configuration to a decondensed chromosome morphology (Bate and Martinez Arias, 1993; Dej and Spradling, 1999) . Before mid-oogenesis arrest, the only visible chromatin-configuration defect in su(Hw) mutant egg chambers is a delayed chromatin dispersal of nurse cell polytene chromosomes at stage 7 or 8. The prolonged development of defective egg chambers is eliminated by mid-oogenesis arrest resulting in egg chamber degeneration around stages 9 to 10 (Baxley et al., 2011; Harrison et al., 1993; Klug et al., 1968) . This defective chromatin dispersal is a common trait among a large number of unrelated mutants, such as genes encoding the splicesosome component prp22 (Klusza et al., 2013) , the piwi-interacting RNAs (piRNAs) related protein, and rhino (Klattenhoff et al., 2009; Volpe et al., 2001) , which complicates the identification of the mechanisms associated with Su(Hw) activity in the genome of egg chamber cells.
In addition to defects resulting from mutations in oogenesis genes, uncontrolled transposon activity may also cause a severe disruption of development during oogenesis.
piRNAs are critical molecules involved in suppression of transposable element activity. These RNA sequences were originally called repeat-associated siRNAs (rasi-RNAs) as they are derived from repetitive sequences in the genome (Aravin et al., 2003; Brennecke et al., 2007; Feschotte, 2008; Yin and Lin, 2007) . Mutations in genes involved in piRNA production such as aubergine (aub), spindle-E (spnE), and maelstrom (mael) result in transposable element overexpression and mobilization that create double strand breaks (DSBs) in the host genome (Chen et al., 2007; Cook et al., 2004; Klattenhoff et al., 2007; Sienski et al., 2012) . The unleashed retrotransposon activation triggers the DNA damage response, causing defects in microtubule polarization in early oogenesis that further disrupt Gurken signaling in later stages that are essential for dorsoventral determination in embryonic development (Chen et al., 2007; Khurana and Theurkauf, 2010; Klattenhoff et al., 2007) . Interestingly, the DNA damage response resulting from over-activation of retrotransposons has been known to utilize the same pathway triggered by unrepaired DSBs in mutants of spindle class genes encoding meiotic DNA damage repair proteins. These unrepaired DSBs activate meiotic checkpoints mediated by mei-41 (ataxia telangiectasia-related-ATR-ortholog) and mnk (checkpoint kinase 2-chk2-ortholog) (Abdu et al., 2002; Ghabrial and Schupbach, 1999; LaRocque et al., 2007; Staeva-Vieira et al., 2003) .
In this study, we conclude that the loss of Su(Hw) creates an accumulation of nonmeiotic DNA damage in germline cells of ovaries, thereby activating DNA damage checkpoints.
We show that DNA damage is independent of transposable element activity and that mutations in meiotic checkpoint genes mei-41 (ATR) and grapes (chk1), but not mnk (chk2, loki), result in the rescue of the su(Hw) mutant spindle phenotype in ovaries. We conclude that the lack of su(Hw) expression in ovaries provokes an excess of unrepaired DNA breaks in germline cells, and propose that this DNA damage is caused by replication stress. Our data supports that replication stress occurs in dividing somatic cells as well, in su(Hw mutants. upon loss of Su(Hw) was previously unreported and overall, the irregular nurse cell number in su(Hw) mutant ovaries compared with wildtype is statistically significant (p<0.05, Fisher's exact test). Counting the number of ring canals connecting nurse cells with the oocyte provides an indication of whether egg chambers with an irregular nurse cell number originate from incomplete cell divisions, extra cell divisions, or by egg chamber fusions (Wang et al., 2010) .
After counting the number of ring canals contacting the oocyte in egg chambers with less than 15 nurse cells, we found that 33% contained less than 4 rings, suggesting incomplete nurse cell divisions. In 67% of egg chambers with less than 15 nurse cells, the number of rings was 4, suggesting cell death may have occurred in these chambers. Finally, supernumerary egg chambers always have more than 4 ring canals in their oocyte, suggesting these chambers had extra cell divisions ( Figure S1 ).
To confirm that this phenotype is caused by the lack of Su(Hw) in the germline, we quantified nurse cell number in su(Hw) v/e04061 trans-heterozygous females expressing a Su(Hw):eGFP transgene in the germline driven by a nanos-GAL4 driver (P{nos-GAL4::VP16}).
The nanos-GAL4 driver directs the expression of GAL4 throughout all the stages of oogenesis (Rorth, 1998; Van Doren et al., 1998) . We have previously shown that su(Hw)::eGFP completely rescues insulator activity in su(Hw) mutants (Schoborg et al., 2013) and partially rescues the fertility of these mutants when expressed in the germline (Hsu et al., 2015) . Our results show that all 65 egg chambers analyzed in ovaries from females expressing the rescue construct contain the expected number of 15 nurse cells (Figure 1 E) . Taken together, these observations suggest that loss of su(Hw) causes an irregular number of nurse cells.
