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

描述(申请人提供)：干细胞既能自我更新，又能产生大量分化的细胞。Piwi家族的ArgAerte蛋白(这里称为Piwi蛋白)是多种生物干细胞维持所必需的，但其分子功能尚不清楚。Piwi蛋白与一类不同的小RNA关联，并在表观遗传和转录后水平上调节基因表达。这项提议将利用Hydra作为一个模型系统来进一步了解干细胞中的Piwi功能，这些功能在系统发育中是保守的。九头蛇是一种基本的后生动物，有一个特征良好的干细胞谱系，即1-细胞谱系，其中有两个Piwi同源物被特异性地表达。在1-细胞谱系中表达GFP的转基因Hydra允许在体内观察干细胞，因此，第一次将在活的有机体的背景下观察Piwi扰动对干细胞行为的影响。此外，因为九头蛇是无性系繁殖的，所以很容易获得大量的、遗传纯净的种群来进行生化研究。因此，pIwi在干细胞中的分子功能将通过鉴定含有plw1的蛋白质复合体和识别pIwi直接基因调控的靶点来研究。最后，九头蛇有大量假定的piRNAs，它们的身份和功能将被探索。重要的是，像九头蛇这样的蛇类基因组，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所做的功能发现将应用于更复杂的生物体。这些研究将促进我们对PIwi在干细胞中保守的分子功能的理解，从而有助于基本理解在治疗中操纵干细胞所需的干细胞分子电路。 与公共卫生相关：干细胞既能自我更新，又能分化成身体细胞类型，因此在修复丢失或受损的组织方面具有巨大的治疗潜力。Piwi基因是不同生物体干细胞自我更新所必需的；它们编码与小RNA相关的蛋白质，在多个水平上调节基因表达。Hydra可在活体环境中操纵干细胞，为探索Piwi蛋白调节干细胞自我更新和分化的分子机制提供了一个有效的模型。

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.