Coelectroporation of pshCTIP1 and pshTBR1 rescues SCPA defects. A, Western-blot analysis of TBR1 protein in HEK293T cells transfected with pTBR1 (pCAGIG-TBR1) plus pshControl or pshTBR1. B, Coronal sections of brains electroporated in utero with the indicated plasmids at E13.5 and processed at P0. MZ, marginal zone; CP, cortical plate; WM, white matter. C, Quantification of cell position in the cortex of experiments showed in B. The results are expressed as the percentage of GFP-positive cells distributed in different cortical layers. Error bars represent ± SD. Student's t test, ***p < 0.001; **p < 0.01; n = 6 brains per condition. D, Cortical sections of brains electroporated in utero at E13.5 and fixed at P0, showing an accumulation of cells expressing high levels of CTIP2 in layer 6 in the pshCTIP1 plus pshTBR1 coelectroporation condition. E, Coronal sections of brains electroporated as before. Ei, Brains with comparable amounts of electroporated cells and GFP labeling intensity in the cerebral cortex are shown for adequate comparison. Eii, Coronal sections of the brainstems obtained from the same brains shown in Ei. Eii, Right, Higher-magnification of the areas within the white rectangles. The oval and arrow indicate the approximate location of the pyramidal tract. F, Proposed role of CTIP1 in deep cortical layers. Diagrammatic representation of the transcriptional regulatory network regulating PN identity in deep cortical layers. Interactions among different TFs are depicted according to literature and our own results. The arrows and bar-end lines indicate direct positive and negative regulation, respectively. The dashed arrows and dashed bar-end lines indicate proposed indirect positive and negative regulation, respectively. The TFs weakly colored indicate that are expressed at lower levels. The differential dosage of CTIP1 expression in deep cortical layers (high in layer 5 and low in layer 6) determine the degree of repression of Tbr1. We propose that this regulatory relationship is critical to specify differential neuronal identities in deep cortical layers. Scale bars: B, 100 μm; D, (low-magnification) 100 μm, (high-magnification) 25 μm; Di, 500 μm; Dii, 250 μm; magnification in Dii, 100 μm.