Abnormal nurse cell number in egg chambers occurs in loss of function mutants of genes encoding proteins involved in piRNA-related pathways, including rihno and maelstrom (mael) (Sato et al., 2011; Volpe et al., 2001) . mael encodes a g-tubulin associated protein involved in the proper positioning of the MTOC, which is required to determine oocyte polarity and the precise localization of specific mRNAs within the Drosophila oocyte (Clegg et al., 2001; Clegg et al., 1997; Sato et al., 2011) .
Microtubule organization is critical at various stages of oogenesis. In stage one, formation of the MTOC, a structure with concentrated a-tubulin at the posterior of the oocytes, is required for oocyte differentiation. In stages 3 through 6, a microtubule array is extended from the MTOC through ring canals to the neighboring nurse cells. This polarized network of microtubules is required for intercellular transport from nurse cells to the oocyte. During stage 7 the microtubule network is reorganized, causing a shift in the polarity of the MTOC from posterior to anterior, and the growing microtubule network positions the oocyte nucleus to the anterior corner (Steinhauer and Kalderon, 2006; Theurkauf et al., 1992) .
To assess whether su(Hw) mutants also show microtubule disorganization in addition to an irregular number of nurse cells, we used an anti a-tubulin antibody that allows detection of microtubule networks in egg chambers (Theurkauf et al., 1992) . We found that, under our experimental conditions, MTOCs form properly in wildtype ovaries, exhibiting the typical concentration of a-tubulin at the posterior of the oocyte in the germarium (Figure 2 C and G).
However, in su(Hw) mutants, the a-tubulin signal is weaker, more diffuse, and less concentrated at the MTOC (Figure 2 A and E). This phenotype was specific to su(Hw) mutants, as we did not observe the same phenotype in the mod(mdg4) u1 mutant (Figure 2 B and F).
Additionally, we were able to rescue the MTOC defect in the germarium of su(Hw) mutant ovarioles by ectopic expression of su(Hw)::eGFP driven by the nanos-GAL4 driver ( Figure 2 D and H) . These data suggest that proper formation of the MTOC is impaired upon loss of Su(Hw) and imply that the microtubule network is disorganized and may not function efficiently to facilitate egg chamber development. For example, it is well established that defects in MTOC regulation that affect establishment of polarity in early oogenesis result in the disruption of Grk signaling in later stages of oogenesis (Khurana and Theurkauf, 2010) .
patterning is determined by the key axis-determining mRNAs of gurken (grk), oskar (osk), and bicoid (bcd), which are transported along microtubules to specific locations within the oocyte (Kugler and Lasko, 2009) . To test whether defective microtubule organization in su(Hw) mutants could also affect axis determination, we used anti-Grk antibodies to detect the localization of Grk, a Drosophila transforming growth factor a (TGF a) protein, which is important for dorsoventral determination of embryos (Neuman-Silberberg and Schupbach, 1993) . grk mRNA requires transportation to the oocyte prior to translation, and Grk protein specifically localizes at the posterior of the oocyte during stage 6 ( Figure 3 A and B) .
Later, as the oocyte relocates to the anterior-dorsal corner, Grk gradually moves to the corner and forms a crescent shape around the oocyte nucleus at stage 9 (Figure 3 C). Our data shows that in su(Hw) mutants, Grk fails to translocate to the anterior-dorsal corner of the oocyte in 95% (N=20) of the egg chambers at stage 9, compared with 100% correct localization in wildtype (N=16) (p<0.001, Fisher's exact test) ( Figure 6 ). Mislocalization of Grk normally leads to failure of the oocyte to signal follicle cells to determine dorsal fate, consequently interrupting the axis plan of the developing egg chamber. Our data suggest that loss of Su(Hw) causes defective formation of the microtubule network in developing egg chambers and the subsequent mislocalization of Grk.
Non-meiotic DNA damage occurs during oogenesis in su(Hw) mutants: Disorganized microtubules and mislocalized Grk are oogenesis phenotypes observed in mutants of the spindle class genes (Ghabrial et al., 1998) and the piRNA pathway (Chen et al., 2007; Klattenhoff et al., 2007; Sato et al., 2011) . In these mutants, cells lose the ability to repair DNA damage or to repress retrotransposon activity, generating an excess of DNA double-strand breaks (DSBs) such that unrepaired DSBs activate the ATR/Chk2 dependent DNA-damage signaling pathway in the female germline (Khurana and Theurkauf, 2010). Therefore, our results suggest that DNA damage signaling pathways are activated in su(Hw) mutants, resulting in disorganization of the MTOC and Grk mislocalization in the oocyte.
