Induction of a novel piRNA response in zebrafish

生殖细胞的特点是强大的转座子控制系统。这些机制之一被称为PIWI-piRNA途径，其中小RNA（皮尔纳）与PIWI蛋白结合，并作为序列特异性辅因子将PIWI蛋白带到特定靶标（主要是转座子）。该领域的一个主要问题是新序列如何进入皮尔纳生物发生机制。特别是在皮尔纳群体作为RNP母系传递的生物体中，目前尚不清楚新入侵的转座子如何开始被PIWI-piRNA系统识别。我们建议在斑马鱼中研究这一点，斑马鱼是一种脊椎动物，其中皮尔纳介导的沉默是母系传递的。我们最近开发了一套工具，使我们能够解决所提出的问题。该提议的一个重要工具是piRNA靶向EGFP沉默的系统。这是通过将许多含EGFP的转座子拷贝随机插入基因组中来开发的，直到我们击中一个诱导存在于遗传背景中的转基因沉默的位点，并且在生殖细胞中表达EGFP。追踪这种转座子整合的位置将揭示皮尔纳产生的诱导。该系统通过雄性的杂交绕过了通常如此占主导地位的母体贡献，使我们能够研究皮尔纳群体在世代过程中的积累。该提案提出了实现这一目标的实验，以及一般分析这些piRNA表达位点如何发挥作用。该提案还提出了利用所开发的系统来研究亚型皮尔纳途径突变体中的皮尔纳缺陷的实验。特别地，旨在研究皮尔纳前体转录物的核输出的实验是该应用的重要方面。这种转录物的输出知之甚少，特别是在脊椎动物中，并且皮尔纳途径的这个方面实际上可能包含一些队列，关于哪些转录物被漏斗状地引入皮尔纳途径，哪些转录物不是：决定途径的特异性并防止靶向需要在种系中表达的基因的重要方面。最后，基于EGFP-piRNA系统，我们建议开发一种遗传工具包，可以在生殖细胞中同时沉默一个或多个基因，从而可以分离体细胞与生殖细胞系的功能。

Germ cells are characterized by potent transposon-control systems. One of these mechanisms is known as the PIWI-piRNA pathway, where small RNAs (piRNA) bind to PIWI proteins, and serve as sequence specific co-factors to bring the PIWI proteins to specific targets (mostly transposons). A major question in this field is how novel sequences can enter the piRNA biogenesis mechanisms. Especially in organisms where piRNA populations are passed on maternally as RNPs, it is currently unclear how newly invading transposons can start to be recognized by the PIWI-piRNA system. We propose to study this in zebrafish, a vertebrate animal in which piRNA mediated silencing is transmitted maternally. We recently developed a set of tools that allows us to tackle the proposed question. An important tool for this proposal is a system in which piRNAs target the silencing of EGFP. This was developed by randomly inserting many EGFP-containing transposon copies into the genome, until we hit a locus that induced the silencing of transgene that was present in the genetic background, and that expresses EGFP in the germ cells. Tracing where this transposon integrated will shed light on the induction of piRNA production. Crossing of this system via the male bypasses the normally so dominantly present maternal contribution, allowing us to study the build-up of a piRNA population over the course of generations. The proposal proposes experiments to achieve this, as well as to generally analyze how such piRNA-expressing loci function. The proposal also brings forward experiments to exploit the developed system to study piRNA defects in hypomorphic piRNA pathway mutants. In particular, experiments aimed at the study of the nuclear export of piRNA precursor transcripts are an important aspect of this application. Export of such transcripts is poorly understood, notably in vertebrates, and this aspect of the piRNA pathway may in fact hold some queues as to which transcripts are funneled into the piRNA pathway, and which transcripts are not: an important aspects that determines the specificity of the pathway and prevents the targeting of genes that need to be expressed in the germline. Finally, based on the EGFP-piRNA system, we propose to develop a genetic tool-kit to can silence one or more genes simultaneously in germ cells specifically, allowing the dissection of somatic versus germ line functions.