1. Foxf2 in Intestinal Fibroblasts Reduces Numbers of Lgr5+ Stem Cells and Adenoma Formation by Inhibiting Wnt Signaling  
Ali Moussavi Nik, Azadeh Reyahi, Fredrik Pontén, Peter Carlsson 
Gastroenterology 
Volume 144, Issue 5, Pages (May 2013)
DOI: /j.gastro
Copyright © 2013 AGA Institute Terms and Conditions

2. Figure 1
Foxf2 inhibits intestinal adenoma formation and growth in ApcMin mice. (A) Adenomas (arrowheads) in distal ileum and colon from 12-week-old ApcMin/+ mice with different Foxf2 gene dosages. (B) Adenomas per ApcMin/+ mouse (average ± SEM) for different Foxf2 genotypes: Foxf2−/+, 66.2 polyps/mouse (n = 17); wild-type, 42.8 polyps/mouse (n = 24); Tg(FOXF2), 18.5 polyps/mouse (n = 14) (ANOVA, P < 10−6; pairwise P = .02, P < 10−4, and P = 2 × 10−7, respectively). (C) Diameter of adenomas in distal ileum from 12-week-old ApcMin/+ mice with different Foxf2 genotypes (average ± SEM of 10log of diameter in millimeters): Foxf2−/+, 1.22 mm (n = 216); wild-type, 0.81 mm (n = 133); Tg(FOXF2), 0.65 mm (n = 52) (ANOVA, P = 10−22; pairwise P < 10−14, P = .005, and P < 10−16, respectively). (D) Histograms of polyp size for the 3 genotypes.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

3. Figure 2
Intestinal Foxf2 is restricted to nuclei of nonepithelial cells. (A and B) Immunohistochemistry with Foxf2 antibody on sections of small intestine from (A) wild-type and (B) Foxf2−/− E18.5 embryos. Labeling of enteric neurons is nonspecific (ns), that is, identical in wild-type and Foxf2−/−. Nuclear staining of mesenchymal fibroblasts and smooth muscle progenitors is absent from Foxf2−/− and represents Foxf2. Black arrowheads indicate Foxf2+ and white arrowheads indicate Foxf2− fibroblasts. (C) Foxf2 in adult small intestine. A mixture of Foxf2+ and Foxf2− fibroblasts is seen in the lamina propria, with density of Foxf2+ cells decreasing toward the base of crypts. (D) Foxf2 in adenoma of ApcMin/+ mice. A mixture of Foxf2+ and Foxf2− fibroblasts is seen in the stroma. Diffuse staining of epithelium in all panels is nonspecific background.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

4. Figure 3
Foxf2 controls crypt cell proliferation and Lgr5+ stem cell number. (A) Anti-BrdU staining of longitudinal sections from distal ileum of 12-week-old Foxf2−/+, wild-type, and Tg(FOXF2) mice exposed to a 1-hour BrdU pulse. (B) BrdU+ nuclei per crypt (average ± SEM): Foxf2−/+, 9.9; wild-type, 8.0; Tg(FOXF2), 6.7 (ANOVA, P = 10−20; pairwise P < 10−9, P < 10−4, and P < 10−20, respectively). Each data point is the number of BrdU+ nuclei divided by the number of crypts in one microscopic field (n = 62, n = 48, and n = 41, respectively). (C) Clusters of epithelial cells in Foxf2−/+ ileal villi at the age of 12 weeks. (D) qPCR of Lgr5 mRNA from (left) E16.5 intestine (average ± SEM; P = .007; n = 5 wild-type, 6 mutants), and (right) adult small intestine (average ± SEM; P = .08; n = 5 per genotype). (E) Number of Lgr5+ stem cells per crypt in adult small intestine (average ± SEM; ANOVA, P < 10−5; Foxf2−/+, ± 0.32 [n = 49]; wild-type, ± 0.37 [n = 59]; Tg(FOXF2), ± 0.32 [n = 83]; pairwise P = .03, P = .004, and P < 10−4, respectively). (F) Two-dimensional projections of confocal 3-dimensional reconstructions of ileal crypts from mice carrying the Lgr5-EGFP-ires-CreERT2 allele.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

