Onecut1 and Onecut2 Play Critical Roles in the Development of the Mouse Retina

Abstract

The entire repertoire of intrinsic factors that control the cell fate determination process of specific retinal neurons has yet to be fully identified. Single cell transcriptome profiling experiments of retinal progenitor cells revealed considerable gene expression heterogeneity between individual cells, especially among different classes of transcription factors. In this study, we show that two of those factors, Onecut1 and Onecut2, are expressed during mouse retinal development. Using mice that are deficient for each of these transcription factors, we further demonstrate a significant loss (∼70–80%) of horizontal cells in the absence of either of these proteins, while the other retinal cells appear at normal numbers. Microarray profiling experiments performed on knockout retinas revealed defects in horizontal cell genes as early as E14.5. Additional profiling assays showed an upregulation of several stress response genes in the adult Onecut2 knockout, suggesting that the integrity of the retina is compromised in the absence of normal numbers of horizontal cells. Interestingly, melanopsin, the gene coding for the photopigment found in photosensitive ganglion cells, was observed to be upregulated in Onecut1 deficient retinas, pointing to a possible regulatory role for Onecut1. Taken together, our data show that similar to Onecut1, Onecut2 is also necessary for the formation of normal numbers of horizontal cells in the developing retina.

Single cell transcriptome profiling of developing chick retinal cells

“…Using the NCBI BLAST tool, this probe sequence produced a 99% alignment to predicted chick sequence ChEST736n4, as well as high alignment scores (>90%) for the gene Onecut2 (OC2) in a variety of animals. Onecut2, which has been shown to be expressed by HCs in the mouse, results in a loss of 50% of the HCs when removed from mice (Goetz, Martin, Chowdhury, & Trimarchi, 2014;Sapkota et al, 2014). To examine the expression pattern further in the chick retina, we completed ISH and found expression of ChEST736n4 within a subset of cells in the ONBL at E4 (Figure 8l), much like the expression of Cath5 and Fgf19 at that timepoint.…”

“…Initially discovered in subsets of developing murine single cells, studies of OC1-and OC2-deficient mice revealed that these genes are required for the generation of HCs (Goetz et al, 2014;Sapkota et al, 2014;Wu et al, 2013). However, the Onecut factors are not purely limited to expression in mature HCs, as further investigation showed that OC1 is a part of a regulatory network that biases Olig21 RPCs toward a HC fate (Emerson et al, 2013;Hafler et al, 2012).…”

The identification of effectors of retinal cell fate determination through single cell transcriptomics

“…Sequences (between 650 and 800bp in length) were amplified by PCR from isolated mouse cDNA (see supplemental table for details). Probes were visualized using an α-DIG-AP antibody (Roche) and subsequent exposure using BCIP and NBT Goetz et al 2014b). The 3'-targeted Plk3 probe used in this study was from the BMAP collection, accession number AW488956, the sequence for which was confirmed before use.…”

“…Microarray hybridization was performed as described previously (Goetz et al, 2014b further analysis was performed as described . The…”

“…To better understand the role that Plk3 may play in retinal development, we looked for the presence of more subtle changes in adult retinas that were either wildtype or null for Plk3. Full transcriptomic analysis of murine retinas not only reveal phenotypes that are not immediately apparent upon gross morphological analysis, but also provides an unbiased determinant of changes in even small neural subpopulations (Goetz and Trimarchi, 2015;Goetz et al, 2014b). Therefore, microarray analysis was performed on adult Plk3-KO retinas and their littermates (n=3 Plk3-KO, n=3 WT).…”