Fig. 3. The interaction between Otx2 and Gbx2 induce the isthmus. Embryos were injected with different mRNAs (5 ng of Otx2, 2 ng of Gbx2, 0.3 ng of ß-galactosidase, 1 ng of XFD) at the one-cell stage. Animal caps were dissected at stage 10 and cultured as conjugates. (A,B) No Fgf8 expression was detected in conjugates of control uninjected animal caps with Gbx2-injected ones (A, 0%, n=20) or with Otx2-expressing caps (B, 0%, n=23) at stage 17. (C-E) Conjugates of Otx2- with Gbx2-expressing caps performed at stage 10 can induce Fgf8 (C; 69%, n=45), En2 (D; 93%, n=109) and Wnt1 (E; 65%, n=17) (arrowheads) at stage 17. ß-Galactosidase (arrow) was co-injected with Otx2 (C) or with Gbx2 (D). Fgf8 was induced in the Gbx2 injected cap and En2 in the Otx2-expressing cap as shown by the X-Gal staining. (F) Conjugate of Otx2+XFD- and Gbx2-expressing caps. En2 induction was blocked when XFD was co-expressed with Otx2 (arrow in F shows X-Gal staining in the Otx2+XFD animal cap, 22% of expression, n=37). (G) Conjugate of Otx2- and Gbx2+XFD-expressing caps. XFD co-injected with Gbx2 did not block the induction of En2 (arrowhead, 95% of expression, n=47). Arrow in G shows X-Gal in the Gbx2+XFD cap.