Volume 136, Issue 7, Pages 2195-2203.e7 (June 2009) Depletion of the Colonic Epithelial Precursor Cell Compartment Upon Conditional Activation of the Hedgehog Pathway  Willemijn A. van Dop, Anja Uhmann, Mark Wijgerde, Esther Sleddens–Linkels, Jarom Heijmans, G. Johan Offerhaus, Marius A. van den Bergh Weerman, Guy E. Boeckxstaens, Daan W. Hommes, James C. Hardwick, Heidi Hahn, Gijs R. van den Brink  Gastroenterology  Volume 136, Issue 7, Pages 2195-2203.e7 (June 2009) DOI: 10.1053/j.gastro.2009.02.068 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Localization of Hh pathway components in the adult mouse colon by in situ hybridization and immunohistochemistry. (A, left) Ihh mRNA was expressed in the epithelial cells of the midcrypt region. (A, right) Shh mRNA was not detectable in the colon, while the neural tube of an 11-day-old embryo showed the expected ventral staining (inset in A, right). (B) Ihh protein was expressed by the differentiated cells at the top of the crypts. Expression of (C) Ptch1 and (D) Gli1 mRNA was restricted to the mesenchyme underlying the epithelium at the upper part of the crypts in wild-type mice. Many of the Ptch1-positive cells had the typical characteristics of subepithelial myofibroblasts (arrows in C). Gastroenterology 2009 136, 2195-2203.e7DOI: (10.1053/j.gastro.2009.02.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Successful recombination of Ptch1 and activation of Hh signaling in conditional mutant mice. (A) An in situ probe directed against exons 8 and 9 of Ptch1 mRNA showed successful deletion of these 2 exons in the Ptch1 mutant animals. (B) Recombination efficiency at the Ptch1flox locus in the colon was 99% ± 0.3%. A probe against exons 1–6 of Ptch1 mRNA (not excised upon activation of the Cre enzyme) showed that the Ptch1 mutation resulted in up-regulation of this Hh target in the mesenchyme (C). This was confirmed by quantitative reverse-transcription PCR (D, P < .001, n = 12 wild-type vs 11 mutant mice). Gli1 was uniquely expressed in the mesenchyme at the upper half of the crypts in wild-type mice and was increased in Ptch1 mutant animals (E and F, P < .0001, n = 12 wild-type vs 11 mutant mice). Original magnification: 160× (A–D). Gastroenterology 2009 136, 2195-2203.e7DOI: (10.1053/j.gastro.2009.02.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Hypoplastic crypts and premature commitment to the enterocyte lineage in Ptch1 mutant animals. H&E staining of colon of (A, left) wild-type and (A, right) Ptch1 mutant animals showed large areas of colonic crypt hypoplasia in the mutant mice at 19 days after the first tamoxifen injection. The bases of the crypts were broadened and contained more highly polarized cells. (B, left) Electron microscopy revealed that the base of the crypts of the control mice contained undifferentiated cells with large round dark-stained nuclei and little cytoplasm. (B, right) In the Ptch1 mutant mice, these cells were partly replaced by more differentiated cells with smaller nuclei and more cytoplasm filled with small vacuoles. Original magnification: A, 160×; B, 800×. Gastroenterology 2009 136, 2195-2203.e7DOI: (10.1053/j.gastro.2009.02.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Premature differentiation of the epithelial precursor cells. Immunohistochemistry for (A) carbonic anhydrase II (Ca II) and (C) Cdx2 showed that the expression of these differentiation markers was increased in the Ptch1 mutant mice and extended more toward the base of the crypts. This was confirmed by quantitative reverse-transcription PCR on colonic homogenates from wild-type (n = 12) and mutant (n = 11) mice for (B) Ca II (P = .047) and (D) Cdx2 (P = .02). (E and F) No difference was observed in expression of Villin protein or mRNA (P = .27). Original magnification: 100× (A, C, E). Gastroenterology 2009 136, 2195-2203.e7DOI: (10.1053/j.gastro.2009.02.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Accumulation of myofibroblasts in Ptch1 mutant animals. (A) Immunohistochemistry for α–smooth muscle actin (α-Sma) showed accumulation of myofibroblasts in the Ptch1 mutant mice. (B) This was confirmed by quantitative reverse-transcription PCR on colonic homogenates from wild-type (n = 12) and mutant (n = 11) mice (P = .007). Gastroenterology 2009 136, 2195-2203.e7DOI: (10.1053/j.gastro.2009.02.