Extended Data Fig. 5: AKNA knock down elicits cell death, and delamination defects persist upon cell-death rescue by p53 reduction.

a, Confocal micrographs showing staining of E14 cortex one day after co-IUE of membrane-tagged mKO2 (monomeric Kusabira-Orange2) and cytoplasmic GFP. Note that the vast majority of cells electroporated with one plasmid also express the marker of the second plasmid (n = 4 embryos). b, Line graph illustrating the distribution of GFP⁺ cells in the cerebral cortex after IUE for control shRNA (n = 6 embryos), Akna shRNA no. 1 (n = 5 embryos) and co-IUE of Akna shRNA and AKNA overexpression (0.2 μg μl⁻¹) (n = 3 embryos). Note that the effect of AKNA knockdown is rescued with the appropriate amount of Akna expression, and is therefore specific. c–e, Micrographs showing TUNEL staining in E15 cerebral cortex, indicating cell death two days after IUE with Akna shRNA no. 1 (d, n = 3 embryos) or Akna shRNA no. 2 (d, n = 3 embryos) but not with control plasmids (c, n = 4 embryos). f, Dot and box plot showing the TUNEL⁺ area per electroporated (GFP⁺) area with control shRNA (n = 4 embryos) and Akna shRNA (n = 6 embryos, Akna shRNA no. 1 and Akna sRNA no. 2). g, h, Micrographs showing the distribution of GFP⁺ cells in E15 cerebral cortex two days after IUE with control shRNA (g) or Akna shRNA no. 2 plus p53 miRNA plasmids (h). Note that p53 downregulation rescues the apoptotic effect of AKNA knockdown. i, Line graph illustrating the distribution of GFP⁺ cells in the cerebral cortex after control shRNA (n = 6 embryos), p53 miRNA (n = 4 embryos), and Akna shRNA no. 2 and p53 miRNA (n = 4, embryos) IUE, showing the delamination defect that occurs upon AKNA knockdown is also present when apoptosis is blocked (Akna shRNA no. 2 and p53 miRNA). Note that p53 knockdown on its own does not alter the distribution of GFP⁺ cells. j, Dot and box plot showing the decrease of GFP⁺TBR2⁺ cells after IUE of Akna shRNA-positive, p53 miRNA, as compared to control; this shows that defects in delamination are accompanied by retaining an NSC fate (control shRNA, n = 6; Akna shRNA no. 2 and p53 miRNA, n = 8 embryos). k, Dot and box plot showing a decrease in proliferating (KI67⁺, n = 4 embryos each), and a concomitant increase in differentiated, NEUN⁺ cells analysed at E14 after IUE at E13 (GFP, n = 3; AKNA overexpression, n = 5 embryos). l, Box plot showing the fraction of PAX6⁺, KI67⁺ and NEUN⁺ in E14 primary cortical cells 48 h after transfection in vitro (n = 3 embryos each). b, i, Line graphs show mean ± s.e.m. as a transparent band in the same colour; control shRNA and Akna shRNA no. 1 data in b and control shRNA in i are the same data as shown in Fig. 1e. Box plots show median, quartiles (box) and range (whiskers); b, f, i–k, two-sided Mann–Whitney U test; l, two-sided Students t-test. Scale bars, 50 μm.

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