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Several eye-field transcription factors (EFTFs) are expressed in the anterior region of the vertebrate neural plate and are essential for eye formation. The Xenopus EFTFs ET, Rx1, Pax6, Six3, Lhx2, tll and Optx2 are expressed in a dynamic, overlapping pattern in the presumptive eye field. Expression of an EFTF cocktail with Otx2 is sufficient to induce ectopic eyes outside the nervous system at high frequency. Using both cocktail subsets and functional (inductive) analysis of individual EFTFs, we have revealed a genetic network regulating vertebrate eye field specification. Our results support a model of progressive tissue specification in which neural induction then Otx2-driven neural patterning primes the anterior neural plate for eye field formation. Next, the EFTFs form a self-regulating feedback network that specifies the vertebrate eye field. We find striking similarities and differences to the network of homologous Drosophila genes that specify the eye imaginal disc, a finding that is consistent with the idea of a partial evolutionary conservation of eye formation.
Fig. 2. Comparison of EFTF expression patterns by double whole-mount in situ hybridisation. Otx2 expression at stage 12 (A) and 13 (B). In C-I and K-T, the dark blue stain is the expression pattern of the gene named on the left, while the magenta stain is the expression pattern of the gene named on the right, at the stages shown. For example, in C, Otx2 is dark blue and Rx1 is magenta. (J) Both Emx1 and Rx1 stain dark blue. (J-L) The Rx1 (J), Pax6 (K) and Six3 (L) expression borders are indicated by a broken line. A schematic summary of the overlapping expression patterns of the eye field transcription factors at stage 12.5/13 (U) and 15 (V) is shown. Scale bars: in A, 300 μm for A-L; in M, 300 μm for M-T.
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 β-gal RNAs were injected into one cell of two-cell stage embryos. β-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 eyetissue induction. Animals were scored according to severity of phenotype - from ectopic pigment/eyetissue (most severe) to normal animals. When all the factors were present, most embryos developed ectopic pigment or eyetissue (Ect. Pig./EyeTissue). When Pax6 was left out of the cocktail, for example, the frequency of ectopic pigment or eyetissue was greatly reduced and 20% of the embryos were unaffected (Normal).
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 βgal RNA to identify the injected side. In B and C, the embryos were not stained forβ gal 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 βgal 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.
Fig. 6. Otx2 and noggin potentiate the induction of Rx1 by ET. (A,B) RT-PCR was used to detect changes in Rx1 and XAG expression in ectodermal explants from Xenopus embryos injected with noggin, Otx2 and ET. ET (100 pg) was injected alone, with 50 or 100 pg of Otx2 (A), or 5 pg noggin (B). (A) Lane 1, uninjected; lane 2, ET (100 pg); lane 3, Otx2 (50 pg); lane 4, Otx2 (100 pg); lane 5, ET (100 pg) + Otx2 (50 pg); lane 6, ET (100 pg) + Otx2 (100 pg); lane 7, embryo, no reverse transcription; lane 8, embryo, XAG induction was used as a positive control for Otx2 activity. (B) Lane 1, uninjected; lane 2, ET (100 pg); lane 3, noggin (5 pg); lane 4, ET (100 pg) + noggin (5 pg). (C-G) Rx1 expression was normalised to Histone H4 then set relative to uninjected controls. Otx2 potentiates the ET induced expansion of Rx1 expression in the anterior neural plate. Whole-mount in situ hybridisation was used to detect Rx1 expression at stage 13 in embryos injected with βgal alone (C), or in combination with 25 pg Otx2 (D), 10 pg ET (E) or both Otx2 and ET (F). (G) The rostrocaudal diameter of the Rx1 expression domain on the injected side (βgal-positive) was measured and compared with the uninjected (βgal-negative) side of the embryo (see F for an example).
