Abstract
Purpose :
microRNAs (miRNAs) are translational repressors and are required for proper cell type generation and specification in early (embryonic) stages of retinal development. However, there is not much known about the role of miRNAs in early postnatal (P0-14) development.
Methods :
We created a conditional knock out (CKO) of Dicer1 (Dicer-CKORPC) specific for late (postnatal day P2) retinal progenitors cells (RPCs) at early stages in development (Sox2CreER: Dicerfl/fl : Stopfl/fl tdTomato; wild type: Sox2CreER: Stopfl/fl tdTomato). We injected tamoxifen intraperitoneally at time points ranging from P0-6 and P11-14 to activate the CreER. Retinal cross sections of eyes were used to analyze the Dicer-CKO phenotype 2 and 4 weeks after early Dicer deletion in RPCs, and 8 and 20 weeks in young Muller glia and amacrine cells by means of immunofluorescent labeling and confocal microscopy. For RNA analysis, P2 RPCs from wild type mice were purified by FACS and analyzed using the NanoStrings nCounter® System. Transfection of RPC miRNAs mimics was performed on P12 retinal explants. EdU was added to the medium to track proliferation. Explant cross sections were stained and analyzed after 7 days ex vivo.
Results :
Two weeks after Dicer deletion in RPCs, we found significant disruptions in the retinal architecture such as formation of clusters of rosettes (2-6) and folded layers causing an increase in the thickness of the retina by approximately 25%. Although ratios of Muller glia and amacrine cells did not change, 51% of the Sox2Cre-Tomato+ cells in the DicerCKO co-expressed Sox9 and Otx2 (controls: 16%). In the second approach (P11-14), we found structural disruptions within the retina and ruptures in the outer limiting membrane due to Muller glia migration.
Profiling of RPC miRNAs showed that miR-9, miR-16, miR-720, miR-204, miR-20a+b, miR-25, miR15b, let-7g, let-7d, let-7i, miR-106a/17, and miR-96 are highly expressed. Supplementation of RPC miRNAs in P12 retinal explants led to increased proliferation of Muller glia (8%).
Conclusions :
Structural disruptions indicate that late RPC miRNAs are required for proper formation of the retinal layers, tissue polarity and cell ratio in early postnatal development. Supplementation of RPCs miRNAs to P12 retinal explants led to increased proliferation in Muller glia and therefore might play an important role in the regulation of RPC proliferation.
This is a 2020 ARVO Annual Meeting abstract.