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Brian S Clark, Thuzar Thien, Cristina Zibetti, Erik Aranda-Michel, Fion Shiau, Seth Blackshaw; Identification and characterization of long noncoding RNAs in retinal progenitor cell competence. Invest. Ophthalmol. Vis. Sci. 2017;58(8):118.
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© ARVO (1962-2015); The Authors (2016-present)
Despite extensive profiling of retinal progenitor cells (RPCs), only a few RPC-expressed transcription factors have been identified that both directly regulate and whose expression patterns track with changes in cellular competence. Therefore, we hypothesize that dynamically expressed long noncoding RNAs (lncRNAs) are likely candidates to regulate the temporal specification of cell fates as they can regulate both transcription factor activity and chromatin conformation. We tested this hypothesis through the identification and characterization of lncRNAs during retinal development.
RNA-seq was performed on sorted RPC and post-mitotic cells from E14 and P2 Chx10:GFP mouse retinas. Differential expression analysis was performed and validated through in situ hybridization and qRT-PCR. The regulation of cell fate specification by candidate lncRNAs was tested through in vivo electroporation into P0 mouse retinas.
We have identified >100 lncRNAs that are enriched in RPCs and display differential expression between early and late-stage RPCs. We have validated the spatial and temporal expression patterns of >30 lncRNAs within the developing retina and have begun examining the consequence of altered lncRNA expression on cell fate specification. In particular, we have identified that the intergenic lncRNA Gm11454 is expressed in the neuroblast layer of the postnatal retina. Overexpression of Gm11454 results in decreased expression of the neighboring protein-coding gene, Tox2, and results in an increased production of photoreceptors at the expense of amacrine, bipolar and Müller glial cells. CRISPRi/dCas9-KRAB mediated knockdown of Tox2 expression phenocopies the observed changes in cell fate resulting from Gm11454 overexpression. Additional experiments are addressing the mechanisms by which Gm11454 functions to control cell fate decisions.
These experiments indicate that we can readily identify and characterize lncRNAs whose expression tracks with changes in RPC competence, and, therefore, are positioned to contribute to the temporal specification of retinal cell fates. Studies of the lncRNA Gm11454 suggest that Gm11454 regulates RPC cell fate specification by controlling Tox2 expression. Together, these results provide support for novel, RNA-mediated mechanisms that impact the instructive competence program governing retinal cell fate decisions.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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