Rasmussen JT et al. (2001), Regulation of eye development by frizzled signa...

XB-ART-9318

Proc Natl Acad Sci U S A 2001 Mar 27;987:3861-6. doi: 10.1073/pnas.071586298.

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Regulation of eye development by frizzled signaling in Xenopus.

Rasmussen JT , Deardorff MA , Tan C , Rao MS , Klein PS , Vetter ML .

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Eye development in both invertebrates and vertebrates is regulated by a network of highly conserved transcription factors. However, it is not known what controls the expression of these factors to regulate early eye formation and whether transmembrane signaling events are involved. Here we establish a role for signaling via a member of the frizzled family of receptors in regulating early eye development. We show that overexpression of Xenopus frizzled 3 (Xfz3), a receptor expressed during normal eye development, functions cell autonomously to promote ectopic eye formation and can perturb endogenous eye development. Ectopic eyes obtained with Xfz3 overexpression have a laminar organization similar to that of endogenous eyes and contain differentiated retinal cell types. Ectopic eye formation is preceded by ectopic expression of transcription factors involved in early eye development, including Pax6, Rx, and Otx2. Conversely, targeted overexpression of a dominant-negative form of Xfz3 (Nxfz3), consisting of the soluble extracellular domain of the receptor, results in suppression of endogenous Pax6, Rx, and Otx2 expression and suppression of endogenous eye development. This effect can be rescued by coexpression of Xfz3. Finally, overexpression of Kermit, a protein that interacts with the C-terminal intracellular domain of Xfz3, also blocks endogenous eye development, suggesting that signaling through Xfz3 or a related receptor is required for normal eye development. In summary, we show that frizzled signaling is both necessary and sufficient to regulate eye development in Xenopus.

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Species referenced: Xenopus
Genes referenced: fzd3 gipc1 otx2 pax6 rax rho

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	Figure 1 Overexpression of Xfz3 promotes ectopic eye formation and causes proximal eye defects. (a) Stage 42 embryo showing an ectopic eye in the dorsal head region (white arrow). (Inset) A higher magnification view of the ectopic eye. (b) Stage 45 embryo with an ectopic eye in the dorsal midline of the head. (c) Stage 42 embryo showing streaming of the retinal pigment epithelium from the endogenous eye toward the midline. (d) Stage 45 embryo with abnormally positioned eye on the injected side, marked with Î²-galactosidase (blue). (e) Section through an embryo similar to the one shown in d, showing the eye adjacent to the neural tube on the injected side (Left). (f) Section through a stage 45 embryo, showing an ectopic eye in the roof of the fourth ventricle that is fully labeled with the tracer Î²-galactosidase (blue). (Inset) A high-magnification view from an adjacent section, showing a row of morphologically distinct photoreceptor outer segments in the ectopic eye (arrowheads). (g and h) Immunolabeling of a section through a normal eye (g) and ectopic eye (h) with anti-Pax6 antibodies (red) and anti-rhodopsin antibodies (green). Nuclei are labeled with Hoechst (blue). The ectopic eye in h shows an arrangement of retinal layers similar to that in the normal eye in g.
	Figure 2 Overexpression of Xfz3 promotes ectopic expression of genes regulating eye development. A whole-mount in situ hybridization analysis was made of stage 14 embryos (a) that were either uninjected (a, c, and e) or Xfz3 injected (b, d, and f) and then labeled for Xpax6 (a and b), Xrx (c and d), or Xotx2 expression (e and f). Arrows indicate modest expansion of Xrx or Xotx2 expression (d and f). Stage 28 embryos (g) are either uninjected (g, i, and k) or Xfz3 injected (h, j, and l) and then labeled for Xpax6 (g and h), Xrx (i and j), or Xotx2 expression (k and l). Arrows indicate ectopic expression in injected embryos (h, j, and l). Î²-Galactosidase (sky blue) marks the regions of the embryo derived from the injected blastomere.
	Figure 3 Overexpression of Nxfz3 suppresses expression of genes regulating eye development. A whole-mount in situ hybridization analysis was made of stage 180 embryos that are either uninjected (a, c, and e) or Nxfz3 injected (b, d, and f) and then labeled for Xpax6 (a and b), Xrx (c and d), or Xotx2 expression (e and f). Î²-Galactosidase (sky blue) marks the regions of the embryo derived from the injected blastomere.
	Figure 4 Overexpression of Nxfz3 or Kermit inhibits endogenous eye formation. (a) Uninjected embryo at stage 41. (b and c) Nxfz3-injected embryos at stage 41 showing a reduced (b) or absent eye (c) on the injected side (white arrow). (d) Section through an embryo with a missing eye, showing the loss of normal eye structure on the injected side with only residual disorganized pigment remaining (black arrow). The tissue derived from the injected blastomere is marked with Î²-galactosidase (sky blue). (e) Uninjected embryo at stage 43. (f) Kermit-injected embryo at stage 43 showing a reduced eye on the injected side (arrow).

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