Enright, Jade1; Blackshaw, Seth2; Clark, Brian1
1Washington University School of Medicine in St. Louis John F. Hardesty, MD Department of Ophthalmology and Visual Sciences; 2Johns Hopkins University School of Medicine Solomon Snyder Department of Neuroscience
Purpose: In the developing retina, long noncoding RNAs (lncRNAs) are often expressed in a cell type specific and temporally dynamic manner. We propose that lncRNAs, known to regulate neurogenesis, influence the gene regulatory networks dictating cell cycle exit and cell fate specification during retinal development. In this study, we investigate the functions of the lncRNA Gm11454, which is enriched in late retinal progenitor cells (RPCs), in the regulation of retinal development.
Methods: Gm11454 was overexpressed through in vivo and ex vivo electroporation of P0 mouse retinas. We generated a Gm11454 deletion mouse line using CRISPR/Cas9f. RPC cell cycle exit and cell fate were evaluated using immunohistochemistry. Knockout retinal structure and function were assessed using H&E staining and electroretinograms (ERGs). Gene expression changes to Notch pathway genes were detected by qPCR and further evaluated by electroporation of the CBF1 responsive reporter element (CBFRE) driving GFP expression.
Results: P0 overexpression of Gm11454 promoted cell cycle exit and biased RPCs towards a photoreceptor cell fate. Conversely, Gm11454 knockout retinas have decreased cell cycle exit and decreased photoreceptors, as well as decreased inner layer neurons, compared to heterozygote. ERGs show impeded retinal function of Gm11454 knockout mice. Notch signaling was decreased in Gm11454 overexpressing cells with a corresponding increase in Gm11454 knockout retinas.
Conclusions: Taken together, our data suggest that the lncRNA Gm11454 functions in late RPCs to inhibit Notch signaling, thereby promoting cell cycle exit as well as photoreceptor cell fate, both of which require Notch downregulation. Gm11454 loss of function thus results in altered retinal development and function. Our results implicate lncRNAs as novel regulators of retinal development and retinal cell fate specification.
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