Genetic deletion of afadin causes hydrocephalus by destruction of adherens junctions in radial glial and ependymal cells in the midbrain

Abstract

Adherens junctions (AJs) play a role in mechanically connecting adjacent cells to maintain tissue structure, particularly in epithelial cells. The major cell-cell adhesion molecules at AJs are cadherins and nectins. Afadin binds to both nectins and α-catenin and recruits the cadherin-β-catenin complex to the nectin-based cell-cell adhesion site to form AJs. To explore the role of afadin in radial glial and ependymal cells in the brain, we generated mice carrying a nestin-Cre-mediated conditional knockout (cKO) of the afadin gene. Newborn afadin-cKO mice developed hydrocephalus and died neonatally. The afadin-cKO brain displayed enlarged lateral ventricles and cerebral aqueduct, resulting from stenosis of the caudal end of the cerebral aqueduct and obliteration of the ventral part of the third ventricle. Afadin deficiency further caused the loss of ependymal cells from the ventricular and aqueductal surfaces. During development, radial glial cells, which terminally differentiate into ependymal cells, scattered from the ventricular zone and were replaced by neurons that eventually covered the ventricular and aqueductal surfaces of the afadin-cKO midbrain. Moreover, the denuded ependymal cells were only occasionally observed in the third ventricle and the cerebral aqueduct of the afadin-cKO midbrain. Afadin was co-localized with nectin-1 and N-cadherin at AJs of radial glial and ependymal cells in the control midbrain, but these proteins were not concentrated at AJs in the afadin-cKO midbrain. Thus, the defects in the afadin-cKO midbrain most likely resulted from the destruction of AJs, because AJs in the midbrain were already established before afadin was genetically deleted. These results indicate that afadin is essential for the maintenance of AJs in radial glial and ependymal cells in the midbrain and is required for normal morphogenesis of the cerebral aqueduct and ventral third ventricle in the midbrain.

Figure 1. Gross phenotypes of the nestin-Cre-mediated cKO mice of the afadin gene.

(A) Appearance of the control and afadin-cKO mice at P14 from the third backcross (N3) generation. (left panel) The control mouse; and (right panel) the afadin-cKO mouse. Scale bar: 1 cm. (B) Brain sections stained with HE in the control and afadin-cKO mice at P14 from the N3 generation. (left panel) The control mouse; and (right panel) the afadin-cKO mouse. LV, lateral ventricle. Scale bar: 1 mm. (C) Kaplan–Meier survival curves of the N3 generation of the control (n = 39) and afadin-cKO (n = 13) mice and the N10 generation of the control (n = 10) and afadin-cKO (n = 12) mice. (D) Appearance of the control and afadin-cKO mice at P0 from the N10 generation. (left panel) The control mouse; and (right panel) the afadin-cKO mouse. Scale bar: 1 cm.

Figure 2. Histological phenotypes of the afadin-cKO brain.

Sections were stained by HE. (A) the sagittal sections of the whole brain; (B) the coronal sections of the whole brain; (C) the sagittal sections of the fourth ventricle and the cerebral aqueduct; (D) the coronal sections of the fourth ventricle and the cerebral aqueduct; (E) the coronal sections of the cerebral aqueduct and the third ventricle; (F) the sagittal sections of the cerebral aqueduct; and (G) the coronal sections of the third ventricle in the control and afadin-cKO mice at P0 (A–E) and E13.5 (F–G). 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 (A and B) and 200 µm (C–G).

Figure 3. Ependymal cells of the third ventricle and the cerebral aqueduct in the afadin-cKO midbrain.

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

Figure 4. Distribution of radial glial cells and neurons in the afadin-cKO midbrain.

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

Figure 5. Localization of afadin and AJ proteins in the afadin-cKO cerebral aqueduct and third ventricle.

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

Figure 6. Gradual deletion of the afadin protein in the afadin-cKO brain.

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, afadin ^+/f; Con2, afadin ^+/f; nestin-Cre; cKO, afadin ^f/f; nestin-Cre.