Fig. 3. Coordinated expression of EFTFs induces ectopic Lhx2 expression and ectopic eye-like structures outside the nervous system. (A-D) In situ hybridisation for Lhx2 expression (violet) in stage 20 embryos. (A) Uninjected embryo shows the normal expression pattern of Lhx2. (B-D) Otx2, ET, Pax6, Six3, Rx1, tll, Optx2 and ß-gal RNAs were injected into one cell of two-cell stage embryos. ß-gal staining (light blue) shows the injected side. Arrow indicates to ectopic Lhx2 expression (violet). (E-H) Embryos injected with Otx2, ET, Pax6, Six3, Rx1, tll and Optx2 RNAs, and grown to stage 45. Arrows indicate ectopic eyes and arrowheads point to lens. (I,J) Sections through ectopic eyes reveal the layering of ganglion (GCL), inner nuclear (INL) and outer nuclear (ONL) cell layers. (I) The retinal ganglion cells are detected using the marker, hermes (violet). Rod photoreceptors are identified in the outer nuclear layer, by the detection of opsin (green, J). Opsin also stains a rosette of cells between the GCL and the lens. Lens was detected using anti-crystalline antibodies and stains red in J. (K) Cocktail subsets reveal the relative importance of EFTFs for eye tissue induction. Animals were scored according to severity of phenotype - from ectopic pigment/eye tissue (most severe) to normal animals. When all the factors were present, most embryos developed ectopic pigment or eye tissue (Ect. Pig./Eye Tissue). When Pax6 was left out of the cocktail, for example, the frequency of ectopic pigment or eye tissue was greatly reduced and 20% of the embryos were unaffected (Normal).