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Nicolas Lonfat, Constance L Cepko; Dissection of the mechanisms restricting specific retinal progenitor cells to the production of cones and horizontal cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1770.
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© ARVO (1962-2015); The Authors (2016-present)
The development of the retina into a highly organized structure occurs via the production of over 60 cell types from a pool of retinal progenitor cells (RPCs). While RPCs are generally multipotent, recent studies have shown that some terminally dividing progenitors are restricted to the production of specific types of daughter cells. How specific RPCs make any specific type of neuron is currently not understood. To address this question, we investigated the molecular mechanisms that restrict a specific subset of RPCs to the production of only cones and horizontal subtypes.
Otx2 and Onecut transcription factors have been shown to promote the cone and horizontal cell fates. We dissected the transcriptional regulation of these two key transcription factors. To this end, we used a combination of ChIP-seq and ATAC-seq analysis on chick retinal cells to identify putative regulatory elements of Otx2 and Onecut. Candidate regions are tested for enhancer activity using ex vivo electroporation, along with morphological and immunohistochemical identification of cells that express such constructs. Bioinformatic analyses of the positive sequences, combined with RNA-seq transcriptomes, provide us with a list of potential upstream factors driving cone or horizontal cell fates, which are then tested for function. Furthermore, the identification of Otx2 and Onecut enhancers allows for FACS-sorting of RPCs, as well as cones and horizontal cells that express these reporters, to assess the homogeneity of the RPCs and better characterize them.
We identified several putative regulatory elements of Otx2 and Onecut genes capable of driving reporter activity in the retina. Minimal sequences were further refined and mutations within these fragments allowed us to identify putative binding sites for the upstream regulating transcription factors. Bioinformatic analysis of these enhancers using rVista and TRANSFAC programs provided us with a promising list of transcription factor candidates.
We discovered several regulatory elements of Otx2 and Onecut genes in the chick retina and we identified putative upstream transcription factors that may play roles in promoting the fate of cones and horizontal cells during development. Progress on understanding how to generate cones will contribute to the development of therapies for retinal diseases.
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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