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

人类视网膜包含三种亚型的视锥感光器(蓝色、红色和绿色)，它们使 三色视觉。视锥发育和视锥维持的中断会导致色觉缺陷和 视网膜病变，然而，人们对指定人类视锥细胞亚型的机制知之甚少。锥体亚型 分两步确定：首先，在蓝色或红色/绿色锥形命运之间，然后在绿色或红色命运之间。在……里面 人胎视网膜，蓝色锥体规格先于红色/绿色锥体规格。约翰斯顿实验室先进 人类视网膜器官技术来研究这种发育决定是如何发生的。使用人类视网膜 有机化合物，我们之前表明，甲状腺激素信号促进红色/绿色锥体命运，抑制蓝色 人类视网膜中的锥体命运。 我的项目解决了视网膜如何内在地调节甲状腺激素信号来控制 通过动态表达脱碘酶决定蓝色和红色/绿色锥体命运的时间 和运输机MCT8。DIO3，失活酶，降解T3和T4，以及DIO2，激活 酶，将失活的甲状腺激素(T4)转化为活性的三碘甲腺原氨酸(T3)。利用有机体中的RNA测序， 我们发现，dio3(失活)在蓝锥指定的早期、之前和期间都有高表达， 而DIO2(激活)的表达较晚，是在红色/绿色锥体规格开始的时候。我的初选 研究表明，Dio3早期在视网膜前体细胞(RPC)中表达，DIO2在末期表达 发育后期的视锥细胞分化，MCT8(转运蛋白)在视网膜器官中广泛表达 在整个发展过程中。 我的数据提出了以下假设：RPC早期表达Dio3抑制甲状腺激素 发信号。随着视网膜发育的进展，RPC分化逐渐降低dio3水平。DIO2 蓝锥体的表达增加了局部甲状腺激素的信号，直到达到阈值水平 红色/绿色锥体规格开始。红/绿锥体也表达DIO2以增强高甲状腺激素 信号和红色/绿色锥体选择。我预测MCT8(转运蛋白)对甲状腺激素是必不可少的 信令，但在动态调节中不起作用。我将使用IHC和RNA FISH来验证这一假设 确定每个甲状腺激素调节因子的时间和细胞类型特异性表达(目标1)，然后 通过功能损失率和增益率实验确定每种In锥亚型规格的功能 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和 用于功能丧失实验的药物抑制剂和过度表达甲状腺激素调节剂 使用病毒结构的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，锥体细胞亚型命运选择受到调节，这在很大程度上有助于 我们对基因调控和视网膜疾病机制的一般理解。

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.