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。在早期开发过程中，三个原始细菌层的规范，外胚层，中胚层，和内胚层是定义胚胎组织的第一步，该过程的失调导致发展缺陷。生殖层形成是发育生物学的基本重点。但是，几乎没有关于新兴概念的知道，其表达的启动遵循了时间层次结构。这订购在不同的胚胎模型中进行了辩论，重要的是，尚不清楚是否正确协调时机对于发展至关重要。伴随细菌层形成的细胞分化很大程度上发生在胃肠道中。然而，许多指定单个细菌层的组织特异性基因启动其表达基因组激活后的囊泡阶段，需要方法来跟踪其新生转录的发作。我们开发了一种使用5-乙基尿苷（EU）来量化囊泡新生转录组的新方法掺入和隔离，称为EU-RNA-SEQ。此方法相对于常规的灵敏度提高了 RNA-seq并可以精细测量母体 - 杂种转录本。通过分析细菌在模型脊椎动物Xenopus laevis中的层表达，我们发现表达了外胚层基因比内胚层基因早，表明生殖层表达的分层发作与在鼠标中进行的最新观察结果。但是，生殖层分层发作的基础机制表达在很大程度上未知。有趣的是，分阶段的发作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，首先是在动物极处的细胞（AP，推定外胚层），并延迟植物极（VP，推定性内胚层）的细胞胚胎。已经显示了表观遗传调节，包括染色质可及性和组蛋白修饰在早期发展中在基因调节中发挥核心作用。我们假设差异表观遗传学调节可能是这些基因组激活模式和生殖层诱导的差异时间的基础。我们发现新生的转录和转录因子结合位点（TFBS）之间存在很强的正相关性先锋因子FOXA4和POU3F1的富集以及与组蛋白脱甲基酶的负相关性 KDM2B。为了获得对分层生殖层起源的更深入的洞察力，我们旨在探究胚胎胚胎中假定细菌层的不同区域的染色质可及性并确定 FOXA4和POU3F1的结合。此外，了解失调的发展后果细菌层时机，我们实施了一种方法来改变空间的细胞分裂时序，并改变了囊泡胚胎中的基因组激活。我们将表征细菌层基因表达程序的时间安排在干燥的胚胎中，确定胃发育的表型。揭开层次生殖层表达及其功能重要性的机制将为您提供新的见解我们了解细胞命运决策和组织规格中早期发展中的第一个事件。