HSCs undergo apoptotic cell death in grechetto mutants. (A-P) Confocal microscope images of anti-GFP (in green) and anti–activated caspase3 (in red) double whole-mount immunostaining of the CHT in 3 dpf (A-B,E-F,I-J,M-N) and 4 dpf (C-D,G-H,K-L,O-P) WT siblings (A,E,I,M,C,G,K,O) or grechetto mutant (B,F,J,N,D,H,L,P) cpsf1^zdfl8a12;Tg(c-myb:EGFP) embryos. (A-D) Merged green/red/4′,6-diamidino-2-phenylindole, dihydrochloride/bright-field extended focus images (20×). (E-H) Single-slice images of cells expressing EGFP under the control of the c-myb promoter: grechetto mutants show a normal number of EGFP⁺ cells at 3 dpf (F) compared with their WT siblings (E), but reduced numbers at 4 dpf (G-H). (I-L) Single-slice images of cells expressing activated caspase3, labeled in red, showing increased numbers in grechetto mutants compared with their WT siblings both at 3 dpf (J) and 4 dpf (L). (M-P) Merged single-slice images of cells expressing EGFP and activated caspase3 show that more EGFP⁺ cells undergo apoptosis in grechetto mutants compared with their WT siblings both at 3 dpf (N) and 4 dpf (P). (Q-R) Quantification of the experiments in panels A through P performed by plotting the ratio of EGFP⁺ cells that express activated caspase3 to the total number of EGFP⁺ cells at 3 dpf (Q) and 4 (R) dpf. Error bars represent SEM. **P ≤ .005 by Student t test. Cells were counted from 4 single slices from 3 embryos per condition. (S-Z) Propidium iodide cell-cycle analysis of sorted EGFP⁺ cells (S-V) or whole tails (W-Z) in 3 dpf (S-T,W-X) or 4 dpf (U-V,Y-Z) cpsf1^zdfl8a12;Tg(c-myb:EGFP), WT (S,W,U,Y), or mutant (T,X,V,Z) embryos. Whereas the cell-cycle profile of sorted EGFP⁺ cells at 3 dpf is not different between WT siblings and mutant embryos (S-T), grechetto mutants at 4 dpf show a sub-G1 apoptotic peak not present in WT siblings (red arrows in U-V). The cell-cycle profile of whole tails shows no difference between mutants and WT siblings at 3 dpf (W-X) and 4 dpf (Y-Z).