An enhancer located in a Pde6c intron drives transient expression in the cone photoreceptors of developing mouse and human retinas

• Published in: Developmental biology Vol. 488; pp. 131 - 150
• Main Authors: Bachu, Vismaya S., Kandoi, Sangeetha, Park, Ko Uoon, Kaufman, Michael L., Schwanke, Michael, Lamba, Deepak A., Brzezinski, Joseph A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2022
• Subjects: Animals; Animals, Genetically Modified; Cone photoreceptor; Cyclic Nucleotide Phosphodiesterases, Type 6 - genetics; Development; Enhancer; Eye Proteins - genetics; Humans; Induced Pluripotent Stem Cells; Introns - genetics; iPSC; Mice; Pde6c; Retina; Retina - metabolism; Retinal Cone Photoreceptor Cells - metabolism; Retinal organoid; Transcription Factors - metabolism; Enhancer; Cone photoreceptor; iPSC; Pde6c; Development; Retina; Retinal organoid
• ISSN: 0012-1606; 1095-564X
• DOI: 10.1016/j.ydbio.2022.05.012

How cone photoreceptors are formed during retinal development is only partially known. This is in part because we do not fully understand the gene regulatory network responsible for cone genesis. We reasoned that cis-regulatory elements (enhancers) active in nascent cones would be regulated by the same upstream network that controls cone formation. To dissect this network, we searched for enhancers active in developing cones. By electroporating enhancer-driven fluorescent reporter plasmids, we observed that a sequence within an intron of the cone-specific Pde6c gene acted as an enhancer in developing mouse cones. Similar fluorescent reporter plasmids were used to generate stable transgenic human induced pluripotent stem cells that were then grown into three-dimensional human retinal organoids. These organoids contained fluorescently labeled cones, demonstrating that the Pde6c enhancer was also active in human cones. We observed that enhancer activity was transient and labeled a minor population of developing rod photoreceptors in both mouse and human systems. This cone-enriched pattern argues that the Pde6c enhancer is activated in cells poised between rod and cone fates. Additionally, it suggests that the Pde6c enhancer is activated by the same regulatory network that selects or stabilizes cone fate choice. To further understand this regulatory network, we identified essential enhancer sequence regions through a series of mutagenesis experiments. This suggested that the Pde6c enhancer was regulated by transcription factor binding at five or more locations. Binding site predictions implicated transcription factor families known to control photoreceptor formation and families not previously associated with cone development. These results provide a framework for deciphering the gene regulatory network that controls cone genesis in both human and mouse systems. Our new transgenic human stem cell lines provide a tool for determining which cone developmental mechanisms are shared and distinct between mice and humans. [Display omitted] •An intronic enhancer within Pde6c drives expression in developing cones.•Transgenic retinal organoid systems show that the enhancer is active in human cones.•The Pde6c enhancer is activated by cells deciding between rod and cone fates.•The enhancer likely has five or more essential transcription factor binding regions.•Moderate conservation suggests flexibility of enhancer function between species.