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First published online 15 July 2008
doi: 10.1242/jcs.025528

Journal of Cell Science 121, 2493-2502 (2008)
Published by The Company of Biologists 2008
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Laminin {alpha}5 influences the architecture of the mouse small intestine mucosa

Zhen X. Mahoney1, Thaddeus S. Stappenbeck2 and Jeffrey H. Miner1,3,*

1 Department of Internal Medicine/Renal Division, Washington University School of Medicine, St Louis, MO 63110, USA
2 Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA
3 Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA


Figure 1
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  		Fig. 1. Laminin {alpha}5 is greatly reduced in the small intestine of postnatal KO/Tg mice. (A) Schematic diagram of the Mr5 transgene. (B,C) Frozen sections of small intestine from newborn Het/Tg (B) and KO/Tg (C) mice. Immunostaining for laminin {alpha}5 revealed similar levels in the epithelial BM. (D-I) Representative pictures of laminin-{alpha}5 staining in the proximal (PSI), middle (MSI) and distal (DSI) small intestine of adult mice. (D,F,H) Laminin {alpha}5 was abundantly deposited in the subepithelial BM of control villi (denoted by arrowhead), with almost none in the crypts. (E,G,I) Laminin {alpha}5 was greatly reduced in the villus BM of KO/Tg mice but was detectable in mesenchymal structures within villi (arrows) and in intestinal smooth muscle (asterisks) of both Het/Tg and KO/Tg mice. Bars, 100 µm.

 

Figure 2
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  		Fig. 2. Villus coalescence in adult KO/Tg distal small intestine. (A-D) Whole-mount views of distal small intestine mucosa. Compared with the villi of Het/Tg mice (A), the KO/Tg villi (B-D) showed varying degrees of villus coalescence, from a widened phenotype (B) to a `cerebroid' pattern (C) to a `mosaic' pattern (D). (E-G) Scanning electron micrographs confirmed the findings in (A-D). Bars, 200 µm.

 

Figure 3
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  		Fig. 3. Crypt-villus architecture is disrupted in adult KO/Tg distal small intestine. (A-D) H&E-stained sections of distal small intestines. KO/Tg showed varying degrees of loss of normal crypt-villus architecture (A-C) and resembled colon (D). (E, F) Villus coalescence was clearly seen in cross sections. Note the crypt-like structures trapped in the center (arrow in F). The approximate positions of cross-sectioning are indicated by dashed lines in A and C. (G,H) Whole-mount views of intestine mucosa. KO/Tg small intestine (G) develops local flat epithelial surfaces (boxed area) with visible crypt mouths (arrow) that were also observed in normal colon (arrow and boxed area in H). (I-L) H&E stained sections of intestinal grafts. Lama5–/– small intestine grafts (J,K) failed to develop a normal crypt-villus architecture (I) but instead presented a colon-like architecture (L). Bars, 100 µm.

 

Figure 4
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  		Fig. 4. KO/Tg distal small intestines lose small intestine features and exhibit characteristics of colon. (A-C) High iron diamine staining of intestine. Most control distal small intestine goblet cells contained sulfomucin (dark brown in A), whereas most KO/Tg distal small intestine goblet cells contained sialomucin (blue in B), which is characteristic of colonic goblet cells (blue in C). (D-F) Transmission electron micrographs of goblet cells. The goblet cell in the KO/Tg distal small intestine (E) and wild-type colon (F) are similar; both contain larger and more electron lucent granules than those in goblet cells from normal distal small intestine (D). Bars, 50 µm (A-C); 4 µm (D-F).

 

