Hierarchical Onset of Germ Layer Specification

胚层规范的分层开始

• 批准号: 10590736
• 负责人: Hui Chen
• 金额: $ 4.18万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-15 至 2023-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10590736
• 关键词: ATAC-seq; Amphibia; Animals; Architecture; Binding; Binding Sites; Bioinformatics; Cell Cycle; Cell Differentiation process; Cell Nucleus; Cell division; Cells; ChIP-seq; Chromatin; Data; Defect; Dependence; Development; Developmental Biology; Devices; Ectoderm; Embryo; Embryonic Development; Endoderm; Event; Gastrula; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Germ Layers; Histones; Image; Individual; Knowledge; Lysine; Mesoderm; Methodology; Methods; Modeling; Mus; Neurula; Organ; Pattern; Phase; Phenotype; Play; Process; Regulatory Element; Role; Site; Specific qualifier value; Tissues; Transcript; Transcription Initiation; Uridine; Xenopus; Xenopus laevis; blastocyst; egg; embryo stage 2; embryo tissue; epigenetic regulation; experimental study; gastrulation; histone demethylase; histone modification; insight; programs; spatiotemporal; temporal measurement; transcription factor; transcriptome; transcriptome sequencing; unpublished works; vertebrate embryos; zygote

Summary. During early development, specification of the three primordial germ layers, ectoderm, mesoderm, and endoderm, is the first major step in defining embryonic tissues, and dysregulation of this process leads to developmental defects. Germ layer formation is a fundamental focus of developmental biology; however, little is known about an emerging concept that the initiation of their expression follows a temporal hierarchy. The ordering is debated in different embryonic models and importantly it is not known whether proper coordination of timing is essential for development. The cell differentiation that accompanies germ layer formation largely occurs in gastrulation. Yet, many tissue-specific genes that specify individual germ layers initiate their expression in blastula stages following genome activation, requiring methods to track the onset of their nascent transcription. We developed a new method for quantifying the blastula nascent transcriptome using 5-ethynyl uridine (EU) incorporation and isolation, termed EU-RNA-seq. This method has increased sensitivity relative to conventional RNA-seq and enables delicate measurement of time-of-onset for maternal-zygotic transcripts. By analyzing germ layer expression in the model vertebrate Xenopus laevis, we discovered that the ectoderm genes are expressed earlier than endoderm genes, suggesting a hierarchical onset of germ layer expression that is consistent with recent observations made in mouse. However, the mechanisms underlying the hierarchical onset of germ layer expression remain largely unknown. Interestingly, the phased onset is consistent with our recent finding that large-scale zygotic transcription initiates in a spatially graded manner, first in cells at the animal pole (AP, the presumptive ectoderm) and delayed in cells at the vegetal pole (VP, the presumptive endoderm) in blastula embryos. Epigenetic regulation, including chromatin accessibility and histone modifications, have been shown to play a central role in gene regulation in early development. We hypothesize that differential epigenetic regulation may underlie these patterns of genome activation and differential timing of germ layer induction. We found a strong positive correlation between nascent transcription and transcription factor binding sites (TFBS) enrichment for pioneer factors FOXA4 and POU3F1 and a negative correlation with the histone demethylase KDM2B. To gain deeper mechanistic insights into the origin of hierarchical germ layer initiation, we aim to probe chromatin accessibility in distinct regions for the presumptive germ layers in the blastula embryos and determine binding of FOXA4 and POU3F1. Moreover, to understand the developmental consequences of dysregulated germ layer timing, we implemented an approach to spatially alter cell division timing and alter the pattern of genome activation in blastula embryos. We will characterize the timing of germ layer gene expression programs in the desynchronized embryos and determine the phenotypes of gastrula development. Uncovering the mechanisms of hierarchical germ layer expression and its functional importance will provide new insights into our understanding the first events in cell fate decisions and tissue specification in early development.

摘要在早期发育过程中，三个原始胚层，外胚层，中胚层， 和内胚层，是确定胚胎组织的第一个主要步骤，这一过程的失调导致 发育缺陷胚层形成是发育生物学的一个基本焦点;然而， 我们知道一个新兴的概念，即它们的表达的开始遵循时间层次。的 排序在不同的胚胎模型中存在争议，重要的是，目前尚不清楚是否适当协调 时机对发展至关重要。伴随着胚层形成的细胞分化主要发生在 在原肠胚形成中。然而，许多组织特异性基因，指定个别胚层启动他们的表达， 基因组激活后的囊胚阶段，需要方法来跟踪其新生转录的开始。 我们开发了一种新的方法来定量囊胚新生转录组使用5-乙炔基尿苷（EU） 将其掺入和分离，称为EU-RNA-seq。该方法相对于常规方法具有更高的灵敏度。 RNA-seq和能够精确测量母合子转录本的发病时间。通过分析细菌 层表达的模式脊椎动物非洲爪蟾，我们发现，外胚层基因表达 早于内胚层基因，表明胚层表达的分级起始，这与 最近在老鼠身上观察到。然而，胚层分层发病的机制， 表达方式仍基本未知。有趣的是，阶段性发作与我们最近的发现一致， 大规模合子转录以空间分级的方式起始，首先在动物极的细胞中（AP， 在囊胚中，在植物极的细胞中（VP，推定的内胚层）， 胚胎表观遗传调控，包括染色质可及性和组蛋白修饰，已被证明是 在早期发育的基因调控中发挥核心作用。我们假设差异表观遗传 调控可能是这些基因组激活模式和胚层诱导的不同时间的基础。我们 发现新生转录和转录因子结合位点（TFBS）之间存在很强的正相关性 FOXA 4和POU 3F 1的富集以及与组蛋白去甲基化酶的负相关 KDM2B。为了更深入地了解分层胚层启动的起源，我们的目标是探索 染色质可及性在不同的区域为推定的胚层在囊胚期胚胎，并决定 FOXA 4和POU 3F 1的结合。此外，为了了解失调的发育后果， 胚层的时间，我们实施了一种方法，在空间上改变细胞分裂的时间，并改变模式， 囊胚期胚胎的基因组激活。我们将描述胚层基因表达程序的时间 并确定原肠胚发育的表型。揭开 分层胚层表达的机制及其功能的重要性将提供新的见解， 我们对细胞命运决定和早期发育中组织特化的第一个事件的理解。