Fig. 2. vhnf1 and Fgfs cooperate to drive val expression. (A-C) vhnf1 requires Fgfs to drive val expression in the hindbrain. At 18 hpf val (blue) is normally expressed in r5 and r6 (A); krox20 expression in r3 and r5 and eng3 at the mid-hindbrain boundary (MHB) is in red. After injection of vhnf1 mRNA (50 pg), val expression expands anteriorly to approximately the level of r2 (B). By contrast, vhnf1 overexpression in fgf3^–;fgf8^– embryos does not drive val expression (C). (D-I) vhnf1 and fgfs cooperate to drive val expression. val (blue) is not normally expressed at 8.25 hpf (D), and injection of fgf3 mRNA (25 pg) alone is not sufficient to induce val (E). vhnf1 mRNA alone will induce a low level of val (F), while vhnf1 and fgf3 together induce val at a high level (G). Injection of noggin-gfp mRNA (20 pg) causes a dorsalization similar to fgf3 but when injected alone (H) or with vhnf1 mRNA (I) it does not induce val. (D'-I') Val protein is upregulated similarly to transcript levels at 8.25 hpf following mRNA injection (as in D-I), as detected by anti-Val immunoblot of lysed embryos. (J,K) Overexpression of caMek mRNA (20 pg) alone does not induce val expression (J) but like fgf3 can cooperate with vhnf1 to do so (K). (L,M) Robust upregulation of val downstream of fgf3 and vhnf1 requires val autoregulation. 12 hpf val^+/+ and val^+/– embryos expressing vhnf1 and fgf3 exhibit robust val expression (L), while little or no val is detected in val^–/– embryos (M). A-C, are dorsal views with anterior to the left. D-M are optical cross sections near the dorsal midline.