In order to further investigate whether the ATR-mediated DNA damage pathway is activated by DNA damage in su(Hw) mutants, we performed immunostaining in ovaries using antibodies against phosphorylated Histone 2A variant (gH2Av), a marker for DNA damage. In Drosophila oocytes, DNA damage can be produced by three major sources: DNA damage induced by transposable element activity, DNA damage induced by replication stress, and meiotic DSB induced by Spo11, a nuclease required for meiotic recombination encoded by the mei-w68 gene in Drosophila. These meiotic DSBs are normally repaired before developing egg chambers reach stage one (Jang et al., 2003; McKim and Hayashi-Hagihara, 1998; Mehrotra and McKim, 2006) .
To characterize whether the loss of su(Hw) induces DNA damage and whether the DNA damage is associated with the meiotic recombination pathway, we used gH2Av to compare the DNA damage in wildtype, su(Hw) mutants, mei-w68 mutants, and mei-w68; su(Hw) double mutants. In wildtype germaria, we observed naturally occurring DNA damage in dividing cystocytes (which later become nurse cells and pro-oocytes) in region 2, where homologous recombination takes place (Figure 4 A and B). As expected, mei-w68 (Spo11) homozygous mutants showed no DSBs in region 2 of the germarium, indicating a lack of Spo11 activity and the absence of other significant factors inducing DNA damage. Remarkably, we found an excess of non-meiotic DSBs in region 2 nurse cells from the germarium of su(Hw) and mei-w68 double mutants, demonstrating that DNA damage in su(Hw) mutants is not produced by Spo11, and is therefore of non-meiotic origin (Figure 4 B and D). Additionally, we observed that in wildtype ovaries, gH2Av signal is absent after stage 5, as described in an earlier study (Joyce et al., 2011) .
Interestingly, the gH2Av signal remains present until later stages in su(Hw) mutant egg chambers (Figure 4 I and J) . This result strongly indicates that loss of Su(Hw) leads to formation and accumulation of non-meiotic DNA damage in female germline cells.
Drosophila germline: Su(Hw) strongly regulates transcription of the gypsy retrotransposon, and loss of the insulator activity reverses the phenotype of gypsy induced mutations at several loci in Drosophila (Harrison et al., 1993; Parkhurst and Corces, 1985; Parkhurst and Corces, 1986; Parkhurst et al., 1988) . Additionally, global activation of transposable elements (TEs) in the germline triggers the activation DNA damage checkpoints most likely due to an excess of DNA damage, producing phenotypes similar to those described here (Chen et al., 2007; Khurana and Theurkauf, 2010; Klattenhoff et al., 2007; Mohn et al., 2014) . Our data demonstrating the accumulation of non-meiotic DNA damage in female germline cells of su(Hw) mutants led us to ask whether TEs are over-expressed in the germline of these mutants.
We performed real-time RT-PCR to quantify the transcripts of TEs produced in wildtype and su(Hw) e04061 homozygous mutant ovaries. In both genotypes, egg chambers stage 9 and older were removed manually to eliminate differences in expression levels that could originate from differences in developmental stages between the two samples. Total RNA extracted from mutant and wildtype ovaries was used for real-time RT-PCR. We used 17 primer sets specifically designed to determine the expression of 17 different TEs. Additionally, we used primers for rp49 as an internal control. Our selection of TEs includes germline and somatic specific transposons and covers TEs with long-terminal repeats (LTR) as well as non-LTR elements.
The expression levels of 17 TEs in mutant ovaries were compared to wildtype in a foldchange graph ( Figure 5 ). Consistent with previous studies, our data show that the transcription levels of gypsy as well as stalker4 are reduced significantly in su(Hw) mutants. Compared with the transcription levels of TEs in spindle mutants, which can reach levels of more than 100-fold increase, and considering that the majority of TEs tested showed no significant change in transcription levels, it seems unlikely that TEs are the cause of significant DNA damage in su(Hw) mutants.
