ethylenediaminetetraacetic acid-mediated removal of the epithelium, (B) X-gal-stained intestinal section showing predominantly epithelial staining with occasional positive muscle fibers (arrows), (C) Southern blot analyzed by imaging software showing recombination (3.6-kb band) in (track 1) denuded intestine, (track 2) purified epithelium, and (track 3) intact intestine. Bars are 25 μm. Gastroenterology 2004 126, 1236-1246DOI: (10.1053/j.gastro.2004.03.020)

Figure 5 Timing and localization of Cre recombination in AhcreR26R mice. (A, B) X-gal-stained proximal SI after 1 dose IP of βNF (80 mg/kg): (A) 1 day postinduction, (B) 3 days postinduction. (C–F) X-gal-stained SI after oral feeding of βNF; (C, D) at the end of a 3-day period of feeding, (E, F) 9 months after feeding. (A–C, F) Frozen sections, (D, E) whole-mounts. (C) β-galactosidase expression largely is restricted to the villus and upper crypt. (E) Whole-mount showing that β-gal-positive recombinant epithelium forms a distinct branching pattern. Sections of such whole-mounts (F) show that this stained epithelium overlays blood vessels (arrows). Bars are (A–C, F) 25 μm and (D, E) 5 mm. Gastroenterology 2004 126, 1236-1246DOI: (10.1053/j.gastro.2004.03.020)

Figure 6 Response of the SI to loss of β-catenin. (A–O) Proximal SI analyzed with time after Cre induction. (A, D, H) Control sections from β-cateninflox/wt/Ahcre mice: (B, C, E, F, J–O) from β-cateninflox/flox/Ahcre mice. (A–E) β-catenin staining. At 4 days postinduction there is extensive β-catenin-deficient epithelium present on (B) intestinal villi and (B, E, P) reduction in the number of glandular crypts (A, D) compared with controls. (C) By day 5, the β-catenin-deficient epithelium is restricted to the villus tip and β-catenin immunoreactive epithelium occupies enlarged crypts and extends up the side of villi. (F) Staining for caspase 3 shows increased numbers of apoptoses within crypts at 4 days postinduction. (G) Staining for lysozyme shows mislocalization of Paneth cells above the crypt base (arrows). (H–J) Staining for muc2. (I) Reduced numbers of goblet cells on villi at 4.5 days accompanied by detachment of the epithelium as intact sheets, (H) compared with controls. (I) Remaining points of attachment are mediated by muc2-reactive cells. (J) By 5 days goblet cells in β-cateninflox/flox/Ahcre mice are concentrated at the villus tip. (K–M) Double staining for β-catenin (green) and muc2 (red) at (K, L) 4.5 days and (M) 5 days confirms that the underrepresentation of (K) muc2-positive cells, (K, L) epithelial detachment (white arrows), and (M) goblet cell clustering occurs within β-catenin-deficient epithelium. (L) Close-up of attached goblet cell within a detached villus epithelial sheet showing stretching of the basal region of the cell. Strong immunoreactivity for (N) villin and (O) E-cadherin within the affected villus epithelium of β-cateninflox/flox/Ahcre mice at 4 and 5 days, respectively. (P) Quantitative analysis of crypt loss, (Q) apoptosis, (R) mitoses (white bars, β-cateninflox/wt/Ahcre; grey bars, β-cateninflox/flox/Ahcre), and (S) goblet cell number. Error bars are SE. Bars are (A–K) 25 μm, (L) 5 μm. Gastroenterology 2004 126, 1236-1246DOI: (10.1053/j.gastro.2004.03.020)