Specification of green and red cone cells in the human eye

人眼通过视网膜中的视锥细胞检测颜色。人类的色觉是 通过由光检测视蛋白（L-视蛋白/红，M-视蛋白/红）的表达定义的三种类型的视锥细胞来实现。 视蛋白/绿色和S-视蛋白/蓝色）在视网膜中以随机图案排列。三色色觉包括 视网膜中的红色、绿色和蓝色视锥细胞是人类、猿和旧大陆猴所独有的。突变 在视锥细胞发育和维持中引起色盲、色素性视网膜炎、视锥细胞营养不良， 黄斑变性 本项目的目标是确定控制视锥细胞亚型规格的机制 使用一个强大的系统，我们已经适应了区分人类视网膜类器官在人眼中的命运 从干细胞。这些“培养皿中的视网膜”在基因表达方面再现了人类感光细胞的发育， 发育时间和形态学。以前的数据表明，红色、绿色和蓝色的产生 视锥细胞通过两步决策过程发生。第一，选择蓝色或 红/绿色命运，然后第二次决定成为红色或绿色视锥细胞。决定成为 红色或绿色视锥细胞出现在X染色体上的基因座水平，其中基因座控制区 (LCR)增强子元件位于红色视蛋白和绿色视蛋白基因的上游。我们分析了RNA 来自人胎儿样品和人干细胞衍生的视网膜类器官的测序数据，并显示， 绿色视蛋白表达首先发生在发育中。我们假设：（1）在绿色和 红视锥细胞在人类发育过程中是暂时的，绿色视锥细胞在红视锥细胞之前产生 (Aims（1和2），（2）人视网膜中的视锥细胞分布图将揭示在视网膜上更多的绿色视锥细胞。 早期出生的中央视网膜和更多的红色锥体在晚出生的周边（目的1），以及视黄酸的时间 和甲状腺激素信号水平负责红锥细胞的产生（目的2）。我们将 使用RNA原位杂交技术标记人和胎儿眼中绿色和红色视锥细胞， 成功区分绿色和红色视蛋白表达，并使用计算建模来分析图 整个视网膜（目标1）。我们将确定视黄酸和甲状腺激素如何影响 绿色和红色视锥细胞通过调节类器官中视黄酸的时间和甲状腺激素的水平， 并测试这些信号影响绿色和红色视锥细胞产生的机制（目的2）。这 该项目将在约翰霍普金斯大学生物系罗伯特J。 小约翰斯顿申请人将接受约翰霍普金斯大学合作者的额外培训。 詹姆斯泰勒，博士以利亚罗伯茨）和医学院（博士唐扎克）。本项目将阐明 视锥细胞亚型命运选择背后的时间机制在人视网膜发育期间受到调节， 有助于我们对基因调控和疾病机制的全面理解。

The human eye detects color through cone photoreceptor cells in the retina. Human color vision is enabled by three types of cone cells defined by expression of light-detecting opsin proteins (L-opsin/red, M- opsin/green, and S-opsin/blue) arranged in a random pattern in the retina. Trichromatic color vision including red, green, and blue cone cells in the retina is exclusive to humans, apes, and Old World monkeys. Mutations in cone cell development and maintenance cause color blindness, retinitis pigmentosa, cone dystrophy, and macular degeneration. The goal of this project is to determine the mechanisms controlling the specification of cone subtype fates in the human eye using a powerful system that we have adapted to differentiate human retinal organoids from stem cells. These “retinas in a dish” recapitulate human photoreceptor development in gene expression, developmental timing, and morphology. Previous data suggests that the generation of red, green, and blue cone cells occurs through a two-step decision process. First, there is a decision to choose either the blue or red/green fate, and then a second decision to become either a red or green cone cell. The decision to be either a red or green cone cell occurs at the gene locus level on the X chromosome, where a locus control region (LCR) enhancer element lies upstream of the red opsin and green opsin genes. We have analyzed RNA sequencing data from human fetal samples and human stem cell-derived retinal organoids and showed that green opsin expression occurs first in development. We hypothesize that: (1) the decision between green and red cones is temporal during human development, with green cone cells generated before red cone cells (Aims 1 and 2), (2) a map of cone cell distributions in the human retina will reveal more green cones at the early born central retina and more red cones at the late born periphery (Aim 1), and that timing of retinoic acid and levels of thyroid hormone signaling are responsible for the generation of red cone cells (Aim 2). We will label green and red cone cells in human and fetal eyes using an RNA in situ hybridization technique that successfully distinguishes green and red opsin expression, and use computational modeling to analyze a map of the whole retina (Aim 1). We will determine how retinoic acid and thyroid hormone affects specification of green and red cones by modulating the timing of retinoic acid and the levels of thyroid hormone in organoids, and test a mechanism by which these signals affect the generation of green and red cone cells (Aim 2). This project will be carried out at Johns Hopkins University in the Department of Biology in the lab of Robert J. Johnston Jr. The applicant will receive additional training from collaborators at Johns Hopkins University (Dr. James Taylor, Dr. Elijah Roberts) and Medical School (Dr. Don Zack). This project will elucidate how the temporal mechanisms behind cone cell subtype fate choice are regulated during human retinal development, contributing to our general understanding of gene regulatory and disease mechanisms.