Fig. 2. Pd1 deficiency results in abnormal proliferation and differentiation of neural progenitor cells.

A Illustration for the generation of Pd1 knockout mice. To insert two loxP sites into the flank of Pd1 exon2-4, a single long single-stranded oligonucleotide including the homology arms, loxP sites, and Pd1 gene sequence was designed as the donor vector. After cleaving the target DNA by sgRNA and Cas9, the donor vector including loxP sites was introduced into the target gene by homology-directed repair (HDR) to acquire the Pd1 ^fl/fl mice. Then the Pd1 ^fl/fl mice were crossed with the Nestin-Cre mice to generate Pd1^cKO mice. B Western blot analysis shows that Pd1 is reduced in the brains of Pd1^cKO mice. Pd1^fl/fl and Pd1^cKO are referred to as WT and cKO respectively. n = 4 individual experiments and p = 0.003. C Cell positioning defects are investigated in the cerebral cortex of Pd1^cKO mice. GFP plasmid was electroporated into the brains of Pd1^fl/fl and Pd1^cKO mice at E13. The mice were sacrificed at E16. D Graph shows the percentage of GFP⁺ cells in the VZ/SVZ, IZ, and CP. n = 5 mice, p = 0.00003 for VZ/SVZ; p = 0.562 for IZ; p = 0.0005for CP. E BrdU incorporation is increased in the sections of Pd1^cKO mice at E16. The brains of Pd1^fl/fl or Pd1^cKO mice were electroporated with a GFP plasmid at E13. BrdU was injected 2 hours before the pregnant mice were sacrificed at E16. F The quantification of BrdU⁺ cells per 100 μm length in VZ/SVZ. n = 6 mice and p = 0.014. G Mitotic index is increased in the brain of Pd1^cKO mice at E13. H Graph shows the number of pH3 positive cells in VZ/SVZ. n = 6 mice and p = 0.026. I Neurospheres generated from Pd1^cKO mice are larger than those generated from Pd1^fl/fl mice. NPCs isolated from Pd1^cKO mice and Pd1^fl/fl mice were seeded respectively at a low density (200 cells/ml) for culture in proliferation medium for 3 days. J Graph shows the statistics of neurosphere diameters. n = 15 neurospheres and p = 0.039. K Coronal brain slices of Pd1^fl/fl mice and Pd1^cKO mice at E16 is stained with anti-Tbr2 and anti-Tuj1. L Relative numbers of Tbr2⁺ and Tuj1⁺ cells in VZ of Pd1^fl/fl mice and Pd1^cKO mice. n = 3 mice and p = 0.049 for Tbr2; p = 0.025 for Tuj1. M The number of Ctip2⁺ cells is decreased in Pd1^cKO mice. N Graph shows the number of Ctip2⁺ cells per 100 μm length. n = 7 mice and p = 0.039. Error bars represent means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**), n.s., not significant. Scale bars represent 100 μm (C, E, G, K, M), 200 μm (I).