Spatiotemporal regulation of thyroid hormone signaling in cone subtype specification in human retinal organoids

人视网膜类器官视锥亚型规范中甲状腺激素信号传导的时空调节

• 批准号: 10474346
• 负责人: Christina Lynne McNerney
• 金额: $ 4.68万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10474346
• 关键词: Address; Adult; Animal Model; Carrier Proteins; Cell Differentiation process; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Color Visions; Color blindness; Color vision defect; Cone; Data; Development; Enzymes; Eye Development; Failure; Gene Expression Regulation; Genes; Goals; Health; Human; Immunohistochemistry; Instruction; Iodide Peroxidase; Knock-out; Lead; Light; Macular degeneration; Maintenance; Mitotic; Mosaicism; Opsin; Organoids; Pharmacology; Proteins; RNA; Regulation; Retina; Retinal Cone; Retinal Diseases; Retinitis; Role; SW opsin; Signal Transduction; Specific qualifier value; Specificity; Technology; Testing; Thyroid Gland; Thyroid Hormone Receptor Beta; Thyroid Hormones; Thyroxine; Time; To specify; Triiodothyronine; Viral; Vision; base; cell type; experimental study; fetal; gain of function; gene function; hormonal signals; hormone regulation; inhibitor; loss of function; mutant; overexpression; precursor cell; promoter; spatiotemporal; stem cell differentiation; stem cells; transcriptome sequencing

The human retina contains three subtypes of cone photoreceptors (blue, red, and green), which enable trichromatic vision. Disruptions in cone development and cone maintenance lead to color vision defects and retinopathies, yet the mechanisms that specify cone subtypes in humans are poorly understood. Cone subtypes are specified in two steps: first, between blue or red/green cone fates, and then between green or red fates. In human fetal retinas, blue cone specification precedes red/green cone specification. The Johnston lab advanced human retinal organoid technology to study how this developmental decision occurs. Using human retinal organoids, we previously showed that thyroid hormone signaling promotes red/green cone fate and inhibits blue cone fate in the human retina. My project addresses how the retina intrinsically regulates thyroid hormone signaling to control the temporal decision between blue and red/green cone fates through dynamic expression of deiodinase enzymes and the transporter MCT8. DIO3, the inactivating enzyme, degrades both T3 and T4, and DIO2, the activating enzyme, converts inactive thyroxine (T4) into active triiodothyronine (T3). Using RNA sequencing in organoids, we found that DIO3 (inactivating) is highly expressed early, preceding and during blue cone specification, whereas DIO2 (activating) is expressed late, during the onset of red/green cone specification. My preliminary data indicate that DIO3 is expressed in retinal precursor cells (RPCs) early, DIO2 is expressed in terminally differentiating cones later in development, and MCT8 (transporter) is broadly expressed in retinal organoids throughout development. My data suggest the following hypothesis: early DIO3 expression in RPCs suppresses thyroid hormone signaling. As retinal development progresses, RPC differentiation gradually decreases DIO3 levels. DIO2 expression by blue cones increases local thyroid hormone signaling until a threshold level is reached and red/green cone specification commences. Red/green cones also express DIO2 to reinforce high thyroid hormone signaling and the red/green cone choice. I predict that MCT8 (transporter) is essential for thyroid hormone signaling but does not play a role in dynamic regulation. I will test this hypothesis by using IHC and RNA FISH to determine the temporal and cell-type-specific expression of each thyroid hormone regulator (Aim 1), and then determine the function of each in cone subtype specification through loss- and gain-of-function experiments that will assess the global, spatial and temporal roles of each regulator (Aim 2). I will utilize CRISPR and pharmacological inhibitors for loss-of-function experiments and overexpress thyroid hormone regulators with temporal or cell-type specificity using viral constructs. This project will elucidate how the temporal mechanisms behind cone cell subtype fate choice are regulated during human retinal development, broadly contributing to our general understanding of gene regulation and retinal disease mechanisms.

人类视网膜包含三种亚型的视锥光感受器（蓝色、红色和绿色），其使得能够 三色视觉视锥细胞发育和视锥细胞维持的中断导致色觉缺陷， 视网膜病变，但机制，指定锥亚型在人类知之甚少。视锥细胞亚型 在两个步骤中指定：首先，在蓝色或红色/绿色视锥命运之间，然后在绿色或红色命运之间。在 人胎儿视网膜，蓝色视锥规格先于红色/绿色视锥规格。约翰斯顿实验室 人类视网膜类器官技术来研究这种发育决定是如何发生的。使用人视网膜 在类器官中，我们先前表明甲状腺激素信号促进红/绿色视锥细胞的命运，抑制蓝色视锥细胞的命运。 视锥细胞的命运 我的项目解决了视网膜如何内在地调节甲状腺激素信号，以控制 通过脱碘酶动态表达在蓝色和红色/绿色视锥细胞命运之间的时间决定 和转运蛋白MCT 8灭活酶DIO 3降解T3和T4，活化酶DIO 2降解T3和T4。 酶，将无活性的甲状腺素（T4）转化为活性的三碘甲状腺原氨酸（T3）。利用类器官中的RNA测序， 我们发现DIO 3（失活）在蓝视锥特化的早期、之前和期间高度表达， 而DIO 2（活化）在红/绿色视锥细胞规格开始期间表达较晚。我的初步 数据表明，DIO 3在视网膜前体细胞（RPC）中早期表达，DIO 2在终末表达。 在发育后期分化视锥细胞，并且MCT 8（转运蛋白）在视网膜类器官中广泛表达 在整个发展中。 我的数据提示了以下假设：RPCs中DIO 3的早期表达抑制甲状腺激素 信号随着视网膜发育的进展，RPC分化逐渐降低DIO 3水平。Dio2 蓝色视锥细胞的表达增加了局部甲状腺激素信号传导，直到达到阈值水平， 红色/绿色圆锥规格开始。红/绿色视锥细胞也表达DIO 2以增强高甲状腺激素 信号和红/绿色锥选择。我预测MCT 8（转运蛋白）对甲状腺激素至关重要 信号，但不发挥作用的动态调节。我将通过使用IHC和RNA FISH来验证这一假设 确定每种甲状腺激素调节因子（Aim 1）的时间和细胞类型特异性表达，然后 通过功能丧失和获得实验来确定视锥细胞亚型规格中每一种的功能， 将评估每个监管机构的全球、空间和时间作用（目标2）。我将利用CRISPR， 功能丧失实验的药理学抑制剂和过表达甲状腺激素调节剂， 时间或细胞类型特异性。这个项目将阐明时间机制是如何 在人类视网膜发育过程中，视锥细胞亚型的命运选择受到调节，广泛地有助于 我们对基因调控和视网膜疾病机制的一般理解。