Because of the similarity between spindle class and piRNA mutants with su(Hw) mutant phenotypes and the elevated levels of gH2Av seen in su(Hw) mutant egg chambers, we hypothesize that su(Hw) mutations activate a DNA damage checkpoint during oogenesis. We first tested whether su(Hw) mutants activate a DNA damage signaling pathway by asking whether oocyte development is restored in females double mutant for su(Hw) v/e04061 and the Drosophila ATR allele, mei-41 D5 (Brodsky et al., 2004; Laurencon et al., 2003) . Results show that although double mutant females remained sterile, 54% of egg chambers recovered correct positioning of Grk around the oocyte nucleus at stage nine, (N=24, p<0.001, Fisher exact test), and had proper enlargement of the developing oocyte in stage nine and ten ( Figure 6 C, H, and I). These results show that loss of ATR function partially recovers oocyte development in su(Hw) v/e04061 mutants and suggests that loss of Su(Hw) function triggers a DNA-damage response through an ATR-dependent pathway. Chk1 (Grp) and Chk2 (Mnk, Lok), two highly conserved downstream kinases in ATR/ATM DNA damage signaling, are both phosphorylated in response to DNA damage and participate in cell cycle checkpoint activation (Cimprich and Cortez, 2008) . However, mutations of chk2, but not chk1, recover the dorsoventral patterning defects associated with mutants of spindle and piRNA pathway genes (Chen et al., 2007; Ghabrial et al., 1998; Klattenhoff et al., 2007) . To determine the role of DNA repair pathways in su(Hw) mutant ovaries, we first tested the ability of chk1 (grp z5170/209 ) chk2 (lok 30 ) double mutant flies to rescue the mutant phenotypes of su(Hw) v/e04061 ovaries. We found that grp z5170/209 lok 30 double mutants were able to rescue defective Grk localization, but were unable to rescue the lack of fertility in su(Hw) mutants (Figure 6 D). To test whether the loss of Su(Hw) activates a DNA damage response specifically mediated by ATR/Chk2, we generated double mutant flies using su(Hw) v/e04061 and chk2 (lok 30 , mnk 6006 ) mutations (Brodsky et al., 2004) and tested whether a mutation in chk2 is able to rescue Grk localization in su(Hw) mutants. The results showed that neither fertility nor Grk localization were rescued in these double mutants ( Figure 6 E and F). Finally, we generated double mutant flies using su(Hw) v/e04061 and chk1 (grp z5170/209 ) mutations. grp z5170/209 ; su(Hw) v/e04061 double mutants were able to rescue the Grk localization phenotype in 80% of egg chambers analyzed, but were unable to rescue the sterility phenotype ( Figure 
Our results have revealed that Su(Hw) may have a role in maintaining genome integrity by preventing replication stress in nurse cells. Accordingly, loss of Su(Hw) causes a significant increase in DNA damage, followed by defects in microtubule organization, egg chamber formation, and mislocalization of Grk. These phenotypes can be rescued by mei-41 D5 and grp z5170/209 , mutant alleles of ATR and Chk1 respectively, suggesting that the defects originate from the activation of checkpoints in response to DNA damage.
In addition to these phenotypes, homozygous mutant females for spindle class genes produce embryos with dorsoventral defects in their eggshells which result from mislocalization of Grk and other morphogens in the developing oocyte (Ghabrial et al., 1998; Ghabrial and Schupbach, 1999) . Egg chamber development in su(Hw) mutants is arrested at mid-oogenesis, preventing the opportunity to test eggshells for the formation of dorsoventral defects.
Interestingly, we have shown that su(Hw)::eGFP expression driven by nanos-GAL4 (P{nanos-GAL4::VP16}) not only can rescue MTOC in egg chambers, (Figure 2 ) but also partially restores fertility in mutant flies. These rescued females laid a reduced number of eggs (~41% of normal females) (Hsu et al., 2015) , and only less than 25% of these embryos produced viable adults, indicating that the spatio-temporal expression of nanos-GAL4; su(Hw)::eGFP is not sufficient to fully restore Su(Hw) activity in all egg chambers from mutant ovaries.
Eggshells produced by nanos-GAL4; su(Hw)::eGFP partially rescued females provide an opportunity to test whether loss of Su(Hw) also induces the formation of dorsoventral defects.
We performed a quantitative analysis of the eggshell morphology produced by nanos-GAL4; su(Hw)::eGFP females. Results show that all embryos that failed to develop displayed eggshells with dorsoventral axis defects ( Figure 7A ). We categorized these phenotypes as previously described in (Ghabrial et al., 1998) : Type I: eggshells with two wildtype appendages, Type II: eggshells showing two short appendages, Type III: eggshells showing a single appendage, and Type IV: eggshells with no appendages. The number of each type of eggshell is shown in Figure   7B . Type I eggshells are only found in viable embryos and have no noticeable developmental defects through adult stage. These results show that in a large number of egg chambers expressing su(Hw)::eGFP driven by nanos-GAL4, oogenesis defects are only partially rescued, and suggest that these chambers activate a spindle-like checkpoint that leads to dorsoventral transformations.
Surprisingly, it has been recently shown that heterozygous mutations in the Rbp9 gene (Rbp9 P2775/+ , su(Hw) 2/v ) are able to rescue the sterility of su(Hw) mutant females (Soshnev et al., 2013) . However, embryos produced by these females show dorsoventral transformations in their eggshells. Soshnev and collaborators conclude that transformations are produced because of the lack of Su(Hw) expression in follicle cells, suggesting that expression of Su(Hw) in follicle cells is necessary to allow establishment of communication between Gurken and follicle cell receptors. In experiments described here, we used a nanos-GAL4 driver to express Su(Hw) in nurse cells. This driver induces transgene expression exclusively in the germline, showing expression peaks at the germarium and at stage 9, but does not activate expression in follicle cells (Rorth, 1998; Van Doren et al., 1998) . We tested expression of Su(Hw)::eGFP in follicle cells using antibodies against GFP, showing no detectable expression, yet 20% of the embryos were able to develop into normal adult flies, suggesting that expression of su(Hw) is not required in follicle cells to support correct dorsoventral patterning during oogenesis (Hsu et al., 2015) .
