Figure 6

Stimulation of Wnt signaling in intestinal epithelial cells. (A) An increased number of Wnt-activated cells are detected in an intestinal adenoma from a progeny of a BatGal and Min mouse mating. β-gal-positive cells are in red (arrows). Wnt signaling is stimulated in response to gamma-irradiation-induced injury. (B) Intestinal tissue sections from lethally irradiated TOPGAL mice harvested at various timepoints were stained with antibodies to β-gal (red) and quantified. At 24 h post-irradiation, the number of crypts harboring Wnt-receiving cells significantly increased (p = 0.004; asterisk) relative to non-irradiated controls. (C-D) Comparison of representative intestinal tissue sections from lethally irradiated TOPGAL mice stained with antibodies to β-gal (red) and counterstained with Hoechst dye (blue) at 1 h post-irradiation (C) and 24 h post-irradiation (D). (E) Wild type mice, 24 h post-irradiation, were examined with antibodies to β-catenin (brown) and counterstained with Hematoxylin (purple). Solid line marks the epithelial-mesenchymal boundary of the intestinal crypts. At 1 h post-irradiation, the number of β-gal-expressing cells was similar to the 0 h control, but increased in 24 h post-irradiated tissues. Asterisks denote β-gal or nuclear β-catenin positive crypts; black arrowheads denote apoptotic cells. Bar = 25 μm. (F) The number of β-gal-positive cells per crypt was scored in both non-irradiated (Non-IR) and 24 h post-irradiated (post-IR) intestines. The percentage of crypts with 1, 2 or greater than 2 β-gal-positive cells are shown. (G) qRT-PCR performed on mRNA from small intestinal crypt fractions of TOPGAL mice 24 h post-irradiation revealed an increase in the lacZ reporter gene compared to non-irradiated samples. In addition, three Wnt ligands known to be expressed in the intestinal epithelium (Wnt3, Wnt6, and Wnt9b) and a Wnt target gene (c-Myc) increased in response to the irradiation stimulus.