Hey2 is essential for HSC formation. (A-B) The number of runx1-expressing HSCs (black arrow and arrowhead) was severely reduced in hey2 morphants at 28 hpf. Embryos injected with a control MO had normal runx1 expression at 28 hpf (n = 24/24; data not shown). Ikaros⁺ (C-D) and c-myb⁺ (E-F) blood progenitors were missing in the DA (black arrow and arrowhead) and in the CHT (blue arrow and arrowhead) of hey2 morphants at 48 hpf. (G-H) Homozygous hey2/grl^m145 mutants possessed fewer runx1⁺ HSCs (black arrow and arrowhead) than wt control embryos. (I-K) In the CHT, the number of cd41-gfp⁺ cells was significantly lower in hey2 morphants than in uninjected embryos (Student t test, P < .001). Error bars represent the standard error of the mean for both groups of embryos. In panels A-H, numbers of embryos with normal gene expression are given as a fraction of the number of embryos analyzed. All views of embryos are lateral, with anterior at the left and dorsal at the top. The embryos in panels C-D were treated with phenylthiourea to prevent pigmentation. All morphants were injected with 5 ng of hey2 or control MO.