Fig. 1. vhnf1 acts downstream of RA to activate val. (A,B) vhnf1 (blue) is expressed posterior to the r4/5 boundary at about 10.7 hpf in control EtOH-treated embryos (A, krox20 expression in r3 and r5 is in red), and is expanded in its AP extent in the hindbrain of embryos treated with 10^–7 M all-trans RA (B). (C,D) Treating embryos with 10^–5 M AGN193109, a pan-RAR antagonist, from 4.5-11 hpf blocks vhnf1 expression and r5 specification (D) compared with DMSO-treated controls (C). (E,F) Wild-type embryos (E) exhibit robust vhnf1 expression by 9.5-10 hpf compared with their nls^– siblings (F). (G-J) Embryos were treated with 2% DMSO or 10^–5 M AGN193109, either alone or in combination with overexpression of vhnf1 (35 pg mRNA) as shown. (G) DMSO-treated controls show normal val expression (blue) in r5 and r6 at approximately 11.7 hpf and this expression is inhibited by treatment with AGN 193109 (H). Overexpression of vhnf1 causes a slight expansion of val expression in untreated embryos (I) and rescues val expression in AGN193109-treated embryos (J). A-F are shown as dorsal views with anterior to the left (A-D) or to the top (E,F).