Additionally, we found that the source of ventralized phenotypes in nanos-GAL4; su(Hw)::eGFP eggshells can be attributed to mislocalization of Grk, since 29% of egg chambers from ovaries of these females also show mislocalization of Grk ( Figure 7C and D).
We also generated nos-GAL4, Su(Hw)::eGFP flies, in which the driver is P{GAL4nos.NGT}40), that highly express Su(Hw)::eGFP through oogenesis. In these flies, the sterility phenotype of su(Hw) mutant females was poorly rescued, but a large number of egg chambers achieved normal oocyte enlargement and were able to reach the latest stages of oogenesis. nos-GAL4, Su(Hw)::eGFP; su(Hw) v/e04061 flies laid 6.1% of expected eggs, compared to wildtype (Hsu et al., 2015) . Nanos-GAL4::VP16 includes the nos 5' and 3' UTRs translation regulation sequences and appears to be a stronger driver than GAL4-nos.NGT, which includes a 3′UTR region from a tubulin mRNA (Tracey et al., 2000) . Embryos from nos-GAL4, Su(Hw)::eGFP females never developed into larvae and all eggshells showed ventralization phenotypes with the more severe classes more frequently represented than in embryos from nanos-GAL4::VP16 driver ( Figure 7B ). Importantly, the severity of the ventralization phenotypes correlates with a higher frequency of mislocalization of Grk (82%) ( Figure 7C and E), suggesting that defects stem from misexpression of Su(Hw) in oocytes and not from the lack of su(Hw) expression in follicle cells. Altogether, this data further supports our findings that expression of su(Hw) is required for genome stability during oogenesis and that loss of Su(Hw) results in the generation of DNA damage, followed by the activation of cell cycle checkpoints, mislocalization of Gurken, and dorsoventral patterning defects in eggshells.
Monomethylated H4K20 and gH2Av accumulate in su(Hw) mutant ovaries: We have shown that loss of Su(Hw) causes an accumulation of DNA damage in nurse cells, but its origin remains uncertain since the damage is not derived from meiotic recombination and a substantial activation of TEs was not observed in these mutants. Given that, in addition to Spo11 and TE activity, a major contributing source of intrinsically produced DNA damage under normal conditions is replication stress (Zeman and Cimprich, 2014) and, because the spindle-like phenotype we observed is ATR/Chk1 dependent, we used monomethylated Histone 4 at lysine 20 mutants is related to irregularities in replication. H4K20me1 has an important role in maintaining genome stability (Beck et al., 2012b; Jorgensen et al., 2013; Wu and Rice, 2011) .
Monomethylation of H4K20 is mediated by the PR-Set7/ SET8 methyltransferase, and removal of PR-Set7 results in DNA damage and S phase arrest (Jorgensen et al., 2007) . Conversely, constant expression of PR-Set7 causes accumulation of H4K20me1 at replication origins and results in replication stress due to re-replication (Jorgensen et al., 2007; Tardat et al., 2010b) . In Drosophila S2 cells, depletion of PR-Set7 affects chromosome compaction and higher-order chromatin organization, also triggering the DNA damage response (Sakaguchi et al., 2012; Sakaguchi and Steward, 2007) . Additionally, highly proliferating tissues, such as wing discs and salivary glands, are smaller in size and contain fewer cells in pr-set7 mutants, due to improper cell division during development (Karachentsev et al., 2007; Karachentsev et al., 2005) .
Together, this evidence suggests that misregulation of H4K20me1 can alter chromatin organization and lead to genome instability in a manner dependent on the cell cycle and DNA replication.
We performed western blotting in ovaries using both anti-gH2Av and anti-H4K20me1 antibodies. Our results show that, as expected, the amount of gH2Av and the amount of H4K20me1 increased in su(Hw) mutant ovaries compared to wildtype (Figure 8 B) . The increase in gH2Av levels revealed by western blots confirms our earlier observation that high levels of non-meiotic DNA damage occur in su(Hw) mutant ovaries (Figure 8 A) . We also showed that an additional mutation in ATR (mei-41 D5 ), which partially rescues oogenesis in su(Hw) v/e04061 , does not significantly reduce the levels of gH2Av in mutant ovaries (Figure 8 A) , suggesting that H2Av is likely phosphorylated also by ATM in su(Hw) mutant ovaries. In addition, the elevated amounts of H4K20me1 in mutant ovaries support the notion that DNA damage may originate from replication stress, which we propose is caused by the loss of Su(Hw). Altogether, our results suggest that DNA damage in su(Hw) mutants is caused by replication stress, which disrupts genome stability and eventually leads to checkpoint activation and contributes to the oogenesis phenotype of these mutants.
