© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 259-268 doi:10.1242/dev.101410

RESEARCH ARTICLE STEM CELLS AND REGENERATION

Zfrp8/PDCD2 is required in ovarian stem cells and interacts with the piRNA pathway machinery Svetlana Minakhina*,‡, Neha Changela* and Ruth Steward‡

ABSTRACT suppress the activity of transposable elements (TEs) and protect The maintenance of stem cells is central to generating diverse cell genome integrity during germ stem cell (GSC) differentiation and populations in many tissues throughout the life of an animal. oocyte development (reviewed by Siomi et al., 2011; Guzzardo et Elucidating the mechanisms involved in how stem cells are formed al., 2013; Peng and Lin, 2013). Different sets of TEs are active in and maintained is crucial to understanding both normal the ovarian germline and surrounding somatic cells and are developmental processes and the growth of many cancers. regulated by piRNA mechanisms that partially overlap (Malone et Previously, we showed that Zfrp8/PDCD2 is essential for the al., 2009). The pathway used in the somatic cells is called the maintenance of Drosophila hematopoietic stem cells. Here, we show primary piRNA processing pathway. The primary single-stranded that Zfrp8/PDCD2 is also required in both germline and follicle stem RNAs are transcribed from piRNA clusters and exported into the cells in the Drosophila ovary. Expression of human PDCD2 fully cytoplasm. Their maturation requires the RNA helicase Armitage rescues the Zfrp8 phenotype, underlining the functional conservation (Armi) (Klattenhoff et al., 2007; Olivieri et al., 2010; Saito et al., of Zfrp8/PDCD2. The piRNA pathway is essential in early oogenesis, 2010; Qi et al., 2011), co-chaperone Shutdown (Shu) (Munn and and we find that nuclear localization of Zfrp8 in germline stem cells Steward, 2000; Olivieri et al., 2012; Preall et al., 2012), and their offspring is regulated by some piRNA pathway genes. We endoribonuclease Zucchini (Zuc) (Pane et al., 2007; Nishimasu et also show that Zfrp8 forms a complex with the piRNA pathway protein al., 2012), and soma-specific Tudor domain-containing RNA Maelstrom and controls the accumulation of Maelstrom in the nuage. helicase, Yb [Fs(1)Yb – FlyBase] (Olivieri et al., 2010; Saito et al., Furthermore, Zfrp8 regulates the activity of specific transposable 2010; Qi et al., 2011). Then primary piRNAs complex with Piwi and elements also controlled by Maelstrom and Piwi. Our results suggest are targeted to the nucleus (Cox et al., 2000; Ishizu et al., 2011; that Zfrp8/PDCD2 is not an integral member of the piRNA pathway, Darricarrère et al., 2013). Current studies suggest that Piwi silences but has an overlapping function, possibly competing with Maelstrom TEs at the transcriptional level by inducing chromatin changes at and Piwi. genomic TE sites (Brower-Toland et al., 2007; Klenov et al., 2007; Sienski et al., 2012; Huang et al., 2013; Le Thomas et al., 2013; KEY WORDS: Somatic stem cells, Germline stem cells, piRNA Rozhkov et al., 2013). pathway, Drosophila TE silencing in the germline requires two additional Piwi family proteins, Aubergine (Aub) and Argonaute 3 (AGO3) (Harris and INTRODUCTION Macdonald, 2001; Vagin et al., 2004; Brennecke et al., 2007; Gene expression in multicellular organisms is regulated at many Gunawardane et al., 2007; Li et al., 2009). Unlike Piwi, Aub and levels. Complex transcriptional control is followed by processing, AGO3 are cytoplasmic proteins. They mainly localize to the transport and translation of all mRNAs. Small RNAs function in all germline-specific perinuclear structure called the nuage (Harris and of these steps by guiding the regulatory protein complexes to Macdonald, 2001; Brennecke et al., 2007; Lim and Kai, 2007; Patil specific RNA and DNA targets. One class of small RNAs is the and Kai, 2010). The nuage is thought to serve as a docking site for repeat-associated small interfering RNAs (rasi-RNAs) or, more assembly of the piRNA machinery and as a site of ‘ping-pong’ commonly, Piwi-interacting RNAs (piRNAs) (Saito et al., 2006; piRNA amplification (Gunawardane et al., 2007; Lim and Kai, Vagin et al., 2006; Brennecke et al., 2007). These 23-31 nt small 2007; Ishizu et al., 2011; Siomi et al., 2011). The nuage contains RNAs are produced by Dicer-independent mechanisms and many other conserved components of the piRNA pathway, including associate with Piwi family Argonaute (AGO) proteins. Both RNAs Vasa (Vas), Spindle E (Spn-E) and Maelsrom (Mael) (Findley et al., and protein components of the piRNA pathway are most abundantly 2003; Vagin et al., 2004; Klenov et al., 2007). expressed in the gonads of all animals, but recent studies suggest Zinc finger protein RP-8 (Zfrp8), PDCD2 in vertebrates, is a that the pathway may similarly function in other somatic stem and conserved protein with unknown molecular function (Minakhina et progenitor cells (reviewed by Juliano et al., 2011; Siddiqi and al., 2007). All Zfrp8/PDCD2 proteins share a zinc finger, Myeloid, Matushansky, 2012; Mani and Juliano, 2013; Peng and Lin, 2013). Nervy and Deaf1 (MYND) domain, present in a large group of Much of the progress in understanding piRNA pathway proteins and involved in protein-protein interactions (Matthews et mechanisms comes from Drosophila ovaries, where piRNAs al., 2009). Mammalian PDCD2 is most prevalent in the cytoplasm, but is also detected in the nucleus, where it is associated with chromatin (Scarr and Sharp, 2002; Mu et al., 2010). Rutgers University, Department of Molecular Biology, Waksman Institute, Cancer We showed previously that Zfrp8/PDCD2 is essential in fly Institute of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA. *These authors contributed equally to this work hematopoietic stem cells (HSCs), but is largely dispensable in more mature cells (Minakhina and Steward, 2010). PDCD2 is highly ‡ Authors for correspondence ([email protected]; expressed in human HSCs and precursor cells (Kokorina et al., [email protected]) 2012; Barboza et al., 2013). Zfrp8/PDCD2 is also essential in mouse

