Abstract
Purpose: :
Despite evidence showing the neurogenic potential of Muller cells, only a fraction of cells turn on neuronal genes. The repressor element (RE)-1 silencing transcription factor (REST) represses neuronal genes and neuronal differentiation in non-neuronal tissues. The purpose of this study is to examine Rest mRNA and protein expression in cultured mouse Muller cells and the mouse retina, and to target Rest mRNA for RNAi knockdown using an inducible shRNA lentiviral construct.
Methods: :
Quantitative RT-PCR was used to analyze Rest expression in embryonic (E17), neonatal (P0) and adult mouse retinas and conditionally immortalized Muller cells (ImM10) under conditions that promote growth, neurosphere formation and neuronal differentiation. REST and Co-REST protein expression in ImM10 cells and adult retina was analyzed by immunocytochemistry. For RNAi, 3 shRNAs targeted against Rest and control inserts containing Gapdh shRNA or lacking a shRNA insert were subcloned into a Tet-inducible, lentiviral construct with a red fluorescent protein (RFP) reporter. Transduced Muller cells were puromycin selected (6µg/ml) and promoter activity was induced by 1µg/ml doxycycline.
Results: :
Developing and mature retina and cultured Muller cells expressed Rest mRNA. Expression was decreased in neurospheres, but returned to original levels in differentiation conditions. REST antibody prominently labeled nuclei and filamentous processes of Muller cellsin culture and in the mature retina, where they also labeled nuclei in the INL, fibers in the nerve fiber layer, and both plexiform layers. Monoclonal Co-REST antibody labeled Muller cell nuclei in vitro and nuclei of Muller cells, rod bipolar cells and horizontal cells in the mature retina, where perinuclear Co-REST staining was also present throughout the ONL, particularly in cones. In transduced, puromycin selected Muller glia, 100% of cells expressed the RFP reporter following doxycycline induction, but the 3 shRNA constructs tested to date did not significantly knockdown REST mRNA.
Conclusions: :
REST is expressed in Muller glia and expression persists under conditions previously shown to upregulate neuronal gene expression in Muller-derived stem cells. This suggests that cells maintaining a non-neuronal phenotype are also present under current culture conditions. Ongoing studies are focused on developing effective shRNAs to knock-down Rest mRNA using RNAi to promote increased neuronal differentation of Muller glia derived stem cells.
Keywords: Muller cells • gene/expression • immunohistochemistry