Transcriptional regulators of iris muscle cell development

虹膜肌细胞发育的转录调节因子

• 批准号: 9895360
• 负责人: Timothy A. Blenkinsop
• 金额: $ 25.3万
• 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 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895360
• 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; 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 虹膜肌肉是一种罕见的神经管来源的肌肉，令人惊讶的是，人们对这种情况知之甚少。 对胚层规则进行了显著的打破。虽然我们知道PAX6在虹膜肌肉中的表达 规范和ACTA2的存在，虹膜肌肉细胞如何在视杯尖端被指定是未知的。 长期目标是了解激活肌肉命运程序的信号事件 显影视杯。一系列的命运决定使这个罕见的肌肉分化例子成为可能 从神经管的发展是本提案拟考察的过程，也是本提案的目的。 我们预测将涉及许多平滑肌细胞分化所必需的候选细胞，但 将是虹膜肌肉发育所必需的不同过程，这是独一无二的，因为它们的规格来自 神经管而不是神经脊或中胚层。在炎性细胞因子刺激下的视网膜 分化为肌肉细胞的命运，尽管在增殖性玻璃体视网膜病变等疾病中是病理的 增殖性糖尿病视网膜病变、老年性黄斑变性、黄斑皱褶等。成虫 人视网膜色素上皮(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 肌肉收缩膜的形成，一旦收缩，就会导致视网膜脱离和视力丧失。 中心假说是视网膜转化为肌肉表型的能力起源于 与虹膜肌肉有共同的发育起源。这一建议背后的理性是：虹膜肌肉 发育还不是很清楚，了解虹膜肌肉的发育可能会让我们洞察到 在眼科疾病中，视网膜发育成可收缩的膜。核心假说将通过追查来检验 这些特定的目的：1)测试肌肉相关基因OLFM2和MYOCD是否在患者中被识别 在眼器官模型中，解剖的收缩膜是虹膜肌肉分化所必需的。2)至 成人虹膜肌肉基因特征与人眼类器官单细胞表达的比较 RNA序列3)通过对现有的KO小鼠进行评估，确定OLFM2和MYOCD的作用。虹膜肌肉 分化于视杯顶端，是从神经管发育而来的具有RPE的连续双层。我们会 使用分析和实验技术的创新组合来实现这些目标。其中包括 使用我们先前建立的方法从成人身体组织中提取RNA 单细胞RNA序列的存活率和产量。此外，我们还发现了眼器官的分化 产生假定虹膜肌肉细胞的协议，这是第一次。这项研究提案意义重大，因为 结果将开始描述参与虹膜肌肉发育的因素，我们将深入了解 眼科疾病中的收缩膜形成。这项工作的预期成果是一个更加完整的 了解神经外胚层衍生的肌肉规格和收缩膜的形成。结果是 将产生积极影响，因为获得的新见解将指明预防视力丧失的新目标。