L(3)mbt and the LINT complex safeguard tissue identity in the Drosophila ovary

Maintenance of cellular identity is essential for tissue development and homeostasis. At the molecular level, cell identity is determined by the coordinated activation and repression of defined sets of genes. Defects in the maintenance of the genetic programs required for identity can have dire consequences such as organ malformation and cancer. The tumor suppressor L(3)mbt was shown to secure cellular identity in Drosophila larval brains by repressing germline-specific genes. Here we interrogate the temporal and spatial requirements for L(3)mbt in the Drosophila ovary, and show that it safeguards the integrity of both somatic and germline tissues. L(3)mbt mutant ovaries exhibit multiple developmental defects, which we find to be largely caused by the inappropriate expression of a single gene, nanos, a key regulator of germline fate, in the somatic cells of the ovary. In the female germline, we find that L(3)mbt represses testis-specific and neuronal genes. Molecularly, we show that L(3)mbt function in the ovary is mediated through its cofactor Lint1 but independent of the dREAM complex. Together, our work uncovers a more complex role for L(3)mbt than previously understood and demonstrates that L(3)mbt secures tissue identity by preventing the simultaneous expression of original identity markers and tissue-specific misexpression signatures.


Introduction
Development requires tight control of gene expression as differentiating cells must express lineage-specific genes while repressing genes that promote other fates. Mechanisms ensuring the maintenance of cellular identity must be robust, as changes in cell fate appear to be fairly uncommon in wild-type conditions, with only one documented case of regulated, complete fate switch being described in C. elegans (Jarriault et al., 2008). However, rare cases of transdifferentiation have been observed in mutants affecting chromatin complexes, suggesting a role for chromatin structure in the maintenance of cellular identity (Petrella et al., 2011;Tursun et al., 2011). A notable example is given by mutations affecting the Drosophila lethal (3) malignant brain tumor (l(3)mbt) gene, which cause malignant brain tumors that ectopically express germline-specific genes and have been proposed to be soma-to-germline transformations (Gateff et al., 1993;Janic et al., 2010). Gene expression profiling of l(3)mbt brain tumors and L(3)mbt-depleted cultured somatic cells identified a group of upregulated genes known as the Malignant Brain Tumor Signature (MBTS) that is enriched for factors specifically expressed in germ cells (Georlette et al., 2007;Janic et al., 2010;Meier et al., 2012;Sumiyoshi et al., 2016).
Mutations of germline-specific genes, including those impairing the piRNA factors piwi, aub, and vasa, as well as the translational repressor nanos, were found to suppress the neural overgrowth induced by loss of L(3)mbt (Janic et al., 2010). A subsequent study provided evidence that the Hippo growth control pathway is critical for l(3)mbt mutant brain overgrowth, suggesting an alternative cause of tumorigenesis (Richter et al., 2011). Furthermore, our lab showed that strong l(3)mbt mutations cause a maternal, germline autonomous phenotype that precludes normal embryonic development including primordial germ cell formation (Yohn et al., 2003). Together, these studies suggest that L(3)mbt may impart many functions in regulation of tissue identity. L(3)mbt encodes a 1477 amino-acid protein that is ubiquitously expressed in Drosophila and is conserved from worms to humans. L(3)mbt is thought to be a chromatin reader and harbors three