Journal: Nature medicine

Article Title: An AKT3-FOXG1-Reelin Network Underlies Defective Migration in Human Focal Malformations of Cortical Development

doi: 10.1038/nm.3982

Figure Lengend Snippet: ( a ) Quantitative real-time PCR was performed to measure RELN expression in hNPCs expressing AKT3 E17K and compared to vector-expressing hNPCs. Three independent experiments were quantified in triplicate. ( b ) Mouse embryos at E14.5 were electroporated with indicated DNA constructs. Brains isolated at E18.5 used for reelin expression. Arrowheads: reelin immunostaining in pericellular area of ectopic neurons (intensity-per-pixel: 622.55 ± 45.80 compared to 4092.77 ± 9.46, marginal zone). ( c ) E14.5 embryos were co-electroporated with indicated constructs or RNAs. ( d ) Localization of GFP + neurons at E18.5 was quantified in each cortical region ( n = 3 for each). ( e, f ) Developing cortices were in utero electroporated (IUE) at E14.5 with RFP vector. After 15 min, embryos were sequentially electroporated with either GFP vector ( n = 3) or GFP also expressing AKT3 E17K (A3) with or without Reln siRNA ( n = 6 and 4, respectively). At E18.5, brains were sectioned for imaging. Localization of RFP + GFP − cells was quantified in each cortical region. Values: mean ± s.d. n.s., not significant; **, P < 0.01; ***, P < 0.001, Student’s t -test ( a ); G-test of goodness-of-fit ( d, f ). Scale bars: 25 μm ( b ); 100 μm ( c, e ). ( g ) Enrichment of FOX-binding site. See Online Methods for details. ( h ) Model of effect of AKT3 mutation on surrounding cells. Mutant cell, pink; surrounding cells, gray. AKT3* activating somatic mutation leads to mTOR activation and phosphorylation and cytoplasmic sequestration of FOXG1, relieving repression of RELN , which is then secreted by mutant cells to act in an autocrine (cell autonomous) and paracrine (non-cell autonomous) fashion.

Article Snippet: Paraffin embedded tissues were sectioned, rehydrated and retrieved for antigen (citrated buffer, pH 6.0) before immunostaining for GFP (1:200; Rockland; cat. no. 600-106-215) followed by detection by DAB (Vector Labs).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Construct, Isolation, Immunostaining, In Utero, Imaging, Binding Assay, Mutagenesis, Activation Assay, Phospho-proteomics