Hierarchical Onset of Germ Layer Specification

胚层规范的分层开始

基本信息

批准号：
10982692
负责人：
Hui Chen
金额：
$ 3.62万
依托单位：
UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-15 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10982692
关键词：
Hierarchical Onset Germ Layer Specification

项目摘要

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.

概括。在早期发育过程中，三个原始胚层（外胚层、中胚层、和内胚层，是定义胚胎组织的第一个主要步骤，这个过程的失调会导致发育缺陷。胚层形成是发育生物学的一个基本焦点。然而，很少有人们知道一个新兴的概念，即它们的表达的启动遵循时间层次结构。这在不同的胚胎模型中，排序存在争议，重要的是，尚不清楚是否正确协调时机对于发展至关重要。伴随着胚层形成的细胞分化大量发生原肠胚形成中。然而，许多指定单个胚层的组织特异性基因在囊胚阶段在基因组激活后，需要方法来追踪其新生转录的开始。我们开发了一种使用 5-乙炔基尿苷 (EU) 定量囊胚新生转录组的新方法掺入和分离，称为 EU-RNA-seq。该方法相对于传统方法提高了灵敏度 RNA-seq 能够精确测量母体合子转录本的起始时间。通过分析细菌在模型脊椎动物非洲爪蟾的层表达中，我们发现外胚层基因得到表达早于内胚层基因，表明胚层表达的分层起始与最近在小鼠中进行的观察。然而，胚层分层起始的机制表达仍然很大程度上未知。有趣的是，分阶段发生与我们最近的发现是一致的：大规模合子转录以空间分级的方式启动，首先在动物极的细胞中（AP，假定的外胚层）和囊胚植物极细胞（VP，假定的内胚层）的延迟胚胎。表观遗传调控，包括染色质可及性和组蛋白修饰，已被证明在早期发育的基因调控中发挥核心作用。我们假设表观遗传差异调控可能是这些基因组激活模式和胚层诱导差异时间的基础。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先驱因子 FOXA4 和 POU3F1 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起始的起源，我们的目标是探究囊胚胚胎中假定胚层不同区域的染色质可及性，并确定 FOXA4 和 POU3F1 的结合。此外，要了解失调的发育后果胚层计时，我们实施了一种在空间上改变细胞分裂计时并改变细胞分裂模式的方法囊胚胚胎中的基因组激活。我们将描述胚层基因表达程序的时序在不同步的胚胎中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重要性将为我们提供新的见解我们了解早期发育中细胞命运决定和组织规范的最初事件。