MBT repeats that bind methylated histone tails in vitro as well as a Zinc-Finger domain (Bonasio et al., 2010). L(3)mbt is enriched at the promoters of repressed genes, suggesting a direct role in transcriptional repression but its binding sites overlap with insulator elements, indicating that L(3)mbt might also function as an insulator accessory factor (Richter et al., 2011;Van Bortle et al., 2014). Notably, L(3)mbt was purified in two non-enzymatic repressive chromatin complexes: the Drosophila RBF, E2F2, and Myb-interacting proteins (dREAM complex, also called Myb-Muv B) as well as the L(3)mbt-Interacting complex (LINT complex) (Lewis et al., 2004;Meier et al., 2012). DREAM is a large multi-subunit complex that controls gene expression throughout the cell cycle but also represses developmental genes. L(3)mbt associates at sub-stoichiometric levels with dREAM and is strictly found in its repressive forms (Georlette et al., 2007;Lewis et al., 2004). The LINT complex is composed of L(3)mbt, the novel transcriptional repressor Lint1 as well as the corepressor CoREST, and was shown to silence developmental genes in cultured cells (Meier et al., 2012). Interestingly, dREAM and LINT complexes repress overlapping sets of genes in somatic cells, including genes that are normally expressed in the germline. Despite extensive biochemical studies, we still know little about which chromatin complex mediates L(3)mbt's role in tissue identity.
Drosophila melanogaster ovaries are composed of 16 to 20 egg assembly chains called ovarioles ( Fig. 1A,B). At the tip of each ovariole a region called the germarium houses germline stem cells (GSCs), which divide asymmetrically to generate a new GSC and a differentiating daughter cell.
The differentiating GSC daughter undergoes four rounds of mitosis with incomplete cytokinesis to form a sixteen-cell germline cyst in which sibling germ cells remain interconnected through cytoplasmic bridges called ring canals. GSCs are marked by a spectrin-containing spherical Endoplasmic Reticulum-derived vesicle known as spectrosome, which fuses into a branched fusome connecting the cells of the same cysts through the ring canals (Huynh, 2006). Only one of the cyst germ cells develops into an oocyte, while the other 15 cells become supportive, polyploid nurse cells. Somatic cells of the ovary play important roles in supporting oogenesis: they compose the GSC niche that promotes cyst divisions and differentiation, and the follicle cells enclose and individualize egg chambers facilitating oocyte-nurse cell development.
To understand how L(3)mbt secures tissue identity, we combined genetic and genomic approaches to characterize the functions of L(3)mbt in Drosophila melanogaster ovarian development. We find that L(3)mbt affects gene expression in a tissue-specific manner. In somatic cells of the ovary, L(3)mbt represses germline genes, whereas in the female germline it controls genes normally expressed in the testis and the nervous system. Mutant ovarian tissues continue to express signatures of the tissue of origin, indicating that loss of L(3)mbt does not induce transdifferentiation. Remarkably, we show that ectopic expression of a single gene in the somatic ovarian cells, the translational repressor and key regulator of germline fate nanos, is largely responsible for aberrant development. Using a genetic approach, we find that in the ovary L(3)mbt function requires its cofactor Lint1 but is independent of the dREAM complex.
Together, our experiments provide insight into the role of L(3)mbt in securing tissue identity by repressing expression signatures characteristic of other tissues that are incompatible with normal development.