Received 17 July 2013; Accepted 15 October 2013 embryonic stem cells (Mu et al., 2010), and profiling of mouse Development

259 RESEARCH ARTICLE Development (2014) doi:10.1242/dev.101410 embryonic, neural and hematopoietic stem cells showed an morphology and numbers of wild-type and Zfrp8 escort cell clones enrichment of PDCD2 mRNA (Ramalho-Santos et al., 2002). were similar to those observed previously (Fig. 1A-C,E) (Kirilly et To investigate if the requirement of Zfrp8 is restricted to al., 2011). The presence of Zfrp8 escort cells suggests that in Drosophila hematopoiesis and to obtain insight into the molecular contrast to the FSCs, Zfrp8 function is dispensable in these somatic function of the gene, we studied the Zfrp8 phenotype in ovaries and self-duplicating cells. Using the same approach we also looked for found that loss of Zfrp8 protein results in the abnormal development transient Zfrp8 clones 2 and 5 days ACI. At each time point, the of germline and somatic stem cell-derived cells. Importantly, we number and morphology of wild-type and mutant clones were found that Zfrp8 is essential in stem cells, as both somatic and similar. Together, our results show that loss of Zfrp8 only affects germline mutant stem cells stop dividing and are ultimately lost. The cells derived from mutant FSCs and the FSCs themselves. The phenotype can be rescued by the expression of human PDCD2, requirement of Zfrp8 in ovarian stem cells is in agreement with our demonstrating that the molecular function of Zfrp8/PDCD2 is prior observations that the gene is required in lymph gland stem conserved. We discovered genetic interactions of Zfrp8 with piRNA cells but not in prohemocytes (Minakhina and Steward, 2010). pathway genes and confirmed their close connection at the cellular and molecular levels. We show that Zfrp8 complexes with Mael and Zfrp8 function in the germline is required for perinuclear localization of Mael in GSCs and To investigate the requirement of Zfrp8 in the germline and GSCs, cystoblasts. In turn, the nuage components Spn-E, AGO3 and Vas we induced clones by flippase recognition target (FRT)-mediated are required for nuclear localization of Zfrp8. Zfrp8 regulates a site-specific mitotic recombination (see Materials and Methods) (Xu subset of germline-specific TEs, which are also regulated by Piwi and Rubin, 1993). Phenotypes were analyzed 10 and 20 days ACI. and Mael. Although Zfrp8 has weaker effects on transposon At both time points, we detected similar numbers of wild-type and expression than piwi and mael, it causes stronger phenotypes, Zfrp8 clones (45% of ovarioles 10 days ACI and 15% of ovarioles especially in stem cells. We propose that Zfrp8, though not an 20 days ACI). But Zfrp8 clones showed strong phenotypes that integral component of the piRNA pathway, does have overlapping became more pronounced in older clones. We did not detect Zfrp8 function with Piwi and Mael and is particularly important in the egg chambers older than stage 8 (Fig. 2A-D) and ovarioles maintenance of stem cell identity and survival. containing only mutant germ cells accumulated several egg chambers of similar size (stage 4 to 5; Fig. 2A, all egg chambers in RESULTS 2D). In mosaic ovarioles, smaller Zfrp8 egg chambers were located Zfrp8 is essential in follicle stem cells posterior to developmentally more advanced heterozygous cysts Zfrp8 was originally identified by its strong hematopoietic (Fig. 2B). These phenotypes were observed in 40% of ovarioles 10 phenotype and lethality (Minakhina et al., 2007). Clonal analysis in days ACI and 35% 20 days ACI (small egg chambers in Fig. 2D). lymph glands showed that Zfrp8 is essential in stem cells, as no Twenty days ACI, 7% of Zfrp8 clones were restricted to the persistent clones were recovered, but is dispensable in more mature germarium compared with none in control GSC clones. Thus, cells, as transient Zfrp8 clones had the same developmental potential similar to the phenotype observed in the soma, depletion of Zfrp8 in as wild-type clones (Minakhina and Steward, 2010), for clone the germline stem cells results in germline cysts that cannot support nomenclature see Fox et al. (Fox et al., 2008). To investigate normal development. whether Zfrp8 may be required in other stem cells, we studied its To investigate the function of Zfrp8 in GSCs further, we obtained function in the Drosophila ovary, which contains two distinct a short hairpin RNAi line (TRiP at Harvard Medical School, Boston, populations of stem cells: GSCs and somatic or follicle stem cells MA, USA) and expressed Zfrp8 RNAi under the nos-Gal4 germline- (SSCs or FSCs) located in the germarium (reviewed by Kirilly and specific driver (see Materials and Methods). Ovaries from flies Xie, 2007; Morrison and Spradling, 2008; Losick et al., 2011; carrying either the RNAi or the driver alone did not show any Spradling et al., 2011). phenotype, but the Zfrp8 knockdown (KD) ovaries displayed an To determine the function of Zfrp8 in follicle stem cells, we array of phenotypes that worsened with the age of the flies (Fig. 3). induced mosaic analysis with a repressible cell marker (MARCM) In young flies (<2 days old) the organization of the germarium was clones in wild type and Zfrp8/+ (see Materials and Methods) (Lee close to normal and contained GSCs that gave rise to a few egg and Luo, 2001), and analyzed persistent clones in adult females 10 chambers (Fig. 3H,J). Ovarioles of older flies (7-10 days) either and 20 days after induction (ACI) (Fig. 1) when transient clones contained a large cyst of cells instead of a well-organized germarium were no longer detectable in the ovary (Margolis and Spradling, and egg chambers (Fig. 3A) or ovarioles that lost most or all of the 1995). In wild-type ovaries 10 days ACI, we observed persistent germline cells (Fig. 3A, arrow). clones in nearly 60% of ovarioles (Fig. 1E), whereas the number of To verify that the observed phenotype was specific to the loss of ovarioles with Zfrp8 clones was only ~20%. Twenty days ACI, 38% Zfrp8, we complemented the nos-Gal4 Zfrp8 KD by expressing the of ovarioles contained wild-type clones but only 2% contained Zfrp8 green fluorescent protein (GFP)-tagged human homolog of Zfrp8, clones. Wild-type persistent clones usually included one stem cell PDCD2, under the same driver. The rescued GSCs gave rise to and ~50% of the follicle cells in one ovariole (Fig. 1A,B, arrow). By normal egg chambers, and the flies were fertile (Fig. 3C,D). These contrast, mutant clones were often found in only few egg chambers results underline the strong functional conservation of the fly Zfrp8 and were smaller (Fig. 1D), suggesting that mutant FSCs initially and human PDCD2 proteins. undergo a few divisions but that their progeny cannot sustain normal In both Zfrp8 germline clones and Zfrp8 KD ovaries, we observed development. The absence of mutant stem cells or persistent clones abnormalities in the stem cells and disruption of egg chamber in older flies demonstrates that Zfrp8 stem cells stop dividing and development, but depletion of Zfrp8 due to KD had a stronger are lost. phenotype. To assess the effect of loss of Zfrp8 on GSCs, we stained Interestingly, in the absence of Zfrp8 persistent clones Zfrp8 ovaries with P-MAD, a marker for TGFβ signaling between the niche mutant escort cells were observed with similar frequency as wild- and GSCs (Kai and Spradling, 2003), and an antibody (1B1) that type controls [~70% of ovarioles 10 days ACI, and ~40% of stains spectrosomes and fusomes (de Cuevas et al., 1996). In wild-

