Genetic Factors Mediating Thyroid Hormone-Induced Opsin Switching in Zebrafish Cone Photoreceptors

介导斑马鱼视锥细胞中甲状腺激素诱导的视蛋白转换的遗传因素

• 批准号: 10456801
• 负责人: Ashley Alice Farre
• 金额: $ 2.6万
• 依托单位: UNIVERSITY OF IDAHO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-16 至 2023-02-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10456801
• 关键词: Adolescent; Biological; CRISPR/Cas technology; Candidate Disease Gene; Cell Extracts; Color; Color Visions; Color blindness; Communities; Cone; Data; Data Set; Defect; Degenerative Disorder; Disease; Exhibits; Fishes; Follow-Up Studies; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Goals; Hormone use; Human; Human X Chromosome; In Situ Hybridization; In Vitro; Individual; Injury; Knock-out; Knowledge; Locus Control Region; Maintenance; Mediating; Methods; Modeling; Neurons; Opsin; Organism; Pattern; Photoreceptors; Phototransduction; Population; Process; Public Health; RXR; Regenerative Medicine; Regulation; Research; Retina; Retinal Cone; Retinal Pigments; Role; Testing; Thyroid Hormone Receptor; Thyroid Hormones; Validation; Zebrafish; base; cell replacement therapy; cell type; chromatin remodeling; combinatorial; differential expression; gain of function; gene therapy; hormonal signals; insight; loss of function; member; receptor; response; single-cell RNA sequencing; synaptogenesis; tool; transcriptome; transcriptome sequencing; treatment strategy; vision science

Specific vertebrate cone opsins with distinct peak spectral sensitivities are expressed in separate cone populations, which provide differential input to downstream neurons. This information is interpreted as color. In humans, the opsins that sense red and green light (LWS/MWS) are encoded by an array of tandemly replicated genes sharing a single locus control region (L/M array). Defects in this array result in color blindness and more severe cone degenerative disorders. Zebrafish possess an orthologous tandemly replicated long wavelength- sensitive array (lws array, red-sensing). The process by which tandemly replicated opsins are differentially regulated is poorly understood, and filling this knowledge gap would allow the vision science community to develop treatments for disorders related to defects in the L/M array, as well as establish conditions for differentiating human cones in vitro for cell replacement therapies that permit high acuity color vision. The prevalent model for control of the human array predicts stochastic regulation. However, the presence of topographic gradients in LWS:MWS cone ratios in human retinas (and LWS1:LWS2 ratios in zebrafish) suggests that a trans regulatory mechanism is involved. The Stenkamp lab demonstrated that thyroid hormone (TH) regulates opsin expression in LWS cones, supporting the presence of a trans regulatory mechanism. The proposed research aims to characterize TH-mediated biological differences between the LWS cones and to investigate the genetic mechanisms underlying the switch in LWS cone opsin expression upon TH exposure in zebrafish. We will use single-cell RNA sequencing (scRNA-Seq) to obtain comprehensive transcriptional signatures of LWS cones from control and TH-treated zebrafish. We will also use previously collected bulk RNA-Seq and single-cell RNA-Seq (scRNA-Seq) data to identify genes that are differentially expressed (DE) in LWS1 vs LWS2 cones, and, after validation, we will determine whether the candidate genes are coordinately regulated by TH. Thirdly, we will use CRISPR/Cas9-mediated cell type-selective gene disruption strategies to elucidate the roles of TH receptors (TRs) and retinoid X receptors (RXRs) in TH-induced opsin switching. This project will provide insight into zebrafish LWS cone differentiation and ultimately into the mechanisms by which cones expressing one member of a tandemly duplicated opsin gene array differentiate in general.

具有不同峰值光谱敏感性的特定脊椎动物视锥蛋白表达于 分离的视锥细胞群，为下游神经元提供差分输入。这 信息被解释为颜色。在人类中，感知红色和绿色光的视蛋白 (LWS/MWS）由共享单个位点的串联复制基因阵列编码 控制区（L/M阵列）。这种排列的缺陷会导致色盲和更严重的视锥细胞 退行性疾病斑马鱼拥有一个正交串联复制的长波长- 敏感阵列（LWS阵列，红色传感）。串联复制视蛋白的过程是 差异调节是知之甚少，填补这一知识空白将允许 视觉科学界开发与L/M阵列缺陷相关的疾病的治疗方法， 以及建立体外分化人类视锥细胞的条件， 允许高敏锐度色觉的疗法。控制人体阵列的流行模型 预测随机调节。然而，LWS：MWS中地形梯度的存在 人类视网膜的视锥细胞比率（以及斑马鱼的LWS 1：LWS 2比率）表明， 涉及监管机制。斯滕坎普实验室证明，甲状腺激素（TH） 调节视蛋白在LWS视锥细胞中的表达，支持存在反式调节蛋白。 机制拟议的研究旨在表征TH介导的生物学差异 之间的LWS锥，并研究遗传机制背后的开关， 斑马鱼TH暴露后LWS视锥蛋白的表达。我们将使用单细胞RNA 测序（scRNA-Seq），以获得LWS锥细胞的综合转录特征 来自对照和TH处理的斑马鱼。我们还将使用先前收集的批量RNA-Seq， 单细胞RNA-Seq（scRNA-Seq）数据，以鉴定在哺乳动物中差异表达（DE）的基因。 LWS 1与LWS 2视锥细胞，并且，在验证之后，我们将确定候选基因是否是 由TH协调调节。第三，我们将使用CRISPR/Cas9介导的细胞类型选择性 基因破坏策略，以阐明TH受体（TRs）和维甲酸X受体的作用 （RXR）在TH诱导的视蛋白转换中的作用。该项目将提供深入了解斑马鱼LWS锥 分化，并最终进入表达一个成员的锥体的机制， 串联重复视蛋白基因阵列一般可区分。