Results showing spindle like phenotypes in su(Hw) mutant ovaries, and a partial rescue of this phenotype by mutations in mei-41 and grapes, suggest Su(Hw) may play a role in the maintenance of genome stability and that lack of Su(Hw) function may lead to replication stress.
However, these data did not show whether Su(Hw)'s role in genome maintenance is general or if this role is specific to the female germline. To address this question, we obtained metaphase spreads from third instar larval neuroblasts mutant for su(Hw), and chromosomes were scored for the presence of chromosomal aberrations (CABs). Dividing neuroblasts provide an ideal opportunity to score a large number of cells and have previously been used to quantify the occurrence of CABs (Gatti and Goldberg, 1991) . Screening larval brains from homozygous su(Hw) e04061 larvae reveals neuroblasts with either normal karyotypes (Figure 9 , A1) or karyotypes showing CABs (Figure 9 , A2-6). Different types of aberrations were noted in this genetic background, including many with chromatid or isochromatid deletions. Numerous cells of this genotype carried more than one aberration (Figure 9 , A4), with some genomes appearing largely fragmented (Figure 9 , A5-6). The frequency of CAB occurrence per brain between genotypes was quantified over multiple brains, revealing a significant increase in the average ratio of cells per brain that contain CABs in the homozygous su(Hw) e04061 background (11.9%, Figure 9 B and C) as compared to the wildtype strain Oregon R (OR) (p=0.004). The frequency of CABs observed in OR is 0.7%, close to previously reported values for wildtype CAB frequency (Mengoli et al., 2014; Merigliano et al., 2017) , and is close in value to that obtained from the su(Hw) e04061 /TM6B heterozygote. Examination of brains from a trans-heterozygous su(Hw) v/e04061 genotype showed a similar increase in CAB frequency over OR as the su(Hw) e04061 homozygote (p=0.0298), implying that this phenotype is not due to second-site mutations in the su(Hw) e04061 genetic background. This suggests that su(Hw) has a role in maintaining chromosome integrity and that half the wildtype dosage of su(Hw) is sufficient to prevent chromosomal aberrations from occurring.
This study has uncovered a novel role for Su(Hw) in the maintenance of genome stability. This Ataxia Telangiectasia-Related (ATR) and Ataxia Telangiectasia-mutated (ATM) are two highly conserved kinases with central roles in DNA repair, cell-cycle checkpoint progression, and cell fate determination (Sancar et al., 2004) . In the DNA damage response, ATM primarily acts in repair of DSBs, whereas ATR is activated in response to various DNA lesions, particularly those generated by replication stress (Zeman and Cimprich, 2014) . Both kinases are able to phosphorylate unique downstream effectors as well as a number of common targets, such as the human H2AX or the Drosophila equivalent H2Av. Phosphorylated H2AX (gH2AX) in humans, or phosphorylated H2Av (gH2Av) in Drosophila, is an indicator for ATR/ATM activity, and crosstalk between these two pathways occurs in response to DNA damage (Cimprich and Cortez, 2008; Sirbu and Cortez, 2013) . Although the detailed mechanism of how ATR and ATM coordinately function during oogenesis in Drosophila is still unclear, it seems that both proteins have distinct functions. Specifically, ATM is primarily involved in DNA damage repair, whereas ATR is also involved in cell-cycle checkpoint regulation (Joyce et al., 2011).
Due to the fundamental role that gH2AX (or gH2Av) has in all DNA damage repair pathways, the recognition of this modification has become a standard assay for DSB and DNA damage detection. In this study, we find a strong accumulation of gH2Av in mutant nurse cells, and reveal that mutation of the checkpoint gate keeper ATR/mei-41 rescues the spindle-like phenotype in su(Hw) mutants. Previous observations have shown that an elevated frequency of DSBs is generally produced by mutations in DNA repair genes or by retrotransposon activity induced by mutations in components of the pi-RNA pathway. Both phenomena can lead to DNA damage and the activation of checkpoint repair pathways mediated by ATR and Chk2, which are phenotypically characterized by microtubule disorganization and translational repression of Grk (Chen et al., 2007; Klattenhoff et al., 2007; Mohn et al., 2014; Sienski et al., 2012) . Our data, however, shows that su(Hw) mutant egg chambers display microtubule disorganization and Grk mislocalization, and that mutants for ATR and Chk1 (grp), but not Chk2 (mnk, lok), partially rescue this phenotype. This important difference suggests that an alternative pathway, mediated by ATR and Chk1 is activated by a response to accumulation of DNA damage in su(Hw) mutant ovaries ( Figure 10 ). This conclusion is further supported by our observation that DNA damage in su(Hw) mutant ovaries results from a pathway independent from transposable element activity.
