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 虹膜肌肉是神经管衍生肌肉的罕见例子，令人惊讶的是，很少知道 生殖层规则的明显中断。虽然我们知道PAX6表达在虹膜肌肉中起作用 规格和ACTA2存在，尚不清楚如何在光学杯尖端指定虹膜肌肉细胞。 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。命运决策的顺序使这一罕见的肌肉分化例子 从开发神经管中，该提案打算检查和该提案的目的。 我们预测将涉及平滑肌细胞分化所需的许多候选者，但是 虹膜肌肉的发展是独一无二的，这将是独特的过程，因为它们的规格来自 神经管代替神经rest或中胚层。视网膜用炎性细胞因子刺激 尽管在诸如增生性玻璃体病变之类的疾病中，但在病理上区分了肌肉细胞命运 （PVR），增殖性糖尿病性视网膜病，与年龄相关的黄斑变性黄斑皱纹等。成人 人RPE（AHRPE），视网膜神经胶质和视网膜周细胞表达收缩设备，导致 在收缩后，肌收集膜的发展会导致视网膜脱离和视力丧失。 中心假设是视网膜转变为肌肉表型的能力源自 与虹膜肌肉具有共同的发育起源。该提议的理性基础是：虹膜肌肉 发展尚不清楚，了解虹膜肌肉的发育可能会提供有关如何如何 视网膜发展为眼部疾病的收缩膜。中心假设将通过追求来检验 这些具体目的：1）测试患者中是否识别出肌肉相关的基因OLFM2和MYOCD 解剖的收缩膜对于眼球模型中的虹膜肌肉分化是必需的。 2）到 比较成年人类虹膜肌肉基因特征与单细胞表达人眼器官的表达 RNA-seq。 3）通过评估现有的KO小鼠来确认OLFM2和MYOCD的作用。虹膜肌肉 通过神经管开发的光学杯的尖端，是带有RPE的连续双层。我们将 通过分析和实验技术的创新组合来追求这些目标。这些包括 使用我们先前建立的方案从人类成人尸体组织中分离出RNA 生存和产量以实现单细胞RNA-seq。此外，我们已经开发了眼器官分化 产生推定的虹膜肌肉细胞的协议，第一个。研究建议很重要，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 收缩膜形成眼科疾病。这项工作的预期结果更完整 了解神经外胚层衍生的肌肉规格和收缩膜形成。结果 由于获得的新见解将指出预防视力丧失的新目标，因此会产生积极的影响。