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V. Canto Soler, R. Adler; Cellular and Molecular Mechanisms Regulated by Pax6 in Early Eye Development . Invest. Ophthalmol. Vis. Sci. 2005;46(13):568.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Developmental abnormalities caused by down–regulation of Pax6 expression in chick embryos are very severe when treatment is applied at Hamburger–Hamilton (HH) stages 9–10, but considerably milder when treatment is applied 7 hours later, at HH 11–12 (Canto Soler and Adler, 2004 ARVO). The purpose of the present study is to investigate cellular and molecular events involved in these stage–dependent abnormalities. Methods: Fluorescein–labeled anti–PAX6 or control morpholinos (MPs) were microinjected and electroporated into embryonic optic vesicles at HH stages 9 to 12, and analyzed 10, 24 or 96 hours later in toto, and in crysections processed for immunocytochemistry, in–situ hybridization, or the TUNEL reaction. Quantitative analysis was performed using ImageJ (NIH). Results: Control MPs caused no detectable changes in Pax6 expression or eye development at any of the stages studied. Conspicuous downregulation of Pax6 immunoreactivity was seen in eye tissues containing anti–Pax6 MPs, independently of the stage when treatment was applied. No significant differences in proliferation were observed by phosphohistone 3 immunocytochemistry at any of the stages studied. TUNEL (+) apoptotic cells were significantly increased in embryos treated with anti–Pax6 MPs, and much more so in embryos treated at stage 10 than in those treated at stage11. Transcription factor expression also changed in a stage–dependent manner. Embryos treated at HH 9–10 showed an expansion of the Pax2 domain, no detectable Chx10 expression, and no apparent changes in cVax. Embryos treated at HH 11–12 showed expansion of Pax2 and cVax domains to dorsal retina, but no apparent changes in Chx10 expression. Conclusions: These results show a hitherto unknown effect of Pax6 on the survival of optic vesicle cells which, together with transcription factor mis–expression, is likely involved in the genesis of ocular abnormalities triggered by anti–Pax6 MPs. The data also support the hypothesis that there are narrow time windows during which Pax6 is critical for different aspects of optic vesicle patterning and development.
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