Initiation of shoot meristem stem cells during Arabidopsis embryogenesis

生物体如何建立多能干细胞？植物和动物发育的这一中心步骤是终生生产细胞和组织的基础。然而，虽然胚胎后干细胞维持的调控已经被详细研究，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为植物一生中形成叶、侧枝或花蕾提供细胞。在我们的准备工作中，我们证明了茎分生组织干细胞程序的启动是由WOX2转录因子控制的，部分是通过平衡植物激素细胞分裂素和生长素。我们发现，在干细胞建立阶段，WOX2只在胚胎最外层的细胞层表达，这表明该层作为一个信号源，将底层细胞切换到干细胞程序。最后，我们分离了WOX2的蛋白相互作用因子，这可能解释了WOX2如何在干细胞启动中发挥作用。这些结果为揭示植物茎分生组织干细胞启动的基本分子机制提供了一个令人兴奋的机会。这项建议的目标是深入了解WOX2如何调控拟南芥胚胎发育中干细胞的启动。首先，我们将使用全基因组染色质和转录组分析来识别和表征干细胞启动过程中直接的WOX2靶基因。其次，我们将研究WOX2调控转录的分子机制，并检验它是表观遗传调节因子的假设。第三，我们将探索WOX2是通过外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。第四，我们将研究已经分离的WOX2蛋白相互作用因子，以分析哪些机制使WOX2能够启动干细胞。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，我们已经为这一提议做了广泛的准备工作，包括蛋白质相互作用研究和分子工具的准备，使拟议的实验能够迅速开始。我们期待这项工作将为理解拟南芥茎分生组织干细胞的启动提供重要的进展。由于WOX2的类似序列在受调控的根和形成层干细胞中是保守的元件，而且WOX蛋白对干细胞的调控至少在高等植物中是保守的，我们希望我们的发现将有助于我们对植物中干细胞启动的一般理解。此外，除了阐明植物干细胞发展的基本过程外，我们还与国际同行合作，将我们的结果纳入植物再生战略，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.