ovarioles 20 days ACI (Fig. 1B′,C′ arrowheads, 1E)]. The location, type GSCs round spectrosomes are located on the anterior side next Development

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Fig. 1. Zfrp8 is required for somatic stem cell maintenance. Wild-type MARCM FSC persistent clones (green) in ovariole (A,A′) and germarium (B,B′). The number of Zfrp8 FSC clones (C-D′) is strongly reduced 10 and 20 days ACI (E), whereas the number of wild-type and Zfrp8 escort cell clones (arrowheads in A′-C′) is similar (E). Abnormal, persistent Zfrp8 clones are observed 10 days ACI. FSC clones encompass less than 50% of the follicle cells (compare D and A). See also inset (D′ and D″, MAX projection). The number and appearance of wild-type and Zfrp8 transient clones (2 and 5 days ACI) are similar (F). MARCM clones are labeled with CD8-GFP (green), 1B1 (red) stains follicle cells, fusomes and spectrosomes, DNA is shown in blue. Scale bars: 10 μm. WT, wild type.

to the cap cells that form the niche, and in dividing GSCs it forms an Orb in early oogenesis. In normal ovarioles, both proteins become asymmetric extension reminiscent of an ‘exclamation mark’ localized to the oocyte as soon as the 16-cell cyst is formed and (Fig. 3E,E′, arrows). The majority of Zfrp8 GSC clones (60% in 65 control the establishment of the polar axes (Lantz et al., 1994; Oh total mutant stem cells analyzed) contained morphologically abnormal and Steward, 2001). In Zfrp8-depleted egg chambers BicD failed to spectrosomes. Frequently, there was no spectrosome visible on the localize to the oocyte and remained distributed throughout all 16 cap-side, and only a short dumbbell-shaped spectrosome connecting cyst cells, forming aggregates, whereas Orb accumulated and was the GSC to a daughter cystoblast was detected (Fig. 3F′, arrow). We abnormally distributed within the oocyte. Oocyte specification and did not detect the same abnormalities in wild-type GSCs. In Zfrp8 KD polarity is disrupted in a similar way in ovaries with defects in the GSCs the spectrosomes were also abnormal and frequently replaced dynein motor complex and in piRNA pathway mutants, such as spn- by a fusome connecting several cells, a hallmark of more advanced E, aub, armi, vas and mael (Paré and Suter, 2000; Navarro et al., cysts (Fig. 3B,D). Interestingly, these abnormal GSCs kept their 2009). The abnormal distribution of the BicD and Orb proteins was connection to the cap cells and were positive for P-MAD, indicating reminiscent of our findings several years ago in shutdown (shu) that TGFβ signaling between the niche and GSCs was not affected mutants when we identified shu as a gene regulating BicD (Fig. 3B, arrowhead) (Kai and Spradling, 2003). localization within the egg chamber and the oocyte (Munn and To explore how Zfrp8 affects egg chamber formation and oocyte Steward, 2000). Shu protein has recently been shown to be a part of

development, we studied the distribution of Bicaudal D (BicD) and the piRNA biogenesis machinery, acting as a co-chaperone (Olivieri Development

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Fig. 2. Zfrp8 function in germline. Germline Zfrp8 FRT clones (A-B′) in Zfrp8/Ubi-GFP.nls background. Mutant stem cells and their progeny cysts are recognized by the absence of GFP. Similar numbers of wild-type and mutant clones were observed 10 and 20 days ACI (C), but Zfrp8 mutant cysts show delay in growth and development that worsen with time (D). In both mosaic ovarioles and ovarioles in which all egg chambers were mutant, cyst development stopped before stage 8. Undersized mutant cysts were frequently observed posterior to more mature wild-type cysts (B, arrows in B′, ‘Small egg chambers’ in D). In ovarioles with all mutant egg chambers (‘All egg chambers’ in D), cysts appeared to reach mostly stage 4 to 5 (A,A′). 20 days ACI 7% (compare with 0% in wild type) of mutant clones were confined to the germarium (‘Germarium only’ in D) and showed a strong GSC phenotype (see Fig. 3F,F′). 1B1 (red), Vasa (red), DNA (blue). Scale bars: 10 μm.

