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

项目摘要/摘要 神经脊和外胚层胎盘是脊椎动物胚胎的独特和决定性特征，起源于 外胚层的区域称为神经板边界，它并列在神经板和神经板之间。 原肠胚的非神经性外胚层。它们构成了头面部结构的起源，以及 脑神经节，以及其他不同类型的细胞和组织。这些细胞或其衍生物的畸形 可能会对胚胎发育产生毁灭性的影响，导致包括颅面在内的病理 综合症、先天性心脏缺陷和癌症。尽管如此，人们对基因和基因的了解非常少 神经脊和外胚层胎盘细胞具体化的表观遗传机制。这样做的目的是 建议研究神经板规范过程中微小RNAs(MiRNAs)的表观遗传调控 边界细胞，以了解这种不同的祖细胞池如何变得对 获得截然不同的命运。重要的是，我们将在发展至关重要的背景下解决这一问题 维生素、叶酸和维生素C，它们是DNA甲基化和上游去甲基化的重要辅助因素 MiRNA的表达。我们将把先进的测序技术与经典的技术相结合 胚胎学，以验证维生素C和叶酸是建立适当DNA所必需的假说 MiRNA调控元件上的甲基化模式对神经板边缘细胞的命运起作用 规格。具体来说，我们将(1)在转录组范围内对miRNAs的前体进行分析 不同发育时间点的神经板和神经板边界揭示时空miRNA 基于差异规范的签名，(2)执行SCATAC-SEQ以识别差异染色质 可达性(即假定的调节元件)，并执行oxBS-seq以揭示不同的测量状态 这些元素，以及(3)决定必需DNA(去)甲基化辅助因子叶酸和 维生素C在调节命运特定期间miRNA调节元件上的甲基化状态。这项建议 旨在提供对miRNAs和相应的mRNAs靶标的第一个转录组范围的分析 规范，并首次全面评估染色质景观(即揭示了染色质的可及性 ATAC-seq和oxBS-seq揭示的5hmC/5mC)在神经板和神经板边缘组织中表达。此外， 拟议中的实验结果将有助于了解复杂机制的基础知识 调节神经板边界的命运规范。我们的发现也将对 神经管、神经脊和外胚层畸形所致出生缺陷的认识 安慰剂。拟议的项目将在领导玛丽安·布朗纳博士的指导下进行 他是发育生物学领域的专家，也是神经脊发育方面的专家。她是加州理工学院的主任 贝克曼研究所，她的实验室拥有卓越的核心设施(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。

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.