Analysis of transcriptional priming of progenitors in the vertebrate neural plate border

脊椎动物神经板边缘祖细胞的转录启动分析

• 批准号: 10452502
• 负责人: Brittany Megan Edens
• 金额: $ 6.91万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-11-01 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452502
• 关键词: ATAC-seq; Activities of Daily Living; Affect; Ascorbic Acid; Ascorbic Acid Deficiency; Binding; CRISPR/Cas technology; Cardiovascular system; Cells; Central Nervous System; Cephalic; Chick Embryo; Chromatin; Complex; Congenital Abnormality; Congenital Heart Defects; Core Facility; CpG Islands; DNA; DNA Methylation; DNA Modification Methylases; Defect; Development; Developmental Biology; Dioxygenases; Disease; Disparate; Ectoderm; Elements; Embryo; Embryology; Embryonic Development; Epidermis; Epigenetic Process; Folic Acid; Functional disorder; Ganglia; Gastrula; Gene Expression; Genetic; Genetic Transcription; Genome; Goals; Health; Human; Individual; Investigation; Knowledge; Malignant Neoplasms; Mediating; Messenger RNA; Methylation; MicroRNAs; Microscopy; Molecular; Multipotent Stem Cells; Neural Crest; Neural tube; Neuroblastoma; Pathology; Play; Process; Regulatory Element; Role; Sensory; Site; Small RNA; Specific qualifier value; Structure; Syndrome; Techniques; Technology; Territoriality; Testing; Tissues; Vitamins; cell fate specification; cell type; cofactor; congenital deafness; craniofacial; craniofacial structure; demethylation; embryonic stem cell; epigenetic regulation; experimental study; folate-binding protein; genetic approach; in vivo; insight; knock-down; mRNA Expression; malformation; melanoma; methyl group; methylation pattern; miRNA expression profiling; nerve stem cell; neural; neural plate; progenitor; promoter; single cell sequencing; spatiotemporal; transcription factor; transcriptome; transcriptome sequencing; vertebrate embryos

Project Summary/Abstract The neural crest and ectodermal placodes, unique and defining features of the vertebrate embryo, arise from the region of the ectoderm termed the neural plate border, which is juxtaposed between the neural plate and the non-neural ectoderm of the gastrula. They constitute the origins of the craniofacial structures as well as the cranial ganglia, in addition to other diverse cell and tissue types. Malformation of these cells or their derivatives can have devastating effects on embryonic development, resulting in pathologies including craniofacial syndromes, congenital heart defects, and cancers. Nonetheless, exceedingly little is known of the genetic and epigenetic mechaniasms by which neural crest and ectodermal placode cells become specified. The goal of this proposal is to investigate the epigenetic regulation of micro RNAs (miRNAs) during specification of neural plate border cells in order to understand how this diverse pool of progenitors becomes differentially primed for the acquisition of distinct fates. Importantly, we will address this question in the context of developmentally critical vitamins, folate and Vitamin C, which are essential cofactors for DNA methylation and demethylation upstream of miRNA expression. We will combine advanced sequencing technologies with classical techniques in embryology to test the hypothesis that Vitamin C and folate are required for establishing proper DNA methylation patterns on miRNA regulatory elements to prime neural plate border cells for fate specification. Specifically, we will (1) perform transcriptome-wide profiling of miRNAs in progenitors of the neural plate and neural plate border at different developmental timepoints to uncover spatiotemporal miRNA signatures underlying differential specification, (2) perform scATAC-seq to identify differential chromatin accessibilty (i.e. putative regulatory elements), and perform oxBS-seq to reveal differential mehtyation states of these elements, and (3) determine the respective roles of the essential DNA (de)methylation cofactors folate and Vitamin C in regulating methylation states on miRNA regulatory elements during fate specifcation. This proposal seeks to provide the first transcriptome-wide analysis of miRNAs and the corresponding mRNA targets that they regulate, and the first comprehensive assessment of chromatin landscape (i.e. chromatin accessibility revealed by ATAC-seq and 5hmC/5mC revealed by oxBS-seq) in neural plate and neural plate border tissues. Moreover, results from the proposed experiments will contribute fundamental knowledge of the complex mechanisms regulating fate specification at the neural plate border. Our findings will also have important implications for the understanding of birth defects arising from malformation of the neural tube, neural crest, and ectodermal placodes. The proposed project will be carried out under the guidance of Dr. Marianne Bronner who is a leader in the field of developmental biology and an expert in neural crest development. She is the director of Caltech’s Beckman Institute where her lab has access to outstanding core facilities (e.g. advanced microscopy and sequencing centers) that will be instrumental to the successful completion of the proposed study.

项目概要/摘要 神经嵴和外胚层基板是脊椎动物胚胎的独特和决定性特征，源自 外胚层区域称为神经板边界，位于神经板和神经板之间 原肠胚的非神经外胚层构成了颅面结构和颅面结构的起源。 颅神经节，以及其他不同的细胞和组织类型，这些细胞或其衍生物的畸形。 可能对胚胎发育产生破坏性影响，导致包括颅面畸形在内的病理学 然而，对于遗传和癌症的了解却非常少。 神经嵴和外胚层基板细胞被指定的表观遗传机制。 提案是研究神经板规范过程中微小 RNA (miRNA) 的表观遗传调控 细胞，以了解这个边界多样化的祖细胞库如何针对 重要的是，我们将在发展关键的背景下解决这个问题。 维生素、叶酸和维生素 C，它们是上游 DNA 甲基化和去甲基化的重要辅助因子 我们将先进的测序技术与经典技术相结合。 胚胎学检验维生素 C 和叶酸是建立正确 DNA 所必需的假设 miRNA 调控元件的甲基化模式为神经板边缘细胞的命运做好准备 具体来说，我们将 (1) 对祖细胞中的 miRNA 进行全转录组分析。 不同发育时间点的神经板和神经板边界揭示时空 miRNA 差异规范背后的特征，(2) 执行 scATAC-seq 来识别差异染色质 可访问性（即假定的调控元件），并执行 oxBS-seq 以揭示不同的甲基化状态 这些元素，以及 (3) 确定必需 DNA（去）甲基化辅因子叶酸和 维生素 C 在命运规范过程中调节 miRNA 调控元件的甲基化状态。 旨在提供第一个 miRNA 及其相应 mRNA 靶标的全转录组分析 调节，并对染色质景观进行首次全面评估（即染色质可及性揭示 通过 ATAC-seq 和 oxBS-seq 显示的 5hmC/5mC）在神经板和神经板边缘组织中。 拟议实验的结果将有助于了解复杂机制的基础知识 我们的发现也将对神经板边界的命运规范产生重要影响。 了解由神经管、神经嵴和外胚层畸形引起的出生缺陷 拟议的项目将在领导者 Marianne Bronner 博士的指导下进行。 她是发育生物学领域的专家，也是加州理工学院神经嵴发育的专家。 贝克曼研究所，她的实验室拥有出色的核心设施（例如先进的显微镜和 测序中心）这将有助于成功完成拟议的研究。