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Depletion of proliferating precursor cells and reduced Wnt signaling in Ptch1 mutant mice. (A) Immunohistochemistry for Ki67 in control (n = 14) and Ptch1 mutant (n = 11) mice showed loss of Ki67-positive nuclei in the mutant mice. In some crypts, the whole precursor cell compartment was depleted of Ki67-positive cells (arrows in A). Ki67-positive cells were counted per crypt in (B) the distal colon and (C) the proximal colon 19 days after the first tamoxifen injection. A significant reduction in proliferating precursor cells was observed in both segments (P = .0002 and P = .003, respectively). (D) Analysis of proliferation at different time points showed that the reduction in proliferation was first apparent at 10 days after recombination. (E) Immunohistochemistry for β-catenin showed staining of membrane-bound β-catenin all throughout the crypt in both wild-type and mutant mice. In the control mice (n = 6), β-catenin accumulated in the cytoplasm and nucleus of cells at the base of the crypts (arrows in E), the hallmark of active Wnt signaling. (E and F) Nuclear staining of β-catenin was strongly reduced (P = .0006) in the mutant mice (n = 6). Original magnification: 200× and 400×. Gastroenterology 2009 136, 2195-2203.e7DOI: (10.1053/j.gastro.2009.02.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Mesenchymal induction of Hh signaling results in a reciprocal increase of epithelial Bmp signaling. (A) Immunohistochemistry for pSmad1,5,8 showed increased phosphorylation of the Bmp-specific Smad1,5,8 in the epithelium of Ptch1 mutant mice. Whereas the base of the crypts of wild-type animals showed no or very little Smad1,5,8 phosphorylation (A, left), phosphorylation was increased throughout the epithelium but most notably at the base of the crypts in mutant mice (A, right). (B) A similar pattern was observed for Id2. (C) Quantitative reverse-transcription PCR for Id2 and Id4, both targets of Bmp signaling, confirmed the increase in Bmp signaling (P = .03 and P = .01, respectively). Original magnification: 200× and 400×. Gastroenterology 2009 136, 2195-2203.e7DOI: (10.1053/j.gastro.2009.02.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 (A) In situ hybridization for Bmp2 showed that Bmp2 mRNA was expressed by the differentiated epithelial cells at the top of the crypts. (B) Bmp4 mRNA was expressed in a graded pattern from the base to the top of the crypt and in the mesenchyme. (C) Bmp7 was expressed by the mesenchyme underlying the epithelium at the upper parts of the crypts. (D) Quantitative reverse-transcription PCR on colonic homogenates from wild-type (n = 12) and mutant (n = 11) mice showed an induction of Bmp2 (P = .01), Bmp4 (P = .04), and Bmp7 (P = .03). (E) Proposed model of negative feedback signaling in the intestinal epithelial crypt. Ihh secreted by the differentiated cells acts on the myofibroblasts, which secrete factors such as Bmps that subsequently negatively regulate precursor cells at the base of the crypt. Original magnification: 200×. Gastroenterology 2009 136, 2195-2203.e7DOI: (10.1053/j.gastro.2009.02.068) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 Myofibroblasts are mesenchymal targets of Hh signaling. Adjacent tissue sections were stained for Ptch1 by in situ hybridization and for α–smooth muscle actin by immunohistochemistry. Due to the thickness of the sections required for in situ hybridization, not all cells are present in both sections. The arrows show examples of cells that express both Ptch1 and α–smooth muscle actin. Original magnification: 200×. Gastroenterology 2009 136, 2195-2203.e7DOI: (10.1053/j.gastro.2009.02.068) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 2 Epithelial apoptosis does not contribute to colonic crypt hypoplasia in Ptch1 mutant mice. No cleaved caspase-3–positive cells were observed in the epithelium at any time point in our experiments. Intestine of Apc+/min mice was used as a positive control for active caspase-3 staining (arrows in right panel). Original magnifications: 200× (2 left panels) and 400× (right panel). Gastroenterology 2009 136, 2195-2203.e7DOI: (10.1053/j.gastro.2009.02.068) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 3 Down-regulation of Wnt targets. In the control mice, Wnt targets EphB2 (A, left), EphB3 (B, left), and CD44 (C, left) are expressed at the base of the crypts, where Wnt signaling is active in the precursor cells under physiologic conditions. In the mice with constitutively active Hh signaling, expression of EphB2 (A, right), EphB3 (B, right), and CD44 (C, right) was reduced. Original magnification: 200× (overview pictures in A–C), 400× (enlargements in A–C). Gastroenterology 2009 136, 2195-2203.e7DOI: (10.1053/j.gastro.2009.02.068) Copyright © 2009 AGA Institute Terms and Conditions