Figure 5.

Loss of HSCs in grechetto mutants. (A-B,E-F,I-J,M-N) WISH for c-myb in 36 hpf (A-B), 2 dpf (E-F), 3 dpf (I-J), 5 dpf (M-N) grechetto mutants (B,F,J,N), and their WT siblings (A,E,I,M). Lateral view, anterior to the left, dorsal upward. grechetto mutants specify normal numbers of c-myb–positive HSCs at 36 and 2 dpf (B,F), but this number decreases by 3 dpf (J), and c-myb–positive cells were almost undetectable at 5 dpf (N) compared with their WT siblings. Arrows in panels A and B and E and F indicate the aorta-gonad-mesonephros region. Arrows in panels I and J and M and N indicate the CHT. (C-D,G-H,K-L) Anti-GFP whole-mount immunostaining of the body region (CHT magnified in the inset) of cpsf1^zdfl8a12;Tg(c-myb:EGFP) embryos at 3 dpf (C-D) and 4 dpf (G-H), and 5 dpf (K-L) grechetto mutants (D,H,L) and their WT siblings (C,G,K). Lateral view, anterior to the left, dorsal upward. EGFP⁺ cells are normally represented at 3 dpf (D) but decrease at 4 dpf (H) and are almost undetectable at 5 dpf (L) compared with their WT siblings. (O-P) quantifications of the experiments in panels A through N. (O) c-myb–positive cells as counted in 15 WT and mutant WISH-stained embryos. Cells were counted from the aorta-gonad-mesonephros region of 36 and 2 dpf animals and from the CHT of 3, 4, and 5 dpf animals (36 and 2 dpf embryos were genotyped after counting to identify mutants). The number of cells in mutants was plotted as a ratio to the number of cells in WT siblings at the same stage normalized to 1. (P) Percentage of GFP⁺ cells in single-cell suspensions from the dissected tails of cpsf1^zdfl8a12;Tg(c-myb:EGFP) embryos at 3, 4, and 5 dpf plotted as a ratio to the percentage of cells in WT siblings at the same stage normalized to 1. Error bars represent SEM. *P ≤ .05; **P ≤ .005; ***P ≤ .0005 by Student t test.