Regulation of the Timing and Spatial Patterning of Zygotic Genome Activation During Embryogenesis

摘要 转录最初在胚胎发育早期被抑制，然后在经过加工的 称为合子基因组激活(ZGA)。ZGA的精确时机对胚胎发育至关重要：延迟 ZGA会阻碍原肠形成和细胞分化；过早的ZGA诱导会扰乱正常发育。 然而，ZGA调节的性质和控制其发生的因素的身份仍然难以捉摸。 已经提出了三种模型来解释ZGA的发生：1)胚胎含有计时器或时钟，2)大小 它测量细胞体积，或3)跟踪细胞分裂的事件计数器。计时器可以由母级 编码蛋白质的mRNAs，如多能性因子，在受精和 它的积累诱导了ZGA。大小假说假设，当细胞达到足够小的 它们达到了一个阈值DNA：细胞质的比率，克服了合子转录的整体障碍。 组蛋白和DNA复制因子与这一阻断有关。构成这些分子的 细胞周期计数器是未知的。时间、细胞大小和细胞周期数相互交织，因此 很难确定这些参数中的一个或所有参数是否控制ZGA。最终，我们的理解是 由于无法测量细胞间的可变性，胚胎基因组激活的研究一直受到阻碍 胚胎，缺乏基因工具来耗尽ZGA调控中涉及的母体因素，以及 改变细胞或胚胎的尺寸。我的实验室最近开发了一种最先进的技术来对ZGA进行成像 标记新合成的RNA在整个卵裂期胚胎单细胞分辨率上的时间和空间 含5-乙炔基尿苷(5-Eu)。使用这项技术，我们区分了ZGA调节机制 完全基于定时器、定尺器或计数器。在目标1中，我们将构建一个基因组激活的空间地图 非洲爪哇和斑马鱼囊胚的单细胞。此外，通过缩小胚胎尺寸，我们 将产生迷你胚胎，以区分由细胞周期计数器启动的基因组激活 细胞体积传感器。在目标2中，我们将描述核心组蛋白调节ZGA启动的机制， 使用茎环结合蛋白2突变斑马鱼胚胎，该胚胎含有显著降低的核心水平 组蛋白。在目标3中，我们将确定多能性因子的翻译是否构成计时器 触发ZGA。这一应用的前提是单一模型不能解释精确的时间和 ZGA的图案。具体地说，我们假设细胞必须达到一个阈值大小才能释放基于组蛋白的 抑制，并含有足够水平的多能因子来诱导合子转录。这项研究 本文的提出将为胚胎基因组激活提供一种新的机制--一种普遍的 包括人类在内的所有脊椎动物的发育特征--以及调控的具体组合 范式决定了合子转录的模式和时间。

Abstract Transcription is initially repressed during early embryo development and then globally activates in a processed called zygotic genome activation (ZGA). The precise timing of ZGA is critical for embryo development: delaying ZGA blocks gastrulation and cell differentiation; premature ZGA induction disrupts normal development. However, the nature of ZGA regulation and the identity of factors that control its onset have remained elusive. Three models have been proposed to explain the onset of ZGA: 1) embryos contain a timer or clock, 2) a sizer that measures cell volume, or 3) an event counter that tracks cell divisions. A timer could consist of maternal mRNAs encoding proteins, such as pluripotency factors, that are steadily translated after fertilization and whose accumulation induces ZGA. The sizer hypothesis supposes that when cells achieve a sufficiently small volume they reach a threshold DNA:cytoplasm ratio that overcomes a global block of zygotic transcription. Histones and DNA replication factors have been implicated in setting this block. The molecules that constitute a cell cycle counter are unknown. Time, cell size, and cell cycle number are intertwined, therefore it has been challenging to determine whether one or all of these parameters controls ZGA. Ultimately, our understanding of embryonic genome activation has been hampered by an inability to measure cell-to-cell variability in an embryo, a lack of genetic tools to deplete maternal factors implicated in ZGA regulation, and the challenge of altering cell or embryo dimensions. My lab recently developed a state-of-the-art technique to image ZGA in time and space at single-cell resolution in whole cleavage-stage embryos by labeling newly synthesized RNA with 5-ethynyl uridine (5-EU). Using this technique, we distinguish between ZGA regulatory mechanisms based exclusively on a timer, sizer or counter. In Aim 1, we will construct a spatial map of genome activation in single-cells of Xenopus and zebrafish blastula embryos. Additionally, by constricting embryo dimensions, we will generate mini-embryos to distinguish genome activation that is initiated by a cell cycle counter from or a cell volume sensor. In Aim 2, we will characterize the mechanisms by which core histones regulate ZGA onset, using a stem loop binding protein 2 mutant zebrafish embryos that contain significantly reduced levels of core histones. In Aim 3, we will determine whether translation of pluripotency factors constitutes a timer for triggering ZGA. The premise of this application is that a single model cannot explain the precise timing and patterning of ZGA. Specifically, we hypothesize that cells must reach a threshold size to relieve histone-based repression, and contain a sufficient level of pluripotency factors to induce zygotic transcription. The research proposed here will provide a new mechanistic understanding of embryonic genome activation – a universal feature of developmental in all vertebrates, including humans - and how specific combinations of regulatory paradigms dictate patterning and timing of zygotic transcription.