Fig. 4

Laminar distribution and fate of neurons born at E12.5, E14.5 and E16.5 in WT and Zbtb20 ^lacZ/lacZ neocortex. BrdU was injected at each of the above indicated embryonic stages and the distribution of the BrdU-tagged cells was evaluated in the postnatal SS cortex. a1-e3 Distribution and fate of cells born at E12.5 and investigated at P8. (a1-a3) Overview images of the localization of BrdU⁺ cells to bins 1–10 and a statistical analysis showing no significant differences between WT and mutant in any of the bins. b NeuN IHC providing an overview of the cortical layers and the position of frames where counting was done in c1-e3 (red squares with respective letters). (c1-e2) Cell fate of E12.5-generated neurons in L5 (co-stained for Neurofilament-H/Smi32 and Ctip2) and L6 (co-labeled for Tbr1), and a statistical analysis (c3,d3,e3, *, P < 0.05, n = 3 per genotype). Double-labeled cells are depicted by arrows. f1-j3 Distribution and fate of cells born at E14.5 and investigated at P12. (f1-f3) Overview images of the localization of BrdU⁺ cells to bins 1–10 and a statistical analysis showing a significant increase of the proportion of BrdU⁺ cells distributed in the lower bins (6–7), and a reduction of cells localized in upper bins (3–4) in the mutant cortex as compared to WT. (g) NeuN IHC providing an overview of the cortical layers and the position of frames where counting was done in h1-j2 (red squares with respective letters). (h1-j3) Fate of E14.5-generated neurons with L5 identity (co-stained for Neurofilament-H/Smi32 and Ctip2) and L6 (co-labeled for Tbr1), and a statistical analysis (H3,I3,J3, *, P < 0.05, n = 3 per genotype). Double-labeled cells are depicted by arrows. k1-o3 Distribution and fate of cells born on E16.5 in P12 SS cortex. (k1-k3) Overview images of the localization of BrdU⁺ cells to bins 1–10 and a statistical analysis showing a significant increase of the proportion of BrdU⁺ cells localized in bins 4–6 in mutant, while a reduction of the percentage of cells localized in uppermost position in bin 1 as compared to WT. (l) NeuN IHC providing an overview of the cortical layers and the position of frames where counting was done in m1-o2 (red squares with respective letters). (m1-o3) Fate of E16.5-generated neurons. (m1-m2) In L5, none of the BrdU+ cells in either WT or mutant co-expressed Neurofilament-H/Smi32. (n1-n3) Co-labelling of BrdU+ cells with UL marker Cux1 in L6 shows an increased number of retained in the LLs Cux1⁺ cells most of which express BrdU (arrows). (n3) Statistical analysis (*, P < 0.05, n = 3 per genotype). (o1-o3) Double-labelling for BrdU and ROR demonstrates the strongly increased numbers of E16.5-born L4 ROR⁺ cells in the mutant compared to WT cortex (arrows). (n3) Statistical analysis (*, P < 0.05, n = 3 per genotype). All countings of the laminar distribution of BrdU⁺ cells (A1/A2,F1/F2,K1/K2) were performed within frames sized 800 μm (h) × 400 μm (w) spanning the entire cortical thickness, subdivided into 10 equally-sized bins. BrdU/Ctip2 and BrdU/NF colabaling was evaluated in 200 μm (h) × 200 μm (w) frames, while BrdU/Tbr1, BrdU/Cux1 and BrdU/ROR co-staining was evaluated within 300 μm (h) × 100 μm (w) frames, positioned as presented in panels B/G/L. b/g/l depict NeuN immunostaining of a WT cortex. Scale bars: k2/b/g/l, 200 μm, o2, 20 μm