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Fig. 2 | Acta Neuropathologica Communications

Fig. 2

From: MUNC18–1 gene abnormalities are involved in neurodevelopmental disorders through defective cortical architecture during brain development

Fig. 2

Role of Munc18–1 in neuronal migration during mouse brain development. a Characterization of shMunc vectors. pCAG-Myc-mMunc18–1 was transfected into COS7 cells with pSuper-H1.shLuc (Cont), sh-Munc#1 or #2. After 48 h, cells were harvested and subjected to western blotting (10% gel) with anti-Myc (Munc18–1). Anti-Sept11 was used for a loading control. b Knockdown of endogenous Munc18–1 in cortical neurons. pCAG-GFP was transfected with pSuper-H1.shLuc (Cont), shMunc#1 or #2 into dissociated neurons obtained at E14 and cultured for 48 h. Then, cells were fixed and immunostained for GFP (green) and Munc18–1 (red). Merged images were also shown. Bar, 10 μm. The fluorescent signals of Munc18–1 in the cell body enclosed with dotted lines were measured by ImageJ software. The ratio of Munc18–1 signal of knockdown cell to that of control one was calculated (n = 30 cells each). **p < 0.01 by Student’s t-test. c Migration defects of Munc18–1-deficient cortical neurons. pCAG-RFP was electroporated in utero with pSuper-H1.shLuc (Cont), sh-Munc#1 or #2 into E14.5 embryonic brains. Coronal sections were prepared at P2 and stained with anti-RFP (white) and DAPI (blue). Bar, 100 μm. d Quantification of the distribution of Munc18–1-deficient neurons in distinct parts of the cortex (bin 1–5, and IZ) for each condition shown in (c). Error bars indicate SD (Control, n = 5; shMunc18–1#1, n = 8; shMunc18–1#2, n = 4); **p < 0.01 *p < 0.05 by Tukey-Kramer LSD. e Morphology of Munc18–1-deficient migrating neurons at E18. After transfection with shMunc#1 with pCAG-RFP at E14.5, coronal sections were stained for nestin (green) and RFP (red). Images of the indicated areas in i and ii were shown at higher magnification in i’ and ii’, respectively. Bar, 5 μm. f Effects of sh-Munc#1 and #2 on Munc18–2 and Munc18–3 expression. pCAG-Myc-mMunc18–1, −mMunc18–2 or -mMunc18–3 was transfected into COS7 cells with pSuper-H1.shLuc (Cont), sh-Munc#1 or #2. Analyses were done as in (a)

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