Human Retinal Progenitor Cells Grown as Neurospheres Demonstrate Time-Dependent Changes in Neuronal and Glial Cell Fate Potential

During a developmental period that extends postnatally in the mouse, proliferating multipotent retinal progenitor cells produce one of 7 major cell types (rod, cone, bipolar, horizontal, amacrine, ganglion, and Müller glial cells) as they exit the cell cycle in consecutive waves. Cell production in the retina is tightly regulated by intrinsic, extrinsic, spatial, and temporal cues, and is coupled to the timing of cell cycle exit. Arsenic-resistance protein 2 (ARS2, also known as SRRT) is a component of the nuclear cap-binding complex involved in RNA Polymerase II transcription, and is required for cell cycle progression. We show that postnatal retinal progenitor cells (RPCs) require ARS2 for proper progression through S phase, and ARS2 disruption leads to early exit from the cell cycle. Furthermore, we observe an increase in the proportion of cells expressing a rod photoreceptor marker, and a loss of Müller glia marker expression, indicating a role for ARS2 in regulating cell fate specification or differentiation. Knockdown of Flice Associated Huge protein (FLASH), which interacts with ARS2 and is required for cell cycle progression and 3′-end processing of replication-dependent histone transcripts, phenocopies ARS2 knockdown. These data implicate ARS2–FLASH-mediated histone mRNA processing in regulating RPC cell cycle kinetics and neuroglial cell fate specification during postnatal retinal development.

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

10.1101/538710 ◽

2019 ◽

Cited By ~ 2

Author(s):

Miruna G. Ghinia Tegla ◽

Diego F. Buenaventura ◽

Diana Y. Kim ◽

Cassandra Thakurdin ◽

Kevin C. Gonzalez ◽

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Keyword(s):

Single Cell ◽

Progenitor Cells ◽

Cell Fate ◽

Gene Editing ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Conditional Knockout ◽

Proliferative Potential ◽

Retinal Progenitor Cells ◽

Retinal Progenitor

AbstractDevelopment 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.

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