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

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.

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

“…We first tested the effect of Notch inhibition in retinal development using DNA reporters, including reporters with enrichment in multipotent RPCs (VSX2ECR4; Buenaventura et al, 2018), cone/HC restricted RPCs (ThrbCRM1; Emerson et al, 2013), subsets of cones (ThrbCRM2; Emerson et al, 2013; Schick et al, 2019) and HCs/RGCs (OC1ECR22; Patoori et al, 2020 and manuscript in preparation). E5 chicken retinas were electroporated with a co-electroporation control, with or without the MAML-DN, and assessed by confocal microscopy after two days culture.…”

“…CAG::TdT was described in (Schick et al, 2019). OC1ECR22::GFP was described in (Patoori et al, 2020 and manuscript in preparation). VSX2ECR4::GFP reporter and CAG::iRFP were described in (Buenaventura et al, 2018).…”

“…CAG::Nucβgal was obtained from the Cepko lab (Cepko Lab, Harvard Medical School). Previously reported plasmids include CAG::TdT (Schick et al, 2019), OC1ECR22::GFP (Patoori et al, 2020; Schick et al, 2020), VSX2ECR4::GFP and CAG::iRFP (Buenaventura et al, 2018), ThrbCRM1::GFP and ThrbCRM2::GFP (Emerson et al, 2013), ThrbCRM1::PhiC31 and CaNa::GFP (Schick et al, 2019) and CAG::AU1Gapdh (Emerson and Cepko, 2011). pCAG is vector with a CAG promoter with no protein coding region downstream.…”

“…CAG::TdT was described in (Schick et al, 2019). OC1ECR22::GFP was described in (Patoori et al, 2020 and manuscript in preparation).…”

Building a Mammalian Retina: An Eye on Chromatin Structure

“…Classically, strategies to pinpoint cell-type specific cisregulatory elements in the developing retina have exploited DNA conservation and enrichment of lineage-specific transcription factor motifs coupled with in vivo screening for enhancer activities. This method was successful in the identification of numerous distal regulatory elements near genes essential for retinal development and cell-type specification such as Vsx2 and Grm6 (bipolar cells), Nrl, Otx2 and Prdm1 (photoreceptors), Atoh7 (ganglion cells), Onecut1 and Thrb (cones/horizontal cells), and Pax6 (RPCs, amacrine cells), among others (Kleinjan et al, 2004;Rowan and Cepko, 2005;Riesenberg et al, 2009;Willardsen et al, 2009;Emerson and Cepko, 2011;Kautzmann et al, 2011;Emerson et al, 2013;Mills et al, 2017;Goodson et al, 2020;Patoori et al, 2020).…”