Transposon silencing in the Drosophila female germline is essential for genome stability in progeny embryos

Suppression of transposons by the Piwi-interacting RNA biogenesis factor Vasa in the supporting nurse cells is essential to prevent their accumulation in the developing oocyte, ensuring proper Drosophila embryonic development.


Introduction
Transposons and other selfish genetic elements are found in all eukaryotes and comprise a large fraction of their genomes. Although transposons are thought to be beneficial in driving evolution (Levin & Moran, 2011), their mobilization in the germline can compromise genome integrity with deleterious consequences: insertional mutagenesis reduces the fitness of the progeny, and loss of germ cell integrity causes sterility. Therefore, it is of great importance for sexually reproducing organisms to firmly control transposon activity in their germ cells. Metazoans have evolved a germline-specific mechanism that, by relying on the activity of Piwi family proteins and their associated Piwi-interacting RNAs (piRNAs), suppresses mobile elements.
Drosophila harbors three PIWI proteins: Piwi, Aubergine (Aub), and Argonaute 3 (Ago3), which, guided by piRNAs, silence transposons at the transcriptional and posttranscriptional levels (reviewed in Guzzardo et al [2013]). Besides PIWI proteins, other factors such as Tudor domain proteins and RNA helicases are involved in piRNA biogenesis and transposon silencing. Mutations in most piRNA pathway genes in Drosophila females cause transposon up-regulation that leads to an arrest of oogenesis. This effect can be rescued by suppression of the DNA damage checkpoint proteins of the ATR/ Chk2 pathway (Chen et al, 2007;Klattenhoff et al, 2007;Pane et al, 2007). By contrast, inhibition of DNA damage signaling cannot restore embryonic development (Chen et al, 2007;Klattenhoff et al, 2007;Pane et al, 2007). Recent studies suggest that PIWI proteins might have additional roles during early embryogenesis independent of DNA damage signaling (Khurana et al, 2010;Mani et al, 2014). However, functions of the piRNA pathway during early embryonic development remain poorly understood.
One of the essential piRNA pathway factors with an important role in development is the highly conserved RNA helicase Vasa. First identified in Drosophila as a maternal-effect gene (Schüpbach & Wieschaus, 1986;Hay et al, 1988;Lasko & Ashburner, 1990), vasa (vas) was subsequently shown to function in various cellular and developmental processes (reviewed in Lasko [2013]). In the Drosophila female germline, Vasa accumulates in two different cytoplasmic electron-dense structures: the pole (or germ) plasm at the posterior pole of the oocyte, and the nuage, the perinuclear region of nurse cells. In the pole plasm, Vasa interacts with the pole plasm-inducer Oskar (Osk) (Markussen et al, 1995;Jeske et al, 2015) and ensures accumulation of different proteins and mRNAs that determine primordial germ cell (PGC) formation and embryonic patterning (Hay et al, 1988;Lasko & Ashburner, 1990). In the nuage, Vasa is required for the assembly of the nuage itself (Liang et al, 1994;Malone et al, 2009) and facilitates the transfer of transposon RNA intermediates from Aub to Ago3, driving the piRNA amplification cycle and piRNA-mediated transposon silencing (Xiol et al, 2014;Nishida et al, 2015). As Vasa's involvement in many cellular processes renders it difficult to analyze its functions in each process individually, it remains unknown whether Vasa's functions in development and in the piRNA pathway are linked or independent.
In this study, we address the role of Vasa in transposon control in Drosophila development. We find that failure to suppress transposons in the nuage of nurse cells causes DNA double-strand breaks (DSBs), severe nuclear defects, and lethality of progeny embryos. Even transient interruption of Vasa expression in early oogenesis de-represses transposons and impairs embryo viability. Depletion of the Drosophila Chk2 ortholog maternal nuclear kinase (mnk) restores oogenesis in vas mutants, but does not suppress defects in transposon silencing or DSB-induced nuclear damage and embryonic lethality. We show that up-regulated transposons invade the maternal genome, inducing DNA DSBs that, together with transposon RNAs and proteins, are maternally transmitted and consequently cause embryogenesis arrest. Our study thus demonstrates that Vasa function in the nuage of Drosophila nurse cells is essential to maintain genome integrity in both the oocyte and progeny embryos, ensuring normal embryonic development.

