Maternally inherited piRNAs direct transient heterochromatin formation at active transposons during early Drosophila embryogenesis

The PIWI-interacting RNA (piRNA) pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. In Drosophila, piRNAs are intergenerationally inherited through the maternal lineage, and this has demonstrated importance in the specification of piRNA source loci and in silencing of I- and P-elements in the germ cells of daughters. Maternally inherited Piwi protein enters somatic nuclei in early embryos prior to zygotic genome activation and persists therein for roughly half of the time required to complete embryonic development. To investigate the role of the piRNA pathway in the embryonic soma, we created a conditionally unstable Piwi protein. This enabled maternally deposited Piwi to be cleared from newly laid embryos within 30 min and well ahead of the activation of zygotic transcription. Examination of RNA and protein profiles over time, and correlation with patterns of H3K9me3 deposition, suggests a role for maternally deposited Piwi in attenuating zygotic transposon expression in somatic cells of the developing embryo. In particular, robust deposition of piRNAs targeting roo, an element whose expression is mainly restricted to embryonic development, results in the deposition of transient heterochromatic marks at active roo insertions. We hypothesize that roo, an extremely successful mobile element, may have adopted a lifestyle of expression in the embryonic soma to evade silencing in germ cells.


Introduction
indication that the piRNA pathway could play roles also in the developing soma, for 116 example helping to establish its epigenetic landscape (Gu and  Here, we exploit a conditional protein degradation strategy to explore the function of 131 maternally deposited piRNAs during Drosophila embryonic development. We find that 132 Piwi-piRNA complexes present in the embryo are primarily derived from the oocyte, 133 whereas  The roo expression peak at 4-6h AEL could be due to transcription from germ cell 243 precursors, which become transcriptionally active around 3.5h AEL (stage 8) (Van 244 Doren et al., 1998;Zalokar, 1976). However, the sheer abundance of roo and other 245 transposon transcripts argued strongly that they must emanate at least in part from 246 somatic nuclei, as these vastly outnumber the germ cell precursors. To test directly 247 the origin of roo transcripts during embryogenesis, we performed RNA fluorescence 248 in situ hybridisation (RNA-FISH). In agreement with our RNA-seq data, roo transcripts 249 were detected as early as stage 6 (in gastrulating embryos ~3h AEL) and localised 250 predominantly to yolk cell nuclei ( Figure 1D, Figure 1-figure supplement 2B). Stage 251 11 embryos (~5h AEL) showed strong roo RNA signal in somatic cells of the 252 mesoderm, similar to earlier reports (Bronner et al., 1995;Ding and Lipshitz, 1994 as a propagation mechanism. To determine whether roo-encoded proteins are 262 expressed in embryos, we mined quantitative proteomic data from three 263 developmental intervals ( Figure 1A). The first, 0-2h AEL, represents the time before 264 ZGA when the proteome is derived from maternal protein deposition and zygotic 265 translation of maternal mRNAs. The second, 5-7h AEL, represents an interval where 266 zygotic roo expression had become robust, and the third, 10-12h AEL, is a time at 267 which roo RNA levels had substantially declined.

279
Compared to the early time point (0-2h AEL), 5-7h AEL embryos showed significant 280 accumulation of roo peptides (p<0.01) corresponding to its expression peak. roo 281 encodes a single ORF (with a predicted protein weight of 272 kDa), which contains a 282 Group-specific antigen-like protein (gag), a reverse transcriptase (RT/pol), an 283 envelope protein (env), two peptidases-like domains (Pep), and a zinc finger ( Figure  284 1-figure supplement 2E). We detected peptides corresponding to the gag, pol and 285 env proteins (Figure 1-figure supplement 2E, bottom), indicating potential 286 competence for retrotransposition. We additionally detected proteins derived from 287 other transposons including copia and 297. Of note, roo ORFs remained detectable 288 at 10-12h AEL (Figure 1-figure supplement 2F), possibly suggesting substantial 289 stability, as this was a time at which roo mRNA levels had diminished.

