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During vertebrate eye development, the expression of the homeobox gene Six6 is restricted to the neural retina and is initiated later than Rx and Pax6 in the presumptive retina field. We show here that overexpression of mouse Six6 in Xenopus embryos can induce transformation of competent tissue of the anterior neural plate into retinal tissue. In Six6 injected embryos, the molecular identity of the presumptive midbrain and rostral hindbrain regions was lost, as shown by the absence of XEn-2 and Xpax2 expression, being replaced by the ectopic expression of the retinal markers Xpax6 and Xrx. When allowed to grow further, Six6 injected embryos developed ectopic eye-like structures in the rostral brain and showed a transformation of the midbrain into retina. Similar results were obtained upon overexpression of Six3 or Xsix3, revealing a possible redundance of Six3 and Six6 activities. Taken together, results obtained suggest that during normal retina development, the relatively late expressed Six6 gene becomes part of a network of retinal homeobox genes that are linked together by positive feedback loops. Furthermore, our results demonstrate that the primitive neural ectoderm of the future midbrain and rostral hindbrain is competent to form retinal tissue.
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Fig. 1. RNA injection of Six6, Six3 or Xsix3 at low concentrations induces an increase in retina size. Vibratome sections of tailbud stage Xenopus embryos after whole-mount in situ hybridization. (A) Injection of 12.5 pg of Six6 RNA, or (B) 10 pg of Six3 RNA, or (C) 25 pg of Xsix3 RNA leads to enlargement of the neural retina. Overall eye shape and lens formation are not affected. (A,B) Xpax6 riboprobe. (C) Xrx riboprobe. rpe, retinal pigmented epithelium; nr, neural retina; ls, lens; is, injected side; nis, non-injected side.
Fig. 2. RNA injection of Six6, Six3 or Xsix3 at high concentrations leads to retinal expansion by midbrain transformation. Whole-mount in situ hybridization of neural groove stage embryos (stage 18; anterior view). (A,E,I,M) Non-injected control embryos. (B,F,J,N,Q,R) Six6 injected embryos. (C,G,K,O,S) Six3 injected embryos. (D,H,L,P) Xsix3 injected embryos. The injected side is always on the right (except for Q). (B–D) Ectopic expression of Xpax2 in the optic area extends into the presumptive midbrain territory (red arrowhead). Expression at the midbrain/hindbrain boundary is lost (black arrowhead). (F–H) Enlarged Xpax6 expression domain in the eyeprimordia and ectopic expression in the presumptive midbrain area (red arrowhead). The forebrain/midbrain boundary (black arrowhead) normally demarcated by Xpax6 is lost. (J–L) Expanded Xrx expression in the optic area and ectopic expression in the presumptive midbrain territory (red arrowhead). Normal limit of Xrx posterior expression domain (black arrowhead). (N–P) Xen2 expression at the midbrain/hindbrain boundary is absent (red arrowhead), while Xkrox20 expression in the hindbrainrhombomeres 3 and 5 is maintained. Normal Xen2 expression domain at the midbrain/hindbrain boundary (black arrowhead). (Q) Six6 injected mRNA is still present in the injected side of the embryos at the neurula stage, as visualized with an antisense Six6 riboprobe (arrowhead). (R) Xsix3 expression domain (black arrowhead) remains limited to the optic area upon Six6 injections (red arrowhead). (S) Moderate expansion of Xsix3 expression domain into the midbrain area upon Six3 injections (red arrowhead).
Fig. 3. Six6 injection results in the repression of RPE formation and in the expansion of the expression domain of retinal markers. Left and right views of the same tadpoleembryo (stage 36–38) are shown side by side. (C,F,I) Dorsal view of the head; anterior is at the top. (A–C) Loss of RPE and reduction of body pigmentation in the injected side (B). (D–F) Ectopic Xpax6 expression expanding caudally and disruption of the normal eye and forebrain expression domains. (G–I) Enlarged Xrx expression domain in the optic area. is, injected side; nis, non-injected side.
Fig. 4. Retinal hyperplasia and ectopic expression of Xpax6 and Xrx as a result of Six6 overexpression. Serial sections of tadpole embryos (stage 36–38) after whole-mount RNA in situ analysis are shown in a rostral to caudal direction within each row; the injected side is to the right. Note the strong reduction or absence of RPE on the injected side (A–C) in Six6 injected embryos (100 pg). Xpax2 expression in the optic stalk is normal in the injected side (A,B), but expression is absent at the presumptive midbrain/hindbrain boundary (C). Xpax6 expression is broader in the optic area and expands into the presumptive midbrain and rostral hindbrain areas (D–F). Xrx expression is normally restricted to the ciliary margin of the retina. At the injected side of the embryo, Xrx expression extends up to the presumptive rostral hindbrain region (G–I). fb, forebrain; mb, midbrain; m/h, midbrain/hindbrain boundary; os, optic stalk; rpe, retinal pigment epithelium; cm, ciliary margin.
Fig. 6. A model for neural retina determination in vertebrates. (A–C) Sequential activation of genes involved in eye development from the early neural plate to the optic vesicle stages as observed normally. The diagram represents an anterior view of a schematized vertebrate embryo; the dorsal part is at the top. (A) Rx, Pax6 and Six3 demarcate the retina field and Otx2 the anteriorneuroectoderm of the future rostral head. (B) The retina field is divided by the activity of a morphogen (Shh) emanating from the midline region, concomitant with the appearance of the optic stalk. (C) Six6 is expressed in the presumptive neural retina. (D–F) Six6 directs competent tissue towards the retinal program. (D,E) Overexpression of Six6 activates a regulatory feedback loop leading to ectopic expression of Pax6 and Rx in the Otx2 territory. (F) Six6 overexpression leads to retinal expansion by midbrain transformation.
