Transcriptional regulators of iris muscle cell development

虹膜肌细胞发育的转录调节因子

• 批准号: 10077562
• 负责人: Timothy A. Blenkinsop
• 金额: $ 20.43万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10077562
• 关键词: Adult; Age related macular degeneration; BMP4; BMP7 gene; Binding; Blindness; CRISPR/Cas technology; Cadaver; Cell Differentiation process; Cells; Cytokine Signaling; Data; Development; Disease; Event; Eye; Eye Development; Eye diseases; Fostering; Future; Gene Expression; Genes; Genetic Transcription; Genomics; Germ Layers; Goals; Grant; Health; Human; Image; Immunofluorescence Immunologic; Inflammatory; Iris; Knock-out; Knockout Mice; Knowledge; Lead; Macular degeneration; Membrane; Mesoderm; Mission; Modeling; Muscle; Muscle Cells; Muscle Development; Mutation; Myoblasts; Neural Crest; Neural Tube Development; Neural tube; Neuroectoderm; Neuroglia; Organoids; Outcome; Pathologic; Patients; Pericytes; Phenotype; Play; Population; Prevention; Primary Open Angle Glaucoma; Process; Proliferative Vitreoretinopathy; Protocols documentation; Public Health; RNA; Research; Research Proposals; Retina; Retinal Detachment; Risk; Role; Signal Pathway; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Source; Specific qualifier value; Structure; Structure of retinal pigment epithelium; System; TNF gene; Techniques; Testing; Time; Tissues; Transforming Growth Factor beta; United States National Institutes of Health; Visual Fields; Western Blotting; Work; Zebrafish; cell fate specification; cytokine; differentiation protocol; experimental study; gene induction; genetic signature; improved; inhibitor/antagonist; innovation; insight; knock-down; novel; olfactomedin; optic cup; overexpression; prevent; programs; proliferative diabetic retinopathy; public health relevance; single-cell RNA sequencing; therapy development; transcriptome sequencing

Project Summary Iris muscle is a rare example of a neural tube derived muscle and surprisingly little is known how this remarkable break in germ layer rules is carried out. While we know PAX6 expression plays a role in iris muscle specification and that ACTA2 is present, how iris muscle cells are specified at the optic cup tip is not known. The long-term goal is to understand the signaling events that activate the muscle fate program from the developing optic cup. The sequence of fate decisions that enable this rare example of muscle differentiation from developing neural tube is the process this proposal intends to examine and the objective of this proposal. We predict many of the candidates necessary for smooth muscle cell differentiation will be involved, but there will be distinct processes necessary for iris muscle to develop that is unique, due to their specification from the neural tube instead of neural crest or mesoderm. The retina when stimulated with inflammatory cytokines differentiates towards muscle cell fate, albeit pathologically in diseases such as Proliferative Vitreoretinopathy (PVR), Proliferative Diabetic Retinopathy, Age-related Macular Degeneration Macular Pucker and more. Adult human RPE (ahRPE), retinal glia and retinal pericytes express contraction apparatus, leading to the development of myocontractile membranes, which upon contraction, cause retinal detachment and vision loss. The central hypothesis is the ability of the retina to transdifferentiate into a muscle phenotype originates from having a shared developmental origin with the iris muscle. The rational underlying this proposal is: iris muscle development is not well known and understanding iris muscle development may provide insight into how the retina develops into contractile membranes in eye diseases. The central hypothesis will be tested by pursuing these Specific Aims: 1) To test whether muscle associated genes OLFM2 and MYOCD identified in patient dissected contractile membranes are necessary for iris muscle differentiation in an eye organoid model. 2) To compare the adult human iris muscle gene signature with expression of human eye organoids by single cell RNA-seq. 3) To confirm role of OLFM2 and MYOCD by evaluating existing KO mice. The iris muscle differentiates at the tip of the optic cup, a continuous bilayer with RPE, developed from the neural tube. We will pursue these aims using an innovative combination of analytical and experimental techniques. These includes using our previously established protocol to isolated RNA from human adult cadaver tissue with sufficient survival and yield to enable single cell RNA-seq. Moreover, we have developed an eye organoid differentiation protocol that generates presumptive iris muscle cells, a first. The research proposal is significant, because the results will begin to describe factors participating iris muscle development and we will gain insight into contractile membrane formation in eye disease. The expected outcome of this work is a more complete understanding of neuroectoderm derived muscle specification and contractile membrane formation. The results will have a positive impact as the new insight gained will point to new targets for the prevention of vision loss.