Vasa-dependent transposon control is not essential for oogenesis
Vasa is required for piRNA biogenesis and transposon silencing in Drosophila, as in vas mutants piRNAs are absent and transposons are up-regulated (Vagin et al, 2004;Malone et al, 2009;Zhang et al, 2012;Czech et al, 2013;Handler et al, 2013). To investigate the importance of transposon control in Drosophila development, we expressed WT GFP-Vasa fusion protein (GFP-Vas WT ; Fig S1A) in the female germline of loss-of-function (vas D1/D1 ) vas flies using two promoters with distinct expression patterns (Fig S1B and C): the vas promoter is active at all stages of oogenesis, whereas the nos promoter is attenuated between stages 2 and 6 ( Fig S1B and C).
We first assessed the ability of GFP-Vas WT fusion protein to promote transposon silencing in the female germline, and examined the effect of GFP-Vas WT on the level of expression of several transposons in vas mutant ovaries. We chose the LTR retrotransposons burdock and blood, and the non-LTR retrotransposon HeT-A, which were previously reported to be up-regulated upon Vasa depletion (Vagin et al, 2004;Czech et al, 2013). The LTR retrotransposon gypsy, which belongs to the so-called somatic group of transposons and is not affected by Vasa depletion, served as a negative control (Czech et al, 2013). Loss-of-function vas D1/D1 ovaries contained elevated levels of burdock, blood, and HeT-A RNA ( Fig 1A). Remarkably, silencing of transposons by GFP-Vas WT in vas D1/D1 flies depended on which Gal4 driver was used (Fig S1B and C): When driven by nos-Gal4, GFP-Vas WT had no effect on transposon levels, whereas when driven by vas-Gal4, it led to the re-silencing of transposons ( Fig 1A). This differential effect presumably reflects the stages of oogenesis at which the nos and vas promoters are active, and suggests that lack of Vasa between stages 2 and 6 of oogenesis ( Fig  S1B) leads to transposon de-repression. Importantly, independent of Gal4 driver used, expression of GFP-Vas WT restored oogenesis (Figs 1B and S1D) and egg-laying (Fig S1E and F). The fact that in spite of transposon up-regulation oogenesis and egg-laying rates were largely restored in vas D1/D1 flies (Fig 1A, indicated by + and − and Fig S1D-F) is consistent with the notion that transposon activation affects but does not completely block oogenesis unless the level of activation is so high as to cause its arrest.

Loss of Vasa during early oogenesis affects viability of progeny embryos
Concentration of Vasa protein at the posterior pole of the embryo is essential for PGC and abdomen formation during embryogenesis (Schüpbach & Wieschaus, 1986;Hay et al, 1988;Lasko & Ashburner, 1990). We analyzed the number of PGC-positive embryos and the hatching rate of eggs produced by vas D1/D1 flies expressing GFP-Vas WT either under control of the nos or the vas promoter (vas D1/D1 ; nos-Gal4>GFP-vas WT and vas D1/D1 ; vas-Gal4>GFP-vas WT embryos). Embryos from vas D1/D1 mutant flies could not be included in these and all the other experiments on embryos, as vas D1/D1 females arrest oogenesis early and do not lay eggs. PGC formation was restored in approximately 50% of vas D1/D1 ; nos-Gal4>GFP-vas WT and vas D1/D1 ; vas-Gal4>GFP-vas WT embryos ( Fig 1C) (Table S1). However, DAPI staining revealed nuclear damage in some vas D1/D1 ; nos-Gal4>GFP-vas WT embryos (see below), which we excluded from the quantification.
Expression of GFP-Vas WT also partially rescued the hatching of eggs produced by vas D1/D1 flies ( Fig 1D). However, expression of GFP-Vas WT led to a significantly lower hatching rate in vas D1/D1 ; nos-Gal4>GFP-vas WT than in vas D1/D1 ; vas-Gal4>GFP-vas WT flies ( Fig 1D) (Table S2). Expression of GFP-Vas WT in heterozygous loss-of-function vas D1/Q7 females led to a low hatching rate similar to vas D1/D1 (Fig S2A and B) (Table S3), excluding a possible secondary mutation as the cause of the low hatching rate. The fact that in spite of comparable GFP-Vas WT levels ( Fig S2C), the hatching rate of vas D1/D1 ; vas-Gal4>GFP-vas WT embryos was higher than that of vas D1/D1 ; nos-Gal4>GFP-vas WT embryos, suggests that transient loss of vas expression during early oogenesis impairs viability of progeny embryos ( Fig 1D).

