Emerson, M. M. | bioRxiv Science

bioRxiv2019

Identification of genes with enriched expression in early developing mouse cone photoreceptors

Cone photoreceptors are the critical first cells that mediate high acuity vision. Despite their importance and their potential use in cell-based therapies for retinal diseases, there is a lack of knowledge about the early developmental stages of these cells. Here we characterize the expression of the homeobox transcription factor Lhx4 as an early and enriched cone photoreceptor expressed gene in both chicken and mouse. A Lhx4 GFP reporter mouse was found to recapitulate this early cone photoreceptor expression and was used to purify and profile embryonic mouse cone photoreceptors by single cell RNA sequencing. This enrichment in cone photoreceptors allowed for the robust identification of genes associated with the early cone transcriptome and also identified subpopulations of these cells. A comparison to previously reported datasets allowed the classification of genes according to developmental timing, cell type specificity, and whether they were regulated by the rod transcription factor Nrl. This analysis has extended the set of known early cone enriched genes and identified those that are regulated independently of Nrl. This report furthers our knowledge of the transcriptional events that occur in early cone photoreceptors.

developmental biology↗

bioRxiv2019

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

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.

developmental biology↗