UC San Diego

Retinogenesis, the development of the retina, is regulated by dynamic changes in transcriptional expression and the chromatin landscape. While model organisms have contributed significantly to understanding retinal development, they do not allow studies of human-specific conditions. We utilized a SIX6-eGFP/VSX2-tdTomato dual fluorescent pluripotent stem-cell (PSC) line and grew retinal organoids (ROs) through 45 days. RNA- and ATAC-seq analyses through 45 days revealed transcriptomic and chromatin-landscape regulatory changes that drive retinal development and the activation of retinal specific pathways. To study an early modifier of retinal development we also investigated the Sonic Hedgehog (Shh) signaling pathway by modulating the timing and concentration of a smoothened agonist (SAG) in SIX6-eGFP/POU4F2-tdTomato dual reporter ROs. Early SAG treatment led to the development of non-retinal tissue while later treatment favored retinal development, indicated by robust POU4F2-tdTomato expression. To investigate factors governing the formation of retinal ganglion cells (RGCs; the first cell born in the eye), we developed an inducible transgene cassette expressing the transcription factors (TFs) NEUROG2-ATOH7-ISL1-POU4F2 which, in combination with Bone Morphogenic Protein (BMP) inhibition, led to robust formation of RGC-like neurons in as little as one week. An RGC-like identity was validated via fluorescent reporter expression, transcriptomic analysis, and electrophysiology. Our findings provide a better understanding of the complex interplay of transcriptional regulation, chromatin dynamics, and microenvironmental cues in RO development and advancing our understanding of retinal biology. Generating RGC-iNs illuminates factors governing RGC formation and provides insight into the early formation of the eye.