Control of epidermal self-renewing populations by bHLH and MAPK regulatory system

摘要 自我更新是创造和维持包括人类在内的许多组织和器官不可或缺的一部分 上皮、肌肉和血液系统。这些组织中的每一个都建立了自我更新的种群 (干细胞)，并必须确保这些干细胞分裂并在 适当的速率和适当的位置；没有足够的细胞或分裂，组织将 如果病情恶化，可能会形成太多的分裂和肿瘤。我们的长期目标是了解 分散的自我更新种群的建立机制(S)及其划分 分化受到与邻近细胞相互作用的影响。一组强大的基因， 分子和功能基因组工具，结合可视化和跟踪细胞分裂的能力 使拟南芥气孔发育成为研究这一系列问题的一个有吸引力的系统。 在我们之前的工作中，我们发现了三个bHLH转录因子和一个MAPK的组件 在气孔发育的不同阶段，途径调节分裂和分化的选择。 基于遗传、生化和功能分析的数据，我们认为 在这三人组中，最早行动的bHLH，无言(SPCH)，控制着产生不对称分裂的 自我更新的种群。我们证明SPCH是MAP依赖的直接靶点 体外磷酸化，这种磷酸化改变了SPCH在体内的行为。我们的特定 这个建议的目的是：(1)阐明SPCH活性的分子机制。 调节(2)创建气孔谱系细胞的分子图谱，并鉴定和功能 鉴定SPCH在这些细胞中的转录靶点，以及(3)利用SPCH诱导的 从基因上剖析抑制干细胞所需的内源性信号网络的表型 差异化。因为bHLH类转录调节因子和MAPK通路都是 这些研究是普遍保守的，不仅有助于理解自我更新，而且 将有助于我们理解MAPK和bHLH信号机制的多样性 回应。

Summary Self renewal is integral to the creation and maintenance of many tissues and organs including human epithelia, muscle and blood systems. Each of these tissues establishes populations of self-renewing (stem) cells and must ensure that these stem cells divide and create new differentiated cells at the appropriate rate and in the appropriate place; not enough cells or divisions and the tissue will deteriorate, too many divisions and tumors can form. Our long term goal is to understand the mechanism(s) by which dispersed self-renewing populations are established and how their division and differentiation is influenced by interaction with neighboring cells. A powerful set of genetic, molecular and functional genomic tools, combined with the ability to visualize and track cell divisions makes Arabidopsis stomatal development an attractive system for investigating this set of questions. In our previous work we found that a trio of bHLH transcription factors and components of a MAPK pathway modulate division vs. differentiation choices at discrete stages in stomatal development. Based on data from genetic, biochemical and functional assays, we propose that activity of the earliest-acting bHLH in this trio, SPEECHLESS (SPCH), controls the asymmetric divisions that create the self-renewing populations. We show that SPCH is a direct target of MAP kinase-dependent phosphorylation in vitro, and that phosphorylation alters the behavior of SPCH in vivo. Our specific aims in this proposal are to: (1) elucidate the molecular mechanisms by which SPCH activity is regulated (2) Create a molecular profile of stomatal lineage cells and identify and functionally characterize transcriptional targets of SPCH in these cells, and (3) Take advantage of SPCH-induced phenotypes to genetically dissect the endogenous signaling network required to repress stem-cell differentiation. Because both the bHLH class of transcriptional regulators and the MAPK pathway are universally conserved, these studies may contribute not only to an understanding of self-renewal, but will contribute to our understanding of the diversity of MAPK and bHLH signaling mechanisms and responses.