Feedback between multiple signaling modes in control of a plant stem cell niche

描述（由申请人提供）：了解干细胞群体的维持和分化机制是实现干细胞在人类疾病治疗中的全部治疗潜力的核心科学问题。表征维持和调节干细胞群的细胞信号将导致对生物体控制生长和发育的原理的新认识，并开发新技术，将干细胞用于包括组织和器官再生在内的健康应用。该研究通过在模型系统中描述调节干细胞增殖、分化和规范的因素，将揭示干细胞生态位组织的新原理。提出的研究旨在回答干细胞生物学中悬而未决的核心问题：在干细胞群体中，细胞间通讯指导细胞行为的机制是什么？拟南芥（Arabidopsis thaliana）的茎尖分生组织（shoot apical merisystem， SAM）具有较强的再生能力，可应用于现代系统和基因组生物学的全谱研究。我们将研究两个关键的信号通路及其相互作用：CLAVATA-WUSCHEL肽信号通路和激素细胞分裂素信号通路。实现这一目标的结果将是对干细胞壁龛的理解达到一个新的水平，以及反馈、信号和局部细胞类型反应的原理，这些原理使它们能够作为永久性的、受调节的细胞储存库，用于创建组织。这些实验将使我们对发育和植物干细胞调控的理解达到一个新的复杂水平；因此，对植物和动物干细胞调控的原理有了更全面的理解和比较的看法。每个目标都将使用非侵入性的实时成像技术，这种技术在植物中特别有效，因为它们的干细胞靠近表面，以及转基因报告基因，以及大量改变干细胞行为调节的突变。第一个目标的目标是充分表征激素细胞分裂素水平的变化通过肽信号通路的调节直接影响SAM干细胞生态位的不同结构域的机制，以及通过激素信号系统自身的多重反馈。第二个目标是一个系统的方法来表征从受体到反应调节转录的细胞分裂素信号通路。利用新的TRAP-seq和完好无损的方法，细胞分裂素靶基因将在细胞和分子分辨率上进行表征。第三个目标是一个无偏倚的酵母- 1杂交筛选，以确定细胞分裂素信号的转录调节因子和细胞分裂素生物合成基因。第四个也是最后一个目标是使用通过这些方法产生的数据来参数化包含系统中复杂反馈的计算模型，以测试细胞间通信调节SAM干细胞生态位的机制的新假设。

DESCRIPTION (provided by applicant): Understanding the mechanism of maintenance and differentiation of stem cell populations is a central scientific issue on the path to realizing thefull therapeutic potential of stem cells in the treatment of human disease. Characterizing the cellular signals that maintain and regulate stem cell populations will lead to a new appreciation of the principles by which living organisms control growth and development and to the development of new technologies to utilize stem cells in health applications including tissue and organ regeneration. The proposed study, by characterizing the factors that regulate stem cell proliferation, differentiation, and specification in a model system, will reveal new principles of stem cell niche organization. The research proposed is designed to answer the central unanswered question in stem cell biology: what is the mechanism by which cell-cell communication instructs cellular behavior in stem cell populations? Because of its amenability to the full spectrum of methods from modern systems and genomic biology, as well as the remarkable regenerative abilities of plants, the shoot apical meristem (SAM) of the flowering plant Arabidopsis thaliana will be used as a model system. Two key signaling pathways, and their interactions, will be studied: the CLAVATA-WUSCHEL peptide signaling pathway and the hormonal cytokinin signaling pathway. The result of accomplishing the aims of the proposal will be a new level of understanding of stem cell niches, and the principles of feedback, signaling, and local cell type response that allow them to serve as permanent and regulated reservoirs of cells for creation of tissues. The experiments will lead to a new level of sophistication in our understanding of development, and of stem cell regulation in plants; and therefore to a more complete understanding of, and comparative view of, the principles of stem cell regulation in both plants and animals. Each aim will use non-invasive live imaging techniques that are particularly effective in plants, because their stem cells are near the surface, and transgenic reporter transgenes, as well as an extensive array of mutations that change the regulation of stem cell behavior. The goal of the first aim is a full characterization of the mechanisms by which alterations in the levels of the hormone cytokinin affect the different domains of the SAM stem cell niche, both directly, via regulation of the peptide signaling pathway, and by the multiple feedbacks of the hormone signaling system on itself. The second aim is a systems approach to characterize the cytokinin signaling pathway from receptor to response regulator transcription. Using the novel TRAP-seq and INTACT methods, cytokinin target genes will be characterized at cellular and molecular resolution. The third aim is an unbiased yeast-one hybrid screen to identify transcriptional regulators of cytokinin signaling, and cytokinin biosynthetic genes. The fourth and final aim is to use the data generated through these approaches to parameterize computational models that encapsulate the complex feedbacks in the system, to test new hypotheses of the mechanism by which cell-cell communication regulates the SAM stem cell niche.