项目摘要 虹膜肌肉是神经管衍生肌肉的一个罕见的例子，令人惊讶的是，很少有人知道这是如何发生的。 显着打破了胚层规则。虽然我们知道PAX 6表达在虹膜肌肉中起作用， 说明和ACTA 2存在，虹膜肌细胞如何在视杯尖端被指定尚不清楚。 长期目标是了解激活肌肉命运程序的信号事件， 开发光学杯。这种命运决定的顺序使得这种罕见的肌肉分化的例子 从发展神经管的过程中，这是本建议打算审查和本建议的目标。 我们预测许多平滑肌细胞分化所需的候选细胞将参与其中，但 将是虹膜肌肉发展所必需的独特过程，这是独一无二的，由于它们的规格来自 神经管而不是神经嵴或中胚层。当受到炎性细胞因子刺激时， 尽管在病理学上在疾病如炎症性玻璃体视网膜病变中 (PVR)糖尿病性视网膜病变、视网膜病变相关性黄斑变性、黄斑皱褶等。成人 人RPE（ahRPE）、视网膜神经胶质细胞和视网膜周细胞表达收缩装置，导致视网膜细胞收缩。 肌收缩膜的发展，其在收缩时引起视网膜脱离和视力丧失。 核心假设是视网膜转分化成肌肉表型的能力来源于视网膜的细胞分化。 与虹膜肌有共同的发育起源。这一建议背后的理性是：虹膜肌肉 虹膜的发育并不为人所知，了解虹膜肌肉的发育可能会提供对虹膜如何发育的深入了解。 视网膜在眼病中发育成可收缩的膜。中心假设将通过以下方式进行检验： 这些具体目的：1）检测患者中是否存在肌肉相关基因OLFM 2和MYOCD， 解剖的收缩膜对于眼类器官模型中的虹膜肌分化是必需的。2)到 通过单细胞比较成人虹膜肌基因签名与人眼类器官表达 RNA测序3)通过评估现有KO小鼠确认OLFM 2和MYOCD的作用。虹膜肌肉 在视杯的尖端处区分，具有RPE的连续双层，从神经管发育而来。我们将 利用分析和实验技术的创新组合来实现这些目标。这些包括 使用我们先前建立的方案，从人成人尸体组织中分离RNA， 存活率和产量以实现单细胞RNA-seq。此外，我们已经开发了一种眼类器官分化 第一个产生假定虹膜肌肉细胞的协议。这项研究计划意义重大，因为 结果将开始描述参与虹膜肌肉发育的因素， 眼部疾病中的收缩膜形成。这项工作的预期成果是一个更完整的 了解神经外胚层衍生的肌肉特化和收缩膜形成。结果 将产生积极的影响，因为获得的新见解将为预防视力丧失指出新的目标。

项目成果

SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium.

• DOI: 10.1016/j.stem.2021.04.028
• 发表时间: 2021-07-01
• 期刊: Cell stem cell
• 影响因子: 23.9
• 作者: Eriksen AZ;Møller R;Makovoz B;Uhl SA;tenOever BR;Blenkinsop TA
• 通讯作者: Blenkinsop TA

Protocols for SARS-CoV-2 infection in primary ocular cells and eye organoids.

• DOI: 10.1016/j.xpro.2022.101383
• 发表时间: 2022-06-17
• 期刊: STAR PROTOCOLS
• 作者: Eriksen, Anne Zebitz;Moller, Rasmus;Makovoz, Bar;tenOever, Benjamin R.;Blenkinsop, Timothy A.
• 通讯作者: Blenkinsop, Timothy A.

Multi-species single-cell transcriptomic analysis of ocular compartment regulons.

• DOI: 10.1038/s41467-021-25968-8
• 发表时间: 2021-09-28
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Gautam P;Hamashima K;Chen Y;Zeng Y;Makovoz B;Parikh BH;Lee HY;Lau KA;Su X;Wong RCB;Chan WK;Li H;Blenkinsop TA;Loh YH
• 通讯作者: Loh YH