Single cell profiling of CRISPR/Cas9-induced OTX2 deficient retinas reveals fate switch from restricted progenitors

Abstract

Development of the vertebrate eye, like many developmental systems, depends on genes that are used iteratively in multiple distinct processes. The OTX2 transcription factor is one such gene, with a requirement for eye formation, photoreceptor formation, and retinal pigment epithelium specification, among others. Recent evidence has suggested that OTX2 is also expressed in subsets of retinal progenitor cells with restricted fate choices. However, given the multiple roles for OTX2 and limitations of conventional conditional knockout strategies, the functional significance of this expression is unknown. Here we use CRISPR/Cas9 gene editing to produce mutations of OTX2, identifying similar phenotypes to those observed in human patients. In addition, we use single cell RNA sequencing to determine the functional consequences of OTX2 gene editing by CRISPR/Cas9 on the population of cells derived from OTX2-expressing retinal progenitor cells. We not only confirm that OTX2 is required for the generation of photoreceptors, but also for maintaining the proliferative potential of cells and suppressing the formation of specific retinal fates. These include subtypes of retinal ganglion and horizontal cells normally associated with these progenitor types, suggesting that in this context OTX2 functions to repress sister cell fate choices. Upregulation of key transcription factors involved in the formation of these cells was observed suggesting that OTX2 is upstream of critical nodes of gene regulatory networks of these alternative fates.