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. 2012 Aug 14;23(11):2632–2643. doi: 10.1093/cercor/bhs252

Figure 4.

Figure 4.

Brn1/2 are proneural genes that are required for neurogenesis in cortex at E13.5. (A and B) Besides prematurely instructing upper-layer identity and migration, Brn1/2 electroporations at E12.5 promoted neural differentiation at the expense of progenitor maintenance, seen at P0. Some of the strongest-expressing Brn1 (A) or Brn2 (A′)-electroporated cells remained in the VZ or SVZ at P0, presumably due to precocious differentiation. Almost no Sox2+(blue) progenitors remained in either Brn1 or Brn2 electroporation (insets), compared with electroporation of EGFP alone (A″). Tbr1 (red), which weakly labels a subpopulation of SVZ cells at P0, strongly labeled many of the neurons remaining in the VZ with Brn1/2 electroporation. A quantification of these findings in P0 brains electroporated at E12.5 is shown in (B) § denotes 1-factor ANOVA P < 0.01. (C and F) When compared with control EnR electroporations performed at E13.5 and examined at E15.5 (C″ and C), Brn2-EnR (D″ and D) electroporations exhibited fewer GFP+(green)/Tbr2+(red) intermediate neural precursors, and fewer GFP+ migrating neurons. This was apparently due to the blockade of early step in neural differentiation by Brn2-EnR; quantifications of electroporated cells demonstrated less Ngn2 and Tbr2 labeling (E), and an increase in the proportion of GFP remaining in the VZ(F). (G and M) Impaired neural differentiation by Brn2-EnR electroporation at E13.5 persisted to P0 (G′), whereas EnR electroporation alone (G) resulted in normal cortical development. Weak forced Satb2 expression was partially able to rescue defects associated with Brn2-EnR (G″), whereas weak forced dnMAML expression was nearly sufficient to overcome impairment of the initial step in neural differentiation by Brn2-EnR (G″′). Numerous Pax6+(blue) or Ctip2+(red) cells were noted in subcortical white-matter positions at P0 in Brn2-EnR electroporations at E13.5 (H); similar cells experienced a failure in migration and/or differentiation in dnMAML-rescued Brn2-EnR electroporations (H′); these cells were largely negative for Sox2 (J and J′) and Brn2 (K and K′) in either case. Quantifications in several sections from each electroporation condition are shown in (L and M: overlap of GFP(green) with Ctip2 (red) or Pax6 (blue)) in (L), while the proportional location of GFP labeling is shown in (M). § denotes 1-factor ANOVA P < 0.001. Pairwise t-test P-values for (L and M) are provided, along with replicate charts, in Supplementary Fig. 6C′–D′, respectively. UL = upper layer and DL = deep layer.