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First published online 12 January 2009
doi: 10.1242/jcs.033720

Journal of Cell Science 122, 324-334 (2009)
Published by The Company of Biologists 2009
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Vav proteins are necessary for correct differentiation of mouse cecal and colonic enterocytes

John Y. Liu, Hiroshi Seno, Ana V. Miletic, Jason C. Mills, Wojciech Swat and Thaddeus S. Stappenbeck*

Department of Pathology and Immunology, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, MO 63110, USA


Figure 1
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  		Fig. 1. Vav proteins are expressed in upper zone enterocytes of the mouse cecum and localize near adherens junctions. (A,B) Sections of WT adult mouse cecum stained with hematoxylin and eosin (H+E) (A) and PAS/alcian blue (B). A single crypt-surface epithelial unit is shown. Black dashed lines indicate the basal epithelial surface. White dashed lines demarcate three epithelial zones. In A, the asterisk denotes the crypt lumen, the arrowhead indicates an M-phase cell in the lower zone and the arrow denotes an apoptotic body in the upper zone. In B, the white arrow denotes a goblet cell (purple). (C-E) Sections of a WT mouse cecum double-labeled with rabbit anti-Vav1, Alexa Fluor 594-conjugated donkey anti-rabbit (red), mouse anti-β-catenin, Alexa Fluor 488-conjugated sheep anti-mouse (green) antibodies and bis-benzimide. Vav1 protein expression was most robust in upper zone enterocytes (arrow; the tangential orientation shows a crypt orifice). The white and yellow arrowheads indicate cells with Vav protein expression in the mesenchyme and crypt epithelium, respectively. (F,G) Sections of WT upper zone cecal enterocytes labeled with anti-Vav2 (F, red) and anti-Vav3 (G, green) antibodies. The yellow dashed lines in C-G indicate the epithelial-mesenchymal junction. (H) Immunogold labeling for Vav1 in an apical junctional complex from a WT upper zone enterocyte. The red arrow denotes microvilli. The yellow and blue brackets indicate tight and adherens junctions, respectively. Vav1 was detected near adherens junctions. (I,J) Sections of a WT upper zone cecal enterocyte double-labeled with anti-ZO-1 (I, green) and anti-Vav1 (I, red) or anti-ZO-1 (J, green) and anti-occludin (J, red) antibodies. Scale bars: A-G, 15 µm; H-J, 200 nm.

 

Figure 2
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  		Fig. 2. Surface enterocytes of Vav1/2/3null mice contain abnormal microtubule networks. (A-D) Sections of cecum from (A,C) WT and (B,D) Vav1/2/3null mice stained with rabbit anti-actin and Cy3-conjugated donkey anti rabbit (red) antibodies and bis-benzimide (blue) (A,B), and FITC-conjugated rabbit anti-{alpha}-tubulin (green) and bis-benzimide (C,D). The arrowheads in A and B denote the high level of actin staining in the apex of upper zone enterocytes of both Vav1/2/3null and WT mice. Arrows in C indicate the apical accumulation of polymerized {alpha}-tubulin at the cell apex of the lower and middle zones, which is similar to Vav1/2/3null cells in these regions; the arrowhead denotes increased cytoplasmic microtubules in upper zone enterocytes compared with their counterparts in the Vav1/2/3null mice. (E) Insets of boxed regions in C and D from upper and lower zone epithelial cells. (F) Ratio (± s.e.m.) of mean fluorescent intensity of {alpha}-tubulin staining in the apical cytoplasm (upper zone/lower zone) of WT and Vav1/2/3null mice. *P<0.01, Student's t-test comparing WT and Vav1/2/3null mice. (G-J) Sections of cecums from WT (G,I) and Vav1/2/3null (H,J) mice stained with rabbit anti-PKC{zeta} and FITC-conjugated donkey anti rabbit (green) antibodies (G,H) and rabbit anti-Dlg1 and FITC-conjugated donkey anti rabbit (green) antibodies (I,J). (K,L) Summary of cellular localization and microtubule networks in upper zone enterocytes in WT and Vav1/2/3null mice. Scale bars: A,C,D,G-J, 15 µm; B, 7.5 µm.

 

Figure 3
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  		Fig. 3. Upper zone enterocytes of Vav1/2/3null mice are short. (A-C) H+E-stained sections of (A) WT and (B,C) Vav1/2/3null mouse cecums. The upper zone WT epithelial cells were taller than the corresponding Vav1/2/3null surface epithelial cells. The black brackets in C highlight the epithelial cell heights in all three zones (compare with Fig. 1A for WT). (D,E) Quantification of cell height in all three zones. Asterisks indicate a statistically significant difference between the upper zone enterocytes of (D) WT and Vav1/2/3null mice and (E) Dlg1flox/flox and Dlg1flox/flox; Villin-Cre mice (P<0.001) using a Student's t-test (means for each group ± s.e.m., n=3-4 mice/group, n=100 cells measured per zone per mouse). (F,G) Confocal analysis of the mouse cecal whole mounts from a WT and Vav1/2/3null mouse stained with rabbit anti-ZO-1 to label tight junctions of the upper zone enterocytes at the cell apex. This staining highlights the shape and size of surface epithelial cells present around a single crypt opening (asterisks). A confocal Z-stack is shown above the red line for each image. (H,I) Staining of WT and Vav1/2/3null ascending colons with Cy3-conjugated UEA lectin (Ulex, red) and FITC-conjugated HPA lectin (green). UEA and HPA stain crypt goblet cell mucin. UEA stains the apical surface of enterocytes in WT but not Vav1/2/3null mice (indicated by yellow arrows). (J,K) Staining of WT and Vav1/2/3null ascending colons with Cy3-conjugated CTB lectin. CTB detects a more robust Golgi-type pattern in the enterocytes of WT compared with Vav1/2/3null upper zone enterocytes (yellow arrows). (L,M) Quantification of M-phase cells (L) and apoptotic bodies (M) per 100 crypt-surface units showed no statistically significant differences when comparing WT and Vav1/2/3null mice. (N) PAS/AB stained section of a Vav1/2/3null mouse colon. Scale bars: A-C, 30 µm; F,G, 5 µm; H-K, 30 µm; N, 15 µm.