5. Figure 4
Foxf2 inhibits the Wnt/β-catenin pathway in intestinal epithelium. (A) Anti–β-catenin immunohistochemistry of small intestine with adenomas from ApcMin/+ and ApcMin/+; Foxf2−/+ mice. β-catenin is constitutively nuclear in adenomas (black arrowheads), and normal epithelium (white arrowheads) stains stronger in Foxf2−/+ (contrasts more with negative mesenchyme [asterisk] than with adenoma) than in wild type. (B) Western blot showing more nuclear β-catenin protein in Foxf2−/+ intestine than in wild type (Foxf2−/+ 235% of wild type in nucleus, 96% in cytoplasm; HDAC1 and α-tubulin as loading controls for nuclear and cytoplasmic, respectively). (C) mRNA encoding β-catenin is not elevated in Foxf2−/+ intestine (qPCR; n = 3/genotype). (D) Foxf2−/+ intestine contained 31% more Myc mRNA, and Tg(FOXF2) 62% less, than wild type (qPCR; ANOVA, P = .001; pairwise P = 0.18, P = .009, and P = .0005, respectively; Foxf2−/+, n = 7; wild type, n = 8; Tg[FOXF2], n = 8). (E) Myc Western blot of proteins from small intestine of adult wild type, Foxf2−/+, and Tg(FOXF2) (β-actin as loading control). Quantification (Myc/actin): Foxf2−/+ 178% and Tg(FOXF2) 21% of wild-type level.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

6. Figure 5
Foxf2 activates expression of the extracellular Wnt inhibitor Sfrp1 in lamina propria fibroblasts. (A) Anti-Sfrp1 immunohistochemistry on sections of adult wild-type small intestine. The Sfrp1 protein is localized to the lamina propria mesenchyme, with concentration highest in the villi and decreasing toward the base of the crypts (ie, the same pattern as Foxf2) (Figure 2). (B) qPCR quantification of Sfrp1 mRNA in adult small intestine (left panel) and in vitro cultured intestinal fibroblasts (center and right panels). Wild-type intestine contained 23%, and Tg(FOXF2) 56%, more Sfrp1 mRNA than Foxf2−/+ (ANOVA, P = .005; n = 14 per genotype). In cultured fibroblasts from the same 3 genotypes (center panel), wild type contained 30%, and Tg(FOXF2) 55%, more Sfrp1 mRNA than Foxf2−/+ (P < .05 for Foxf2−/+ vs Tg[FOXF2]; n = 14 per genotype). Cultured intestinal fibroblasts from wild-type E18.5 embryos contained 59% more Sfrp1 mRNA than cells from Foxf2−/− embryos (right panel; P < .005; n = 12 cultures per genotype). (C) qPCR quantification of Foxf2/FOXF2 mRNA in adult small intestine from the 3 Foxf2 genotypes (n = 8 per genotype) and in E18.5 Foxf2−/− gut (n = 2) with primers targeting sites 100% conserved between mouse and human. Foxf2−/− intestines were indistinguishable from negative controls (ND, not detected). Foxf2−/+ contained 40% and Tg(FOXF2) 127% of the wild-type level (ANOVA, P = 10−3 with Foxf2−/− excluded and 2 × 10−4 when included). (D) Scatter plot of 10log Sfrp1 mRNA vs 10log of Foxf2/FOXF2 mRNA for small intestine samples from 24 individuals (8 per genotype; covariance P = .01; r = 0.50). (E) Sfrp1 Western blot of proteins from small intestine of adult wild-type, Foxf2−/+, and Tg(FOXF2) (β-actin as loading control). Quantification (Sfrp1/actin): Foxf2−/+ 16% and Tg(FOXF2) 154% of wild-type level. (F) qPCR quantification of mRNA (left panel) for Sfrp1, 2 mesenchymal markers (Foxf2 and Fn1), and 2 epithelial (Ctnnb1 and Lgr5) markers in RNA prepared separately from epithelium (red bars) and mesenchyme-muscle (blue bars) from adult wild-type small intestine. Western blot of protein (right panel) from epithelium (Ep) and mesenchyme-muscle (Mes) from adult wild-type small intestine with anti-Sfrp1, anti–β-catenin, anti-fibronectin (Fn1), and anti–β-actin.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

7. Figure 6
(A) Schematic illustration of gradients formed by signaling molecules of the crypt-villus axis, and the proposed mechanism for how Foxf2 in fibroblasts limits the stem cell niche for Lgr5+ cells (yellow) by inhibition of Wnt signaling. (B) Simplified summary of paracrine signaling between epithelium (red) and fibroblasts (blue), and a proposed mechanism through which Foxf2 in fibroblasts inhibits Wnt signaling in adjacent epithelial cells. “Wnt” is used as a generic term for different Wnt ligands produced by epithelium and fibroblasts and “Hh” for Shh and Ihh.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions