Figure 3

The labeling index is normal at the beginning of cortical neurogenesis, but decreases at late stages in the perlecan-null neocortex. (A-D) Immunofluorescence for BrdU (green) in the cortical primordium after a 30 min (A, B) and a 24 hours (C, D) survival to a BrdU pulse in wild-type (A, C) and perlecan-null (B, D) E16.5 embryos. In both cases, less BrdU⁺ cells are seen in the perlecan mutants. (E-F) Double immunofluorescence for BrdU (green) and Ki67 (red) in the pallium of E16.5 wild-type and perlecan-null embryos after a 24 h survival to a BrdU pulse at E15.5. Note the panels are high magnification views of the boxed areas in C, D. Observe abundant double-labeled cells (yellow) in the VZ and a thick layer containing BrdU⁺ cells (and no Ki67⁺ cells) in the SVZ of wild-type embryos, corresponding to the newly generated neurons (arrow in C and E). Observe the reduced BrdU incorporation in the perlecan-null dorsal cortex, affecting both the VZ and the SVZ. (G) Labeling index (the percentage of BrdU⁺ cells among Ki67⁺ progenitors) in cortical sections after 30 min, 4 hours and 24 hours survival to a BrdU pulse at E12.5, E13.5 or E16.5. Means ± SEM values are shown. n = 2 embryos for E12.5; n = 4 for E13.5 BrdU 4 h; n = 5 for E13.5 BrdU 24 h; n = 2 for E16.5 BrdU 30 min; and n = 2 for E16.5 BrdU 24 h. ** p < 0.001. No significant changes of labeling index are evident in the perlecan-deficient embryos at E12.5 and E13.5, but at E16.5 the index is significantly reduced. Scale bars: 100 μm (A-D), 40 μm (E, F).