291
The known cohort of piRNA coTGS factors is present during embryogenesis 292 293 The decline in transposon expression from 4-6h to 10-12h intervals of embryogenesis 294 could potentially involve the piRNA pathway. However, piRNA-guided post-295 transcriptional or co-transcriptional silencing also requires a growing list of additional 296 proteins (reviewed in (Czech et al., 2018;Ozata et al., 2019)). We therefore probed 297 the expression of known piRNA pathway components during various stages of 298 embryogenesis in our transcriptomic and proteomic datasets. 299 300 With the exception of Piwi, genes involved in coTGS were both maternally deposited 301 and zygotically expressed during the first ~10h of embryogenesis (Figure 1-figure  302 supplement 2A  In contrast, we noted little or no maternal deposition and low zygotic expression of key 317 components of the piRNA precursor expression and export machinery and of critical 318 piRNA biogenesis factors (Figure 1-figure supplement 2A Piwi during the pre-blastoderm stage (NC1-9, ~0-30min AEL) localized to the posterior 338 pole where it formed a crescent-like structure (Video S1, Figure 2C). 339 340          dynamic data revealed that nuclear Piwi signal strongly decreased during mitotic 390 cycles, with little fluorescence signal overlapping with H2Av-RFP during nuclear 391 divisions (Video S1, Figure 2D). We continued to detect Piwi expression in somatic 392 nuclei throughout the first 10h of embryogenesis; however, signal intensity decreased 393 over time. This observation was consistent with transcriptomic and proteomic 394 measurements taken over a comparable time course (Figure 2A insertions that are absent from the dm6 reference genome, and these were used for 453 our chromatin analyses, as most annotated insertions in the dm6 genome assembly 454 were absent from our strain.

456
In order to determine the fate of transposon loci throughout embryogenesis, we 457 performed H3K9me3 chromatin immunoprecipitation followed by sequencing (ChIP-  To investigate whether this mechanism is specific to roo or more general, we 526 examined the transposon 297, which is also expressed during embryogenesis ( Figure  527 1-figure supplement 1F) and showed high targeting potential by maternally inherited 528 piRNAs ( Figure 2G). Genomic loci in close proximity to euchromatic 297 insertions 529 (n=20) showed dynamic deposition of H3K9me3 similar to roo (Figure 3- figure  530 supplement 1B). However, while H3K9me3 levels at roo insertions peaked between 531 6-10h AEL, 297 insertions showed the maximum H3K9me3 signal intensity between 532 2-8h AEL, suggesting that these loci are targeted by coTGS earlier than roo insertions. 533 In contrast, H3K9me3 occupancy at transposons such as mdg1 and 412 that were 534 expressed during embryogenesis but lacked substantial maternal deposition of 535 piRNAs, retained low H3K9me3 levels throughout embryogenesis, though they 536 showed a strong enrichment in ovaries (Figure 3-figure supplement 1C).

538
To determine whether the deposition of repressive chromatin marks at euchromatic 539 297 and roo insertions was specific, rather than reflecting a general trend of H3K9me3 540 accumulation genome-wide, we analysed genomic regions not targeted by maternally 541 inherited piRNAs. H3K9me3 signal at constitutive heterochromatin remained stable 542 throughout the sampled time points (Figure 3-figure supplement 1D), while 543 H3K9me3 levels on chromosome 4 increased steadily throughout development 544 (Figure 3-figure supplement 1E). Of note, while ovaries showed no coTGS signature 545 at roo insertions, other transposons, such as Doc, showed a clear accumulation of 546 H3K9me3 marks that was absent in embryos during all assayed time points ( Figure  547 3B). Considered together, these results are consistent with piRNA-guided chromatin 548 modification of a subset of transposons that show activity during Drosophila embryonic 549 development.

551
An auxin-inducible degron enables rapid depletion of Piwi in ovaries and early 552 embryos 553 554 Though embryonically repressed transposons bore hallmarks of piRNA-guided 555 heterochromatin formation, the reliance of the pathway on maternally deposited  piRNA complexes prevented a demonstration that silencing depended on the pathway 557 through conventional genetics. Ovaries that lack key piRNA pathway silencing factors 558 show substantial expression changes and produce morphologically altered eggs that 559 yeast paste was sufficient to induce complete degradation of Piwi in ovaries ( Figure  581 4B, C, Figure 4-figure supplement 1A), and this depletion resulted in the de-582 repression of transposons ( Figure 4D). Notably stronger changes were observed 583 following longer treatments, possibly implying a lag between loss of piRNA pathway 584 function and that of repressive chromatin marks. Following a 1-day treatment, embryos 585 laid by Piwi-depleted females developed without defects and showed similar hatching 586 rates as their control treated siblings ( Figure 4E, F). Longer auxin treatments resulted 587 in more frequent deformation of embryos that was accompanied by reduced hatching 588 rates ( Figure 4E, F), likely due to patterning defects as a result of Piwi depletion from 589 follicle cells.

