Fig. 3

Transient expression of Bcl2 rescues the defective hematopoiesis in kri1l^cas002 mutant embryos. (A) Statistic analysis of the proportion of proliferative HSPCs (cmyb and PH3 double positive) in total HSPCs in kri1l^cas002 mutant embryos and wild-type siblings during hematopoietic development (40 hpf-2 dpf). N e 10 for each group. 40 hpf AGM, p = 0.0873; 40 hpf CHT, p < 0.0001; 2 dpf CHT, p<0.0001. Error bars represent SEM. **P ≤ 0.01; ***P ≤ 0.001 (Student′s t-test). (B) Proportion of kri1l^cas002 mutant embryos with classified hematopoiesis phenotype (indicated by cmyb staining) with or without microinjection of p53 MO, treatment with caspase inhibitor (Z-VAD-FMK) or Bcl2 expression, respectively. (C-E) The CHT hematopoiesis is recovered by Bcl2 overexpression in kri1l^cas002 mutant embryos. Embryos from two heterozygous parents were injected with bcl2 mRNA at one-cell stage and harvested for WISH analysis. After WISH and photographing, all embryos were genotyped by genomic DNA sequencing; percentage of rescue was then calculated. 26 out of 33 mutant embryos injected at one-cell stage with bcl2 mRNA are fully rescued, while the rest of embryos are partially rescued. (C′-E′) The enlarged CHT regions. (F) Percentage of the proliferative HSPCs in the CHT of kri1l^cas002 mutant embryos and wild-type siblings with or without Bcl2 overexpression at 3 dpf. N = 12 for each group. Without Bcl2 overexpression, p < 0.0001; With Bcl2 overexpression, p = 0.3136. Error bars represent SEM. **P ≤ 0.01; ***P ≤ 0.001 (Student′s t-test). (G-R) Representative confocal images of double staining of cmyb RNA (red) and pH3 protein (green) in the CHT at 3 dpf. G, K and O are bright-field images overlaid with fluorescent staining. Scale bars, 50 µm.