Figure 6.

Defects in meckelin localization in neuroepithelial cells of Flna^Dilp2 E13.5 mouse embryos mutant for filamin A. (A) Comparable transverse sections of male wild-type and Flna^Dilp2 hemizygote embryonic E13.5 brains stained with haematoxylin and eosin, showing normal morphology of the lateral ventricles (LV) and third ventricles (3V) in wild-type controls. The Flna^Dilp2 brain has a broad mid-line (brace), intraventricular heterotopias (arrows), fusion of the fourth ventricle (4V) with the aqueduct (AQ) and the presence of severe oedema (*). CB, cerebellum; MO, medulla oblongata. Scale bar, 0.3 mm. (B) Immunohistochemical staining for meckelin Ct (brown) in neuroepithelial cells of the lateral ventricles (LV; top panels) and third ventricles (3V; bottom panels) for male wild-type matched controls (left-hand panels) and Flna^Dilp2 (right-hand panels) E13.5 embryo brain sections. Nuclei are stained with Mayer's haematoxylin (blue). Note the diffuse localization of meckelin Ct in Flna^Dilp2 neuroepithelia (magnified insets in black frames). PVH of neurons is indicated by arrowheads. mw, medial wall, lw, lateral wall. Scale bars, 50 μm. (C) Co-immunocytostaining and confocal microscopy of neuroepithelial cells of the third ventricles for adjacent sections to those shown in (B). Stainings are for polyglutamylated tubulin (red), γ-tubulin (green) and ZO-1 (blue) to visualize the cell cortex. The enlarged insets (white border) show cilia in the wild-type controls (arrowheads). Note the disruption of the ZO-1-stained cell cortex in Flna^Dilp2 mutant cells. Scale bar, 10 µm. (D) Upper graph: quantifying cilia length in neuroepithelial cells of the lateral ventricles of male mouse embryo hemizygous for the Flna^Dilp2 mutation in Flna, and male wild-type controls. Mean cilia length was 1.5 μm in wild-type controls and 0.6 μm in Flna^Dilp2 mutated cells. ****P < 0.0001, Student's t-test. Lower graph: quantifying basal body position relative to the apical cell surface (marked by ZO-1 immunostaining) in neuroepithelial cells of the lateral ventricles of a male embryo hemizygous for the Flna^Dilp2, and male wild-type controls. Basal body position was between −1 and 11 μm in wild-type controls and between −5 and +21 μm in Flna^Dilp2 mutated cells. ***P < 0.001, Student's t-test.