A previous study reports that cutting the gene dosage of an RNA-binding protein, Rbp9, in half results in the rescue of female fertility in su(Hw) 2/v mutants (Soshnev et al., 2013) . This study suggests that female sterility in su(Hw) 2/v mutants is due to the derepression of neuronal genes such as rbp9 in the germline that are under the transcriptional control of Su(Hw) binding sites. Our results support the idea that Su(Hw) has roles during oogenesis that extend beyond transcriptional regulation. One possibility against this notion is that Su(Hw) may control the expression of genes involved in DNA damage signaling in the ovaries. However, published reports show no indication that Su(Hw) specifically controls the expression of genes involved in DNA repair or replication pathways (Baxley et al., 2011; Hsu et al., 2015) . Instead, we suggest that null mutations of su(Hw) result in a complex multifactorial oogenesis phenotype. Sterility is caused mostly by the over-expression of neuronal genes such as rbp9, due to the lack of transcriptional repression activity of Su(Hw), whereas the spindle-like phenotype described here would be dependent on the structural activity of Su(Hw) in the genome through its insulator function. Thus, a reduction of the levels of rbp9 is enough to restore oogenesis development but not sufficient to eliminate the ventralization phenotype induced by replication stress and the activation of the ATR/chk1 checkpoint. Likewise, expression of su(Hw)::eGFP driven by nanos-GAL4 can partially rescue fertility but does not completely eliminate the spindle-like phenotype, producing frequent mislocalization of Grk and ventralized eggshells. and damaged DNA has been observed at the junction between replicated euchromatin and underreplicated heterochromatin regions (Hammond and Laird, 1985; Lilly and Spradling, 1996b; Yarosh and Spradling, 2014) . In salivary gland polytene chromosomes, for example, the accumulated gH2Av signals are detected at local underreplication sites (Andreyeva et al., 2008) .
Because Su(Hw) has been associated with heterochromatin-euchromatin boundaries (Khoroshko et al., 2016) , one possibility is that the absence of Su(Hw) in mutants disrupts the endoreplication process and leads to an excess of DNA damage at these sites. DNA breaks may result from stalled replication forks at the boundaries that fail to reinitiate replication (Lilly and Spradling, 1996a; Mirkin and Mirkin, 2007; Peng and Karpen, 2008) . Interestingly, this mechanism has been suggested to explain the five lobes nuclear organization phenotype, which is typical in nurse cells during stages 1 to 5 of oogenesis, but that extends beyond stage 5 in su(Hw) and in other oogenesis mutants (Dej and Spradling, 1999) . The 5 lobes structure is the result of homolog chromosome pairing during re-replication, and in normal nurse cells, these chromosomes separate and disperse in the nucleus after stage 5. The 5 lobes anomalous phenotype at later stages results from failure of chromosomes to disperse, which is likely due to unfinished replication forks that prevent sister chromatids from dispersing after stage 5 (Dej and Spradling, 1999) .
Whereas research on insulator proteins has focused mostly on their roles in transcription, the possibility that chromatin insulators might also be involved in genome replication emerged recently after analysis of the genomic distributions of insulators and origin of replication proteins. This analysis revealed that a number of ORC and MCM2-7 helicase complex binding sites overlap with binding sites of Su(Hw). In addition, Su(Hw) is also capable of altering chromatin accessibility by recruiting the histone acetyltransferase SAGA and the BRAHMA chromatin remodeling complex, thereby creating a platform of low nucleosome density levels favorable for the recruitment of ORC and replication firing (Lu and Tower, 1997; Vorobyeva et al., 2013) . In line with these findings suggesting a role for Su(Hw) in the regulation of DNA replication, we have also found a significant accumulation of H4K20me1 in su(Hw) mutant ovaries. H4K20me1 is highly regulated during the cell cycle and plays an important role in the licensing of origins of replication. PR-Set7 is the histone methyltransferase responsible for the monomethylation of H4K20 (H4K20me1). Mutations in PR-Set7 in mice models suggest that this protein has a role in DNA replication (Abbas et al., 2010; Tardat et al., 2010a) . Specifically, mutations in set7 that prevent PR-Set7 degradation during the cell cycle are lethal due to uncontrolled re-replication of the genome (Centore et al., 2010) , and loss of PR-Set7 enzymatic activity also causes defects in origin of replication firing (Jorgensen et al., 2007; Tardat et al., 2007) . Further experimental evidence has shown that the role of PR-Set7 in origins of replication depends on its specific mono-methyltransferase activity on H4K20. H4K20me1 functions as a substrate for subsequent methylation by Suv4-20h1 and Suv4-20h2 methyltransferases, which further dimethylate and trimethylate H4K20me1, respectively. H4K20me2 and H4K20me3 directly bind ORC and promote firing of replication forks at replication origins (Beck et al., Altogether, our data suggest that DNA damage and genome instability may arise upon loss of Su(Hw) function in both germline and replicating somatic cells. Whether the DNA damage in su(Hw) mutants results from defects in genome organization that lead to replication stress, defects in the DNA repair pathway, or both is still unknown. However, our observation of chromosomal aberrations in dividing neuroblasts in su(Hw) mutants suggest this phenomenon expands also to replicating somatic cells and is not limited to the germline or to polytene chromosomes. Our study opens a new avenue to further understand the role of architectural proteins in genome function and genome stability.
