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Stefanie Gabriele Wohl, Thomas A Reh; Changes in miRNAs in Müller glia after retinal injury and Dicer deletion.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4190.
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© ARVO (1962-2015); The Authors (2016-present)
microRNAs (miRNAs) are negative regulators of gene expression and play roles in retinal development and regeneration (in zebrafish). Less is known about the role of miRNAs in the response to injury in mouse Müller glia (MG). We used NanoString technologies® and quantified miRNAs in 1) mature wild type MG, 2) after light damage (LD), as well as 3) in Dicer conditional knock out (CKO) MG cells.
We isolated the MG from naïve or light damaged adult wildtype mice (Rlbp-CreER: floxed-stop-tdTomato (TdTom)) mice, as well as from Dicer CKO mice (Rlbp-CreER: floxed-Dicer: floxed-stop-tdTomato), by means of fluorescent activated cell sorting (FACS). To activate the CreER/reporter in the wild type mice, we injected Tamoxifen (Tmx) intraperitoneally 2 days before sorting. The LD mice received Tmx 2 days before the LD (8 h) and the cells were collected 1 week later. To delete Dicer from the MG glia, Tmx was given at postnatal days (P) 11-14 and the cells were collected 4 weeks later. We verified the specificity of the MG labeling using histological analysis and immunofluorescent labeling. Samples of at least 5 sorts (4-6 mice each) were pooled the RNA was purified and analyzed.
With multiple Tmx injections the Rlbp1-creER is very efficient: ~ 98% of all MG were tdTom+. For all experiments, the tdTom+ MG were over 90% pure, whereas the reporter negative fractions had no tdTom+ MG. In adult MG, 256 miRNAs were more highly expressed than negative controls. Comparing the TdTom+ cells with the negative fraction allowed us to determine which miRNAs are specifically expressed in MG. After Dicer deletion in the MG, these enriched miRNAs declined between 63-90%. LD caused a number of changes in miRNAs in the MG, with many declining and other increasing in response to injury. Interestingly, there was an increase in the number of tdTom+ MG in the Dicer CKO as assessed by FACS (3.2% versus 1.8%, p = 0.000) or cell counts in sections (18% more MG/ field; p = 0.012). However, the retinas in the Dicer CKO appeared normal and the MG displayed a normal morphology.
Our data shows that specific miRNAs are highly expressed in MG, and that LD caused dramatic changes in many of these. The CKO of Dicer caused a decline in most miRNAs and an increase in MG cell number, though the overall retinal structure and MG morphology is not altered.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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