Transcriptional Regulation of Cone Photoreceptor Genesis

视锥光感受器起源的转录调控

• 批准号: 10219258
• 负责人: MARK M EMERSON
• 金额: $ 38.07万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10219258
• 关键词: Age related macular degeneration; Benchmarking; Binding; Biological Assay; Blindness; CRISPR/Cas technology; Cell Therapy; Cells; Chick; Chickens; Cone; Development; Disease; Elements; Embryo; Event; Flow Cytometry; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Genome; Goals; Human; Knowledge; LIM Domain Protein; Mediating; Methods; Mission; Molecular; Muller' s cell; Mus; Mutagenesis; Mutation Analysis; National Eye Institute; Natural regeneration; Nucleic Acid Regulatory Sequences; Organism; Output; Phenotype; Photoreceptors; Play; Population; Production; Proteins; Protocols documentation; RXRG gene; Regulator Genes; Regulatory Element; Reporter; Repression; Resolution; Retina; Retinal Cone; Retinal Photoreceptors; Retinitis Pigmentosa; Rod; Role; Site; Source; Specificity; Testing; Transcriptional Regulation; Vertebrate Photoreceptors; Vision; Visual; Visual impairment; base; cell replacement therapy; design; human disease; in vivo; innovation; insight; loss of function; loss of function mutation; postnatal; prevent; programs; retinal rods; single-cell RNA sequencing; stem cells; therapy development; transcription factor; transcriptome; transcriptomics; translational impact

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.

项目概要/摘要 视锥细胞是介导高敏锐度和日光视觉的主要感光细胞。作为 因此，它们在人类疾病过程中的丧失会极大地损害视觉功能。基于细胞的疗法 已经提出了改善这些条件的建议，其中引入新的锥体可以替代丢失的锥体 到疾病。这些细胞可以从干细胞等来源外源产生，也可以从体内产生 体内群体，例如穆勒胶质细胞。对于这两种策略，都需要开发新方法来专门 促进视锥细胞的形成，并评估从头形成的视锥细胞是否与那些从头形成的视锥细胞在分子上等效 正常发育过程中形成。然而，目前对基因调控的认识存在显着差距。 促进视锥细胞发育的保守网络。该项目的长期目标是确定 活跃在锥体发生中的基因调控网络，并根据这些知识设计方法来生成 培养新的视锥细胞用于细胞替代疗法。最近，转录因子在 早期发育的视锥细胞已被识别，但这些因素的遗传要求或它们如何相互作用 相互推动锥体命运未知。该提案的中心假设是这些 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 具有明确的监管相互作用。进行这些研究的基本原理是阐明正常的 视锥细胞光感受器形成的遗传机制将指导从茎中定向形成视锥细胞 细胞或内源性视网膜细胞群，例如米勒神经胶质细胞。鉴定具有im-的蛋白质 视锥细胞中重要的调节功能将允许开发新的视锥细胞诱导协议 更有效地生产功能性锥体。为实现这一目标，要实现以下三个目标： 提出：1）早期锥体富集转录因子的功能分析，2）关键表达的鉴定 Sall1 的功能参数和功能参数，Sall1 是一种参与视锥细胞诱导和视杆细胞抑制的转录因子，以及 3) 鉴定抑制视锥细胞中 Nrl 表达的 Nrl 顺式调控元件。第一个目标将使用高效 科学的基因编辑和单细胞转录组学方法来确定新鉴定的转录的必要性 早期视锥细胞中表达的转录因子。第二个目标是研究 Sall1 的内源性作用 锥体形成以及该基因的表达足以诱导锥体转录的程度 程序。第三个目标是确定阻止杆特异性转录的转录机制 因子 Nrl 以锥体表示。这种方法是创新的，因为它将调查早期模式 使用高分辨率和稳健的方法研究锥体形成中的分子事件。完成拟议项目 将产生对促进视锥细胞形成的基因调控网络的重要见解，并将提供信息 开发合理设计且有效的方法来促进从头锥形成。