591
Drosophila embryos develop within a relatively impermeable chorion, and treatment 592 of embryos directly with auxin showed little impact. However, in dechorionated 593 embryos we observed a near complete degradation of Piwi protein following 30min 594 auxin treatment of embryos collected for 0-30min AEL ( Figure 5A, B). To investigate 595 the dynamics of auxin-mediated Piwi depletion in embryos, we used light-sheet 596 fluorescence live microscopy. Early blastoderm embryos treated with 5mM auxin 597 showed rapid degradation of GFP-AID-Piwi signal, which was undetectable after 598 25min of treatment ( Figure 5C, Video S4). Of note, the removal of maternal Piwi in 599 this time window did not significantly affect the embryo hatching rate (Figure 4-figure  600 supplement 1B).     We next investigated the impact of degrading maternal Piwi from early-stage embryos 637 on transposons. Embryos derived from flies expressing GFP-AID-Piwi and OsTIR1 638 were collected across a 30min period and treated for an additional 2.5h with or without 639 5mM auxin before RNA extraction and library generation ( Figure 5A). These embryos 640 corresponded to 2.5-3h AEL, the point at which we began to observe zygotic roo 641 transcripts ( Figure 1C). The majority of transposons showed no significant expression 642 change upon Piwi depletion, however, roo and 297 were significantly de-repressed 643 (p<0.05) by more than 2-fold ( Figure 5D) Trost et al., 2016) but to control for effects of auxin itself on TE regulation, we also 647 evaluated transposon expression in auxin-treated GFP-AID-Piwi embryos that lack 648 OsTIR1. Without OsTIR1, 2.5-3h embryos treated with 5mM auxin showed no 649 significant changes in transposon expression, compared to control siblings treated 650 with PBS ( Figure 5-figure supplement 1A).

652
We additionally examined changes in the repressive chromatin mark H3K9me3 to 653 determine whether these were deposited in a piRNA-dependent fashion, at 654 euchromatic roo and 297 transposon insertions. We again collected embryos for 655 30min and treated with 5mM auxin (or PBS as a negative control) for 6h which yielded 656 embryos 6-6.5h AEL ( Figure 5A) and corresponds to the peak in H3K9me3 signal at 657 roo insertions in control w 1118 embryos ( Figure 3A, Figure 3-figure supplement 1A). 658 Piwi depletion severely impacted H3K9me3 signal over the transposon consensus 659 sequence of roo and 297, but not that of other TEs ( Figure 5E)     ). Yet, to date, the lack of mechanisms to rapidly 750 deplete maternally deposited PIWI proteins specifically from early embryos has 751 hampered our ability to broadly assess their zygotic roles. By fusing a chemically-752 inducible degron to Piwi, we were able to deplete Piwi-piRNA complexes from 753 dechorionated embryos within less than 30min of treatment and well before the 754 nuclear accumulation of Piwi that is observed following activation of zygotic 755 transcription.

757
Though nuclear localisation of Piwi correlates with the appearance of its potential whether it also occurs in germline and follicle cells or is restricted to embryogenesis 772 remains unclear.

774
It has been suggested that the evolution of the abbreviated piRNA pathway in ovarian 775 follicle cells arose as a consequence of the lifestyle adopted by gypsy family elements. While this remains speculative, it does provoke questions of whether a similar strategy 786 is adopted by roo in the embryo. roo is a quite successful element, as indicated by it 787 being the element with the highest copy number of individual insertions in our 788 sequenced strains (9.4% of all identified TE insertions in the w 1118 strain and 9.9% in 789 our degron line). How this is achieved remains mysterious, since roo expression is 790 extremely low in the ovary. Moreover, roo does not appear to be a target of the ovarian 791 piRNA pathway, since its gonadal expression is not increased nor does its HP1a 792 enrichment and H3K9me3 levels change in piRNA pathway mutant animals ( Figure  793 5 giving rise to the adult mesoderm. Previous studies have suggested that roo 796 expression is activated by twist (twi) and snail (sna), which are highly expressed in the 797 embryogenic mesoderm (Bronner et al., 1995), and this is consistent with the spatial 798 expression pattern that we also observe. roo expresses the full repertoire of proteins 799 needed to form virus-like particles, and its high expression levels (exceeding 1% of 800 the transcriptome at its peak) might enable a strategy of propagation by infection in 801 trans, even if rates of transmission to the germ cell precursors are relatively low.

