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. 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. 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. 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, 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.

中文翻译：

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命运限制性视网膜祖细胞中 Onecut1 表达的顺式调控分析

脊椎动物视网膜由六大类神经元细胞组成。在发育过程中，这些细胞由表达 Vsx2 基因的多能视网膜祖细胞 (RPC) 池生成。最近发现了受命运限制的 RPC，与多能 RPC 相比，其有丝分裂潜力和细胞命运可能性有限。一组受命运限制的 RPC，以调节元件 ThrbCRM1 的活性为标志，产生视锥细胞和水平细胞。这些细胞不表达 Vsx2，但共表达与 ThrbCRM1 结合的转录因子 (TF) Onecut1 和 Otx2。控制从多能向受命运限制的基因表达转变的基因调控网络的组成部分尚不清楚。这项工作旨在识别和评估接近 Onecut1 的顺式调控元件，以确定参与 RPC 命运限制的基因调控网络。我们通过 ATAC-seq 和保守性确定了调控元件，然后是报告基因检测，以根据时间和空间标准筛选活性。对感兴趣的调控元件进行缺失和突变分析，以识别功能序列并通过定量流式细胞术检测进行评估。最后，我们将增强子::报告基因检测与候选 TF 过表达相结合，以评估 TF、增强子和早期脊椎动物视网膜发育之间的关系。统计检验包括方差分析、Kruskal-Wallis 或非配对 t 检验。两个监管元素，ECR9 和 ECR65，被确定为活跃在 ThrbCRM1(+) 限制 RPCs。候选 bHLH 结合位点被鉴定为两个元件中的关键序列。候选 bHLH TF 的过度表达揭示了特定的增强子-bHLH 相互作用。Nhlh1 过表达将 ECR65 活性扩展到 Vsx2(+) RPC 人群中，NeuroD1/NeuroG2/NeuroD4 的过表达对 ECR9 有类似的影响。此外，能够激活异位 ECR9 报告基因的 bHLH 能够诱导内源性 Otx2 表达。这项工作报告了一个大型屏幕，用于识别 Onecut1 基因座附近的时空特定调控元件。这些元素用于识别发育中的视网膜中的不同种群。此外，命运限制的调控元件对 bHLH 因子的反应不同，