Genetic Ablation of Afadin Causes Mislocalization and Deformation of Paneth Cells in the Mouse Small Intestinal Epithelium

<div><p>Afadin is an actin filament-binding protein that acts cooperatively in cell adhesion with the cell adhesion molecule nectin, and in directional cell movement with the small G protein Rap1 in a nectin-independent manner. We studied the role of afadin in the organization of the small intestinal epithelium using <i>afadin</i> conditional gene knockout (cKO) mice. Afadin was localized at adherens junctions of all types of epithelial cells throughout the crypt-villus axis. Paneth cells were localized at the base of the crypt in control mice, but not confined there, and migrated into the villi in <i>afadin</i>-cKO mice. The distribution of other types of epithelial cells did not change significantly in the mutant mice. The Paneth cells remaining in the crypt exhibited abnormal shapes, were buried between adjacent cells, and did not face the lumen. In these cells, the formation of adherens junctions and tight junctions was impaired. Rap1 and EphB3 were highly expressed in control Paneth cells but markedly down-regulated in the <i>afadin</i>-deficient Paneth cells. Taken together, the results indicate that afadin plays a role in the restricted localization of Paneth cells at the base of the crypt by maintaining their adhesion to adjacent crypt cells and inhibiting their movement toward the top of villi.</p></div

Impairment of the formation of AJs and TJs in <i>afadin</i>-cKO Paneth cells.

<p>(<b>A</b>) Immunostaining of the small intestine of control and <i>afadin</i>-cKO mice with antibodies against lysozyme (red), E-cadherin (green), and Hoechst33258 (blue). The upper two rows show the crypt side, and the bottom row shows a villus side of the crypt-villus axis from the indicated mice. Scale bars = 50 µm (left column), 10 µm (right column). (<b>B</b>) Immunostaining of the small intestine of control and <i>afadin</i>-cKO mice with antibodies against lysozyme (red), ZO-1 (green), and Hoechst33258 (blue). The upper two rows show the crypt side, and the bottom row shows a villus side of the crypt-villus axis from the indicated mice. Scale bars = 50 µm (left column), 10 µm (right column). (<b>C</b>) Immunostaining of the horizontal section of the crypt in the small intestine of control and <i>afadin</i>-cKO mice with antibodies against lysozyme (red), E-cadherin (green, left column), ZO-1 (green, right column), and Hoechst33258 (blue). Scale bars = 50 µm (left column), 10 µm (right column).</p

Mislocalization of Paneth cells in the small intestines of <i>afadin</i>-cKO mice.

<p>(<b>A–C</b>) Immunostaining of the small intestine of control and <i>afadin</i>-cKO mice with the indicated antibodies. (<b>A</b>) Paneth cell marker lysozyme. Scale bar = 100 µm. (<b>B</b>) Paneth cell marker lysozyme (red), adherens junction marker E-cadherin (green), and nuclear marker Hoechst33258 (blue). Scale bars = 50 µm. (<b>C</b>) Goblet cell marker PAS (left column), enteroendocrine marker chromograinin A (center column), and tuft cell marker Dclk1 (right column). Scale bars = 100 µm. (<b>D</b>) Immunostaining of the crypts in the small intestine of control and <i>afadin</i>-cKO mice with antibodies against EphB2 (green) and Hoechst33258 (blue). Scale bars = 50 µm. (<b>E</b>) Detection of <i>Olfm4</i> mRNA by <i>in situ</i> hybridization in the small intestine of the indicated mice. Scale bars = 100 µm.</p

Enhanced death of the small intestinal epithelial cells in <i>afadin</i>-cKO mice.

<p>(<b>A</b>) TUNEL staining in the small intestine of control and <i>afadin</i>-cKO mice. Scale bars = 100 µm. (<b>B</b>) High magnification images of the TUNEL staining (left panel) and hematoxylin and eosin staining (right panel) of the serial section in the small intestine of <i>afadin</i>-cKO mouse. Scale bars = 50 µm. (<b>C</b>) Histogram showing the number of TUNEL-positive cells in each crypt-villus axis of the control and <i>afadin</i>-cKO mice. Ten axes were examined in each mouse (control, n = 5; <i>afadin</i>-cKO, n = 7). (<b>D</b>) Immunostaining of the small intestine of control and <i>afadin</i>-cKO mice with antibodies against E-cadherin (green), cleaved caspase-3 (cl-casp3: red), and Hoechst33258 (blue). Single channel images for cleaved caspase-3 are shown to the right of the merged pictures. Arrows indicate the apoptotic cells with cleaved caspase-3 signal. Scale bars = 50 µm.</p

Increased length and enhanced proliferation of the crypt-villus axis of the small intestines of <i>afadin</i>-cKO mice.