et al., 2012; Preall et al., 2012). In addition, a number of piRNA (supplementary material Table S1A,B). The egg phenotypes of vas, pathway mutants [piwi, fs(1)Yb, zuc, cuff] cause severe AGO3 and spn-E mutants were notably enhanced by lack of one abnormalities in germline development, reduction in GSC divisions copy of Zfrp8, consistent with their effect on Zfrp8 nuclear and eventual GSC loss (Cox et al., 1998; King et al., 2001; localization. These results suggest that Zfrp8 functions in the Szakmary et al., 2005; Chen et al., 2007; Klenov et al., 2011). These nucleus and that its function is regulated at least in part by piRNA observations lead us to investigate a possible function of Zfrp8 in pathway genes. the piRNA pathway. Mael and Zfrp8 interaction Nuage components alter Zfrp8 protein distribution We further defined the function of Zfrp8 within the piRNA pathway Zfrp8 is expressed at all stages of development (supplementary by looking at the distribution of piRNA pathway proteins in Zfrp8 material Fig. S1B; FlyBase data) (Tweedie et al., 2009; Marygold et ovary clones and Zfrp8 KD germaria. We did not observe notable al., 2013). To study the tissue-specific expression and subcellular changes in Piwi nuclear localization or in the cytoplasmic distribution localization of Zfrp8 we raised antibodies that specifically recognize of Armi (data not shown; supplementary material Fig. S3). Three the protein on western blots and in tissues (see Materials and cytoplasmic proteins, Vas, AGO3 and Aub, which normally Methods; supplementary material Fig. S1). The protein was present accumulate in the nuage of nurse cells, also show similar perinuclear in all cells of the ovary, distributed throughout the cytoplasm and localization in GSCs and cystoblasts. In Zfrp8 KD germaria their nucleus (Fig. 4A,A′). distribution was not disrupted (Fig. 5A-B′, supplementary material A large number of piRNA pathway proteins are enriched in the Fig. S3) suggesting that Zfrp8 does not affect the formation of nuage- cytoplasm, often in the nuage (Lim and Kai, 2007; Pane et al., 2007; like structures in the germarium. By contrast, the distribution of Mael Patil and Kai, 2010). By contrast, Piwi and Mael proteins are was clearly changed in both Zfrp8 KD and Zfrp8 GSC clones detected in the cytoplasm, the nuage and the nucleus (Findley et al., (Fig. 5D-E′″, white arrows). In normal germaria, Mael shows strong 2003; Klenov et al., 2011; Sienski et al., 2012), and their subcellular perinuclear accumulation (Fig. 5C,C′,E-E′″, green arrows) and is also distribution is regulated by the cytoplasmic factors Armi, Aub, Spn- found in the cytoplasm and the nucleus. The nuage-like localization E and Vas (Findley et al., 2003; Sienski et al., 2012). To investigate of Mael was lost in Zfrp8 GSCs and cystoblasts and Mael protein the possible connection between Zfrp8 and the piRNA pathway we distribution appeared more uniform, reminiscent of Mael distribution tested the subcellular localization of Zfrp8 in a number of mutants. previously observed in vas and spn-E mutant ovaries, where the nuage We found that the nuclear localization of Zfrp8 was most strongly is disrupted (Findley et al., 2003). reduced in vas, AGO3, spn-E and squ GSCs (Fig. 4; for all tested In contrast to vas and spn-E, the egg phenotype of mael and the mutants see supplementary material Fig. S2). ovary phenotype of piwi are suppressed by loss of one copy of Zfrp8 To study the relationship between Zfrp8 and piRNA pathway (supplementary material Table S1), suggesting that Zfrp8 has an factors we tested whether lack of one copy of Zfrp8 can modify the opposite function to that of Mael and Piwi and is required phenotypes of mutants with an established function in piRNA downstream of or in parallel with the two proteins. biogenesis, even though Zfrp8/+ ovaries do not show a notable Zfrp8, Piwi and Mael are all found in the cytoplasm and the phenotype. Zfrp8 dominantly enhanced or suppressed the ovary or nucleus. This led us to investigate whether the three proteins are

egg phenotypes of the majority of piRNA pathway mutants physically associated. Co-immunoprecipitation with anti-Mael Development

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Fig. 3. Loss of Zfrp8 results in abnormal GSCs and failed oogenesis. The array of phenotypes observed in Zfrp8 KD driven by nos-Gal4 (A,B,H,J) ovaries was rescued by expression of human Zfrp8 homolog, PDCD2 (37% identical aa sequence) in the same genetic background (nos-Gal4, UAS- Zfrp8 RNAi, UAS-GFP-PDCD2 C,D, green). In 10- day-old females Zfrp8 KD (A,B) leads to fused cysts and eventual loss of germline (arrow in A). Zfrp8 KD GSCs were P-MAD (red) positive (B, arrowhead), indicating that normal niche signaling has occurred. However, instead of normal spectrosomes (red in D), the Zfrp8 KD stem cells contain fusome-like structures (green in B) typical of wild-type cysts. Similarly, Zfrp8 mutant stem cells were often found connected to the cystoblast with symmetric fusomes; compare the dumbbell- shaped spectrosome/fusome in Zfrp8 GSC clone (red in F,F′, arrow) to normal round and exclamation-mark-shaped spectrosomes (arrow and arrowhead) in wild-type clones (red in E,E′). Clones are recognized by the absence of GFP (green). In younger ovaries (<24 hours from eclosure), depletion of Zfrp8 results in abnormal localization of BicD and Orb (G-J). In nos-Gal4 Zfrp8 KD egg chambers BicD (green) is distributed and forms aggregates in the nurse cells instead of accumulating in the oocyte (compare G and H). Orb is mislocalized to the oocyte anterior (green in J) instead of sharp lateral and posterior localization observed in wild type (green in I). nos- Gal4/+ was used as a wild-type control. DNA (blue) Scale bars: 10 μm.

