Defining Novel Molecular Pathways to Totipotency

项目总结 全能性，一种单个细胞在产生一个完整的有机体时的显著细胞可塑性，具有 胚胎和胚胎外的贡献对多细胞和发育是必不可少的。在小鼠身上，完全 全能细胞暂时存在于受精卵和卵裂期的卵裂球中，尽管全能的2细胞(2C)- 类细胞(2CLCs)也可以在培养的胚胎干细胞(ESCs)中零星或遗传诱导产生。 它们是多能性的，只能为胚胎谱系做出贡献。最近，扩增的潜在干细胞和 扩展的多潜能干细胞，统称为EPSCs，具有全能特征的来源和 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 不同品系的培养条件、转录本和核心多能性因子的表达不同，提示 另一种全能态。2CLCs和EPSCs是容易获得的细胞来源，在体外具有很高的价值 了解全能的模型，了解全能对于捕获和稳定 全能2CLC状态，这目前是不可能的。定义了这些不同的分子途径 全能细胞将有助于解开全能的复杂调控机制，并实现 全能2CLCs的捕获/稳定。据报道，全能的2CLCs可以重新激活转录 内源性逆转录病毒(ERV)，特别是MERVL，其激活现已被发现是引起 在我们的初步研究中是全能的。ZSCAN4在2C期胚胎和标记中也有显著表达 这些零星的ESCs从Esc状态向2CLCs转移。通过表征MERVL/ZSCAN4 Double 报道阳性(DR/)ESCs，我们已经开始剖析2CLC背后的分子通路 全能性，并发现miR-344及其直接靶点ZMYM2作为新的正负调控因子， MERVL激活和2CLC全能性。此应用程序的目标是扩展我们的 了解胚胎干细胞培养中不同的细胞潜能状态，并确定MERVL对 全能和探索潜在的全能的转录和转录后机制。我们 假设2CCs代表由新的分子轴调节的另一种全能状态 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)确定EPSCs和2CLCs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 单个MERVL元件在2CLC全能性中的功能作用。3)剖析分子 将miR-344建立为理解2CLC全能性的新范式 哺乳动物的全能性。了解驱动和诱导全能特征的分子机制 体外培养对于理解如何实现最大程度的细胞可塑性是必不可少的 维持，从而为有效地重新编制方案和潜在的治疗途径提供更多的选择。

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.