Hey2 acts upstream of Notch in HSC specification. (A) Expression of runx1 in the DA was lost in cyclopamine (100μM)-treated and hey2 (4 ng of MO)–morphant embryos (i,iii,v). Although 80 ng of vegfA mRNA expression convincingly rescued runx1 expression in cyclopamine-treated embryos (iv), it failed to do so in hey2 morphants (vi). Ectopic expression of runx1 was observed in 4/14 wt (ii) and in 75 of 199 cyclopamine-treated (iv) VegfAa121 mRNA-injected embryos. (B) Expression of runx1 in the DA was lost in cyclopamine-treated (iii; 100μM, treated from 9 hpf), VegfR inhibitor–treated (v; 5μM, treated from 9 hpf), hey2-morphant (vii; 4 ng), scl/tal1-morphant (ix; 5 ng), and etsrp-morphant (xi; 10 ng) embryos. Heat-shock–induced NICD expression at the 17s (17.5 hpf) stage caused ectopic expression of runx1 in control embryos (ii). Furthermore, it rescued and caused ectopic expression of runx1 in cyclopamine-treated (iv), VegfR inhibitor–treated (vi), and hey2-morphant (viii) embryos. By contrast, it failed to induce runx1 expression in tal1/scl-morphant (x) and etsrp-morphant (xii) embryos. All views of embryos are lateral, with anterior left and dorsal up. High-magnification images of the posterior trunk region of all embryos are provided. Numbers of runx1-expressing embryos are given as a fraction of the number of embryos analyzed. inh indicates inhibitor.