Defining Novel Molecular Pathways to Totipotency

项目概要 全能性，单个细胞在产生具有这两种能力的完整有机体时具有显着的细胞可塑性 胚胎和胚胎外的贡献对于多细胞性和发育至关重要。在小鼠中，完全 全能细胞短暂存在于受精卵和卵裂期卵裂球中，尽管全能 2-细胞 (2C)- 类似细胞 (2CLC) 也可以在培养的胚胎干细胞 (ESC) 中偶发或通过基因诱导产生 它们是多能的，只能对胚胎谱系做出贡献。最近，扩大了潜在的干细胞和 衍生出具有全能性特征的扩展多能干细胞，统称为 EPSC 使用两组不同的小分子抑制剂在体外稳定培养。然而，2CLC 和两个 EPSC 细胞系在培养条件、转录组和核心多能性因子的表达方面存在差异，提示 替代全能状态。 2CLC 和 EPSC 代表易于获取的细胞来源，并且在体外具有价值 理解全能性的模型，其知识对于捕获和稳定全能性至关重要 全能2CLC状态，目前不可能。定义这些不同的分子途径 全能细胞将有助于揭示全能性的复杂调节机制并实现 全能 2CLC 的捕获/稳定。据报道，全能 2CLC 可重新激活 内源性逆转录病毒 (ERV)，特别是 MERVL，现已发现其激活是致病原因 在我们的初步研究中寻找全能性。 ZSCAN4 在 2C 期胚胎中也急剧表达，并标志着 那些零星的 ESC 从 ESC 状态退出到 2CLC。通过表征 MERVL/ZSCAN4 双 报告阳性（DR / ）ESC，我们已经开始剖析2CLC潜在的分子途径 全能性并发现 miR-344 及其直接靶标 ZMYM2 作为新型正负调节因子， 分别用于 MERVL 激活和 2CLC 全能性。该应用程序的目的是扩展我们的 了解 ESC 培养中不同的细胞效力状态，并定义 MERVL 对 全能性并探索全能性背后的转录和转录后机制。我们 假设 2CLC 代表一种由新分子轴调节的替代全能状态 包括转录和转录后机制。我们建议进行以下研究 检验我们的假设。 1) 定义 EPSC 和 2CLC 中替代全能状态的分子路线图。 2） 确定 MERVL 元件对 2CLC 全能性的功能贡献。我们将定义和区分 各个 MERVL 元件在 2CLC 全能性中的功能作用。 3）剖析分子 通过建立 miR-344 作为理解的新范式来研究 2CLC 全能性的机制 哺乳动物的全能性。了解驱动和诱导全能特征的分子机制 体外对于理解如何实现最大程度的细胞可塑性至关重要 维持，从而为有效的重编程和潜在的治疗途径提供更多选择。

PROJECT SUMMARY Totipotency, a remarkable cellular plasticity of a single cell in generating a complete organism with both embryonic and extraembryonic contribution, is essential for multicellularity and development. In mice, fully totipotent cells exist transiently in the zygote and cleavage-stage blastomeres, although totipotent 2-cell (2C)- like cells (2CLCs) can also arise sporadically or induced genetically in cultured embryonic stem cell (ESCs) that are pluripotent and can only contribute to embryonic lineages. Recently, expanded potential stem cells and extended pluripotent stem cells, collectively known as EPSCs, with totipotency features were derived and stably cultured in vitro using two distinct sets of small molecule inhibitors. However, 2CLCs and the two EPSC lines differ in their culture conditions, transcriptomes, and expression of core pluripotency factors, suggestive of alternative totipotent states. 2CLCs and EPSCs represent easily accessible cell source and valuable in vitro models for understanding totipotency, the knowledge of which will be critical to capture and stabilize the totipotent 2CLC state, which is not currently possible. Defining molecular pathways underlying these various totipotent cells will facilitate unraveling the complex regulatory mechanisms of totipotency and achieving the capture/stabilization of totipotent 2CLCs. The totipotent 2CLCs were reported to reactivate transcription of endogenous retroviruses (ERVs), in particular MERVL, whose activation has now been found to be causative for totipotency in our preliminary studies. ZSCAN4 is also sharply expressed in 2C-stage embryo and marks those sporadic ESCs exiting from the ESC state towards 2CLCs. By characterizing MERVL/ZSCAN4 double reporter positive (DR+/+) ESCs, we have begun to dissect the molecular pathways underlying 2CLC totipotency and discovered miR-344 and its direct target ZMYM2 as novel positive and negative regulators, respectively, for MERVL activation and 2CLC totipotency. The objective of this application is to extend our knowledge of the divergent cellular potency states in ESC culture, and define the MERVL contribution to totipotency and explore transcriptional and post-transcriptional mechanisms underlying totipotency. We hypothesize that 2CLCs represent an alternative totipotent state that is regulated by a novel molecular axis encompassing the transcriptional and post-transcriptional mechanisms. We propose the following studies to test our hypothesis. 1) Define a molecular roadmap for alternative totipotent states in EPSCs and 2CLCs. 2) Establish the functional contribution of MERVL elements to 2CLC totipotency. We will define and distinguish functional roles of individual MERVL elements in contributing to 2CLC totipotency. 3) Dissect the molecular mechanism underlying 2CLC totipotency by establishing miR-344 as a new paradigm for understanding mammalian totipotency. Understanding the molecular mechanisms that drive and induce totipotent features in vitro is essential to understanding of how a maximum degree of cellular plasticity can be achieved and maintained, thereby providing more options for efficient reprogramming and potential therapeutic avenues.