Loss of the superficial SC neurons in <i>Brn3b</i> mutants occurs around birth.

(A) Schematic diagram illustrating a specific localization of the imaged sagittal section according to a lateral distance from the midsagittal line at E13. (B) (Left) Example of sagittal section. (Right) Magnified view of the boxed area. Brn3b+ neurons (red) were missing in the layer labeled by Ki67, a maker of proliferating cells (cyan) at E13 (n = 3 mice). (C) Schematic diagrams showing the brain area (boxed) of a coronal image used for analysis (top) and a sagittal image depicting the level (dashed line) where each coronal section was obtained (bottom) at E16-E18 (i) and at P1-P5 (ii). (D, E) Brn3b+ neurons, visualized by AP signals, were weakly detectable at E18 and become obvious at P1 and P4 (arrows) in the superficial SC of control (D). However, these neurons were missing at E18, P1 and P4 in the superficial SC of cKO (E) (n = 3 mice/group/development stage). (F) Schematic diagram depicting the brain areas (boxed) analyzed to examine the number of cleaved caspase-3+ cells in the sagittal section. The localization of imaged sections was shown as a lateral distance from the midsagittal line. (G, H) Cleaved caspase-3 immunoreactivity (red) revealed increased cell death at E18 in cKO (H), compared to control (G). DAPI (blue). (I, J) Quantification revealed increased cell death in cKO (35.7 ± 4.9/mm2 for control, 56.6 ± 3.3/mm2 for cKO; n = 5 mice/group), primarily occurring in the superficial and intermediate layers; unpaired two-tailed Student’s t test (mean ± SEM, p = 0.008 [**] in I and p = 0.03 [*] and 0.02 [*] in J). Brackets represent the layer segregation and dashed lines indicate the pial surface. Scale bars: 250 μm (B) and 150 μm (D–E and G–H). The data underlying this figure can be found in S1 Data. AP, alkaline phosphatase; SO, stratum opticum.