Cis-regulatory analysis of Onecut1 expression in fate-restricted retinal progenitor cells

Abstract

Background: The 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. Method: We 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.Results: Two 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. (Continued on next page)Conclusions: This 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, faterestricted 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.

Otx2 and Oc1 directly regulate the transcriptional program of cone photoreceptor development

“…We also looked at the cellular activity of the Oc1 CRM G, which showed a similar chromatin accessibility between ThrbCRM1+ and ThrbCRM1− cells. As described recently for an overlapping CRM, Oc1 CRM ACR8 41 , the activity of Oc1 CRM G activity was found excluded from ThrbCRM1+ cells in what could be other types of RPCs (Figure S2B). Additionally, we found higher expression driven by Oc1 CRM G in cells morphologically resembling RGCs (Figure S2B).…”

“…Multiple peaks were tested (n=13), with 9 of them showing AP activity (Figure 3B, S2A). While Oc1 CRM B, J, K and L have not been previously described as potential CRMs, the other CRMs were recently reported to have activity in the chick retina 41 . The activity of the regions more open in ThrbCRM1+ cells that showed the strongest AP staining was then examined using GFP expression within tissue sections (Figure 3C).…”

“…Negative regulation of bHLH factors by Notch, in part through Hes TFs, likely limits neuronal fates as bHLH TFs positively regulate many retinal cell fates 77 . If these Notch-dependent activities lead to the initiation of Otx2 and Oc1 transcription 41,76 , which then reach a threshold level, they might remain stably expressed via auto-regulation.…”

Notch signaling represses cone photoreceptor formation through the regulation of retinal progenitor cell states

“…We first tested the effect of Notch inhibition on specific cell types using DNA reporters, including reporters with enrichment in multipotent RPCs (VSX2ECR4 18 ), cone/HC restricted RPCs (ThrbCRM1 20 ), subsets of cones (ThrbCRM2 20 , 23 ) and HCs/RGCs (OC1ECR22 31 , 32 ). E5 chicken retinas were co-electroporated with an electroporation control, with or without the MAML-DN, and assessed by confocal microscopy after 2 days culture.…”

“…CAG::Nucβgal was obtained from the Cepko lab (Cepko Lab, Harvard Medical School). Previously reported plasmids include CAG::TdT 23 , OC1ECR22::GFP 31 , 32 , VSX2ECR4::GFP and CAG::iRFP 18 , ThrbCRM1::GFP and ThrbCRM2::GFP 20 , ThrbCRM1::PhiC31 and CaNa::GFP 23 and CAG::AU1Gapdh 19 . pCAG is vector with a CAG promoter with no protein coding region downstream.…”