antibody (Fig. 5F; see Materials and Methods) shows that indeed similar numbers of egg chambers. The levels of TE RNAs in these Zfpr8 protein interacts with Mael, but no interaction between Zfrp8 young wild-type ovaries were 3-5 times higher (P<0.01) than those and Piwi was observed in co-immunoprecipitation experiments in ovaries of 5-day-old flies that are usually the source for this type using anti-Piwi antibody (data not shown). Taken together, our data of experiment (supplementary material Fig. S5) (Li et al., 2013). show that Zfrp8 and Mael are found in the same complex, and that Consequently, piRNA pathway mutants did not show as strong an perinuclear accumulation of Mael depends on Zfrp8. They have increase in the levels of expression of several TEs in our antagonistic functions but do not control each other’s nuclear experiments as noted by others (Klenov et al., 2011) (Fig. 6A). Lack targeting (Fig. 4E; Fig. 5E′″). of one copy of Zfrp8 did not significantly affect the level of expression of TEs in piwi, mael and aub (P>0.05 in Fig. 6A) mutant Zfrp8 and transposon activity ovaries, suggesting that the phenotypic suppression by Zfrp8 we Of all the functions associated with the piRNA pathway, its role in described above did not depend, or only partly depended, on the repression of TEs has been most intensively studied. In many reduction of transposon activity. piRNA pathway mutants the RNA levels of TEs is strongly To assess the germline-specific effects of Zfrp8 on TE expression, increased. We have chosen nine representatives of different TE we used nos-Gal4 Zfrp8 KD that strongly depleted the protein in the classes mainly expressed in somatic cells (mdg1, Gypsy), germline germline (supplementary material Fig. S1D,D′). Out of the seven cells (G-element, roo, HeT-A and TART-A) or in both soma and TEs typically expressed in germline, RNA levels of two TEs, TART- germline (F-element, 297 and 412) for quantitative reverse A and HeT-A, were significantly increased in Zfrp8 KD ovaries (six transcription polymerase chain reaction (RT-PCR) analysis (Fig. 6; to eight times, P<0.01). HeT-A and TART-A are non-long-terminal- see Materials and Methods). repeat (LTR) elements that constitute Drosophila telomeres For these experiments, we used RNA prepared from wild-type (Danilevskaya et al., 1997; Pardue et al., 2005; Shpiz and and mutant ovaries of newly eclosed flies (>10 hours old). At this Kalmykova, 2011; Shpiz et al., 2011). Unlike roo and most other

stage, Zfrp8 KD and wild-type ovaries are both small and contain germline TEs, they have a clear cellular role in maintaining genome Development

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not required in cells with limited developmental potential, as transient wild-type and mutant clones were similar in number and size. We also found no difference in Zfrp8 and wild-type escort cell clones, indicating that Zfrp8 is not required in these cells that multiply by self-duplication (Margolis and Spradling, 1995; Kirilly et al., 2011). Furthermore, Zfrp8 and wild-type MARCM clones induced in third instar larvae were indistinguishable in the adult antenna and legs 20 days ACI (supplementary material Fig. S5). These results support the conclusion that Zfrp8 function is primarily required in stem cells. Despite this functional requirement, Zfrp8 protein was not enriched in Drosophila GSCs and FSCs. This is surprising, because in mice Zfrp8/PDCD2 is enriched in several types of stem cell (Ramalho-Santos et al., 2002; Mu et al., 2010). Zfrp8/PDCD2 is also highly expressed in human bone marrow and cord blood stem and precursor cells with protein levels decreasing significantly as these cells differentiate (Kokorina et al., 2012; Barboza et al., 2013). We observed that loss of Zfrp8 in the Drosophila germline did not affect signaling from the niche to the stem cells. But the stem cells themselves are highly sensitive to loss of Zfrp8. In both Zfrp8germline stem cell clones and Zfr8 KD germaria we observed abnormal spectrosomes reminiscent of fusomes. These phenotypes suggest that these germline stem cells are losing stem identity and show features of a stem cell and a more advanced cystocyte. Germline and somatic stem cells and their daughter cells ultimately stop dividing when depleted of Zfrp8 but continue to survive for several days, as evident from the phenotype of the persistent stem cell clones. Similarly, in leukemia and in cancer cell lines that initially have high levels of the protein, reduction of Zfrp8/PDCD2 correlates with delay or arrest of the cell cycle rather than cell death (Barboza et al., 2013). The most severe abnormalities were observed 10-20 days ACI in Zfrp8 GSC clones induced in larvae (Fig. 2) and adults (data not shown). The phenotype of Zfrp8 KD ovarioles also became more pronounced with age, starting from a relatively normal-looking germarium and a few egg chambers (Fig. 3H,J) in young flies, to ovarioles made up of disorganized cysts (Fig. 3A), and finally, to Fig. 4. Zfrp8 localization in pi-RNA pathway mutants. In the wild-type ovarioles in which germ cells were almost entirely absent (Fig. 3A, germarium (A,A′) Zfrp8 is present in all cells and evenly distributed arrow). The temporal change in phenotype can be explained in two throughout nuclei and cytoplasm. In vas (B,B′), AGO3 (C,C′) and spnE (D,D′) mutants, the nuclear localization of Zfrp8 is strongly reduced. mael (E,E′) ways. First, it is possible that Zfrp8 levels are initially high enough does not affect Zfrp8 nuclear localization in GSCs. Lamin (green) is used to in mutant and KD stem cells to support a few divisions and the visualize the nuclear envelope, Zfrp8 is shown in red and as a separate formation of mutant cysts. However, as Zfrp8 is gradually depleted channel (white). Mutant alleles are indicated. w118 is used as a wild-type the cells stop dividing and are eventually lost. Alternatively, lack of control. Scale bars: 10 μm. WT, wild type. For complete germaria and more Zfrp8 may induce changes in parental cells that affect the mutants see supplementary material Fig. S2. developmental potential of the daughter cells. For instance, chromatin modifications could be affected in the absence of Zfrp8, but it could take several cell generations for these changes to have integrity, and at early stages of oogenesis both are mainly regulated a phenotypic effect. In both these scenarios, loss of Zfrp8 would by Piwi and Mael rather than by Aub (Fig. 6A). predominantly affect cells undergoing constant or rapid divisions, In the Zfrp8 somatic KD ovaries (tj-GAL4 Zfrp8RNAi) the RNA such as stem cells and cancer cells. levels of soma-specific TEs were not specifically affected, but it The loss of asymmetry in the stem cells, the mislocalization of must be noted that in these ovaries the level of protein expression is BicD and Orb proteins to and within the oocyte, the mislocalization not strongly reduced (supplementary material Fig. S1E). We did of Zfrp8 protein in GSCs of several piRNA pathway mutants, and observe increased expression of one germline element, roo, about the genetic interaction of Zfrp8 with piRNA pathway genes twice (P<0.003). Furthermore, in nos-GAL4 Zfrp8 ovaries the suggested a connection between Zfrp8 and the piRNA pathway. The expression of one soma-specific element, mdg1, was increased de-repression of the subset of transposons in Zfrp8 KD ovaries threefold (P<0.001). These results suggest that Zfrp8 has a non-cell- further links the gene with the piRNA pathway. or non-tissue-autonomous effect on the expression of these TEs. We have tested several LTR and non-LTR retroelements that represent three major TE classes based on their tissue-specific DISCUSSION activity in the germline, soma or in both tissues (intermediate) Our studies on Zfrp8 requirement in the Drosophila ovary show that (Malone et al., 2009; Sienski et al., 2012; Rozhkov et al., 2013).

