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

点击翻译按钮获取中文摘要

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.