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: 10308867
• 负责人: Brittany Megan Edens
• 金额: $ 6.56万
• 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/10308867
• 关键词: ATAC-seq; Activities of Daily Living; Address; Affect; Ascorbic Acid; Ascorbic Acid Deficiency; Binding; CRISPR/Cas technology; Cardiovascular system; Cells; Cephalic; Chick Embryo; Chromatin; Complex; Congenital Abnormality; Congenital Heart Defects; Core Facility; CpG Islands; Cytosine; DNA; DNA Modification Methylases; Defect; Development; Developmental Biology; Dioxygenases; Disease; 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; Institutes; Investigation; Knowledge; Malignant Neoplasms; Mediating; Messenger RNA; Methylation; MicroRNAs; Microscopy; Molecular Genetics; Multipotent Stem Cells; Neural Crest; Neural tube; Neuraxis; Neuroblastoma; Pathology; Play; Process; Regulatory Element; Role; Sensory; Site; Small RNA; Specific qualifier value; Structure; Syndrome; Techniques; Technology; Territoriality; Testing; Tissues; Vitamins; base; 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; nerve stem cell; neural plate; progenitor; promoter; relating to nervous system; 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.

项目摘要/摘要 神经rest和外胚层位置，脊椎动物胚胎的独特和定义特征，由 外胚层的区域称为神经元板边界，该区域是在神经元板和 胃的非神经外胚层。它们构成了颅面结构的起源 颅神经节除了其他各种细胞和组织类型外。这些细胞或其衍生物的畸形 可能对胚胎发育产生毁灭性的影响，从而导致包括颅面在内的病理 综合征，先天性心脏缺陷和癌症。但是，对遗传和 表观遗传机制通过指定神经rest和外胚层斑点细胞的表观遗传机制。目标的目标 提案是研究神经板规范期间微RNA（miRNA）的表观遗传调节 边界细胞以了解这种发散的祖细胞如何变化为 收购不同的命运。重要的是，我们将在发展至关重要的背景下解决这个问题 维生素，叶酸和维生素C，它们是DNA甲基化和脱甲基化的必不可少的辅助因子 miRNA表达。我们将结合高级测序技术与经典技术 胚胎学测试维生素C和叶酸的假设是建立适当DNA所必需的 miRNA调节元件上的甲基化模式，用于脂肪的基本神经板边界细胞 具体的。具体而言，我们将（1）在祖细胞中进行miRNA的整个转录组分析 在不同的发育时间点处的神经板和神经板边界，以发现时空miRNA 差异规范的签名，（2）执行SCATAC-SEQ以识别差异染色质 访问权限（即推定的监管元素），并执行OXBS-SEQ以揭示 这些元素，（3）确定必需DNA（DE）甲基化辅助因子叶酸和 在脂肪规范期间，在miRNA调节元件的调节状态中，维生素C。这个建议 试图提供对miRNA的第一个整个转录组分析以及它们的相应mRNA靶标的 调节，以及对染色质景观的首次全面评估（即染色质可及性显示 由ATAC-SEQ和5HMC/5MC在神经板和神经板边界组织中揭示的。而且， 提出的实验的结果将有助于对复杂机制的基本知识 调节神经板边界的命运规格。我们的发现也将对 了解因神经管，神经rest和外胚层畸形引起的出生缺陷 放置。拟议的项目将在领导者玛丽安·布朗纳（Marianne Bronner）博士的指导下进行。 在发育生物学领域和神经rest发育方面的专家。她是加州理工学院的主任 贝克曼学院（Beckman Institute）她的实验室可以使用出色的核心设施（例如高级显微镜和 测序中心）将有助于成功完成拟议的研究。