Specification of green and red cone cells in the human eye

人眼通过视网膜中的视锥感光细胞来检测颜色。人类的色觉是 由光检测视蛋白(L-视蛋白/红，M-视蛋白/红)表达所定义的三种锥体细胞使能 视蛋白/绿色和S-视蛋白/蓝色)在视网膜中随机排列。三原色视觉包括 视网膜中的红色、绿色和蓝色锥体细胞是人类、猿类和东半球猴子所独有的。突变 视锥细胞的发育和维持会导致色盲、视网膜色素变性、视锥细胞营养不良和 黄斑变性。 本项目的目标是确定控制锥体亚型规格的机制 使用一种强大的系统来区分人类视网膜类器官的人眼命运 来自干细胞。这些“盘子里的视网膜”概括了人类光感受器在基因表达方面的发展， 发育时间和形态。先前的数据表明，红色、绿色和蓝色的生成 锥体细胞通过两个步骤的决定过程发生。首先，有一个决定，要么选择蓝色，要么选择 红色/绿色的命运，然后第二次决定成为红色或绿色的锥形细胞。这个决定要么是 红色或绿色的锥体细胞出现在X染色体上的基因座位水平上，在那里有一个座位控制区 (LCR)增强子元件位于红色视蛋白和绿色视蛋白基因的上游。我们已经分析了RNA 从人类胎儿样本和人类干细胞衍生的视网膜有机体中获得的测序数据显示 绿色视蛋白的表达首先发生在发育阶段。我们假设：(1)绿色和绿色之间的选择 红锥体在人类发育过程中是暂时的，绿色锥体细胞先于红锥体细胞生成 (目标1和2)，(2)人类视网膜中的视锥细胞分布图将显示更多的绿色视锥细胞 早期出生中央视网膜和较晚出生的外周较多的红色视锥(目标1)，以及维甲酸的时间 甲状腺激素信号的水平负责红锥细胞的产生(目标2)。我们会 使用RNA原位杂交技术标记人和胎儿眼睛的绿色和红色锥体细胞，该技术 成功区分绿色和红色视觉表情，并使用计算建模分析地图 整个视网膜(目标1)。我们将确定维甲酸和甲状腺激素如何影响 绿色和红色锥体通过调节维甲酸的时间和有机体中甲状腺激素的水平， 并测试这些信号影响绿色和红色锥体细胞产生的机制(目标2)。这 该项目将在约翰·霍普金斯大学的罗伯特·J·霍普金斯大学生物系的实验室进行。 小约翰斯顿。申请者将从约翰·霍普金斯大学的合作者那里接受额外的培训。 詹姆斯·泰勒、以利亚·罗伯茨博士)和医学院(唐·扎克博士)。这个项目将阐明 锥体细胞亚型命运选择背后的时间机制在人类视网膜发育过程中受到调控， 有助于我们对基因调控和疾病机制的总体理解。

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.