the gene is essential in stem cells. Our results suggest that Zfrp8 is When Zfrp8 is depleted in the germline, two out of seven Development

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Fig. 5. Zfrp8 and Mael interaction. (A-E′″) The distribution of AGO3 (red in A,B, white in A′,B′) shows that depletion of Zfrp8 (nos-Gal4 Zfrp8 KD, B,B′) does not disrupt nuage formation in GSCs and cystoblasts. Localization of Mael (red in C,D, white in C′,D′) to nuage of GSCs and cystoblasts in control germaria (C,C′, green arrows), is not observed in Zfrp8 KD germaria (D,D′). Similarly, Mael was mislocalized in Zfrp8 GSC clones 10 days ACI (E-E′″). Compare mutant clone GSC and cystoblast (white arrows) and corresponding heterozygous cells (green arrows). Clones are recognized by absence of GFP (green). Mael is shown in red; DNA is in blue (each channel is shown separately in E′-E′″). (F) Immunoprecipitation from wild-type ovary extracts with anti-Mael antibody show that Zfrp8 is found in a complex with Mael. The upper panel shows western blot of extracts (Input) and co-immunoprecipitates (IP) probed with anti- Mael antibody. The lower panel shows the same western blot probed with anti-Zfrp8. For the negative control, co- immunoprecipitation without antibody (no AB) was performed.

intermediate and germline elements tested, HeT-A and TART, show are also their targets for repression. By contrast, the majority of significant de-repression. These elements are different from the primary piRNAs are derived from piRNA clusters and target TEs majority of Drosophila TEs. The HeT-A, TART and the TAHRE dispersed throughout the genome (Brennecke et al., 2007; Yin and elements are integral components of fly telomere. Their activity is Lin, 2007). Furthermore, the repression of TART and HeT-A in the tightly regulated and is required to protect chromosome ends germline involves an unusual combination of piRNA factors. We (Danilevskaya et al., 1997; George et al., 2010; Shpiz and found that at early stages of oogenesis they appear to be regulated Kalmykova, 2011; Shpiz et al., 2011). These elements, like other by piwi and mael, but not by the germline-specific Piwi family TEs, are controlled by the piRNA machinery, but their primary member Aub (Fig. 6A). This result is in agreement with recent piRNAs are likely to be derived from the same telomeric loci that studies on piwi function in the soma and germline that showed that

Fig. 6. Zfrp8 is involved in TE repression. Quantitative RT-PCR of fold accumulation of TE transcripts in young mutant ovaries relative to w118 controls (log2 scale, mean ± s.d.; n≥3, normalized to RP49/RPL32). (A) Mutations in piwi (piwi06843/piwi2), mael (maelr20/Df(3L)ED230) and aub (aubQC42/aubHN2) leads to TE de-repression. As expected, soma specific TEs (blue) were not affected by aub, but de-repressed in piwi. Germline-specific (yellow) and intermediate (green) TEs were differentially affected in different mutants. Germline-specific telomeric elements HeT-A and TART-A (black and gray) are strongly de- repressed in piwi and mael, but not in aub mutant ovaries. Mutants lacking one copy of Zfrp8 show insignificant changes of TE levels compared with mutants in Zfrp8+ background (P≥0.05). (B) Fold accumulation of transcripts in young ovaries with Zfrp8 KD in the germline (nos>Zfrp8 KD), soma (tj>Zfrp8 KD) and in controls (average nos-Gal4/+ tj-Gal4/+ UAS-Zfrp8 RNAi/+) relative to w118 ovaries (Log2 scale, mean ± s.d.; n≥6, normalized to RP49/RPL32). Major changes were observed for HeT-A and TART-A TEs in nos>Zfrp8 KD ovaries (six to ten times, P<0.01). Other TEs, with the exception of mdg1 in nos>Zfrp8 KD and roo