803
Our data strongly suggests that only maternally deposited piRNAs engage Piwi in the 804 soma of the developing embryo. Since roo is not regulated by the piRNA pathway in Piwi but maintained in a Piwi-independent mechanism throughout adult life (Gu and 864 Elgin, 2013 Fly ovaries were dissected in ice-cold PBS and fixed in 4% PFA diluted in PBS for 930 15min at room temperature while rotating. Following 3 rinses and three 10min washing 931 steps in PBS-Tr (0.3% Triton X-100 in PBS), ovaries were blocked for 2h at RT while 932 rotating in PBS-Tr +1% BSA. Primary antibody incubation was carried out in blocking 933 buffer overnight at 4°C while rotating, followed by three washing steps for 10min each 934 in PBS-Tr. All following steps were performed in the dark. Secondary antibodies were 935 diluted in blocking buffer and incubated overnight at 4°C while rotating. Ovaries were 936 washed 4 times for 10min in PBS-Tr and stained with 0.5µg/ml DAPI (Thermo Fisher 937 Scientific) for 10min. Following two additional washing steps for 5min in PBS, ovaries 938 were mounted in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific) and 939 imaged on a Leica SP8 confocal microscope using a 40x Oil objective.

941
Drosophila embryo immunofluorescence 942 Embryos were collected and dechorionated in 50% bleach for 1min. Embryos were 943 transferred into 1ml fixing solution (600µl 4% PFA in PBS, 400µl n-heptane) and fixed 944 for 20min at RT while rotating. The lower aqueous phase was removed and 600µl 945 methanol added. The tube was vortexed vigorously for 1min to remove vitelline 946 membranes. Embryos were allowed to sink to the bottom of the tube and all liquid was 947 removed, followed by two washes with methanol for 1min each. Embryos were stored 948 at -20°C at least overnight or until further processing. In order to rehydrate embryos, 949 three washes each 5min with PBST (0.1% Tween20 in PBS) were performed and 950 embryos blocked for 1h at RT in PBST + 5% BSA. Primary antibodies were incubated 951 overnight at 4°C while rotating in blocking buffer followed by 3 washes for 15min each 952 with PBST. All following steps were performed in the dark. Secondary antibodies were 953 diluted in blocking buffer and incubated at RT for 2h. Embryos were rinsed 3 times 954 and washed 2 times for 15min. Nuclei were stained with 0.5µg/ml DAPI (Thermo 955 Fisher Scientific) for 10min. Following two additional washing steps for 5min in PBS, 956 embryos were mounted in ProLong Diamond Antifade Mountant (Thermo Fisher 957 Scientific) and imaged on a Leica SP8 confocal microscope using a 40x Oil objective.

980
Light Sheet Fluorescent Microscopy (LSFM) of Drosophila embryos 981 Embryos were collected and dechorionated as described above. 1ml of 1% low melting 982 point (LMP) agarose was prepared and embryos transferred into capillaries (catalogue 983 number 100003476381, Brand) using a fitting plunger. Embryos were attempted to be 984 positioned vertically in the capillary by twisting until agarose solidified. Capillaries were 985 stored in PBS at RT until imaging. LSFM was performed on a Zeiss Lightsheet Z.1 986 (Carl Zeiss, Germany) at 25°C with a 20x/1.0 Plan-Apochromat water-immersion 987 objective lens. Embryos were lowered carefully out of the capillary into the imaging 988 chamber filled with PBS and positioned directly between the light sheet illumination 989 objectives (10x/0.2, left and right). Z-stack images for GFP and RFP (excitation at 488 990 and 561 nm, respectively) were acquired every 2min for >10h with the lowest possible 991 laser intensity (2.5% for GFP and 10% for RFP). Generated data was analysed in 992 Zeiss ZEN Imaging Software and Fiji (ImageJ).

994
ChIP-seq for Drosophila embryos 995 50µl of embryos were collected and dechorionated as described above and 996 transferred in 1ml Crosslinking solution (1% formaldehyde in PBS, 50% n-heptane) 997 and vortexed on high speed for precisely 15min. 90µl 2.5M glycine solution was added 998 to quench excess formaldehyde and incubated for 5min at RT while rotating. Embryos 999 were allowed to sink to the bottom of the tube and all liquid was removed. Embryos 1000 were washed three times for 4min with ice-cold buffer A (60mM KCl, 15mM NaCl, 4mM 1001 ggplot2. Differential expression analysis was performed using DESeq2 (Love et al., of LINE and Retroviral Transposons. Cell 174, 1082-1094 e1012.