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M. M. McNally, C. Gutierrez, V. Canto-Soler; Sustained Pax6 Expression in Retinal Progenitor Cells Induces a Second Wave of Neurogenesis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5937.
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© ARVO (1962-2015); The Authors (2016-present)
The transcription factor Pax6 is important for normal retinal development, but the exact role and timing of its function remain unclear. This study analyzes the possible functions of Pax6 during retinal cell differentiation by overexpressing Pax6 in vivo.
The coding region of chicken Pax6 was amplified by RT-PCR, modified to include an HA-tag and cloned into an RCAS vector. Embryonic day 3 (ED3) chick embryos were injected with RCAS-Pax6 and RCAS-GFP (control) viruses into the right optic cup. Some embryos also received a BrdU injection into the right optic cup at ED9 and were incubated for two additional hours or until they reached ED10. Embryos were fixed at different stages and processed for immunohistochemical analysis. Infected retinal areas were identified by immunostaining against HA (RCAS-Pax6) or GFP. In an additional set of experiments, retinas from embryos infected with RCAS-Pax6 or RCAS-GFP at ED3 were isolated at ED6 and cultured in low-density conditions for four days.
Treated embryos showed normal macroscopic eye phenotypes and normal layered retinas at all stages analyzed. In retinal sections, 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 RA4 expressing cells was observed in RCAS-Pax6 infected regions. These "radially oriented" RA4+ cells were reminiscent of cells observed in the "neurogenic zone" at earlier stages of normal retinal development. "Radially oriented" RA4+ cells were positive for BrdU and co-expressed the neurofilament RMO270, but did not express the Muller glia marker Vimentin. In agreement with our observations at early stages of normal development, RA4/RMO270 positive cells also expressed the transcription factor Prox1, normally present in horizontal cell progenitors. When analyzed in vitro, cells infected with RCAS-Pax6 showed a significant increase in the number of cells expressing RA4 and the transcription factors AP2alpha and Lim1 (normally expressed in amacrine and horizontal cells) compared to those infected with RCAS-GFP. In addition, we observed a concomitant decrease in the number of photoreceptors in the RCAS-Pax6+ cell population.
These results suggest that sustained Pax6 expression in retinal progenitor cells induces a second wave of neurogenesis. Additional experiments including analysis of expression of other transcription factors are currently underway in order to further characterize the phenotype of the newly born neurons.
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