in tj>Zfrp8 KD, were not significantly changed (P≥0.1). Development

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HeT-A and TART elements are among the TEs most strongly into pPGW (Gateway). Transgenic fly lines were created following standard regulated by Piwi in the germline (Rozhkov et al., 2013). Thus, protocols (Rubin and Spradling, 1982; Brand and Perrimon, 1993). For Zfrp8 may target the same TEs as Piwi and Mael but not those RNAi experiments, flies were raised at 29°C. null regulated by Aub. For genetic interaction experiments, the Zfpr8 allele (Minakhina et al., De-repression of TEs caused by Zfrp8 KD could be responsible 2007) was recombined/combined with alleles of each gene tested for the enhancement of developmental defects seen in piRNA (supplementary material Table S1A,B). Eggs were collected from 5- to 7- day-old females and allowed to develop for 2 days at 25°C for examination pathway mutants. For instance, the increase of TE transcripts may of egg phenotypes. Mutant females that produced few or no eggs were enhance dorsoventral patterning defects in armi, AGO3, aub, spnE dissected and 10-15 ovaries of 2-, 5- and 14-day-old females were analyzed. and vas because of the competition between TE transcripts and oocyte polarity factors for the same RNA transport machinery (Van Clonal analysis De Bor et al., 2005; Chen et al., 2007; Klattenhoff et al., 2007; The fly stocks used to induce MARCM clones have been previously Navarro et al., 2009). However, the interaction of Zfrp8 with the described (Lee and Luo, 2001; Minakhina and Steward, 2010). To induce piRNA pathway machinery seems to be more complex. Zfrp8 clones, third instar larvae and 3-day-old adults of the appropriate genotype enhanced the egg phenotype of only three mutants, spnE, AGO3 and were exposed to 38°C for 60 minutes in the morning of two consecutive vas, and in these mutants the nuclear localization of Zfrp8 protein days. Ovaries were dissected, fixed stained and analyzed 2 and 5 days ACI, was also affected. These results suggest that Zfrp8 functions for transient clones, and 10 and 20 days ACI, for persistent clones. downstream of the three factors. The FLP/FRP site-specific recombination system was used to generate Both piwi and mael are dominantly suppressed by Zfrp8. Both mutant clones with a heat-shock promoter (Xu and Rubin, 1993). We induced mutant clones in FRTG13 UAS-mCD8-GFP/FRTG13 Zfrp8null (Zfrp8) these factors have important nuclear functions, regulating chromatin larvae. More than 200 ovarioles of each genotype were analyzed on a Zeiss modifications and controlling TEs at the transcriptional level, and Axioplan-2 microscope. both are required to repress HeT-A and TART-A elements. Zfrp8 could suppress piwi or mael by inducing a competing chromatin Zfrp8 antibody modification at the genomic loci targeted by Piwi or Mael. The C-terminal Zfrp8 sequence coding for amino acids 168 to 347 of Zfrp8 Chromatin modifications are generally stable through several cell was cloned into the pET15 vector expressed, and purified following the generations (Trifonov, 2011; Rando, 2012). Such a function would standard protocols of the Northeast Structural Genomics (NESG) consortium therefore be consistent with the temporal changes of phenotypes in (Acton et al., 2011). Polyclonal antibodies were raised in rabbits and affinity Zfrp8 ovarian clones and KD ovarioles. purified at Pocono Rabbit Farm and Laboratory (Canadensis, PA, USA). Although Piwi and Mael target the same genomic loci, no Antibodies were tested on western blots and by immunostaining ovaries interaction between the two proteins have been detected (Sato et al., (supplementary material Fig. S1). 2011; Le Thomas et al., 2013). Our co-immunoprecipitation experiments suggest that Zfrp8 complexes with Mael but not with Co-immunoprecipitation Co-immunoprecipitation with monoclonal mouse anti-Mael and anti-Piwi Piwi, indicating that the observed genetic interaction between Zfrp8 antibody (Saito et al., 2006; Sato et al., 2011) was performed in the presence and piwi may be mediated by mael. Mael is one of the most of RNase inhibitor RNaseOUT (Invitrogen) as previously described enigmatic proteins in the piRNA pathway. It is found in the (Brennecke et al., 2007). Ovaries from 50 young flies (<20 hours old) were cytoplasm, nuage and nucleus, and has been implicated in diverse used for each experiment. Proteins were analyzed by western blotting using cellular processes including the ping-pong piRNA amplification rabbit polyclonal anti-Mael, anti-Piwi and anti-Zfrp8. cycle in the germline (Findley et al., 2003; Aravin et al., 2009), MTOC assembley in the oocyte (Sato et al., 2011) and Piwi- Antibodies and microscopy dependent chromatin modification in somatic cells (Sienski et al., Rat anti-Vas antibody (1:20) developed by A. Spradling and D. Williams 2012). Zfrp8/PDCD2 is also required in the soma and germline and and mouse anti-1B1 antibody (1:20) developed by H. Lipshitz were obtained may function both in the cytoplasm and in nuclei (Scarr and Sharp, from the Developmental Studies Hybridoma Bank (Iowa University, Iowa 2002; Mu et al., 2010). However, in contrast to mael, Zfrp8 City, IA, USA). Rabbit anti P-MAD (anti-phosphorylated Smad1, PS1) used at 1:3500 was given to us by N. Yakoby (Rutgers University, Camden, NJ, homozygous mutants are lethal and Zfrp8 ovaries show a stronger USA) (Persson et al., 1998; Niepielko et al., 2011). Monoclonal mouse anti- phenotype. Based on the observation that Mael and Zfrp8 are found Mael (1:200), anti-Armi (1:200) and anti-Piwi (1:500) were a gift from M. in the same complex and that Zfrp8 dominantly suppresses Mael, we C. Siomi (University of Tokyo, Tokyo, Japan) (Saito et al., 2006; Sato et al., propose that they act in opposite fashion on a common target, 2011). Polyclonal rabbit anti-Aub (1:2000); anti-AGO3 (1:250) and anti- whether during piRNA biogenesis or chromatin modification. Piwi (1:500) were from T. Schupbach (Princeton University, Princeton, NJ, USA) and G. Hannon (Cold Spring Harbor Laboratory, Cold Spring Harbor, MATERIALS AND METHODS NY, USA) (Brennecke et al., 2007). Polyclonal rabbit anti-Mael (1:200) was Fly lines and genetic interactions a gift from H. Ruohola-Baker (University of Washington, Seattle, WA, We obtained a Zfrp8 RNAi construct in the VALIUM22 vector (line USA) (Findley et al., 2003). Phalloidin-546 (Invitrogen) and secondary GL00541) from the TRiP at Harvard Medical School, Boston, MA, USA. antibodies (Jackson Laboratories) were used at 1:300. Hoechst 33258 The soma-specific Zfrp8RNAi line (P{GD4600}v11521) was obtained from (1:5000) was used to stain DNA. Images were captured using a Leica DM the Vienna Drosophila RNAi Center (Dietzl et al., 2007). The nos-Gal4 IRBE SP2 or Leica TSC SP5 laser scanning confocal microscope (objectives driver (P{GAL4::VP16-nos.UTR}), alleles of aub, cuff, tud, vas and piwi2 40× and 63× oil), analyzed with Leica Microsystems software and further were obtained from T. Schupbach (Princeton, NJ, USA), piwi1 and piwi2 processed using Adobe Photoshop. alleles from H. Lin (New Haven, CT, USA) and tj-GAL4 (tj-GAL4/Cyo; tub- GAL80ts/MKRS) from S. DiNardo (Philadelphia, PA, USA). Other mutant Quantitative RT-PCR alleles, balancer and deficiency stocks were from the Bloomington Stock The level of TE transcripts depends on age and may be dependent on the Center. stage of egg chambers (supplementary material Fig. S4). For comparative UAS-GFP-PDCD2 was made by RT-PCR amplification of the human analysis, we used young virgin ovaries (<10 hours old). RNA was isolated PDCD2 coding region (Barboza et al., 2013). The gene was cloned into the from 10-20 ovaries using RNeasy Plus Mini kit (Qiagen). Quantitative RT-

