Identifying targets of the Piwi/piRNA pathway in stem cells

描述（由申请人提供）：需要成体干细胞来维持组织稳态，干细胞功能的降低导致与衰老相关的健康下降。干细胞衰老的原因知之甚少，探索干细胞调控的基本机制对解决这一问题至关重要。简单的后生动物Hydra vulgaris显然是“不朽的”，而该属的其他成员经历生殖诱导衰老。比较来自两种具有完全不同衰老策略的密切相关动物的干细胞调节的分子机制的能力是一种独特的， 水螅的老化研究有吸引力的功能;其他完善的模式生物没有看似“不朽”的亲戚。此外，水螅干细胞具有用于实验的吸引人的特征，包括：1）干细胞群体被充分表征，2）由于转基因方法的最新进展，干细胞可以容易地大量收集并且可以在体内跟踪和操纵，以及3）水螅干细胞与在更复杂的动物中发现的干细胞共享基因表达谱。这包括Piwi蛋白，这是动物生殖细胞和干细胞功能所必需的。我们的初步数据表明，水螅Piwi蛋白在干细胞特异性途径中运作，以维持组织的稳态和上皮细胞的完整性，以应对营养状况。由于营养状况是寿命的保守调节因子，我们假设Piwi途径是干细胞衰老途径中的关键参与者，我们的目标是了解它在这种情况下如何发挥作用。Piwi蛋白与一类独特的小RNA（piRNA）相关，并且在果蝇和小鼠中，这些Piwi/皮尔纳复合物在表观遗传和转录后水平调节基因表达。水螅干细胞有两个严格的胞质Piwi蛋白，使我们能够专注于识别转录后靶点的途径。我们正在使用转基因方法来敲低Piwi蛋白表达，并在每种Hydra干细胞类型中测试Piwi/皮尔纳通路的功能。为了鉴定途径的靶标，我们：1）将piRNA映射到水螅转录组以鉴定推定的靶标，2）测试piwi敲减动物中推定的靶标的增加的转录物水平，以及3）测试推定的转录物和Piwi/皮尔纳复合物之间的物理相互作用。最后，我们正在测试Piwi/皮尔纳途径通过胰岛素途径对营养状态做出响应以调节干细胞增殖和组织稳态的假设。我们的目标是了解这一途径的上游调控因子和下游靶点。最终，我们希望获得对干细胞衰老调控的重要见解，这将转化为更复杂的生物体。我的长期目标是将Hydra建立为解剖干细胞功能和衰老调控的重要新模型。一旦我建立了自己的实验室，我将开始合作，以测试Piwi/皮尔纳通路在脊椎动物干细胞调控中的保守性。

DESCRIPTION (provided by applicant): Adult stem cells are required to maintain tissue homeostasis and decrease in stem cell function leads to the decline in health associated with aging. The causes of stem cell aging are poorly understood, and exploring basic mechanisms of stem cell regulation is vital to address this issue. The simple metazoan Hydra vulgaris is apparently "immortal", while other members of the genus undergo reproduction-induced senescence. The ability to compare the molecular mechanisms underlying the regulation of stem cells from two closely related animals with starkly different aging strategies is a unique and attractive feature of Hydra for aging studies; other well-established model organisms do not have seemingly "immortal" relatives. Furthermore, Hydra stem cells have attractive features for experimentation including: 1) Stem cell populations are well-characterized, 2) Due to recent advances in transgenic methods, the stem cells can be easily collected in large numbers and can be tracked and manipulated in vivo, and 3) Hydra stem cells share a gene expression profile with stem cells found in more complex animals. This includes the Piwi proteins, which are required for germline and stem cell function in animals. Our preliminary data suggest that Hydra Piwi proteins operate in a stem cell-specific pathway for maintaining tissue homeostasis and epithelial integrity in response to nutritional status. Because nutritional status is a conserved regulator of lifespan, we hypothesize that the Piwi pathway is a key player in the aging pathway of stem cells and we aim to understand how it functions in this context. Piwi proteins associate with a distinct class of small RNAs, piRNAs, and in Drosophila and mouse these Piwi/piRNA complexes modulate gene expression at the epigenetic and post-transcriptional levels. Hydra vulgaris stem cells have two strictly cytoplasmic Piwi proteins, allowing us to focus on identifyin post-transcriptional targets of the pathway. We are using a transgenic approaches to knockdown Piwi protein expression and test the function of the Piwi/piRNA pathway in each Hydra stem cell type. To identify targets of the pathway, we are: 1) Mapping piRNAs to a Hydra transcriptome to identify putative targets, 2) Testing for increased transcript levels of putative targets in piwi knockdown animals, and 3) Testing for a physical interaction between the putative transcript and Piwi/piRNA complexes. Finally, we are testing the hypothesis that the Piwi/piRNA pathway responds to nutritional status through the insulin pathway to regulate stem cell proliferation and tissue homeostasis. We aim to understand both the upstream regulators of the pathway and the downstream targets. Ultimately, we hope to gain significant insights into the regulation of aging in stem cells that will translate to more complex organisms. My long-term goal is to establish Hydra as an important new model for dissecting the regulatory control of stem cell function and aging. Once I have established my own lab I will initiate collaborations in order to test the conservation of the Piwi/piRNA pathway in stem cell regulation in vertebrates.