Fig. 4
LPA signaling disrupts adherens junctions (AJs), basolateral cell–cell adhesion and cellular polarity. a Example of apical mitotic cells with three cleavage plane orientations. A sagittal section from an E13.5 cortical hemisphere stained with DAPI. Apical progenitors at anaphase were classified into three groups according to the angle of mitotic cleavage plane relative to the ventricular surface: vertical, 60°–90°; oblique, 30°–60°; and horizontal, 0°–30°. Ctx, cortex; V, ventricle; R, rostral; D, dorsal; C, caudal. Scale bar 200 µm. b Percentage of apical mitotic cells with non-vertical (oblique or horizontal) cleavage planes in vivo following vehicle (control) or LPA treatment. E13.5 cortical hemispheres were freshly fixed or cultured ex vivo in vehicle (control) or 1 µM LPA-containing medium for 1–6 h. Data represent mean + S.E.M. (n = 4) in vivo, n = 4–5 matched pairs (control vs. LPA), *P = 0.0389, ***P = 0.0072, unpaired t tests). c Neuroepithelial cells in cortical sections double-immunolabeled with N-cadherin (red), and β-catenin (for basolateral cell-to-cell adhesion, green) with nuclei labeled using TO-PRO-3 (blue). E13.5 cortical hemispheres were cultured ex vivo for 6 or 17 h in control or 1 µM LPA-containing medium. LPA-treated cortices displayed rough apical surfaces with disruption of N-cadherin and β-catenin structures at the apical side (arrows) and lower β-catenin immunoreactivity in basolateral cortex, compared to controls (arrowhead). d Cortical sections double-immunolabeled for β-catenin (green), Par3 (for cell polarity, red) and TO-PRO-3 (blue). LPA treatment disrupts Par3 structure at the apical side (arrows). Disrupted sites of Par3 and β-catenin completely overlap. e Effects of LPA on AJ disruption in Lpar1+/−Lpar2−/− vs. Lpar1−/−Lpar2−/− cortex. E13.5 cortical hemispheres from Lpar1+/−Lpar2−/− and Lpa1−/−Lpa2−/− littermates were cultured ex vivo for 17 h with or without 1 µM LPA. Exogenous LPA disrupts structures of neuroepithelial cells labeled with N-cadherin (red) and basolateral cell-to-cell adhesion detected by β-catenin (green) at AJs (arrows) and basolateral cortex (arrowhead) in Lpar1+/−Lpar2−/− cortex but not in the Lpar1−/−Lpar2−/− littermate cortex. c–e Nuclei were counterstained with TO-PRO 3; representative images analyzed by confocal microscopy are shown. VZ ventricular zone, V ventricle; scale bar 10 µm