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First published online 27 August 2003
doi: 10.1242/dev.00723

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Specification of the vertebrate eye by a network of eye field transcription factors

Michael E. Zuber^1,, Gaia Gestri^1,*, Andrea S. Viczian^1,*,, Giuseppina Barsacchi² and William A. Harris^1,

¹ Department of Anatomy, University of Cambridge, Cambridge CB2 3DY, UK
² Laboratorio di Biologia Cellulare e dello Sviluppo, Università di Pisa, Via Carducci 13, 56010 Ghezzano, Pisa, Italy

View larger version (61K): [in a new window]   Fig. 1. Relative timing of EFTF expression. RT-PCR was used to detect the expression of ET, Pax6, Six3, Rx1, tll, Lhx2 and Optx2 in the unfertilised embryo (E) and until stage 18 of development. The transient expression of Six3 and tll prior to stage 10.5 was detected in four independent experiments. PCR amplification of Histone H4 demonstrates that approximately equivalent amounts of cDNA templates were used. A duplicate set of reactions from stage 18 embryo RNA were run without reverse transcriptase to test for contaminating plasmid and genomic DNA (18-RT). The PCR products were subcloned and sequenced to confirm their identities. (B) Schematic showing the results of multiple experiments. Each dot represents the developmental stage at which strong induction was observed.

View larger version (68K): [in a new window]   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.

View larger version (64K): [in a new window]   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).

View larger version (43K): [in a new window]   Fig. 4. Noggin but not Otx2 regulates eye field transcription factor expression while Otx2 blocks the repression of ET by noggin. (A) RT-PCR was used to detect changes in the expression of the EFTFs in response to noggin (10 pg) and Otx2 (200 pg). The effect of Otx2 on its own expression was not determined (ND). The presence of ET and Six3 in uninjected animal caps (U) was not a result of DNA contamination as neither transcript was detected when duplicate samples were amplified in the absence of reverse transcriptase (-RT). Uninjected sibling embryos `E' were used as a positive control for PCR. Histone H4 was used as a loading control. (B) RT-PCR was used to determine the relative expression of ET in ectodermal explants from embryos injected with Otx2, noggin or both. The percent of ET expression relative to uninjected controls is shown above each bar of the graph. (C) Interpretation of the combined results from A and B.

View larger version (48K): [in a new window]   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.

View larger version (52K): [in a new window]   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 ßgal 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 (ßgal-positive) was measured and compared with the uninjected (ßgal-negative) side of the embryo (see F for an example).

View larger version (32K): [in a new window]   Fig. 7. Epigenetic interactions among the eye field transcription factors define a genetic network during eye field formation. (A) ET induces the expression of a subset of EFTFs. RT-PCR was used to detect changes in EFTF expression in ectodermal explants isolated from embryos injected with 200 pg of ET. The fold induction represents the relative expression of the EFTFs when compared with uninjected controls. (B) Epigenetic interactions between noggin, Otx2 and the EFTFs. , induction of target gene; , repression of target gene; NC, no change in target gene expression; *some variability in these inductions was observed. (C) Summary model of eye field induction in the anterior neural plate. Light blue indicates the neural plate, blue shows the area of Otx2 expression and dark blue represents the eye field.