 

Figure 4
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  		Fig. 4. Vav proteins are required for cytoplasmic `nuclei exclusion zones' in mature enterocytes. (A) Supranuclear cytoplasm lengths (distance from the top of the nucleus to the cell apex) were plotted for upper zone of cecal enterocytes (n=3 per genotype). A measurement of zero (y-axis) indicates the apex of a nucleus was located at the cell surface. The red bar denotes the average distance per mouse. The intensity of each X correlates with the number of cells of a given measurement (e.g. darker intensity indicates more cells; n=50 cells measured per zone per mouse). (B) The range of nuclear position in all three zones for WT and Vav1/2/3null mice depicted in a cartoon. The average cell heights (from Fig. 3D) are indicated to the right of each cell. The mean nuclear position (± s.e.m.) is in dark blue. The upper and lower limits of nuclear position are in light blue (abutting the dotted red lines). The apical nuclei free area is hashed. The basal nuclei free area is dotted. The asterisk indicates that both the supra- and subnuclear areas of cytoplasm are statistically significantly different in the upper zone enterocytes of Vav1/2/3null compared with WT mice (P<0.001 determined by Student's t-test).

 

Figure 5
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  		Fig. 5. Vav1/2/3null apical junctions are not obviously altered in the cecal upper zone enterocytes. (A,B) Transmission electron microscopic images of the apical junctional complex from cecal upper zone enterocytes of WT and Vav1/2/3null mice show no detectable differences. Black arrowheads denote adherens junctions and white arrows denote desmosomes. (C) Quantification of junction lengths and plaque widths. (D) Section of Vav1/2/3null cecum stained with anti-β-catenin antibody (red). Scale bars: A,B, 100 nm; D, 15 µm.

 

Figure 6
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  		Fig. 6. Vav1/2/3null cecal surface epithelial cells do not demonstrate defects in polarity. (A,B) Sections of a WT and Vav1/2/3null cecums stained with anti-carbonic anhydrase 4 (Car4; red). (C,D) Sections showing the upper zone epithelium of WT and Vav1/2/3null cecums stained with anti-integrin {alpha}6 (epithelial basal surface, red), anti-villin (apex of the epithelial cells, green) antibodies and bis-benzimide. (E,F) Scanning electron microscopy of WT (E) and Vav1/2/3null (F) cecums. Vav1/2/3null mice contain denser microvilli on their cell surface than WT mice. The yellow dashed lines denote cell borders. (G,H) Transmission EM of cecums from WT (G) and Vav1/2/3null (H) mice showing microvillus morphology (shape, length and caliber) was not significantly different between the two groups of mice. Scale bars: A,B, 10 µm; C-F, 5 µm; G,H, 100 nm.

 

Figure 7
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  		Fig. 7. Spontaneous mucosal ulceration in the cecum and colons of adult Vav1/2/3null mice required Vav expression in bone marrow derived cells. (A,C) Whole-mount of the cecal mucosal surface of an adult Vav1/2/3null mouse showing an ulcer by light microscopy (A) and scanning EM (C). Dashed lines outline ulcer beds surrounded by crypts with enlarged openings at the surface (indicated by arrows). (B) H+E-stained section of an ulcer from a Vav1/2/3null mouse. Arrowhead indicates granulation tissue in the center of the ulcer and arrows indicate enlarged crypts that immediately surround it. (D) H+E-stained section of a cecum from a Vav1/2/3null-WT chimera. The epithelial cells in the upper zone are similar in height to lower zone cells as found in Vav1/2/3null mice. (E) Quantification of ulcers by anatomic region and genotype (n=6-10 mice per group). (F) Quantification of wound healing in biopsy-injured WT and Vav1/2/3null mice. The average surface area of lesions (± s.d.) is plotted for each group at the time of injury, and thereafter every 2 days (n=5 mice per group and 2-3 lesions per mouse were evaluated). Scale bars: A, 200 µm; B, 500 µm; C, 50 µm; D, 15 µm.

 

Figure 8
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  		Fig. 8. Model of the role of Vav proteins in apical maturation. (A,B) The crypt-surface epithelium from a WT and Vav1/2/3null mouse cecum is depicted. The cytoplasmic microtubule networks are depicted in green, actin networks in red and nuclei in blue. The lower and middle zones contain similar distributions and organization of actin and microtubules in WT and knockout mice. The upper zone enterocytes of the Vav1/2/3null mice are shorter and contain less-robust microtubule networks than the WT.

 

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© The Company of Biologists Ltd 2009