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

项目总结/摘要 神经嵴和外胚层基板是脊椎动物胚胎的独特特征， 外胚层的区域称为神经板边界，其并列于神经板和神经板之间。 原肠胚的非神经外胚层。它们构成了颅面结构的起源， 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，导致包括颅面在内的病理学 综合征、先天性心脏缺陷和癌症。尽管如此，人们对遗传和 神经嵴和外胚层基板细胞成为特化的表观遗传机制。这个目标 本研究拟探讨microRNAs（miRNAs）在神经板特化过程中的表观遗传调控 为了了解这种不同的祖细胞库如何为边缘细胞的分化做好准备， 获得不同的命运。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必要辅助因子， miRNA的表达。我们将联合收割机先进的测序技术与经典技术相结合， 胚胎学来检验维生素C和叶酸是建立正确的DNA所必需的假设 miRNA调控元件上的甲基化模式为神经板边缘细胞的命运做准备 规范.具体来说，我们将（1）在转录组范围内对前体细胞中的miRNA进行分析， 不同发育时间点的神经板和神经板边界，以揭示时空miRNA 差异特化的基础签名，（2）进行scATAC-seq以鉴定差异染色质 可接近性（即推定的调控元件），并进行oxBS-seq以揭示 这些元素，和（3）决定了必需的DNA（去）甲基化辅因子叶酸和 维生素C在命运特异性过程中调节miRNA调控元件的甲基化状态。这项建议 试图提供第一个全转录组分析的miRNA和相应的mRNA的目标，他们 调节，并首次全面评估染色质景观（即染色质可及性揭示 通过ATAC-seq和通过oxBS-seq揭示的5 hmC/5 mC）。此外，委员会认为， 实验结果将有助于对复杂机理的基本认识 调节神经板边缘的命运特化。我们的发现也将对 了解神经管、神经嵴和外胚层畸形引起的出生缺陷 基板拟议的项目将在Marianne Bronner博士的指导下进行，他是一位领导者 发育生物学领域的专家和神经嵴发育方面的专家。她是加州理工学院 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。

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.