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Valeria Canto Soler, Christian Gutierrez, Simon Pan, Minda M. McNally; Overexpression Of Pax6 In The Developing Retina Induces A Late Wave Of Neurogenesis Including Most Neuronal Cell Types But Not Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5890.
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© ARVO (1962-2015); The Authors (2016-present)
The dynamic pattern of Pax6 expression during retinal development suggests that Pax6 plays an important role during retinal progenitor differentiation, but the exact role(s) and timing of its function remain unclear. The purpose of this study was to analyze the function of Pax6 during retinal cell differentiation by overexpressing Pax6 in the chick retina.
The coding region of chicken Pax6 was amplified by RT-PCR and cloned into an RCAS vector. Embryonic day (ED) 3 chick embryos were injected with RCAS-Pax6 and RCAS-GFP (control) viruses into the right optic cup. Additionally, some embryos received an injection of the thymidine analog EdU into the right optic cup 5 hours before harvest. Embryos were fixed at different stages of development and processed for immunohistochemical analysis for retinal cell-type specific markers. Infected retinal areas were identified by immunostaining against the RCAS Subgroup A envelope protein.
No differences in the expression of retinal cell markers were seen between control and Pax6-treated embryos at ED6 or ED8. However, at stages 35-36 (ED9-10), a second wave of cells expressing the MAP protein recognized by the RA4 antibody (MAP/RA4) was observed in RCAS-Pax6 infected regions. These "radially oriented" MAP/RA4+ cells were reminiscent of differentiating retinal progenitors characteristic of earlier stages of normal retinal development. MAP/RA4+ cells were observed also at ED12 and ED15 in embryos treated with RCAS-Pax6. MAP/RA4+ cells were positive for EdU and co-expressed the 160kDa intermediate neurofilament protein, but did not express the Muller glia markers Vimentin and Pax2. Interestingly, MAP/RA4+ cells also expressed cell markers specific for ganglion, amacrine, bipolar and horizontal cells, but not for photoreceptors.
These results suggest that overexpression of Pax6 in retinal progenitor cells in vivo is capable of inducing a second wave of neurogenesis, which however does not include photoreceptor progenitors. Additional experiments are currently underway in order to further characterize the mechanisms regulated by Pax6 leading to this phenomenon.
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