Department of Biology, The City College of New York, City University of New York, New York, NY, 10031, USA.
Current Address: The Public Health Laboratory, NYC Department of Health and Mental Hygeine, New York, NY, 10016, USA.
Neural Dev. 2018 Nov 22;13(1):26. doi: 10.1186/s13064-018-0121-x.
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.
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.
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.
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.
视锥细胞和视杆细胞是人类视网膜疾病中主要受影响的两种细胞类型。潜在的治疗策略是使用基因治疗来挽救受损的感光细胞，或产生新的功能性感光细胞来替代病变视网膜中丧失的感光细胞。视锥细胞、视杆细胞或这两种感光细胞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。本研究的目的是鉴定和描述在发育中的感光细胞中具有活性的顺式调控元件的细胞类型特异性和活性。
通过电穿孔将顺式调控元件引入发育中的鸡和鼠视网膜。使用共聚焦显微镜确定报告基因活性与细胞类型标志物的关系。此外，还开发了两种高通量流式细胞术检测方法，以评估这些元件是否在感光细胞特化网络中位于 Onecut1 的下游。
大多数顺式调控元件在视锥细胞和视杆细胞中均具有活性，并且受 Onecut1 显性负性构建体的影响较小。与 Thrb、Nr2e3 和 Rhodopsin 基因相关的元件在视锥细胞或视杆细胞中表现出高度富集的活性，并且受到 Onecut1 信号转导干扰的影响。Rhodopsin 启动子活性受 Onecut1 活性的影响最大，其诱导可被 Maf 家族转录因子 L-Maf 调节。观察到 Nr2e3 元件在视锥细胞中有活性，并且在发育中的视锥细胞中表达 Nr2e3 蛋白，这表明该主要的视杆基因在视锥细胞发育中起作用。
这里呈现的分析为在发育过程中确定特定遗传网络内感光元件的特异性和强度提供了一个实验框架。Onecut1 转录因子是影响视锥细胞和视杆细胞特有的基因调控网络的一个因素，但不是影响两者共有的基因调控网络的因素。
