Determining piwi function using in vivo imaging of stem cells in Hydra.

描述（由申请人提供）：干细胞能够自我更新并产生大量分化细胞。Argonaute蛋白质的Piwi亚家族（本文称为Piwi蛋白质）是多种生物体中干细胞维持所需的，但其分子功能尚未得到很好的理解。Piwi蛋白与一类独特的小RNA（piRNA）结合，并在表观遗传和转录后水平调节基因表达。该提案将利用Hydra作为模型系统，以进一步了解干细胞中的piwi功能，这些功能在整个胚胎发育过程中是保守的。水螅，一种基底后生动物，具有一个特征良好的干细胞谱系，即1-细胞谱系，其中两个piwi同源物特异性表达。在1-细胞谱系中表达GFP的转基因水螅允许在体内观察干细胞，因此，第一次将在活生物体的背景下观察piwi扰动对干细胞行为的影响。此外，由于水螅是无性繁殖的，因此很容易获得大的、遗传纯的种群用于生化研究。因此，将通过表征含有Piwi的蛋白质复合物和鉴定piwi指导的基因调控的靶标来研究piwi在干细胞中的分子功能。最后，Hydra拥有大量假定的piRNA，其身份和功能将被探索。重要的是，刺胞动物基因组，如水螅，表现出与脊椎动物基因组相同的复杂性。因此，在这些相对简单的后生动物中发现的功能将应用于更复杂的生物体。这些研究将推进我们对干细胞中piwi保守分子功能的理解，从而有助于对操纵干细胞用于治疗所需的干细胞分子电路的基本理解。 公共卫生相关性：干细胞既能自我更新，又能分化成身体的细胞类型，因此在替代丢失或受损的组织方面具有巨大的治疗潜力。piwi基因是多种生物体中干细胞自我更新所必需的;它们编码与小RNA相关的蛋白质，以在多个水平上调节基因表达。水螅，其可访问性操纵干细胞在一个活的有机体的背景下，提供了一个有效的模型来探索Piwi蛋白介导的调节干细胞自我更新和分化的分子机制。

DESCRIPTION (provided by applicant): Stem cells are capable of both self-renewal and producing numerous differentiated cells. The Piwi subfamily of Argonaute proteins (herein called Piwi proteins) is required for stem cell maintenance in diverse organisms, yet its molecular function is not well understood. Piwi proteins associate with a distinct class of small RNAs, piRNAs, and modulate gene expression at both epigenetic and post-transcriptional levels. This proposal will take advantage of Hydra as a model system to further understand piwi functions in stem cells that are conserved across phylogeny. The Hydra, a basal metazoan, has a well-characterized stem cell lineage, the 1-cell lineage, in which two piwi homologs are specifically expressed. Transgenic Hydra that express GFP in the 1-cell lineage allow for in vivo observation of stem cells, thus, for the first time, the effect of piwi perturbation on stem cell behavior will be observed in the context of a living organism. In addition, because Hydra propagate clonally it is easy to obtain large, genetically pure populations for biochemical studies. Thus, the molecular function of piwi in stem cells will be investigated by characterizing PlWl-containing protein complexes and identifying targets of piwi-directed gene regulation. Finally, Hydra has a large population of putative piRNAs of which the identity and function will be explored. Importantly, Cnidarian gene sets, such as Hydra, exhibit the same complexity found in vertebrate genomes. Thus, functional discoveries made in these relatively simple metazoans will be applied to more complex organisms. These studies will advance our understanding of the conserved molecular functions of piwi in stem cells, thus contributing to the basic understanding of stem cell molecular circuitry required to manipulate stem cells for use in therapy. PUBLIC HEALTH RELEVANCE: Stem cells both self-renew and give rise to differentiated cell types of the body and therefore have great therapeutic potential in replacing lost or damaged tissues. The piwi genes are required for stem cell self-renewal in diverse organisms; they encode proteins that associate with small RNAs to modulate gene expression at many levels. Hydra, with its accessibility for manipulating stem cells in the context of a live organism, provides an effective model to explore molecular mechanisms mediated by Piwi proteins in regulating stem cell self-renewal and differentiation.