Transcriptional Regulation of Cone Photoreceptor Genesis

项目摘要/摘要 视锥感光细胞是调节高敏锐度和日光视觉的主要感光细胞。AS 因此，它们在人类疾病过程中的丢失极大地损害了视觉功能。基于细胞的治疗方法 已经提出了改善这些条件的建议，其中引入新的圆锥体可以取代那些丢失的圆锥体 为了疾病。这些细胞可以从干细胞等来源外源产生，也可以从居留的 活体种群，如Müler glia。对于这两种战略，都有必要开发新的方法来具体 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 在正常发育过程中形成的。然而，目前对基因调控的认识存在着显著的差距。 促进视锥感光细胞发育的保守党网络。该项目的长期目标是确定 活跃在球果发生中的基因调控网络，并在此基础上设计方法来产生球果发生。 培养新的锥体细胞用于细胞替代治疗。近年来，丰富表达的转录因子在 已经确定了早期发育的锥体，但这些因素的遗传要求或它们如何相互作用 用彼此推动的锥体命运未知数。这一提议的中心假设是，这些 转录因子是视锥感光细胞发生所必需的，是基因调控网络的一部分。 具有明确的监管相互作用。进行这些研究基本原理是阐明正常情况 球果感光细胞形成的遗传机制将促进茎中球果的定向形成 细胞或内源性视网膜细胞群，如穆勒胶质细胞。具有免疫球蛋白的蛋白质的鉴定 锥体的重要调节功能将允许开发新的锥体诱导方案，具有更大的 更高效地生产功能锥体。为了达到这一目标，以下三个目标是有利的： 提出：1)早期锥体富集型转录因子的功能分析；2)关键表达基因的鉴定 以及参与锥体诱导和杆状抑制的转录因子SALL1的功能参数和3) 鉴定抑制视锥细胞中NRL表达的NRL顺式调控元件。第一个目标将使用效果- 有效的基因编辑和单细胞转录方法确定新发现的转基因的必要性 转录因子在早期锥体中表达。第二个目的是研究SALL1的内源性作用 以及该基因的表达足以诱导锥体转录的程度 程序。第三个目标将确定阻止杆状特异转录的转录机制。 因子NRL不在锥体中表达。这种方法是创新的，因为它将调查早期的运动 用高分辨率和稳健性方法观测锥体形成中的耀斑事件。拟建项目竣工 将导致对促进锥体形成的基因调控网络的重大洞察，并将 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.