Volume 133, Issue 2, Pages 539-546 (August 2007) SOX9 Is Required for the Differentiation of Paneth Cells in the Intestinal Epithelium  Yuko Mori–Akiyama, Maaike van den Born, Johan H. van Es, Stanley R. Hamilton, Henry P. Adams, Jiexin Zhang, Hans Clevers, Benoit de Crombrugghe  Gastroenterology  Volume 133, Issue 2, Pages 539-546 (August 2007) DOI: 10.1053/j.gastro.2007.05.020 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 (A and B) Immunohistochemistry of SOX9 in mouse developing gut: (A) E 13.5, and (B) E 15.5. Arrowhead indicates the developing pancreas. (C–F) Immunofluorescent staining of (C, red) β-catenin and (D, green) SOX9 together with (C–E, blue) nuclear staining by ToTo-3, and the (E) merged image of the intestine of a 6-month-old mouse. To better visualize the nuclear colocalization of β-catenin and SOX9 in epithelial cells of the intestinal crypts, fluorescent images in C and D were overlaid as isosurfaces. (F) This colocalization is especially marked in cells in the bottom of the crypts (arrowheads), implying that Paneth cells express SOX9. (G–J) SOX9 immunohistochemical staining shows the presence of SOX9 protein in the crypts of a (G and I) 6-month-old Sox9flox/flox mouse, but not in a (H and J) Sox9flox/flox;VilCre littermate; the deletion of Sox9 is nearly almost complete in the intestine of Sox9flox/flox;VilCre mice. (I and J) Higher magnification of G and H. (I) Arrow shows Paneth cell granules. Note that the nuclei of some epithelial cells in the villi of (G, arrowhead) wild-type mice, but not of Sox9flox/flox;VilCre mice, also stain positive for SOX9. These cells do not show morphologic features of either goblet cells or Paneth cells. Some cells in the stroma and submucosa, which are also positive for SOX9 in both Sox9flox/flox and Sox9flox/flox;VilCre mice, likely are neuronal cells. Gastroenterology 2007 133, 539-546DOI: (10.1053/j.gastro.2007.05.020) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Representative jejunum sections stained with H&E of a (A) 6-month-old Sox9flox/flox mouse and a (B) Sox9flox/flox;VilCre mouse. Characteristic Paneth cell vesicles are absent in Sox9flox/flox;VilCre mutant mice. Bar, 100 μm. Average (C) width and (D) depth of crypts of 6-month-old Sox9flox/flox;VilCre mice and Sox9flox/flox littermates. The average width and the depth of the crypts were determined by measuring about 40 crypts in each of Sox9flox/flox;VilCre mice and 40 crypts in each of Sox9flox/flox littermates in histologic sections generated from the cross-sections of the jejunum about 15 cm below the pylorus using Metamorph software (Molecular Devices). Error bars represent standard errors (N = 3 animals per genotype). Gastroenterology 2007 133, 539-546DOI: (10.1053/j.gastro.2007.05.020) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Absence of Paneth cell markers in Sox9flox/flox;VilCre mice. Jejunum of (A, C, and E) Sox9flox/flox and (B, D, and F) Sox9flox/flox;VilCre 6-month-old-mice. (A and B) Lysozyme immunohistochemical staining, (C and D) MMP7 RNA in situ hybridization, and (E and F) Cryptdin-1 RNA in situ hybridization. Gastroenterology 2007 133, 539-546DOI: (10.1053/j.gastro.2007.05.020) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Goblet cell and enteroendocrine cell numbers are unchanged in the absence of Sox9. (A) PAS–positive cells of Sox9flox/flox, and (B) Sox9flox/flox;VilCre mice. (D) Anti-synaptophysin immunostaining–positive cells in jejunum of Sox9flox/flox. (E) Sox9flox/flox;VilCre. (C) Quantification of goblet cells and (F) enteroendocrine cells per villus in different segments of the small intestine and per crypt of the colon in Sox9flox/flox mice and Sox9flox/flox;VilCre mice by counting periodic acid–Schiff–positive cells and anti-synaptophysin-positive cells, respectively. Open bars represent Sox9flox/flox, and closed bars represent Sox9flox/flox;VilCre mice. Error bars represent standard errors (N = 3 animals per genotype). Gastroenterology 2007 133, 539-546DOI: (10.1053/j.gastro.2007.05.020) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Expansion of the zone of proliferating cells in the bottom of the crypts and increase in proliferating cells in crypts of Sox9flox/flox;VilCre mice. (A and B) Ki67 staining; (C–G) BrdU staining. (A and C) jejunum and (F) colon of 6-month-old Sox9flox/flox mouse and (B and D) jejunum and (G) colon of 6-month-old Sox9flox/flox;VilCre mouse. (E) Average number of BrdU-positive cells per crypt in the different intestinal segments of Sox9flox/flox and Sox9flox/flox;VilCre mice. Gastroenterology 2007 133, 539-546DOI: (10.1053/j.gastro.2007.05.020) Copyright © 2007 AGA Institute Terms and Conditions