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

摘要 转录最初在早期胚胎发育过程中受到抑制，然后在一个过程中被全面激活。 称为合子基因组激活（ZGA）。ZGA的精确时间对于胚胎发育至关重要：延迟 ZGA阻断原肠胚形成和细胞分化;过早的ZGA诱导破坏正常发育。 然而，ZGA调节的性质和控制其发作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）sizer 测量细胞体积，或3）跟踪细胞分裂的事件计数器。定时器可以包括 mRNA编码蛋白质，如多能性因子，在受精后稳定翻译， 其积累诱导ZGA。sizer假说假设，当细胞达到足够小的 它们达到一个阈值DNA：细胞质比，克服了合子转录的全局块。 组蛋白和DNA复制因子与此阻滞有关。组成这些分子的 细胞周期计数器未知。时间、细胞大小和细胞周期数是相互交织的，因此， 确定这些参数中的一个或全部是否控制ZGA具有挑战性。最终，我们的理解 胚胎基因组激活的研究一直受到无法测量细胞间变异性的阻碍， 胚胎，缺乏遗传工具来消除与ZGA调节有关的母体因素，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， 通过标记新合成的RNA在整个卵裂期胚胎中的单细胞分辨率的时间和空间 与5-乙炔基尿苷（5-EU）反应。使用这种技术，我们区分ZGA调节机制， 完全基于计时器、尺寸器或计数器。在目标1中，我们将构建一个基因组激活的空间图， 非洲爪蟾和斑马鱼囊胚胚胎的单细胞。此外，通过压缩胚胎尺寸，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发作的机制， 使用含有显著降低水平的核心的茎环结合蛋白2突变体斑马鱼胚胎， 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这种应用的前提是，单一的模型不能解释精确的时间， ZGA的图案化。具体地说，我们假设细胞必须达到一个阈值大小，以减轻组蛋白为基础的 抑制，并含有足够水平的多能性因子以诱导合子转录。研究 提出这里将提供一个新的机制理解胚胎基因组激活-一个普遍的 包括人类在内的所有脊椎动物的发育特征-以及调控基因的特定组合 范例决定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.