<p>(<b>A</b>) Immunohistochemistry of the small intestine of control and <i>afadin</i>-cKO mice with hematoxylin and eosin. Scale bars = 100 µm. (<b>B</b>) Histogram showing the length of villi (left panel) and crypts (right panel), respectively (n = 6). (<b>C</b>) Immunohistochemistry of the small intestine of control and <i>afadin</i>-cKO mice with anti-Ki-67 antibody. Scale bars = 100 µm. (<b>D</b>) Immunohistochemistry of proliferating cells pulse-labeled with BrdU for 2 hours after peritoneal injection. Scale bars = 50 µm. (<b>E</b>) Histogram showing the number of BrdU-positive cells in each crypt of control and <i>afadin</i>-cKO mice. At least six crypts were counted in each mouse (n = 3).</p

Down-regulation of Rap1 in <i>afadin</i>-cKO Paneth cells.

<p>(<b>A</b>) Immunostaining of the small intestine of control and <i>afadin</i>-cKO mice with antibody against Rap1 (green), the Paneth cell marker UEA-1 (red), and Hoechst33258 (blue). Arrows indicate low UEA cells. Scale bars = 50 µm. (<b>B</b>) Quantification of Rap signal. Relative intensity of Rap signal in crypt cells to that of non-crypt cells is shown. 37 crypts from 11 pictures (control) and 20 crypts from 7 pictures (<i>afadin</i>-cKO) were analyzed.</p

Interaction of afadin with EphB3 in the small intestine.

<p>(<b>A</b>) Western blotting of afadin and EphB3 using immunoprecipitates with the indicated antibodies from the Ls174T colon cancer cell line. (<b>B</b>) Immunostaining of the small intestine of control and <i>afadin</i>-cKO mice with antibodies against EphB3 (green), lysozyme (red), and Hoechst33258 (blue). Scale bars = 50 µm. (<b>C</b>) Relative intensity of EphB3 signal in crypt cells to that of non-crypt cells is shown. 18 crypts from 6 pictures (control) and 24 crypts from 8 pictures (<i>afadin</i>-cKO) were analyzed.</p

Histological phenotypes of the <i>afadin</i>-cKO brain.

<p>Sections were stained by HE. (<b>A</b>) the sagittal sections of the whole brain; (<b>B</b>) the coronal sections of the whole brain; (<b>C</b>) the sagittal sections of the fourth ventricle and the cerebral aqueduct; (<b>D</b>) the coronal sections of the fourth ventricle and the cerebral aqueduct; (<b>E</b>) the coronal sections of the cerebral aqueduct and the third ventricle; (<b>F</b>) the sagittal sections of the cerebral aqueduct; and (<b>G</b>) the coronal sections of the third ventricle in the control and <i>afadin</i>-cKO mice at P0 (<b>A–E</b>) and E13.5 (<b>F–G</b>). Asterisks: stenosed cerebral aqueduct. Arrows: ventral part of the third ventricle. Arrowheads: obliterated third ventricle. LV, lateral ventricle; Aq, aqueduct; 4V, fourth ventricle; 3V, third ventricle; Cb, cerebellum primordium. Scale bars: 1 mm (<b>A and B</b>) and 200 µm (<b>C–G</b>).</p

Gradual deletion of the afadin protein in the <i>afadin</i>-cKO brain.

<p>Lysates from the embryonic telencephalons were subjected to Western blotting using the indicated Abs. Arrows indicate the positions of l-afadin (upper) and s-afadin (lower) proteins. Molecular weight markers (kDa) are shown in the right. Con1, <i>afadin</i>^+/f; Con2, <i>afadin</i>^+/f; nestin-Cre; cKO, <i>afadin</i>^f/f; nestin-Cre.</p

Ependymal cells of the third ventricle and the cerebral aqueduct in the <i>afadin</i>-cKO midbrain.

<p>The coronal sections were stained with the anti-S100β Ab and DAPI in the control and <i>afadin</i>-cKO midbrains at P0; (<b>a1 and a2</b>) with the anti-S100β Ab; and (<b>b1 and b2</b>) with DAPI. (<b>a1–c1</b>) the control midbrain; and (<b>a2–c2</b>) the <i>afadin</i>-cKO midbrain. rAq, rostral aqueduct; v3V, ventral part of the third ventricle. Scale bar: 100 µm.</p