Elevated transposon levels cause DNA and nuclear damage in progeny embryos
Elevated transposon activity leads to DNA damage and ultimately to cell death. During our analysis of PGC formation, we observed nuclear damage in a considerable fraction of vas D1/D1 ; nos-Gal4>GFP-vas WT embryos. Quantification of embryos containing nuclei of aberrant nuclear morphology (Fig 2A, lower panel) compared with the nuclei of WT embryos (Fig 2A, upper panel) revealed a high proportion of such nuclear defects among vas D1/D1 ; nos-Gal4>GFP-vas WT embryos ( Fig  2A). Transposon mobilization causes DSBs in genomic DNA that are marked by the incorporation of a phosphorylated form of the H2A variant (γH2Av), a histone H2A variant involved in DNA DSB repair. Analysis of γH2Av occurrence showed that embryos displaying nuclear damage were γH2Av-positive (Fig 2B), indicating that DNA DSBs cause nuclear defects. The levels of γH2Av were higher in vas D1/D1 ; nos-Gal4>GFP-vas WT embryos compared with WT and vas D1/D1 ; vas-Gal4>GFP-vas WT (Fig 2C) (Table S4).
The correlation between high levels of transposon expression during oogenesis (Fig 1A, nos-Gal4-driven) and a high frequency of nuclear damage and DSBs in vas D1/D1 ; nos-Gal4>GFP-vas WT embryos (Fig 2A-C) suggested that maternally transmitted transposons cause embryonic lethality. To test this, we compared transposon RNA levels in embryos of vas D1/D1 ; nos-Gal4>GFP-vas WT and vas D1/D1 ; vas-Gal4>GFP-vas WT flies, in which transposon RNAs are up-and downregulated, respectively ( Fig 1A). Levels of maternally transmitted transposon RNA were significantly higher in vas D1/D1 ; nos-Gal4>GFP-vas WT embryos ( Fig 2D) suggesting that the increased lethality observed in vas D1/D1 ; nos-Gal4>GFP-vas WT embryos is due to DNA damage ( One of the up-regulated transposons in vas mutants is HeT-A, whose RNA and protein expression is strongly de-repressed in piRNA pathway mutant ovaries (Aravin et al, 2001;Vagin et al, 2006;Zhang et al, 2014;Lopez-Panades et al, 2015). Analysis of HeT-A/Gag protein expression in 0-1 h old embryos showed that the levels of HeT-A/Gag were much higher in vas D1/D1 ; nos-Gal4>GFP-vas WT than in vas D1/D1 ; vas-Gal4>GFP-vas WT embryos ( Fig 2E). In addition, we stained embryos with antibodies against HeT-A/Gag protein and observed that in cellularized WT embryos, HeT-A localized in distinct perinuclear foci (Figs 3A, panel a and S3A, panel a), as previously described for HeT-A/Gag-HA-FLAG fusion protein (Olovnikov et al, 2016). In vas D1/D1 ; nos-Gal4>GFP-vas WT embryos displaying nuclear damage, HeT-A protein accumulated in large foci throughout the embryo ( Altogether, these results show that up-regulation of transposon mRNAs and proteins during oogenesis results in their maternal transmission to the progeny, where they cause DSBs, nuclear damage, and arrest of embryogenesis.

