Hey2 expression in the lateral mesoderm is induced by cloche, scl/tal1, and etsrp and is maintained in the DA by Hedgehog and Vegf signaling. (A) At the 10s stage (14 hpf), hey2 was expressed in the ALM (green arrow), the bilateral heart field (blue arrow), the otic vesicle (orange arrow), parts of the midbrain and hindbrain (purple arrow), the neural crest (turquoise arrow), the somites (yellow bracket), the PLM (black arrow), and the tail bud (red arrow). (B) In clo mutants, hey2 expression was lost in the ALM and PLM (white arrowheads). (C) In scl/tal1 morphants (5 ng of MO), PLM expression was lost (white arrowhead). (D) In etsrp morphants (10 ng of MO), hey2 expression in the ALM was lost (white arrowhead). (E) Inhibition of the Hh pathway with cyclopamine (100μM) from the 2-8 cell stage did not eliminate hey2 expression at the 10s stage in any of the tissues analyzed. (F) Hey2 expression was present in the ALM and PLM of mib^ta52b mutants (green and black arrows) but was absent in the otic vesicle and the tail bud (white arrowheads) and was reduced in the somites (yellow bracket). (G) At 24 hpf, hey2 was expressed in the dorsal aorta (black arrow), the ISVs (red arrow), the somites (yellow arrow), the neural crest (turquoise arrow), and the telencephalon (pink arrow), in addition to parts of the midbrain and hindbrain. In mib^ta52b mutants (H) and rbpja/b morphants (2 ng of MO; I), hey2 expression in the DA (black arrow) and in ISVs (red arrow) was retained, whereas telencephalon expression was lost (white arrowhead). By contrast, VegfR inhibitor–treated (J; 5μM) and cyclopamine-treated (K; 100μM) and etsrp-morphant (L; 10 ng) embryos displayed normal expression of hey2 in the telencephalon (pink arrow) but failed to express hey2 in the DA and the ISVs (white arrowheads). Embryos in panels J-K were treated from 90% epiboly. All views of embryos are lateral, with anterior at the left and dorsal up.