Volume 138, Issue 3, Pages 1079-1090.e5 (March 2010) Secretory Cell Hyperplasia and Defects in Notch Activity in a Mouse Model of Leukocyte Adhesion Deficiency Type II  Christopher C.M. Waterhouse, Steven Johnson, Mia Phillipson, Lori Zbytnuik, Björn Petri, Margaret Kelly, John B. Lowe, Paul Kubes  Gastroenterology  Volume 138, Issue 3, Pages 1079-1090.e5 (March 2010) DOI: 10.1053/j.gastro.2009.10.049 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Unweaned FX−/− mice develop an intestinal phenotype and weight loss when taken off fucose. Litters were either maintained on or taken off fucose at 5 days of age and harvested at 33 days of age for TEM. (A) FX−/− mice on fucose had 1–2 Paneth cells per crypt (arrow; bar, 4 μm) and prominent microvilli on the apical surface of enterocytes (B; bar, 300 nm). Mice off fucose had approximately 4–5 Paneth cells/crypt (C; arrow; bar, 10 μm) and blunted microvilli (D; bar, 300 nm). (E) FX−/− mice on fucose-free diet exhibit poor weight gain compared with those on fucose. (F) Quantitation of microvillus length in animals on versus off fucose (2203 versus 727 nm; P < .001). ***P <.001 compared with on fucose. Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 FX−/− mice develop Paneth and goblet cell hyperplasia. Mice were killed on a fucose-containing diet (0 days) or at intervals after a change to fucose-free diet. (A) FX−/− mice on fucose have normal small intestinal epithelial architecture; bar, 50 μm. Boxes represent areas enlarged in panels B (solid box) and D (hatched box). (B) Paneth cells from FX−/− mice have a normal appearance by light microscopy while on fucose (arrow); bar, 25 μm. (C) Paneth cell hyperplasia occurs in FX−/− mice lacking dietary fucose; bar, 25 μm. (D) Small intestinal goblet cells from FX−/− mice have a normal appearance by light microscopy while on fucose (arrow); bar, 25 μm. (E) Goblet cell hyperplasia occurs in FX−/− mice lacking dietary fucose; bar, 25 μm. (F) Paneth and goblet cell hyperplasia occurs rapidly after switch to a fucose-free diet. Values represent mean ± SEM. *P < .05, ***P < .001 compared with Time 0. Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Goblet and enteroendocrine cell hyperplasia occurs in the colon of FX−/− mice off fucose. Colon tissue was taken from mice on fucose (0 days; A) or fucose-free diet (20 days off fucose; B). Goblet cells were visualized with the use of Alcian blue staining; enteroendocrine cells were identified by immunohistochemistry; bar, 100 μm. (C) Colonic goblet cell hyperplasia occurs within 20 days of starting a fucose-free diet in FX−/− mice. ***P < .001 compared with Time 0. (D) Colonic enteroendocrine cell hyperplasia in FX−/− mice occurs within 20 days of commencing a fucose-free diet. *P < .05, **P < .01, ***P < .001 compared with 0 days off fucose. Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Epithelial cell proliferative index is reduced in FX−/− mice. Epithelial proliferation was measured in FX−/− mice at 0 (A), 7 (B), and 20 (C) days off fucose by BrdU incorporation into small intestinal crypt cells at 2 hours after intraperitoneal injection; bars, 20 μm. Proliferative index (D) was calculated as the number of BrdU-positive cells divided by total cell number in a minimum of 10 well-oriented crypts per animal. ***P < .001 compared with 0 days off fucose. ###P < .001 compared with 7 days off fucose. Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Notch-regulated gene expression is modulated in crypt epithelial cells from FX−/− mice. RT-PCR was performed on RNA isolated from small intestinal crypt cells at various times off fucose, with 2 animals each harvested at 20 or 40 days off fucose. (A) Photographs of each amplicon after electrophoresis are shown. Densitometry measurements of band intensity for Math1 (B) and Ngn3 (C) relative to β-actin are shown. Each bar represents the mean value for the 2 bands shown in panel A. Immunoblot performed on protein from isolated crypts (D) showed up-regulation of Math1 with no change in LGR5 expression. Hes1 was detected by immunoprecipitation of the above lysates with subsequent immunoblotting for Hes1 by using the same antibody as described in Materials and Methods. Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Small intestinal secretory cell expansion is partly independent of FX expression by leukocytes. Chimeric animals expressing FX (C57Bl/6 to FX−/−) or not expressing FX (FX−/− to FX−/−) in leukocytes were either maintained on fucose-containing chow (A and C) or switched to standard diet without fucose (B and D). (E) A small but significant increase in Paneth cell number was seen when mice expressing FX in leukocytes were removed from a fucose-containing diet. *P < .05. ***P < .001. (F) No significant increase in goblet cell number was seen when mice expressing FX in leukocytes were taken off fucose, although a significant increase was seen in FX−/− mice receiving FX−/− bone marrow. **P < .01. Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Colonic secretory cell expansion is independent of FX expression by leukocytes. Chimeric animals expressing FX (C57Bl/6 to FX−/−) or not expressing FX (FX−/− to FX−/−) in leukocytes were either maintained on fucose containing chow (A and C) or switched to standard diet without fucose (B and D). Goblet cell hyperplasia occurred to a similar extent in each case (E) regardless of leukocyte FX expression. *P < .05. Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 8 Transfection with Ad-Notch reverses small intestinal goblet cell hyperplasia in 2-week-old FX−/− mice. (A) Transfection with adenovirus containing GFP results in detectable fluorescence throughout the intestinal epithelium 24–36 hours after transfection. (B) FX−/− mice off fucose have goblet cell expansion. Note that many goblet cells near the villus tips have lost Alcian blue staining, consistent with mucin release. (C) Transfection with Ad-Notch results in a decrease in goblet cell number; bar, 50 μm. (D) Transfection with Ad-Notch causes a statistically significant decline in goblet cell number. (E) Transfection with Ad-Notch increases the number of proliferating (Ki67+) epithelial cells in the intervillus regions from 3.4 to 9.6 cells/intervillus region. A minimum of 10 intervillus regions per mouse were used for quantitation. Ki67-stained sections are included as supplementary data. Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Enteroendocrine cell hyperplasia occurs rapidly after a switch to a fucose-free diet. Enteroendocrine cells were identified by immunohistochemistry as described in Materials and Methods and quantified by microscopy. A minimum of 10 crypt-villus units were counted for each animal, with a minimum of 3 animals for each time point. Values represent the mean ± SEM; **P < .01, ***P < .001 compared with Time 0. Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Secretory cell hyperplasia is reversible with restoration of a fucose-containing diet in FX−/− mice. Mice were taken off fucose for 20 days and then placed back on fucose-containing diet for between 0 and 40 days. Small intestinal Paneth cells (A), goblet cells (B), and colonic goblet cells (C) were quantified as described. Open bars represent the number of cells present on a standard fucose-containing diet as previously assessed in Figures 1 and 5 and are included for comparison. **P < .01, ***P < .001 compared with 0 days on fucose. Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 Secretory cell hyperplasia precedes changes in leukocyte rolling in the small intestine. Intravital microscopy was performed as described in Materials and Methods. All bars represent the mean ± SEM for 3 animals. (A) The number of rolling cells in the ileal microvasculature is unchanged at 20 days but is markedly decreased at 40 and 60 days. *P < .05 compared with 0 days. (B) Rolling at 20 days is P-selectin dependent. Mice on a fucose-containing diet or off fucose for 20 days were treated with either isotype control antibody (solid bars) or anti-P-selectin antibody (open bars), and the number of rolling cells was quantified as described. *P < .05 compared with isotype control. Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 Transfection with Ad-Notch causes increased epithelial proliferation. (A) Proliferating cells (Ki67 positive; brown staining) are detectable in the intervillus region of FX−/− mice on standard diet not containing fucose. (B) Transfection with Ad-Notch increases the number of proliferating (Ki67+) cells in the intervillus regions in FX−/− mice from 3.4 to 9.6 cells/intervillus region (see Figure 8E for quantitation). Gastroenterology 2010 138, 1079-1090.e5DOI: (10.1053/j.gastro.2009.10.049) Copyright © 2010 AGA Institute Terms and Conditions