Hey2 is essential for expression of Notch receptors in the DA angioblast cord. (A) Kdrl was expressed in the DA (black arrow), PCV (blue arrow), and ISVs (red arrow) of the trunk and tail of 24-hpf control embryos. (B) Separate cords of DA and PCV kdrl-expressing angioblasts were present in hey2 morphant embryos. Expression of the arterial genes efnb2a (C-D) and notch1b (E-F) was lost in hey2 morphants. Note that spinal cord expression of notch1b was unaffected (E-F green arrow). VegfAa expression in somites was normal in hey2 morphant embryos (G-H white arrows). The Notch ligand gene dll4 was expressed in the DA of hey2 morphants (Ii-Jii black arrows). Loss of Hey2 protein caused increased endogenous hey2 mRNA expression in the DA and in somites (K-L orange arrow) of hey2 morphants, which suggests the presence of a negative autoregulatory loop. In kdrl- and dll4-stained hey2 morphant embryos, ISV staining was reduced (B,J; n(kdlr) = 41/49; n(dll4) = 51/90). (M) At 24 hpf, flt4 expression was restricted to the PCV in control embryos. (N) In the DA of hey2 morphants, it was retained in addition to normal venous expression. (O-P) Another venous marker, msr, was expressed in the vein of control and hey2 morphant embryos. All views of embryos in panels A-F and I-P are lateral, with anterior left and dorsal up. Dorsal views of embryos with anterior left are provided in panels G-H. Close-ups of the trunk of embryos are shown in C-F. The inserts (Ii and Ji) show transverse sections through the posterior trunk. Numbers of embryos with normal gene expression in the DA (A-F,I-P) or in the somites (G-H) are given as a fraction of the number of embryos analyzed. All morphants were injected with 5 ng of hey2 MO.