Fig 2: RapGEF2 knockdown causes accumulation of ectopic neurons.(a) E14 mouse brains were electroporated in utero using control (pSUPER) or RapGEF2 shRNA (shRapGEF2) together with GFP plasmid. Representative postnatal day (P) 2 cortical sections were stained for GFP, Cux1 (a layer II–IV neuron marker) and TO-PRO3. Arrows denote the heterotopic band of arrested neurons. Scale bar, 200 μm. The experiment was repeated for at least three times. (b) GFP+ neurons at higher magnification. Asterisks indicate representative neurons. Scale bar, 20 μm. (c) Quantification of the percentages of pSUPER- and RapGEF2 shRNA-electroporated neurons in different cortical layers. Error bars indicate the s.e.m. of five different brains containing >1,000 GFP+ cells. (d) Quantification of Cux1+ neurons. Error bars indicate s.e.m. More than 600 cells were analysed from three brains in each group. (e) P20 brain sections were stained for GFP, Cux1 and TO-PRO3. Scale bar, 200 μm. (f) Quantification of the percentages of GFP+ neurons in different cortical layers at P20. Error bars indicate the s.e.m. of five different brains containing >1,000 neurons. *P<0.05, **P<0.01, ***P<0.001 versus pSUPER; Student’s t-test.

Fig 3: RapGEF2 phosphorylation regulates N-cadherin function.(a) Immunostaining of GFP and endogenous N-cadherin in cultured cortical neurons. E14 mouse brains were co-electroporated in utero with vector control (pSUPER) or RapGEF2 shRNA (shRapGEF2) together with GFP plasmid. Cortical neurons from E16 electroporated cortex were cultured in vitro for 2 days and analysed by immunocytochemistry. (b) E16 brain sections electroporated with vector control (pSUPER) or RapGEF2 shRNA (shRapGEF2) together with pCAG-based N-cadherin probe (N-cadherin–HA) were immunostained for GFP and HA. (c) E16 brain sections electroporated with WT or phosphodeficient mutant (S1124A) of RapGEF2 plasmids together with N-cadherin–HA at E14 were immunostained for GFP and HA. Scale bars, 20 μm. The experiments were repeated for at least three times. (d–f) Quantification of the fluorescence intensity profiles of endogenous (d) and ectopically expressed (e,f) N-cadherin across the cell bodies of transfected neurons in the intermediate zone. The red fluorescence intensity across the representative neurons from a to b as indicated in the panels above was measured by ImageJ software. (g) Co-electroporation of E14 mouse brains with vector control (pCAG) or pCAG-based N-cadherin-expressing plasmid together with RapGEF2 shRNA (shRapGEF2) was performed. Representative E17 coronal cortices were stained for GFP and TO-PRO3. Scale bar, 100 μm. The experiment was repeated for at least three times. (h) Quantification of the percentages of GFP+ neurons in different cortical layers. Error bars indicate the s.e.m. of five different brains containing >800 neurons. (i) Quantification of the percentages of neurons with uni- or bipolar morphology, no process and multiple (≥3) processes. Error bars indicate the s.e.m. of three different brains containing >120 neurons. *P<0.05, **P<0.01, ***P<0.001 versus shRapGEF2+pCAG group; Student’s t-test.

Fig 4: Neuronal migration requires the Rap1 activity of RapGEF2.(a) E14 cerebral cortices electroporated with vector control (pSUPER) or shRNAs targeting RapGEF2 or C3G (shRapGEF2 or shC3G) together with GFP plasmid, or shRapGEF2 plus C3G-expressing plasmids were examined at E17. Representative cortical sections at E17 were stained for GFP and TO-PRO3. Scale bar, 100 μm. The experiment was repeated for at least three times. (b) Quantification of the percentages of GFP+ neurons in different cortical layers. Error bars indicate the s.e.m. of four different brains containing >600 neurons. (c) Quantification of the percentages of neurons with uni- or bipolar morphology, no process and multiple (≥3) processes. Error bars indicate the s.e.m. of three different brains containing >120 neurons. **P<0.01, ***P<0.001 versus pSUPER; Student’s t-test. (d) Representative cerebral cortices at E17 electroporated with the indicated combinations of plasmids at E14 were stained for GFP and TO-PRO3. Scale bar, 100 μm. The experiment was repeated for at least three times. (e) Quantification of the percentages of GFP+ neurons in different cortical layers. Error bars indicate the s.e.m. of five different brains containing >1,000 neurons. (f) Quantification of the percentages of neurons with uni- or bipolar morphology, no process and multiple (≥3) processes. Error bars indicate the s.e.m. of three different brains containing >120 neurons. *P<0.05, **P<0.01, ***P<0.001 versus shRapGEF2+pCAG group; Student’s t-test.

Fig 5: Knockdown of RAPGEF2 rescues fear memory deficits in 4‐month‐old 3xTg‐AD mice. A, Time course of stereotaxic delivery of lentiviral particles and behavioural tests. B, Representative image of GFP expression in the hippocampal CA1 area. Lentiviral particles simultaneously expressing shRAPGEF2 and GFP were bilaterally injected into the hippocampal CA1 area. Hoechst dye was used to stain neuronal nuclei (blue). Scale, 10 μm. Right, higher magnification image enclosed in rectangles at the left. A dotted line indicates the CA1 (mo: molecular layer in dentate gyrus). 53% ± 0.9% area of CA1 region was GFP‐positive (virus transduced) (n = 3). Scale, 200 μm. C, Virus‐mediated knockdown extent of RAPGEF2 in vivo. Lentiviral particles expressing either shRAPGEF2 or a control empty vector (pll3.7) were transduced into hippocampal CA1 area in 2 months of wild‐type (WT) and 3xTg‐AD (TG) mice. After 2 months, hippocampal lysates were used for western blot analysis for RAPGEF2. APP‐FL (full length APP) were used as the marker of TG mice. D, Quantification of RAPGEF2 levels normalised to β‐Tubulin. pll3.7 in WT, n = 6 (5 males, 1 female); shRAPGEF2 in WT, n = 8 (7 males, 1 female); pll3.7 in TG, n = 4 (3 males, 1 female); shRAPGEF2 in TG, n = 8 (7 males, 1 female). E, The contextual fear memory test in 4‐month‐old wild‐type (WT) and 3xTg‐AD (TG) mice (n = 6, males). F, Control (pll3.7) or shRAPGEF2‐expressing viral particles were injected into the hippocampal CA1 area in 2‐month‐old WT and 3xTg‐AD mice. After 2 months, the contextual fear memory test was performed (pll3.7 in WT, n = 15 males; shRAPGEF2 in WT, N = 8 males; pll3.7 in 3xTg‐AD mice, n = 22 males; shRAPGEF2 in 3xTg‐AD, n = 17 males). All data are shown as the mean ± SEM. *p < 0.05, **p < 0.01; ****p < 0.0001; two‐tailed unpaired Student's t‐test (E) and Two‐way ANOVA, Tukey's multiple‐comparison test (D and F)