Chk2 mutation restores oogenesis but not transposon silencing and embryogenesis in vas mutants
To test genetically whether DNA damage signaling contributes to the oogenesis arrest of vas loss-of-function mutants (Schüpbach & Wieschaus, 1986;Hay et al, 1988;Lasko & Ashburner, 1990) (Fig 1A), we introduced the mnk P6 loss-of-function allele into the vas D1 background. Genetic removal of mnk ( Fig S3B) suppressed the oogenesis arrest of vas D1/D1 mutants and partially rescued their egg laying (Figs S3C and S4A). Importantly, mnk P6/P6 single mutants expressed Vasa at WT levels and, as expected, the protein was not detected in vas D1/D1 , mnk P6/P6 double mutants (Fig S2C). Taken together, these findings demonstrate that the oogenesis arrest of loss-of-function vas mutants results from activation of the Chk2-mediated DNA damage-signaling checkpoint.
We next examined the distribution of HeT-A RNAs and occurrence of DNA DSBs by FISH and antibody staining of γH2Av, respectively. Damaged nuclei in vas D1/D1 , mnk P6/P6 embryos were γH2Av-positive (Figs 4A and S5A), indicating that DNA DSBs cause nuclear defects. HeT-A RNAs localized in large foci in vas D1/D1 , mnk P6/P6 embryos, and was not detectable in WT embryos (Figs 4A and S5A). Although, we did not detect HeT-A transcripts in the damaged nuclei of vas D1/D1 , mnk P6/P6 embryos, the oocyte nucleus was positive both for HeT-A RNA and γH2Av (Fig 4B), indicating the presence of DNA DSBs. Further analysis showed that HeT-A RNA and HeT-A/Gag protein co-localize in the oocyte cytoplasm and nucleus (Fig 5A) indicating that transposon insertions into the maternal genome begin already during oogenesis. Additional FISH analyses showed that in WT egg-chambers, HeT-A and Burdock transcripts were only detected at sites of transcription in the nurse cell nuclei, whereas in vas D1/D1 , mnk P6/P6 , and ago3 eggchambers, transcripts of both transposons accumulated within the oocyte along the anterior margin, and around and within the nucleus (Figs 5B and S5B). These results show that in vas D1/D1 , mnk P6/P6 double and in ago3 single mutant females up-regulated transposons invade the maternal genome and are transmitted to the progeny, causing severe nuclear defects and embryogenesis arrest. We conclude that tight regulation of transposons throughout oogenesis is essential to maintain genome integrity in the oocyte and in early syncytial embryo, hence for normal embryonic development.

Discussion
Our study shows that a transient loss of vas expression during early oogenesis leads to up-regulation of transposon levels and compromised viability of progeny embryos. The observed embryonic lethality is because of DNA DSBs and nuclear damage that arise as a consequence of the elevated levels of transposon mRNAs and proteins, which are transmitted from the mother to the progeny. We thus demonstrate that transposon silencing in the nurse cells is essential to prevent maternal transmission of transposons and DNA damage, protecting the progeny from harmful transposonmediated mutagenic effects.
Our finding that suppression of Chk2-mediated DNA damage signaling in loss-of-function vas mutant flies restores oogenesis,  (Table S4). P-value was determined by t test. Panel shows an embryo without (top) and an embryo with nuclear damage (bottom). Scale bars indicate 100 μm (embryo) and 10 μm (magnification). (B) Immunohistochemical detection of DNA double-strand breaks using antibodies against H2Av pS137 (γH2Av) in WT (w 1118 ), and vas D1/D1 ; nos-Gal4>GFPvas WT stage 5 embryos. Whole embryos are presented in (A). Scale bars indicate 5 and 2 μm (5× magnification). (C) Western blot analysis using antibodies against H2Av pS137 (γH2Av) showing protein levels in WT (w 1118 ), vas D1/D1 ; nos-Gal4>GFP-vas WT , and vas D1/D1 ; vas-Gal4>GFP-vas WT 1-to 3-h-old embryos. Tubulin was used as a loading control. Table shows  and egg production demonstrates that Chk2 is epistatic to vas. However, hatching is severely impaired, because of the DNA damage sustained by the embryos. The defects displayed by vas, mnk double mutant embryos resembled those of PIWI (piwi, aub, and ago3) single and mnk; PIWI double mutant embryos (Klattenhoff et al, 2007;Mani et al, 2014). Earlier observation that inactivation of DNA damage signaling does not rescue the development of PIWI mutant embryos led to the assumption that PIWI proteins might have an essential role in early somatic development, independent of cell cycle checkpoint signaling (Mani et al, 2014). By tracing transposon protein and RNA levels and localization from the mother to the early embryos, we suggest that, independent of Chk2 signaling, de-repressed transposons are responsible for nuclear damage and embryonic lethality. Our study indicates that transposon insertions occur in the maternal genome where they cause DNA DSBs that together with transposon RNAs and proteins are passed on to the progeny embryos. Transposon activity and consequent DNA damage in the early syncytial embryo cause aberrant chromosome segregation, resulting in unequal distribution of the genetic material, nuclear damage and ultimately embryonic lethality. Our study shows that early Drosophila embryos are defenseless against transposons and will succumb to their mobilization if the first line of protection against selfish genetic elements in the nuage of nurse cell fails.
A recent study showed that in p53 mutants, transposon RNAs are up-regulated and accumulate at the posterior pole of the oocyte, without deleterious effects on oogenesis or embryogenesis (Tiwari et al, 2017). It is possible that the absence of pole plasm in vas mutants (Lehmann & Ephrussi, 1994) results in the release of the transposon products and their ectopic accumulation in the oocyte. Localization of transposons to the germ plasm (Tiwari et al, 2017) may restrict their activity to the future germline and protect the embryo soma from transposon activity. Transposon-mediated mutagenesis in the germline would produce genetic variability, a phenomenon thought to play a role in the environmental adaptation and evolution of species. It would therefore be of interest to determine the role of pole plasm in transposon control in the future.
Transposon up-regulation in the Drosophila female germline triggers a DNA damage-signaling cascade (Chen et al, 2007;Klattenhoff et al, 2007). In aub mutants, before their oogenesis arrest occurs, Chk2-mediated signaling leads to phosphorylation of Vasa, leading to impaired grk mRNA translation and embryonic axis specification (Klattenhoff et al, 2007). Considering the genetic interaction of vas and mnk (Chk2) and the fact that Vasa is phosphorylated in Chk2-dependent manner (Abdu et al, 2002;Klattenhoff et al, 2007), it is tempting to speculate that phosphorylation of Vasa might stimulate piRNA biogenesis, reinforcing transposon silencing and thus minimizing transposon-induced DNA damage (Fig 5C). The arrest of embryonic development as a first, and arrest of oogenesis as an ultimate response to DNA damage, thus, prevents the spreading of detrimental transposon-induced mutations to the next generation.

