Identification of Evolutionarily Conserved VSX2 Enhancers in Retinal Development

Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. Recently, we found that distinct modules within a murine SE regulate gene expression of transcription factor Vsx2 in a developmental stage- and cell-type specific manner. In the present study, we examine the ability of these modules to drive retinal development between species. By inserting the human build of one Vsx2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the implications of these SE modules in a model of human development, we generated human retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module leads to a complete loss of ON cone bipolar neurons. This prototypical SE serves as a model for uncoupling developmental stage- and cell-type specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease. Overall design: Retinal organoids derived from hESC line H9 VSX2-GFP/CRX-TdTomato were generated using an established protocol from the Laboratory of Dr David Gamm. Each organoid was placed in one well of a 24-well plate (1 organoid/ 1 well) with 1mL of 3D Retinal Differentiation Media containing 1:1000 EC23 (3D RDM) and incubated at 37C. Media changes occurred weekly and Mycoplasma testing occurred every 2 months. After 100 days, 3D RDM media changes did not include EC23.