Fig. 5

G2/M cell cycle arrest disrupts HSPC maintenance in the CHT in smc2 mutants. (A) Fluorescence-activated cell sorting (FACS) analysis of DNA content in WT and smc2 mutant embryos at 52 hpf following propidium iodide (PI) staining. Black and white arrows indicate the G1 and G2/M phase peak, respectively. The indicated percentage represents the DNA content in the G2/M phase. Data from two independent biological replicate experiments. (B) Confocal microscopy images of smc2 mutants (n = 9) and WT controls (n = 16) immunohistochemically stained with the H3P antibody in the CHT (red-dotted box) at 52 hpf. Red-dotted box indicates CHT. Right panel: quantification of H3P+ cells in the CHT (***p < 0.001). Data combined from two independent experiments. (C) Confocal imaging of H3P-stained smc2 mutant embryos (n = 29) and WT sibling controls (n = 50) in the CHT at 52 hpf using CD41:GFP transgenic zebrafish. White-dotted box indicates CHT. Right panel: quantification of H3P- and CD41-double positive cells in the CHT (*p < 0.05). The results represent data from two independent experiments. (D) Higher magnification images of the white-dotted box in H3P-stained smc2−/−;CD41:GFP transgenic zebrafish embryos from (C). Yellow arrows indicate H3P- and CD41- double positive cells in the CHT of smc2 mutants. All quantification data were quantified and expressed as the mean ± SEM. Statistical analysis was performed using a t-test to calculate p-values. Scale bar = 200 μm.