Generation of mnk, vas double mutant flies
To generate mnk, vas double mutants, +, +, mnk P6 [P{lacW}] /CyO and b 1 , vas D1 , +/CyO flies were crossed. F1 progeny +, +, mnk P6 [P{lacW}] /b 1 , vas D1 , + females were then crossed to males of the balancer stock CyO/if. F2 progeny were screened for red eyes (mnk P6 marker P {lacW}) and 200 individual red-eyed flies were mated to CyO/if balancer flies. F3 generation stocks were established and screened for non-balanced flies of a dark body color (homozygous for b 1 , a marker of the original vas D1 chromosome). Three lines were obtained and tested for presence of the vas D1 mutation by Western blotting (Fig S2C) and for presence of the mnk mutation by RT-PCR (Fig S3B). A scheme of the crosses and recombination is shown in Table S6 and sequences of primers used for RT-PCR reaction are shown in Table S7.

Fecundity and hatching assays
Virgin females of all genetic backgrounds tested were mated with w 1118 males for 24 h at 25°C. The crosses were then transferred to apple-juice agar plates, and eggs collected in 24 h intervals over 3-4 d.
The number of eggs laid on each plate was counted; the plates were kept at 25°C for 2 d, then the number of hatched larvae counted. Experiments were performed in three independent replicates represented in Tables S2, S3, and S5. w 1118 females were used as a control.

Ovarian morphology and Vasa localization analysis
Ovaries of 3-to 7-d old flies were dissected in PBS. Ovarian morphology was evaluated under an Olympus SZX16 stereo microscope. Vasa localization was assessed in ovaries of 3-to 7-d old flies expressing the GFP-Vasa proteins after fixation in 2% PFA and 0.01% Triton X-100 for 15 min at RT. Fixed ovaries were mounted on glass slides and GFP fluorescence examined under a Zeiss LSM 780 confocal microscope. Vasa localization in WT and vas mutant ovaries and progeny embryos was analyzed by antibody staining (see below). Nuclei were visualized with NucBlue Fixed Cell Stain (Thermo Fisher Scientific).

