Fig. 5. Xiro1 controls the expression of Otx2 and Gbx2 at different developmental stages. Embryos were injected in one blastomere at two-cell stage with 2 ng of Xiro1 mRNA (A-C), 0.5 ng of HD-GR-EnR (D,G,J,M,P,S), 0.5 ng of HD-GR-E1A (E,H,K,N,Q,T) or 0.5 ng of HD-GR (F,I,L,O,R,U) and the expression of Otx2, Gbx2 and Pax2 were analyzed at early neurula stage (stage 14). Activation of the inducible constructs was achieved by adding dexamethasone at stage 9.5-10 (D-L) or at stage 12-12.5 (M-U). Embryos injected with Xiro1 mRNA show a caudal expansion of Otx2 (A, broken lines), expansion and caudal shift of Gbx2 (B, broken lines), and Pax2 is displaced caudally (C, broken lines). (D-I) Otx2 (green) and Gbx2 (purple) were expanded and shifted caudally in embryos injected with HD-GR-EnR mRNA (D,G, broken lines). HD-GR-E1A and HD-GR repressed Otx2 and Gbx2 expression when activated at stage 9.5-10 (E,H and F,I, arrowheads). A caudal shift of Pax2 expression is observed in embryos injected with HD-GR-EnR when activated at stage 9.5-10 (J, broken lines). The injection of both HD-GR-E1A and HD-GR repress Pax2 midbrain expression domain (K,L, arrowheads). (M-O) Otx2 midbrain territory is inhibited and shifted rostrally in embryos injected with HD-GR-EnR mRNA (M, broken lines). A caudal expansion in Otx2 expression is produced by HD-GR-E1A and HD-GR overexpression and activation at stage 12-12.5 (N,O, broken lines). (P-R) Gbx2 expression is expanded anteriorly in embryos injected with HD-GR-EnR mRNA and activated at stage 12-12.5 (P, broken lines), while the injection of HD-GR-E1A and HD-GR mRNAs promote repression of Gbx2 (Q,R, arrowheads). (S-U) Embryos injected with HD-GR-EnR and activated at stage 12-12.5 causes an anterior shift of Pax2 expression (S, broken lines), while HD-GR-E1A and HD-GR produce repression and caudal displacement of Pax2 expression when activated at stage 12-12.5 (T,U, broken lines). Arrowheads indicate the injected sides. Each experiment was performed at least twice with a minimum of 20 embryos. The percentage of effect for each experiment was 
70%.
