Fig. 7. RA and Hh signaling can collaborate in eye ventralization. (A) Embryos were unilaterally injected with 1 pg bhh mRNA and treated with 2 µM ATR from stage 12.5/13. Pax2 is strongly upregulated in the eye by the combination of bhh and ATR, but weakly by bhh or ATR alone in stage 33 Xenopus embryos. Vax2 expression can be activated in the DR by these doses of bhh in the absence of ATR. ET is reduced by bhh or ATR alone, but is strongly repressed by the combination of bhh and ATR. (B) Embryos were unilaterally injected with 0.2 pg bhh mRNA and treated with 5 µM ATR from stage 12.5/13. Vax2 is upregulated in the DR by the combination of bhh and ATR, but not by bhh or ATR alone. These doses of bhh and ATR weakly activate Pax2 expression. ET expression in the DR is partially reduced by bhh or ATR alone, but is strongly repressed by the combination of bhh and ATR. The yellow arrow indicates ET expression domain in the DR. (C) Quantification of the effects of bhh and ATR on Pax2, Vax2 and ET expression. Between 21 and 25 embryos were analyzed for each sample in these experiments. (D) Schematic representation of the results shown in A and B. The combination of 1 pg bhh mRNA with 2-2.5 µM ATR strongly expands the OS. The combination of 0.2 pg bhh with 5 µM ATR ventralizes the DR.