Fluorescent in situ RNA hybridization
All FISH experiments were performed as described in Gáspár et al (2018). In brief, ovaries were dissected in PBS and immediately fixed in 2% PFA, 0.05% Triton X-100 in PBS for 20 min at RT. Embryos (1-3 h) were collected and dechorionated in 50% bleach, fixed for 25 min at RT in the heptane/2% PFA interface and devitellinized by vigorous shaking after adding 1 V of methanol. After washing in PBT (phosphate buffered saline + 0.1% Triton X-100), samples were treated with 2 μg/ml proteinase K in PBT for 5 min and then were subjected to 95°C in PBS + 0.05% SDS for 5 min. Proteinase K treatment was omitted when samples were subsequently to be immunohistochemically stained (see below). Samples were pre-hybridized in 200 μl hybridization (C) In WT flies, the occurrence of DNA DSBs activates Chk2 kinase that regulates several mechanisms that together antagonize deleterious effects of DNA damage. Chk2 might directly or indirectly target Vasa that in turn affects piRNA biogenesis and transposon control, reducing the transposon-induced DSBs. Accordingly, DNA damage induced by high levels of transposons in vas mutants triggers DNA damage-induced apoptosis resulting in oogenesis arrest. Oogenesis can be restored by depletion of Chk2; however, transposon deregulation persists and causes severe nuclear damage and embryogenesis arrest preventing distribution of transposon-induced, detrimental mutations within the population. buffer (300 mM NaCl, 30 mM sodium citrate pH 7.0, 15% ethylene carbonate, 1 mM EDTA, 50 μg/ml heparin, 100 μg/ml salmon sperm DNA, and 1% Triton X-100) for 10 min at 42°C. Fluorescently labeled oligonucleotides (12.5-25 nM) were pre-warmed in hybridization buffer and added to the samples. Hybridization was allowed to proceed for 2 h at 42°C. Samples were washed 3 times for 10 min at 42°C in pre-warmed buffers (1× hybridization buffer, then 1× hybridization buffer:PBT 1:1 mixture, and then 1× PBT). The final washing step was performed in pre-warmed PBT at RT for 10 min. The samples were mounted in 80% 2,2-thiodiethanol in PBS and analyzed on a Leica SP8 confocal microscope.
For simultaneous FISH and immunohistochemical staining, ovaries and embryos were fixed as described above. Samples were simultaneously incubated with fluorescently labeled oligonucleotides (12.5-25 nM) complementary to HeT-A RNA and primary antibodies against γH2Av (rabbit; 1:5,000; Rockland) or HeT-A/Gag (rabbit 1:100; gift of Elena Casacuberta) overnight at 28°C in PBT. Samples were washed 2 times for 20 min at 28°C in PBT and subsequently incubated with secondary Alexa 488 conjugated goat anti-rabbit antibodies (1:1,000; Invitrogen). The samples were mounted in 80% 2,2-thiodiethanol in PBS and analyzed on a Leica SP8 confocal microscope.
Labeling of DNA oligonucleotides for fluorescent in situ RNA hybridization Labeling of the oligonucleotides was performed as described in Gáspár et al (2018). Briefly, non-overlapping arrays of 18-22 nt long DNA oligonucleotides complementary to HeT-A or Burdock RNA (Table  S7) were selected using the smFISHprobe_finder.R script (Gáspár et al, 2018). An equimolar mixture of oligos for a given RNA was fluorescently labeled with Alexa 565-or Alexa 633-labeled dideoxy-UTP using terminal deoxynucleotidyl transferase. After ethanol precipitation and washing with 80% ethanol, fluorescently labeled oligonucleotides were reconstituted with nuclease-free water.
Quantification of relative protein expression levels was performed using ImageJ. A frame was placed around the most prominent band on the image and used as a reference to measure the mean gray value of all other protein bands and the background. Next, the inverted value of the pixel density was calculated for all measurements by deducting the measured value from the maximal pixel value. The net value of target proteins and the loading control was calculated by deducting the inverted background from the inverted protein value. The ratio of the net value of the target protein and the corresponding loading control represents the relative expression level of the target protein. Fold-change was calculated as the ratio of the relative expression level of the target protein in the WT control over that of a specific sample.

RNA extraction and quantitative PCR analysis
Total RNA was extracted from ovaries of 3-to 7-d-old flies or 0-to 1-h-old embryos using Trizol reagent (Thermo Fisher Scientific). For first-strand cDNA synthesis, RNA was reverse-transcribed using a QuantiTect Reverse Transcription Kit (QIAGEN). Quantitative PCR (qPCR) was performed on a StepOne real-time PCR system (Thermo Fisher Scientific) using SYBR Green PCR Master Mix (Thermo Fisher Scientific). Relative RNA levels were calculated by the 2 −ΔΔCT method (Livak & Schmittgen, 2001) and normalized to rp49 mRNA levels for ovaries, and 18S rRNA for embryos. Fold-enrichments were calculated by comparison with the respective RNA levels in w 1118 flies. Sequences of primers used for qPCR reaction are shown in Table S7.