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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

基本信息

批准号：
10687129
负责人：
Christina Lynne McNerney
金额：
$ 4.77万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
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/10687129
关键词：
Address Adult 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 Iodide Peroxidase Knock-out Lead Light Macular degeneration Maintenance Mitotic Opsin Organoids Proteins RNA Regulation Retina Retinal Cone Retinal Diseases Retinitis Pigmentosa Role Science Signal Transduction Specific qualifier value Specificity Technology Testing Thyroid Gland Thyroid Hormone Receptor Beta Thyroid Hormones Thyroxine Time Triiodothyronine Viral Vision cell type experimental study fetal gain of function gene function hormonal signals hormone regulation inhibitor loss of function model organism mosaic mutant overexpression pharmacologic precursor cell promoter spatiotemporal stem cells transcriptome sequencing

项目摘要

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类型的视锥光感受器（蓝色、红色和绿色）