Induction of a novel piRNA response in zebrafish

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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.