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

人类视网膜包含三种视锥细胞亚型（蓝色、红色和绿色），这使得 三色视觉。视锥细胞发育和视锥细胞维护的中断会导致色觉缺陷和 视网膜病变，但人们对人类视锥细胞亚型的机制知之甚少。视锥细胞亚型 分两步指定：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 人类胎儿视网膜，蓝色视锥细胞规格先于红色/绿色视锥细胞规格。约翰斯顿实验室先进 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。使用人类视网膜 类器官，我们之前表明甲状腺激素信号传导促进红/绿视锥细胞的命运并抑制蓝色 人类视网膜中的视锥细胞命运。 我的项目探讨了视网膜如何从本质上调节甲状腺激素信号以控制 通过脱碘酶的动态表达，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MCT8。 DIO3 是一种失活酶，可降解 T3 和 T4，DIO2 是一种活化酶 酶，将非活性甲状腺素 (T4) 转化为活性三碘甲状腺原氨酸 (T3)。在类器官中使用 RNA 测序， 我们发现 DIO3（失活）在蓝锥体规范化的早期、之前和期间高度表达， 而 DIO2（激活）则在红/绿视锥细胞规范出现期间较晚表达。我的初步 数据表明，DIO3 在视网膜前体细胞（RPC）早期表达，DIO2 在终末表达。 在发育后期分化视锥细胞，MCT8（转运蛋白）在视网膜类器官中广泛表达 整个开发过程中。 我的数据提出以下假设：RPC 中的早期 DIO3 表达会抑制甲状腺激素 发信号。随着视网膜发育的进展，RPC 分化逐渐降低 DIO3 水平。二氧化钛 蓝色视锥细胞的表达会增加局部甲状腺激素信号传导，直到达到阈值水平 红/绿锥体规格开始制定。红/绿视锥细胞也表达 DIO2 以增强高甲状腺激素 信号和红/绿锥体选择。我预测MCT8（转运蛋白）对于甲状腺激素至关重要 信号传导但不发挥动态调节作用。我将使用 IHC 和 RNA FISH 检验这个假设 确定每种甲状腺激素调节剂的时间和细胞类型特异性表达（目标 1），然后 通过功能损失和功能增益实验确定每个锥体亚型规格的功能 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 并 用于功能丧失实验的药理学抑制剂和过度表达甲状腺激素调节剂 使用病毒构建体的时间或细胞类型特异性。该项目将阐明时间机制如何 视锥细胞亚型命运选择在人类视网膜发育过程中受到调节，广泛促进 我们对基因调控和视网膜疾病机制的一般了解。

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.