Figure 5
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  		Fig. 5. The laminin composition of KO/Tg small intestine subepithelial BMs resembles that of the normal colon. Intestine sections were stained with antiserum directed against laminin {alpha}-chains, as indicated. (A) Laminin {alpha}5 was detected in the control subepithelial BM of villi but not of crypts. (B) Laminin {alpha}5 staining was greatly reduced in villus BM (arrow) of KO/Tg mice but was detectable in both the mesenchymal structures within villi and the intestinal smooth muscle wall. (C) Low levels of laminin {alpha}1 were detected in the control subepithelial BMs of both villi and crypts. (D) Levels of {alpha}1 were increased in KO/Tg villus BM. (E) In controls, high levels of laminin {alpha}4 were detected in the endothelial BM of blood vessels, but not in the villus subepithelial BM (arrowhead in inset). (F) In KO/Tg, in addition to endothelial BM, laminin {alpha}4 was deposited in the villus subepithelial BM (arrowhead in inset). (G-L) Compared with WT distal small intestine, laminin {alpha}5 was only weakly deposited in the subepithelial BMs of WT colon (arrow in H), whereas laminins {alpha}1 and {alpha}4 were abundant. Arrowheads in the insets of K and L point to the position of epithelial BMs. Dashed horizontal lines indicate the crypt-villus boundary. Bars, 100 µm.

 

Figure 6
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  		Fig. 6. Intestinal epithelial cell behavior is altered in KO/Tg small intestines. (A,B) Sections of distal small intestines from Het/Tg and KO/Tg mice stained with anti-Ki67 (red; Hoechst 33342 stained all nuclei blue) shows the expanded proliferative compartment in KO/Tg mice; this result is quantified in G (four mice per group, ten crypts measured per mouse; P<0.001). (C,D) Staining for BrdU (green) 24 hours after BrdU injection shows that KO/Tg intestinal epithelial cells migrated much further up villi than did controls; arrows denote the leading edge of BrdU-positive cells. (H) Quantification of experiments shown in C and D (four mice per group, 32 villi measured per mouse; P<0.001). (E,F) CldU (red) and IdU (green) staining. The distance between the leading edge of the CldU-labeled and the IdU-labeled epithelial cells is the distance cells have migrated during the 12 hours between CldU and IdU injections. (I-K) Sections of distal small intestines from Het/Tg and KO/Tg mice stained with an antibody against sucrase-isomaltase (SIase). SIase expression was similar in Het/Tg and KO/Tg. (L) Quantification of SIase mRNA levels in small intestine using real-time RT-PCR (n=4, P=0.85). Values shown are fold changes normalized to results from Het/Tg. (M-O) H&E staining of paraffin sections revealed `intermediate' cells on KO/Tg villi (arrow in N). These cells contained apical eosinophilic granules smaller than those in mature Paneth cells (arrow in O) and were absent from controls (arrow in M). The insets in M-O are transmission electron micrographs of granules from a normal goblet cell (M), an intermediate cell (N), and a normal Paneth cell (O). Intermediate cells contain both electron lucent granules as seen in Het/Tg goblet cells (M) and electron-dense granules as seen in Paneth cells (O). (P) Quantification of intermediate cells per 100 crypt-villus units in Het/Tg and KO/Tg distal small intestine (four mice/group, 200 crypt-villus units quantified/mouse; P=0.006). Bar, 100 µm in A-F, I-K; 20 µm in M-O; and 4 µm in the insets of M-O. In graphs, the error bars indicate standard error of the mean; ***P<0.001, **P<0.01.

 

Figure 7
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  		Fig. 7. Reduced Lu/B-CAM expression and p27Kip1 nuclear localization in KO/Tg distal small intestine. (A-D) Sections of distal small intestines (A,B) and proximal small intestines (C,D) from Het/Tg and KO/Tg mice were stained with an antibody against Lu/B-CAM (Lu). Lu/B-CAM expression was high in smooth muscle, medium in the villus core, and very low at the basal surface of intestinal epithelial cells in the Het/Tg DSI (A and inset). Lu/B-CAM expression in the villus core and at the basal surface of intestinal epithelial cells was reduced in the KO/Tg DSI (B and inset). Lu/B-CAM was detected at higher levels on the basal surface of intestinal epithelial cells in Het/Tg PSI than Het/Tg DSI (A,C and insets), and this expression was reduced in the KO/Tg PSI (D and inset). Arrowheads in A and C point to the basal expression of Lu/B-CAM. (E,F) Sections of distal small intestines were stained with an antibody against p27Kip1. p27Kip1 was detected in the nuclei of differentiated intestinal epithelial cells at the bottom of crypts and on the villi in Het/Tg small intestine (E and inset). Nuclear staining was dramatically reduced in KO/Tg distal small intestine (F and inset). Bars, 100 µm.

 

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© The Company of Biologists Ltd 2008