Initiation of shoot meristem stem cells during Arabidopsis embryogenesis

生物体是如何形成多能干细胞的？这是植物和动物发育的核心步骤，是细胞和组织终身生产的基础。然而，尽管对胚胎后干细胞维持的调控已经进行了详细的研究，但对胚胎发生过程中干细胞的启动知之甚少。在高等植物中，茎分生组织的干细胞在植物的整个生命过程中为叶子、侧枝或花蕾的形成提供细胞。在前期工作中，我们发现茎分生组织干细胞程序的启动是由WOX2转录因子控制的，部分通过平衡植物激素细胞分裂素和生长素来实现。我们发现WOX2仅在胚胎干细胞建立阶段的最外层细胞层中表达，这表明该层作为信号来源将底层细胞转换为干细胞程序。最后，我们分离出WOX2的蛋白相互作用物，这可能解释WOX2在干细胞启动中的作用。这些结果为揭示植物茎部分生组织干细胞形成的基本分子机制提供了一个令人兴奋的机会。本提案的目的是深入了解WOX2如何调节拟南芥胚胎发生中的干细胞起始。首先，我们将利用全基因组染色质和转录组分析识别和表征干细胞起始过程中直接的WOX2靶基因。其次，我们将研究WOX2调控转录的分子机制，并验证它是一种表观遗传调节剂的假设。第三，我们将探索WOX2是通过外细胞层的信号机制还是通过表观遗传记忆机制对三层茎分生组织干细胞进行编程的。第四，我们将研究已经分离的WOX2蛋白相互作用物，分析WOX2启动干细胞的机制。我们将结合遗传学、生物化学和分子生物学的方法。除了我们最近发表的发现，我们已经为这个提议做了大量的准备工作，包括蛋白质相互作用的研究和分子工具的准备，使我们的实验能够迅速开始。本研究将为了解拟南芥茎部分生组织干细胞的起始机制提供重要的理论依据。由于WOX2的类似物在受调控的根和形成层干细胞中也是保守的元件，并且由于WOX蛋白对干细胞的调控至少在高等植物中是保守的，我们期望我们的发现将有助于我们对植物中干细胞起始的一般理解。此外，除了阐明植物干细胞发育的基本过程外，我们还与国际同事合作，将我们的结果纳入植物再生策略，这是植物育种的重要工具。

How does an organism establish pluripotent stem cells? This central step in plant and animal development underlies the lifelong production of cells and tissues. However, while the regulation of postembryonic stem cell maintenance has been investigated in detail, relatively little is known about the initiation of stem cells during embryogenesis. In higher plants, the stem cells of the shoot meristem provide the cells for the formation of leaves, side branches, or flower buds throughout the plant's life. In our preparatory work, we showed that the initiation of the shoot meristem stem cell program is controlled by the WOX2 transcription factor, in part through balancing the phytohormones cytokinin and auxin. We found that WOX2 is expressed only in the outermost cell layer of the embryo at the stage of stem cell establishment, suggesting that this layer serves as a signaling source to switch the underlying cells to the stem cell program. Finally we isolated protein interactors of WOX2 that may explain how WOX2 functions in stem cell initiation. These results provide an exciting opportunity to reveal the fundamental molecular mechanisms underlying shoot meristem stem cell initiation in plants. The goal of this proposal is to gain insight into how WOX2 regulates stem cell initiation in Arabidopsis embryogenesis. First, we will identify and characterize direct WOX2 target genes during stem cell initiation using genome-wide chromatin and transcriptome analysis. Second, we will investigate the molecular mechanisms by which WOX2 regulates transcription and test the hypothesis that it is an epigenetic regulator. Third, we will explore whether the three layers of shoot meristem stem cells are programmed by WOX2 through a signaling mechanism from the outer cell layer or through an epigenetic memory mechanism. Fourth, we will investigate already isolated protein interactors of WOX2 to analyze which mechanisms enable WOX2 to initiate stem cells. We will combine genetic, biochemistry and molecular biology approaches. In addition to our recently published finding, we have already done extensive preparatory work for this proposal, including protein interaction studies and preparation of molecular tools, enabling a swift start of the proposed experiments.We expect that this work will provide significant advances in understanding the initiation of shoot meristem stem cell in Arabidopsis. Because paralogs of WOX2 are conserved elements in regulated also root and cambium stem cells and because stem cell regulation by WOX proteins is conserved at least in higher plants, we expect that our findings will contribute to our understanding of stem cell initiation in general in plants. Furthermore, in addition to elucidating a fundamental process in plant stem cell development, we collaborate with our international colleagues to incorporate our results into plant regeneration strategies which are important tools in plant breeding.