Fig. 4

Fig. 4 The defect of definitive hematopoiesis is due to the increased apoptosis in the HSPCs of topbp1^cas003 mutants.

(A-D′) The c-myb in situ hybridization and phospho-histone H3 (pH3) immunostaining at 3.5dpf in sibling and topbp1^cas003 mutant embryos, indicating no significant difference in the number of pH3⁺ HSPCs (pH3 and c-myb double positive cells, white arrows) between wild-type sibling and topbp1^cas003 mutant embryos. The images show the stain in the enlarged CHT region. Scale bars represent 25µm. (E-H′) Double immunostaining of c-myb: EGFP and BrdU at 3.5dpf in the sibling and topbp1^cas003 mutant embryos. The number of BrdU⁺ HSPCs (BrdU and EGFP double positive cells, white arrows) in topbp1^cas003 mutant embryos is comparable to that in wild-type siblings. Scale bars represent 25µm. (I-P′) Double immunostaining of c-myb: EGFP and TUNEL at 3.5dpf (I-L′) and 4dpf (M-P′) in the sibling and topbp1^cas003 mutant embryos. The apoptotic HSPCs (TUNEL and EGFP double positive cells, white arrows) are increased in the topbp1^cas003 mutants comparing to siblings, which is more profound at 4dpf. Scale bars represent 25µm. (Q) Quantification of the number of the pH3⁺ HSPCs in sibling and topbp1^cas003 mutant embryos at 3.5dpf. (R) Statistics result of the percentage of BrdU⁺ HSPCs at 3.5dpf. (Q) and (R) indicate no significant difference in the number of proliferating HSPCs between topbp1^cas003 mutant embryos and siblings at 3.5dpf. (S) Quantitative analysis of apoptotic HSPCs in the CHT region in sibling and topbp1^cas003 mutant embryos at 3.5dpf and 4dpf, showing the increased apoptotic HSPCs in the topbp1^cas003 mutant embryos. For the Quantitative analysis, at least 6 embryos were analysis for each experimental group. Error bars represent SEM. *, p<0.05; ***, p<0.001.