Transcriptional Regulation of Cone Photoreceptor Genesis

项目总结/摘要 视锥光感受器是介导高敏锐度和日光视觉的主要光感受器类别。作为 因此，它们在人类疾病过程中的损失极大地损害了视觉功能。基于细胞的疗法， 已经有人提出了改善这些条件的方法，其中引入新的球果可以取代那些损失的球果 疾病。这些细胞可以从诸如干细胞的来源或从体内、外或体内的细胞中外源地产生。 体内群体如Müller神经胶质。对于这两种策略，有必要开发新的方法， 促进锥体形成，并评估从头形成的锥体是否在分子上等同于那些 在正常发育过程中形成。然而，目前对基因调控的认识存在重大差距， 促进视锥光感受器发育的保守网络。该项目的长期目标是确定 在球果发生中活跃的基因调控网络，并根据这些知识设计方法， 为细胞替代疗法提供新的视锥细胞。近年来，转录因子在细胞中的富集表达， 早期发育的视锥细胞已经被确定，但这些因素的遗传要求或它们如何相互作用， 彼此促进球果的命运未知。这一建议的核心假设是， 转录因子是视锥细胞发生所必需的，并且是基因调控网络的一部分 有明确的监管互动。进行这些研究的基本原理是阐明正常的 视锥光感受器形成的遗传机制将指导视锥从茎的定向形成 细胞或内源性视网膜细胞群，如Muller神经胶质。具有免疫缺陷的蛋白质的鉴定 视锥细胞的重要调节功能将允许开发新的视锥细胞诱导方案，具有更大的和更好的效果。 更有效地产生功能性锥体。为了实现这一目标，以下三个目标是有利的， 提出：1）早期锥富集转录因子的功能分析，2）关键表达的鉴定 和Sall 1的功能参数，Sall 1是一种参与视锥细胞诱导和视杆细胞阻遏的转录因子，和3） 在视锥中抑制Nrl表达的Nrl顺式调节元件的鉴定。第一个目标将使用一个有效的- 高效的基因编辑和单细胞转录组学方法来确定新发现的转基因的必要性， 转录因子在早期球果中表达。第二个目的是研究Sall 1的内源性作用。 以及该基因的表达足以诱导视锥转录的程度 程序.第三个目标是确定阻止杆特异性转录的转录机制 因子Nrl在锥体中表达。这种方法是创新的，因为它将研究早期的 使用高分辨率和鲁棒的方法在锥体形成中的异常事件。完成拟议项目 将导致对促进锥体形成的基因调控网络的重要见解，并将为 开发合理设计和有效的方法来促进从头形成锥体。

PROJECT SUMMARY/ABSTRACT Cone photoreceptors are the primary photoreceptor class that mediates high acuity and daylight vision. As such, their loss during the course of human disease greatly impairs visual function. Cell-based therapies to ameliorate these conditions have been proposed, in which introduction of new cones could replace those lost to disease. These cells could be produced exogenously from sources such as stem cells or from resident, in vivo populations such as Müller glia. For both strategies, it is necessary to develop new methods to specifically promote cone formation, and to assess whether de novo formed cones are molecularly equivalent to those formed during normal development. However, a significant gap exists in the current knowledge of gene regula- tory networks that promote cone photoreceptor development. The long-term goal of this project is to identify the gene regulatory networks active in cone genesis, and to devise methods based on this knowledge to gen- erate new cone cells for cell replacement therapy. Recently, transcription factors with enriched expression in early developing cones have been identified, yet the genetic requirement of these factors or how they interact with one another to promote the cone fate is unknown. The central hypothesis of this proposal is that these transcription factors are required for cone photoreceptor genesis and are part of a gene regulatory network with defined regulatory interactions. The rationale to undertake these studies is that elucidation of the normal genetic mechanisms of cone photoreceptor formation will inform the directed formation of cones from stem cells or endogenous retinal cell populations such as Muller glia. Identification of the proteins that have im- portant regulatory functions in cones will allow development of new cone induction protocols with greater and more efficacious production of functional cones. To accomplish this goal, the following three aims are pro- posed: 1) A functional analysis of early cone-enriched transcription factors, 2) Identification of key expression and functional parameters of Sall1, a transcription factor involved in cone induction and rod repression, and 3) Identification of Nrl cis-regulatory elements that repress Nrl expression in cones. The first aim will use an effi- cient gene editing and single cell transcriptomic approach to determine the necessity of newly identified tran- scription factors expressed in early stage cones. The second aim will investigate the endogenous role of Sall1 in cone formation and the extent to which expression of this gene is sufficient to induce cone transcriptional programs. The third aim will identify the transcriptional mechanisms that prevent the rod-specific transcription factor Nrl from being expressed in cones. This approach is innovative because it will investigate the early mo- lecular events in cone formation using high-resolution and robust methods. Completion of the proposed project will result in significant insights into the gene regulatory networks that promote cone formation and will inform the development of rationally designed and effective methods to promote de novo cone formation.