show Abstracthide AbstractMost piRNAs in the Drosophila female germline are transcribed from heterochromatic regions called dual-strand piRNA clusters. Histone 3 lysine 9 trimethylation (H3K9me3) is required for licensing piRNA production by these clusters. However, it is unclear when and how they acquire this permissive heterochromatic state. Although it has been suggested that piRNA cluster licensing is Piwi-independent, here we show that transient Piwi depletion in Drosophila embryos, using a refined knock-down system, results in H3K9me3 decrease at piRNA clusters. This is accompanied by aberrant maturation of piRNA precursor transcripts, accumulation of transposable element transcripts and female sterility. Conversely, Piwi knock-down at later developmental stages does not disturb piRNA cluster licensing, as previously reported. These results indicate that the identity of piRNA clusters is epigenetically acquired in a Piwi-dependent manner during a limited embryonic development window, which is reminiscent of the widespread genome reprogramming occurring during early mammalian zygotic development. Overall design: Ribo-Zero ribosomal depletion total RNA profil of D. melanogaster ovaries in 2 experimental conditions : control (white-embKD) and piwi-embKD. 2 independant biological replicates for each condition were performed.