Localization of afadin and AJ proteins in the <i>afadin</i>-cKO cerebral aqueduct and third ventricle.

<p>The sections of the cerebral aqueduct and the third ventricle were stained with the indicated Abs in the control and <i>afadin</i>-cKO mice at E13.5 and P0. (<b>A and B</b>) The cerebral aqueduct; (<b>C and D</b>) the third ventricle; (<b>A and C</b>) at E13.5; and (<b>B and D</b>) at P0; (<b>a1 and a2</b>) with the anti-N-cadherin Ab; (<b>b1 and b2</b>) with the anti-nectin-1 Ab; and (<b>c1 and c2</b>) with the anti-afadin Ab. (<b>a1–d1</b>) The control mice; and (<b>a2–d2</b>) the <i>afadin</i>-cKO mice. Aq, aqueduct; 4 V, fourth ventricle. Scale bars: 200 µm.</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

Distribution of radial glial cells and neurons in the <i>afadin</i>-cKO midbrain.

<p>The coronal sections were stained with the anti-Sox2 and anti-class III β-tubulin Abs in the control and <i>afadin</i>-cKO midbrains at E16.5; (<b>a1 and a2</b>) with the anti-Sox2 Ab; and (<b>b1 and b2</b>) with the anti-class III β-tubulin Ab. (<b>a1–c1</b>) The control midbrain; and (<b>a2–c2</b>) the <i>afadin</i>-cKO midbrain. VZ, ventricular zone; IZ, intermediate zone; MZ, mantle zone; Aq, aqueduct. Scale bar: 100 µm.</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

No significant difference in the differentiation and proliferation states between control and afadin cKO mice.

<p>Back skins from embryos at E14.5 were fixed and immunostained with the indicated Abs. Arrowheads in (<b>B</b>) indicate the cells double positive for Prox1 and BrdU. Scale bars in (<b>A</b>) and (<b>B</b>) represent 100 µm and 50 µm, respectively. Bar graphs show the number of Prox1-positive cells in the specific area of lymphatic vessels determined by podoplanin staining (<b>A</b>) and the percentage of BrdU-positive cells among Prox1-positive cells (<b>B</b>). Error bars indicate the mean ±S.D. from at least three independent experiments. N.S., not statistically significant.</p

Enhanced activation of RhoA in LMVECs by inactivation of afadin.

<p>(<b>A</b>) Activation of RhoA in BMVECs and LMVECs. At 2 days after transfection of siRNA or scramble RNA, BMVECs and LMVECs were lysed and then used for the pull-down assay, followed by western blotting with an anti-RhoA pAb. The numbers above the blot represent the relative density of GTP-bound RhoA normalized to the total amount of RhoA by comparing the value of BMVECs transfected with scramble RNA, which is expressed as 1.00. (<b>B, C</b>) Restoration of cell morphology and VE-cadherin-mediated cell-cell junctions by introduction of dominant-negative RhoA in afadin-knockdown LMVECs. LMVECs transfected with the indicated combination of siRNA, scramble RNA, pEGFP and pEGFP-RhoA-N19 were labeled with rhodamine-phalloidin to visualize F-actin (<b>B</b>) and with an anti-VE-cadherin mAb (<b>C</b>). Arrowheads indicate enhanced F-actin assembly (<b>B</b>) and reduced VE-cadherin staining (<b>C</b>). (<b>D</b>) VE-cadherin-mediated cell-cell junctions in control and afadin-knockdown BMVECs. BMVECs transfected with scramble RNA + pEGFP or siRNA + pEGFP were immunostained with an anti-VE-cadherin mAb. Bar graphs show the percentage of cells with VE-cadherin staining at cell-cell junctions. Bar graphs in <b><i>B</i></b>, <b><i>C</i></b> and <b><i>D</i></b> show the percentage of cells with enhanced F-actin assembly (<b>B</b>) and with VE-cadherin staining at cell-cell junctions (<b>C</b>) and (<b>D</b>). Error bars indicate the mean ±S.D. from at least three independent experiments. *, p<0.01 vs. Scramble +EGFP, and †, p<0.01 vs. Afadin siRNA +EGFP. N.S., not statistically significant. Scale bars represent 20 µm.</p

Genotype and number of viable progeny from crosses between afadin^flox/+;Tie2-Cre and afadin^flox/flox mice.

<p>The percentage of each genotype relative to the total progeny at each embryonic and post natal day is shown in parentheses (). The number of mice with an abnormal phenotype is shown in brackets [ ].</p

Disruption of cell-cell junctions on lymphatic, but not blood, vessel walls in afadin cKO mice.

<p>Back skins from control and afadin cKO embryos at E14.5 were fixed and immunostained with the indicated combination of Abs. Magnified images of the dashed boxes in (<b>B</b>) and (<b>E</b>) are also depicted. Arrows in (<b>A</b>) and (<b>D</b>) indicate the afadin-negative and VE-cadherin-positive area with patchy staining of VE-cadherin (<b>A</b>) and the afadin-negative and podoplanin-positive area with abnormal staining of podoplanin (<b>D</b>). White and yellow arrowheads in (<b>C</b>) indicate the arterial and venous vasculatures, respectively. Arrowheads in (<b>E</b>) indicate numerous punctures on lymphatic vessel walls. Bar graphs in (<b>B</b>) and (<b>E</b>) show the blood vessel density determined by VE-cadherin-positive and podoplanin-negative staining in the randomly selected areas (<b>B, left</b>), the percentage of VE-cadherin-positive staining in the podoplanin-positive areas (<b>B, right</b>) and the number of punctures in the LYVE-1-positive areas (<b>E</b>). Error bars indicate the mean ±S.D. at least three different samples. *, p<0.01 vs. Control. N.S., not statistically significant. Scale bars in (<b>A</b>), (<b>B</b>), (<b>C</b>), (<b>D</b>) and (<b>E</b>) represent 100 µm, 200 µm, 20 µm, 20 µm and 200 µm, respectively.</p