BACKGROUND Cone and rod photoreceptors are two of the primary cell types affected in human retinal disease. Potential strategies to combat these diseases are the use of gene therapy to rescue compromised photoreceptors or to generate new functional photoreceptors to replace those lost in the diseased retina. Cis-regulatory elements specific to cones, rods, or both types of photoreceptors are critical components of successful implementation of these two strategies. The purpose of this study was to identify and characterize the cell type specificity and activity of cis-regulatory elements active in developing photoreceptors. METHODS Cis-regulatory elements were introduced into the developing chicken and mouse retina by electroporation. Characterization of reporter activity in relation with cell type markers was determined using confocal microscopy. In addition, two high-throughput flow cytometry assay were developed to assess whether these elements were downstream of Onecut1 in the photoreceptor specification network. RESULTS The majority of cis-regulatory elements were active in both cone and rod photoreceptors and were largely uninfluenced by a Onecut1 dominant-negative construct. Elements associated with the Thrb, Nr2e3, and Rhodopsin genes showed highly enriched activity in cones or rods, and were affected by interference in Onecut1 signaling. Rhodopsin promoter activity was the most highly influenced by Onecut1 activity and its induction could be modulated by the Maf family transcription factor L-Maf. Nr2e3 elements were observed to have activity in cone photoreceptors and Nr2e3 protein was expressed in developing cone photoreceptors, suggesting a role for this predominant rod gene in cone photoreceptor development. CONCLUSIONS The analysis presented here provides an experimental framework to determine the specificity and strength of photoreceptor elements within specific genetic networks during development. The Onecut1 transcription factor is one such factor that influences the gene regulatory networks specific to cones and rods, but not those that are common to both.

中文翻译：

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早期感光体顺式调控元件的鉴定和表征及其与Onecut1的关系。

背景技术锥体和棒状感光体是在人类视网膜疾病中受影响的两种主要细胞类型。对抗这些疾病的潜在策略是使用基因疗法来挽救受损的感光细胞或产生新的功能性感光细胞，以替代在患病视网膜中丢失的感光细胞。视锥细胞，视杆或这两种类型的感光体特有的顺式调控元件是成功实施这两种策略的关键组成部分。这项研究的目的是鉴定和表征在发育中的感光细胞中活跃的顺式调节元件的细胞类型特异性和活性。方法通过电穿孔将顺式调控元件引入发育中的鸡和小鼠视网膜。使用共聚焦显微镜确定与细胞类型标记有关的报道分子活性的特征。此外，开发了两种高通量流式细胞术测定方法，以评估这些元件是否在感光器规格网络中的Onecut1下游。结果大多数顺式调节元件在视锥和视杆感光器中均具有活性，并且不受Onecut1显性负性构建体的影响。与Thrb，Nr2e3和视紫红质基因相关的元素在视锥或视杆中显示出高度丰富的活性，并受到Onecut1信号传导的干扰的影响。视紫红质启动子的活性受Onecut1活性影响最大，其诱导可以受到Maf家族转录因子L-Maf的调节。观察到Nr2e3元素在视锥细胞感光细胞中具有活性，并且Nr2e3蛋白在发育中的视锥细胞感光细胞中表达，提示此主要杆基因在视锥细胞感光细胞发育中的作用。结论本文介绍的分析提供了一个实验框架，可确定发育过程中特定基因网络中感光元件的特异性和强度。Onecut1转录因子就是这样一种因子，它影响视锥和棒特异的基因调控网络，但不影响两者共同的基因。