Fig. 2. Otx2 and Gbx2 participate as transcriptional repressors in the positioning of the isthmus organizer. Embryos were injected in one blastomere of two-cell stage embryos with 2 ng of Gbx2 (A,D,G,J), 2 ng of the Gbx2 repressor fusion (Gbx-EnR) (B,E,H,K) or 0.3 ng Gbx2 activator fusion (Gbx2-E1A) (C,F,I,L) mRNAs. The expression of Otx2, Fgf8, En2 and Pax2 were analyzed at stage 17 and the injected sides were detected by X-Gal stain. (A-C) Otx2 expression is inhibited in embryos injected with Gbx2 or Gbx2-EnR mRNAs (A,B, broken lines), while is displaced caudally in those injected with Gbx2-E1A mRNA (C, broken lines). (D-F) A rostral shift of Fgf8 isthmic expression territory is observed upon Gbx2 or Gbx2-EnR overexpression (D,E, broken lines), and inhibition and caudal shift of this expression domain occurs in Gbx2-E1A-injected embryos (F, arrowhead). (G-I) En2 is displaced anteriorly in Gbx2- or Gbx2-EnR-injected embryos (G,H, broken lines), while is repressed and shift caudally in those injected with Gbx2-E1A mRNA (I, broken lines). (J-L) Pax2 expression is displaced rostrally in embryos injected with Gbx2 or Gbx2-EnR mRNAs (J,K, broken lines), while a caudal shift occurs in Gbx2-E1A-injected embryos (L, broken lines). The injection at the two-cell stage of 5 ng of Otx2 (M,P,S,V), 2 ng of Otx2-EnR (N,Q,T,W) or 1 ng of Otx2-E1A (O,R,U,X). The expression of Gbx2, Fgf8, En2 and Pax2 were analyzed at stage 17. (M-O) Overexpression of Otx2 or Otx2-EnR mRNAs produce repression and caudal shift of Gbx2 (broken lines) and injection of Otx2-E1A mRNA caused an anterior shift and diffusion of Gbx2 (O, broken lines). (P-R) Fgf8 is shifted posteriorly in embryos injected with Otx2 or Otx2-EnR mRNAs (P,Q, broken lines) while injection of Otx2-E1A mRNA causes inhibition of the isthmus expression of Fgf8 (R, arrowhead). (S-U) En2 is shifted caudally in Otx2 and Otx2-EnR injected embryos (S,T, broken lines), while there is a decrease in En2 expression with an anterior displacement in embryos injected with Otx2-E1A mRNA (U, broken lines). (V-X) Pax2 is shifted caudally in Otx2- and Otx2-EnR-injected embryos (V,W broken lines), while its expression decrease in the Otx2-E1A-injected embryos (X, arrowhead). Arrowheads point to the injected sides. Each experiment was performed at least twice with a minimum of 30 embryos. The percentage of effect for each experiment was 
70%.
