Patterning Genes in Retinal Development

视网膜发育中的模式基因--项目总结 这笔赠款的长期目标是确定导致 脊椎动物视网膜中特定细胞类型的产生。在这次更新中，我们关注的是监管机制 视锥细胞视色素基因在双链复制基因阵列上的差异表达。在人类身上 X染色体上的单链复制阵列由一个长波敏感(LWS)基因组成 其次是1-9个中等波长敏感(MWS)基因，它们在光谱上与LWS有差异。这 最近的复制为大多数人类提供了三色视觉，因为LWS、MWS和SWS1 Opsins在不同的锥体种群中唯一表达。LWS/MWS阵列中的可遗传缺陷会导致 各种形式的色盲、X连锁视网膜变性和博恩霍尔姆眼病，一种锥体 与高度近视相关的功能障碍。LWS与MWS差异表达调控的研究进展 Opsins可以通过对基因表达的治疗性操作来治疗这些疾病。此外，未来 治疗其他涉及视锥细胞丢失的视网膜疾病的再生方法(与年龄有关 黄斑变性；Stargardt病)将理想地包括类似的体外或体内操作 以支持高锐度色觉的比率生成视锥表型。 被广泛接受的人类LWS与MWSopin调节模型指出，随机事件 支持上游监管区域与LW或最近端MW的关联。然而， LWS：MWs比率的地形模式表明，一种非随机的、跨调控机制可能是 牵涉其中。对调节机制的追求一直具有挑战性，因为在哺乳动物中，只有灵长类 基因组包含串联视蛋白阵列。相比之下，硬骨鱼的基因组，包括斑马鱼，含有 大量的Opsins串联阵列，它们是独立的基因复制事件和 新功能化。在我们已发表的初步数据中，我们证明了在斑马鱼中， 发育信号分子维甲酸(RA)和甲状腺激素(T3)可以各自控制差异 串联副本的表达，lws1与lws2，与人LWS/MWS阵列同源的阵列。 此外，令人信服的初步数据表明，RA可以促进人类LWS视蛋白的表达 IPSC衍生的3D视网膜有机体，改变LWS：MWs的比例。总之，这些发现奠定了 为理解LWS与MWS锥体亚型的确定方面的巨大突破奠定了基础。 在这一更新中，我们探讨了RA和T3控制差异表达的机制。 复制视蛋白，并将这一知识应用于视网膜再生和人类视网膜器官 发展，有三个特定的目标：1.确定RA和T3信号的相对作用和它们的 受体作为正常复制视蛋白基因差异表达的内源调节因子。2. 确定RA和T3信号调节随机复制的视蛋白基因的机制。3. 确定RA和T3信号在视网膜再生和人类中控制视锥细胞命运的作用 IPSC衍生的3D视网膜有机体。这些研究将揭示差异表达的新机制 并将产生操纵视锥细胞表型所需的信息 命运与人类视网膜再生或细胞替代疗法的应用相一致 堕落的人。

Patterning Genes in Retinal Development – Project Summary The long-range goal of this grant is to determine the cellular and molecular events that lead to the generation of specific cell types in the vertebrate retina. In this renewal we focus upon mechanisms regulating differential expression of the cone visual pigment genes on tandemly-replicated gene arrays. In humans a tandemly-replicated array on the X chromosome consists of one long wavelength-sensitive (LWS) gene followed by 1-9 medium wavelength-sensitive (MWS) genes, which have diverged spectrally from LWS. This recent replication has provided most humans with trichromatic color vision, because LWS, MWS, and SWS1 opsins are uniquely expressed in separate cone populations. Heritable defects in the LWS/MWS array result in various forms of color blindness, X-linked retinal degenerations, and Bornholm Eye Disease, a cone dysfunction associated with high myopia. Insights into regulation of differential expression of LWS vs. MWS opsins could allow therapeutic manipulation of gene expression to treat these disorders. In addition, future regenerative approaches to the treatment of other retinal disorders that involve the loss of cones (age-related macular degeneration; Stargardt’s disease) would ideally include similar in vitro or in vivo manipulations to generate cone phenotypes in ratios that support high-acuity color vision. The widely-accepted model for human LWS vs. MWS opsin regulation states that a stochastic event favors an association of an upstream regulatory region with the LWS or most proximal MWS. However, topographic patterns of the LWS:MWS ratio suggest that a nonrandom, trans regulatory mechanism may be involved. Pursuit of regulatory mechanisms has been challenging because within mammals, only primate genomes contain tandem opsin arrays. In contrast, the genomes of teleost fish, including zebrafish, contain numerous tandem arrays of opsins, which are the consequences of independent gene replication events and neofunctionalization. In our published and preliminary data we demonstrate that in zebrafish, the developmental signaling molecules retinoic acid (RA), and thyroid hormone (T3) can each control differential expression of the tandem duplicates, LWS1 vs. LWS2, an array orthologous to the human LWS/MWS array. Furthermore, compelling preliminary data suggest that RA can promote expression of LWS opsin in human iPSC-derived 3D retinal organoids, changing the ratio of LWS:MWS. Together these findings lay the groundwork for a tremendous breakthrough in understanding determination of LWS vs. MWS cone subtype. In this renewal we pursue mechanisms through which RA and T3 control differential expression of tandemly replicated opsins, and apply this knowledge to retinal regeneration and human retinal organoid development, with three Specific Aims: 1. Determine the relative roles of RA and T3 signaling and their receptors as endogenous regulators of differential expression of tandemly replicated opsin genes. 2. Determine mechanisms through which RA and T3 signaling regulate tandemly replicated opsin genes. 3. Determine roles of RA and T3 signaling for controlling cone fates during retinal regeneration and in human iPSC-derived 3D retinal organoids. These studies will uncover novel mechanisms for the differential expression of tandemly replicated opsin genes, and will generate information necessary to manipulate cone phenotypic fates in concert with the application of regenerative or cell replacement therapies for human retinal degenerations.