Fly genetics: All fly stocks were cultured on cornmeal-agar food with yeast at 25°C. The fly stocks used in this study are: y 2 wct 6 ; su(Hw) v /TM6B, a gift from Victor Corces (Emory University); mei-41 D5 , and w*; P{GAL4-nos.NGT40} (BDSC: 4442), which we refer to as nos-GAL4 through the text, were gifts from Laura Lee (Vanderbilt University). mei-w68 and P{nanos-GAL4::VP16}, which we refer to as nanos-GAL4 throughout the text, were gifts from Documentation of eggshell phenotype: Two-to three-day-old su(Hw) mutant females carrying the su(Hw)::eGFP transgenes driven by P{GAL4-nos.NGT40} or P{nanos-GAL4::VP16}, were crossed to yw male flies. Eggs were collected for three days using grape juice agar plates containing wet yeast paste (Sullivan et al., 2000) . The eggshell morphology was observed and documented using a Leica MZ16FQ stereomicroscope equipped with a Leica DFC420 digital camera.
Western blot: Three to five-day-old female ovaries were dissected and homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors (Roche). In some samples, stages 10 and older were removed manually under the dissecting microscope, previously to homogenization. After removal of stages 9 and older, wildtype and mutant ovaries contain the same developmental stages. Lysates were resolved on a 15% acrylamide gel, transferred overnight at 4°C, and probed with anti-gH2Av (Rockland, 1:1000), rabbit anti-monomethylated H4K20 (Abcam, 1:1000) and mouse anti-lamin Dm0 (DSHB, 1:1000).
Real-time PCR quantification of TE expression was carried out with ABGene (Rockford, IL) SYBR green PCR master mix. PCR conditions for each primer pair were tested to determine the efficiency of amplification and to ensure that the amplification was in the linear range. PCR products for each primer pair were amplified from cDNA using the BioRad iQ5
Multicolor Real-Time PCR detection system (Primers listed in Supporting Information Table 1 ).
cDNA was reverse transcribed from at least three different RNA samples. Threshold Cycle (Ct) values were normalized to the Ct values of the housekeeping gene rp49. Change in expression level was calculated using the ∆∆Ct method based on the Ct value for each PCR reaction (BioRad real-time PCR application guide). Results are presented as fold change in mutant relative to wildtype. The statistical significance of the results was calculated using Student's ttests.
Wandering third instar larvae were quickly dissected in PBS to obtain the brain. Brains were incubated in a small volume of 0.5% sodium citrate, pH 6.00, for 10 minutes. The brains were then fixed in 4% paraformaldehyde (2:1:1 acetic acid, 16% paraformaldehyde, dH 2 O) for 1 minute. The brains were squashed between a slide and a coverslip before staining with 4′, 6diamidino-2-phenylindole (DAPI, 0.5 µg/mL) and were mounted in Vectashield mounting medium (Vector Laboratories). To determine the frequency of metaphasic cells that show chromosomal aberrations (CABs) in larval brains, pictures were taken of approximately fifty fields of view per sample that contained at least one metaphasic cell. Microscopy was performed as described above. Micrographs of neuroblast karyotypes were examined using FIJI (Schindelin et al., 2012) and each metaphasic cell was scored manually for the presence of CABs based on DAPI staining. Comparisons between genotypes were performed using two-tailed Mann-Whitney t-tests. Mutation of genes encoding DSB repair proteins or helicase (spindle mutants, left) disable the mechanisms that repair meiotic DSBs generated at an early stage of oogenesis. Overactivated Transposable Elements (TEs) may produce an excess of DSBs. This type of DSBs is usually observed in piRNA pathway mutants. In both spindle mutants and piRNA pathway mutants, unrepaired DSBs activate an ATR/Chk2 dependent DNA damage checkpoint that leads to a sequence of developmental defects, including microtubule disorganization, Grk mislocalization, Grk repression, and patterning defects in eggshells. In su(Hw) mutant females, replication stress induces DNA damage during endoreplication (red pathway) and activates an ATR/Chk1 checkpoint that leads to Grk mislocalization and dorsoventral transformations. 