Gateway vector pDONR4 (Life Technologies) and subsequently transferred PCR was performed as described in the manufacturer’s instructions using Development

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the QuantiTect SYBR Green kit (Qiagen), Smart cycler II (Cepheid) and the Fox, D., Morris, L., Nystul, T. and Spradling, A. (2008). Lineage analysis of stem relative standard curve method. Primers used for RT-PCT are listed in cells. In Stem Book: Innitiative in Innovative Computing at Harvard University (ed. L. supplementary material Table S2. Transcript levels were normalized to those Girard). Cambridge, MA: Harvard Stem Cell Institute. George, J. A., Traverse, K. L., DeBaryshe, P. G., Kelley, K. J. and Pardue, M. L. of Rp49. nos-GAL4/+, tj-GAL4/+ and UAS-Zfrp8RNAi/+ were used as (2010). Evolution of diverse mechanisms for protecting chromosome ends by control ovaries, and all data were normalized to the transcript levels in w118 Drosophila TART telomere retrotransposons. Proc. Natl. Acad. Sci. USA 107, 21052- ovaries (baseline=1). At least two biological and two technical replicates 21057. were performed for each genotype. Statistical significance (P-value) was Gunawardane, L. S., Saito, K., Nishida, K. M., Miyoshi, K., Kawamura, Y., Nagami, T., Siomi, H. and Siomi, M. C. (2007). A slicer-mediated mechanism for repeat- determined using two-tailed Student’s t-test. associated siRNA 5′ end formation in Drosophila. Science 315, 1587-1590. Guzzardo, P. M., Muerdter, F. and Hannon, G. J. (2013). The piRNA pathway in flies: Acknowledgements highlights and future directions. Curr. Opin. Genet. Dev. 23, 44-52. We thank T. Schupbach, G. Desphande and E. Reilly for helpful comments on the Harris, A. N. and Macdonald, P. M. (2001). Aubergine encodes a Drosophila polar manuscript. The TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) is granule component required for pole cell formation and related to eIF2C. Development 128, 2823-2832. acknowledged for providing the transgenic Zfrp8RNAi fly stock. We also thank L. Huang, X. A., Yin, H., Sweeney, S., Raha, D., Snyder, M. and Lin, H. (2013). A major Nguyen and J. Sussman for expert fly food preparation and stock maintenance, B. epigenetic programming mechanism guided by piRNAs. Dev. Cell 24, 502-516. MacTaggart for the help with the genetic screen, G. Hannon, H. Ruohola-Baker, T. Ishizu, H., Nagao, A. and Siomi, H. (2011). Gatekeepers for Piwi-piRNA complexes to Schupbach, M. C. Siomi, and N. Yakoby for antibodies, and H. Lin, S. DiNardo and enter the nucleus. Curr. Opin. Genet. Dev. 21, 484-490. T. Schupbach for fly stocks. Juliano, C., Wang, J. and Lin, H. (2011). Uniting germline and stem cells: the function of Piwi proteins and the piRNA pathway in diverse organisms. Annu. Rev. Genet. 45, Competing interests 447-469. Kai, T. and Spradling, A. (2003). An empty Drosophila stem cell niche reactivates the The authors declare no competing financial interests. proliferation of ectopic cells. Proc. Natl. Acad. Sci. USA 100, 4633-4638. King, F. J., Szakmary, A., Cox, D. N. and Lin, H. (2001). 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