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E Rungger-Brandle, A Conti, D Rungger; Retinal Deficiencies in Xenopus Tadpoles Carrying Antisense Morpholinos Directed Against Pax6 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2444.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To test the role of Pax6 gene product in retinal development through targeted inhibition by antisense morpholinos. Methods: Anti-Pax6 morpholinos were injected into Xenopus laevis fertilized eggs or the two-cell stage. mRNA levels were measured by real-time PCR and Pax6 protein expression monitored by immunofluorescence microscopy. Phenotypes were scored by external features and morphology. Growing axons were visualized with antibodies to acetylated tubulin and by DiI injection. Differentiation of retinal neurons was monitored using cell-specific antibodies. Results: Antisense injected embryos have normal levels of Pax6 mRNA but protein expression is inhibited up to advanced tadpole stages. As judged by the phenotypes, inhibition is highly specific. In up to 80% of the antisense embryos eye formation is impaired, resulting in small-eyed and eyeless phenotypes that, eventually, may be lethal. Injection into one cell of the two-cell stage causes unilateral effects. The specificity of the antisense effect is underscored by the negligible frequency of aspecific mutants induced by anti-Pax6 and control morpholinos. In Pax6-deficient tadpoles (stage 40), the retinal pigment epithelium (RPE) forms a discontinuous layer. Rosettes of photoreceptor cells, arranged around single RPE cells, are formed inside the neural retina. The inner limiting membrane is poorly defined, pointing to disorganization of Muller cell glia. Moreover, the lens, if present at all, is rudimentary and displaced from the ocular axis. Conclusion: These observations suggest that, in the absence of functional Pax6 protein, retinal dysmorphogenesis may be due to the failure of RPE and Muller cell glia to correctly organize the neurons into a stratified structure. The apparent stability of antisense morpholinos make them a powerful tool to investigate specific gene function during early development and even advanced organogenesis.
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