Fig. 5. ET, Rx1 and Pax6 regulate Otx2 expression. Embryos were injected into one blastomere at the two-cell stage with RNA of the indicated gene. Whole-mount in situ hybridisation was used to detect Otx2 expression in embryos injected with 100 pg ET (B), 400 pg Rx1 (C), 200 pg Pax6 (D), 200 pg Six3 (E) or 500 pg Lhx2 (F) RNA. Embryos in A,D-F were co-injected with ßgal RNA to identify the injected side. In B and C, the embryos were not stained for ßgal expression so that the repression of Otx2 could be more easily visualised. Scale bar: 300 µm. (G) Quantitation of the effect of EFTFs on Otx2 expression. Percent of embryos with an increase (), decrease () or no change (NC) in Otx2 expression. ET induces Rx1 expression. (H,I) Rx1 injection did not effect ET expression, while ET induced Rx1 expression in Xenopus animal caps in a dose-dependent manner. Histone H4 was used as a loading control; U, uninjected; E, parallel, uninjected embryo. (J-M) Whole-mount in situ hybridisation was used to detect ET (J-K) and Rx1 (L-M) expression in stage 13 Xenopus embryos injected with 200 pg Rx1 (K) or ET (M) RNA. In (J,K), embryos were injected with ßgal RNA. In L,M, GFP RNA was used to detect the injected side of the embryo. The right side is the injected side in J-M. Scale bar: 300 µm. (N) Interpretation of the results of Figs 4, 5.

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