We set out to investigate a possible role of IGF-1R in cortical migration by utilizing in utero electroporation of cortical progenitors at embryonic day (E) 15 in order to manipulate and visualize neurons destined to comprise layers II-IV of the cortex, allowing analysis of the location and morphology of the progeny after in vivo differentiation. Electroporation of shRNA-IGF-1R resulted in effective suppression of IGF-1R expression in most electroporated cells, as shown in Fig. 1. In brains co-electroporated with a non-relevant shRNA (control) and a plasmid encoding green fluorescent protein (CAG-GFP) and then immunostained with an antibody to the IGF-1R, 57% of the electroporated cells exhibited a strong staining (Fig. 1a-top and 1b). In contrast only 10% of the cells electroporated with the shRNA-IGF-1R were stained (Fig. 1a middle and 1b). Co-electroporation with resistant IGF-1R construct14 (IGF-1R OPT) rescued IGF-1R expression to control levels (Fig. 1-bottom). Also, the number of proliferating progenitor cells as analyzed by the incorporation of bromodeoxyuridine (BrdU) was found to be unchanged by electroporation of shRNA-IGF-1R compared to control (Supplementary Fig. 1a): 46.7% of the cells electroporated with control shRNA vs. 49,3% of the cells electroporated with shRNA-IGF-1 incorporated BrdU. Staining with doublecourtin to identify migrating neurons15, 16 showed 64.4 or 63.8% of positive cells in controls or cells electroporated with shRNA-IGF-1R, respectively (Supplementary Fig. 1b). We next analyzed the normal differentiation of cells at E19 or postnatal (P) day 4. At E19, about one third of the neurons were located in the ventricular zone/sub-ventricular zone (VZ/SVZ), 25% of cells were found migrating through the IZ and the majority (over 40%) had reached the top of the cortical plate (Fig. 2a-left; quantification shown in Fig. 2b). By P4, almost 100% are found located in layers II-IV of the cortex (Fig. 2c-quantification shown in Fig. 2d), as expected. Cells with knocked-down expression of IGF-1R showed altered distribution and abnormal migration at both E19 and P4. At E19, over 60% of the cells remained arrested at the VZ/SVZ/IZ compared to 30% in the control experiments (Fig. 2a,b). At P4, over 70% of the GFP positive neurons were located at the VZ/SVZ/IZ when knocking down IGF1R, compared to about 10% in controls (Fig. 2c,d). To discard the possibility of nonspecific or off-target effects of the shRNA-IGF-1R, we co-electroporated brains with shRNA-IGF-1R plus IGF-1R OPT. The results of this experiment showed that co-electroporation with IGF-1R OPT cDNA rescued migration to near normal levels, with over 80% of the cells reaching layers II-IV compared to around 20% in the brains electroporated with shRNA-IGF-1R alone (Fig. 2c; quantification shown in Fig. 2d). This demonstrates the specificity of shRNA-IGF-1R-mediated defects in migration and implicates IGF-1R in cortical migration.Figure 1Electroporation with shRNA-IGF-1R (shIGF-1R) significantly reduces expression of IGF-1R. (a) Representative images of brains electroporated at E15 with control shRNA (top), shRNA-IGF-1R (middle) or co-electroporated with IGF-1R OPT and shRNA-IGF-1R (bottom) showing the expression of IGF-1 R (at E17). The ventricular zone (VZ) is labeled for orientation. All brains were co-electroporated with CAG-GFP. (b) Quantification of the number of electroporated cells positive forIGF-1R as shown in A. Note the significant decrease of IGF-1R expression in the cells electroporated with shRNA-IGF-1R. Student’s t test. ***p ≤ 0.0001, ns = not significant. n = 3 independent experiments. At least 100 cells were scored for each condition. Figure 2Expression of IGF-1R regulates neuronal migration. (a) Brains were electroporated with shRNA-IGF-1R (shIGF-1R/CAG-GFP) (right) or control shRNA/CAG-GFP (control-left), at E15 and analyzed at E19. Few GFP positive cells were located in the cortical plate (CP) and the marginal zone (MZ) when IGF-1R expression was knocked down compared to control. Calibration bar = 50 μm. (b) Quantification of the distribution of GFP-positive cells in CP, intermedial zone (IZ) and ventricular/subventricular zones (VZ/SVZ)as indicated in A. Student’s t test *p ≤ 0.05; **p ≤ 0.01; ns = not significant. (c) Brains were electroporated at E15 with shIGF-1R/CAG-GFP (middle), control shRNA/CAG-GFP (left) or co-electroporated with shIGF-1R/CAG-GFP plus cDNA coding for IGF-1R OPT and analyzed at P4. A few GFP positive cells are located in layers V-VI and noticeably fewer GFP positive cells are found in layers II-IV in the IGF-1R suppressed brain compared to the control. In contrast, an important accumulation of GFP positive cells was observed at the (VZ/SVZ/IZ) zones in the shRNA-IGF-1R brain. Note that co-transfection with cDNA coding for IGF-1R OPT (shRNA-refractive cDNA that will express the IGF-1R even in the presence of shRNA-IGF-1R) rescued the morphology. Calibration bar = 100 μm. (d) Quantification of the distribution of GFP positive neurons in VZ/SVZ/IZ, layers II-IV, V and VI as in c. Post hoc Turkey’s ANOVA **p ≤ 0.01 ***p ≤ 0.0001. n = 3 independent experiments. An average of 300 cells (a,b) or 500 cells (c,d) were scored for each condition.