Figure 1: Defects in small intestinal CBC stem cell and Paneth cell gene expression in late-generation mTR^−/− mice.

(a) Representative images of in situ hybridization for Ascl2 transcripts in ileum from WT, G2 and G4 mTR^−/− mice and quantitation of crypts positive for staining (right; n=3); *P<0.05. Scale bars, 100 μm. (b) Representative images of Sox9 immunostaining in ileum from WT (n=5), 2nd generation (G2, n=3) and 4th generation (G4, n=4) mTR^−/− mice and quantitation of crypts positive for staining (right); *P<0.05. Scale bars, 50 μm. (c) Gene Set Enrichment Analysis (GSEA) revealed G4 mTR^−/− ileal crypts to have significantly reduced expression of genes downregulated in β-catenin knockout mouse crypts (FEVR_CTNNB1_TARGETS_DN) (n=3). Enrichment score (ES), normalized enrichment score (NES), P value and false discovery rate (FDR) are as described⁵⁰ (see ‘Methods’ section). (d) Quantitative reverse transcription PCR (qRT-PCR) analyses of gene expression for the Wnt ligand (Wnt3), Wnt co-receptor (Lrp6) and Wnt target gene and co-receptor (Lgr5) in WT and G4 mTR^−/− crypts (n=5). Also, note that Msi1 protein expression is unchanged (Supplementary Fig. 2d). *P<0.05 and ***P<0.005. (e) Immunostaining for Paneth cell lysozyme in WT and G4 mTR^−/− ileum. Enlarged insets show examples of cells at the normal CBC location between lysozyme-positive Paneth cells (asterisks). β-catenin staining marks cell peripheries. Scale bars, 50 μm (top), 25 μm (bottom). (f) Quantitation of Paneth cells as measured by lysozyme staining and (g) of cells intercalated between Paneth cells from WT and G4 mTR^−/− crypts (n=6); *P<0.05. All error bars reflect s.e.m., and P values reflect unpaired two-tailed Student’s t-tests.