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Sui Wang, Cem Sengel, Mark Emerson, Connie L Cepko; Dissection of gene regulatory networks that control retinal cell fate specification. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1686.
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© ARVO (1962-2015); The Authors (2016-present)
Recent advances in stem cell biology offer great potential for the study and treatment of human retinal diseases, yet our knowledge of the molecular mechanisms that direct the specification and generation of various types of retinal cells remains inadequate. This encourages us to put more effort into understanding how different retinal cell types, especially photoreceptors, are specified during development, with the hope of producing functional retinal neurons for the improvement of cell therapies.
Many transcription factors (TFs) are essential individually or in combination for the specification of one or multiple retinal cell types. However, their interactions with each other and with their cognate cis-regulatory modules (CRMs) to form gene regulatory networks (GRNs) have not been systematically explored. Deciphering these GRNs can provide an in-depth understanding of specification events.<br /> <br /> We employed a straightforward strategy to dissect GRNs: 1) We identified enhancers of key TFs that regulate the specification of different retinal cell types; 2) We analyzed the critical sequences within such enhancers, allowing the definition of TFs that bind to and activate such enhancers; 3) Using gene perturbation assays, we established the epistatic relationships among TFs and the GRNs.<br /> <br /> Notably, to facilitate GRN studies, we developed and applied several novel techniques, such as CRISPR in vivo, Single Molecule FISH, in vivo electroporation of plasmids and enhancer screen assays.
We started by dissecting the GRN that controls the binary fate decision of rod and bipolar cells. Based on the identification of a CRM for Blimp1, a gene that is important in this GRN, we discovered that Otx2 and RORb are upstream regulators of Blimp1, while Blimp1 was found to be a negative feedback regulator of Otx2. These TFs together with others form a rod-bipolar GRN to regulate the level of Otx2, which in turn regulates the final ratio of rods vs. bipolar cells.<br /> <br /> We also demonstrated the necessity of CRMs within GRNs by applying CRISPR in the mouse retina in vivo for the first time. We found that CRISPR successfully created homozygous and heterozygous alterations in >50% of the electroporated retinal cells.
1) GRNs provide complex interactions that can specify the many cell types of the neural retina. 2) The CRISPR/Cas9 technique can allow for rapid genomic editing in the mouse retina in vivo.
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