Abstract
BackgroundThe vertebrate retina consists of six major classes of neuronal cells. During development, these cells are generated from a pool of multipotent retinal progenitor cells (RPCs) that express the gene Vsx2. Fate-restricted RPCs have recently been identified, with limited mitotic potential and cell fate possibilities compared to multipotent RPCs. One population of fate-restricted RPCs, marked by activity of the regulatory element ThrbCRM1, gives rise to both cone photoreceptors and horizontal cells. These cells do not express Vsx2, but co-express the transcription factors (TFs) Onecut1 and Otx2, which bind to ThrbCRM1. The components of the gene regulatory networks that control the transition from multipotent to fate-restricted gene expression are not known. This work aims to identify and evaluate cis-regulatory elements proximal to Onecut1 to identify the gene regulatory networks involved in RPC fate-restriction.MethodWe identified regulatory elements through ATAC-seq and conservation, followed by reporter assays to screen for activity based on temporal and spatial criteria. The regulatory elements of interest were subject to deletion and mutation analysis to identify functional sequences and evaluated by quantitative flow cytometry assays. Finally, we combined the enhancer::reporter assays with candidate TF overexpression to evaluate the relationship between the TFs, the enhancers, and early vertebrate retinal development. Statistical tests included ANOVA, Kruskal-Wallis, or unpaired t-tests.ResultsTwo regulatory elements, ECR9 and ECR65, were identified to be active in ThrbCRM1(+) restricted RPCs. Candidate bHLH binding sites were identified as critical sequences in both elements. Overexpression of candidate bHLH TFs revealed specific enhancer-bHLH interactions. Nhlh1 overexpression expanded ECR65 activity into the Vsx2(+) RPC population, and overexpression of NeuroD1/NeuroG2/NeuroD4 had a similar effect on ECR9. Furthermore, bHLHs that were able to activate ectopic ECR9 reporter were able to induce endogenous Otx2 expression.ConclusionsThis work reports a large-scale screen to identify spatiotemporally specific regulatory elements near the Onecut1 locus. These elements were used to identify distinct populations in the developing retina. In addition, fate-restricted regulatory elements responded differentially to bHLH factors, and suggest a role for retinal bHLHs upstream of the Otx2 and Onecut1 genes during the formation of restricted RPCs from multipotent RPCs.
Highlights
The vertebrate retina is comprised of six main classes of neuronal cells and one class of glial cells, organized into three discrete nuclear layers and two plexiform layers
While Otx2 expression is involved in multiple cell fates during retinal development, it has been shown that the combination of Otx2 and Onecut1 activates ThrbCRM1, which is a specific Thrb cis-regulatory element (CRE) active in cone/Horizontal cell (HC) restricted Retinal progenitor cell (RPC) (RPC [CH]) [11]
Guided by both sequence conservation and chromatin accessibility, we identified two regulatory elements near the Onecut1 gene, ECR9 and ECR65, that are preferentially active in Onecut1-expressing ThrbCRM1(+) RPCs
Summary
The vertebrate retina is comprised of six main classes of neuronal cells and one class of glial cells, organized into three discrete nuclear layers and two plexiform layers. While Otx expression is involved in multiple cell fates during retinal development, it has been shown that the combination of Otx and Onecut activates ThrbCRM1, which is a specific Thrb cis-regulatory element (CRE) active in cone/HC restricted RPCs (RPC [CH]) [11]. Loss-of-function mutations in Otx and Onecut affect early cone gene expression, cone number, cone type, and horizontal cell genesis [31, 35, 45], suggesting that these transcription factors (TFs) are critical in the gene regulatory networks of ThrbCRM1 restricted RPCs. The vertebrate retina consists of six major classes of neuronal cells. One population of fate-restricted RPCs, marked by activity of the regulatory element ThrbCRM1, gives rise to both cone photoreceptors and horizontal cells These cells do not express Vsx, but co-express the transcription factors (TFs) Onecut and Otx, which bind to ThrbCRM1. This work aims to identify and evaluate cis-regulatory elements proximal to Onecut to identify the gene regulatory networks involved in RPC fate-restriction
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