Zebrafish germline development in presence and absence of a functional PIWI pathway

Abstract

Germcells are responsible for the transmission of all genetic information from one generation to the next and engage a genome defense pathway that is based upon RNA interference. This socalled PIWI or piRNA pathway protects the genome by targeting transposon transcripts and renders them inactive. PIWI proteins and their interacting small RNAs, the piRNAs, therefore represent an ancient defense system against intergenomic parasites. In zebrafish, where germcells are specified by the inheritance of germplasm, information about maternal transposon activity is passed on via the maternally provided PIWI protein Ziwi, a component of germ plasm. This maternally provided piRNA pool is thought to represent a first line of defense against transposon activity in early germ cells. Little is known about what happens in early germ cells of zebrafish in the time between their arrival at the genital ridge and the time when a functional gonad is established. We find that in this timeframe primordial germ cells (PGCs) are going through a transition from a maternal to a zygotic program. This change is accompanied by massive morphological changes and the induction of a germline specific transcriptional program. This not only leads to the expression of germline genes but also large intergenetic regions, start to get expressed. PiRNAs produced in the adult gonads map to these regions, suggesting that they might represent piRNA clusters, similar to what is described in mouse and drosophila. In the first few days, not only these clusters become transcribed but also the piRNA pathway becomes active leading to the amplification of maternal piRNAs. The second chapter of this thesis is looking at this early germ cell development. What happens in gonads where the PIWI pathway is compromised is subject to chapters 3 and 4. Vasa and Tdrd9 are two conserved proteins involved in the PIWI pathway in other organisms and we find that in zebrafish, absence of either of these two proteins leads to the loss of germ cells and mutants develop as sterile males, a phenotype that is shared with other PIWI pathway mutants. Tdrd9 is required for proper piRNA production and similar to Tdrd1 mutants, mainly small RNAs from RNA transposons are affected. Additionally we observe a nuage phenotype which suggests that Tdrd9 plays a role in the dynamics between different RNA granules. Although we identify many nuage proteins including the two zebrafish PIWI proteins, Zili and Ziwi, as possible interaction partners of Vasa, we don’t see a dramatic effect on piRNA production. This effect might be obscured though by the presence of maternally provided Vasa protein and RNA as maternally provided protein can still be detected in mutant germ cells at 3 weeks post fertilization. Gtsf1, another conserved factor involved in small RNA biology is subject of chapter 4 where we find that both gtsf homologs in zebrafish localize to perinuclear nuage and interact with PIWI proteins. Nevertheless we do not find an effect on piRNA production in gtsf1 mutants, but, similar to the situation in mouse, gtsf1 is required for spermiogenesis and male fertility.