Sterility in l(3)mbt mutant females is associated with aberrant ovarian development
L(3)mbt was previously shown to be required for the development of the nervous system as l(3)mbt mutant flies grown at restrictive temperatures (29˚C) develop malignant brain tumors and die at larval stages (Gateff et al., 1993;Janic et al., 2010;Richter et al., 2011). We observed that, when grown at lower temperature (18˚C or 25˚C), null l(3)mbt mutant females were viable but fully sterile, indicating that L(3)mbt is critically required for germline development. At the macroscopic level, l(3)mbt mutant ovaries were atrophied and adult females did not lay eggs. To characterize the ovarian phenotype in detail, we used antibodies against the germline marker Vasa and α-Spectrin (α-Spe), which labels the membranes of somatic cells and spectrosomes/fusomes. Similar to the wild type, l(3)mbt mutant germaria contained GSCs adjacent to the somatic niche ( Fig. S1 A-B). However, mutant ovarioles contained fewer individualized egg chambers (1.35 egg chambers/ovariole in average vs. 6 in WT, p<10 -4 ), which were highly abnormal and displayed several defects (Fig. 1C, E, G, quantified in J). Such defects included extra-numerous germ / nurse cells within egg chambers ( Fig. 1C; "extra-numerous differentiated germ cells" in Fig. 1J) as well as egg chambers in which somatic follicle cells fail to fully enclose germ cells (Fig. 1E, arrowhead; "packaging defects" in Fig 1J). In the wild type, fusomes degenerate as cysts proceed past the 16 cell stage and are enveloped by somatic follicle cells (Huynh and St Johnston, 2004). In l(3)mbt mutants, however, we observed accumulation of undifferentiated germ cells, which were marked by branched fusomes and enclosed by follicle cells (Fig. 1G, arrow; "extra-numerous undifferentiated germ cells" in Fig 1J).
Another hallmark of the 16-cell wild-type cyst is the specification of a single oocyte, while the remaining fifteen cells develop into polypoid feeder cells, the nurse cells. Using the RNA binding protein oo18 RNA-binding protein (Orb, Christerson and McKearin, 1994) as a marker for the future oocyte (figure 1H, arrowhead), we observed that most mutant egg chambers had multiple Orb positive cells (Fig. 1I, arrowheads). Wild-type oocytes are connected to the adjacent nurse cells by four ring canals, as a product of four divisions (Huynh and St Johnston 2004). We determined whether the additional cells were bona fide oocytes by counting the associated ring canals (stained by F-Actin, Fig. S1C and D, yellow arrows and dotted circles) and found that ectopic Orb-expressing cells contained four or more ring canals, while egg chambers contained multiple oocytes and extra-numerous germ cells. Thus, l(3)mbt loss causes egg chamber fusions and possibly, additional rounds of cyst division. In rare cases (2%), we observed multiple germaria connected to the same aberrant egg chamber (Fig. S1 E,F), suggesting that ovarioles were fused during ovary morphogenesis. Taken together, our results indicate that in addition to its previously reported, conditional requirement in the brain, L(3)mbt has a critical, temperature-independent role required for ovarian morphogenesis and differentiation.

L(3)mbt functions in ovarian somatic cells to safeguard ovary development
Previous experiments had shown that loss of l(3)mbt specifically in germ cells caused developmental defects in the resulting embryos, but allowed oocytes to mature (Yohn et al., 2003). Thus, we wondered whether the gross abnormalities of mutant ovaries were indicative of a role for L(3)mbt in somatic cells of the ovary. To determine the tissue-specific requirement of L(3)mbt, we generated homozygous l(3)mbt GM76 mutant clones in the ovarian somatic cells by using the FRT-FLP system (Harrison and Perrimon, 1993) under the transcriptional control of the Ptc-Gal4 or c587-Gal4 drivers, which drive expression in the somatic cells of the germarium (Hinz et al., 1994;Zhu and Xie, 2003). Interestingly  Fig. S2A,B). These results demonstrate that L(3)mbt is required specifically in the somatic tissues of the ovary to support normal oogenesis.

Mutant larval somatic cells are properly specified but intermingled cells fail to contact with germ cells
Ovarioles lacking L(3)mbt exhibit striking morphological defects. Most of the structures in the adult ovary are established and organized during the third instar larval (L3) stage (Gilboa, 2015), we thus examined ovaries from mid to late L3 larvae to investigate whether l(3)mbt mutation affects ovarian organogenesis. Germ cells and somatic cells associate during late embryonic stages and proliferate during most of larval development. Starting mid L3 stage, the somatic precursors differentiate into distinct populations of somatic cells (Gilboa and Lehmann, 2006;Li et al., 2003). In the apical compartment, post-mitotic terminal filament cells stack to form terminal filaments and associate with sheath cells (Godt and Laski, 1995). In the medial region, the Intermingled cells (IC) are closely associated with germ cells and are thought to give rise to the adult escort cells (Gilboa, 2015). We performed confocal imaging analysis by immunostaining with antibodies against the transcription factor Traffic Jam (TJ), which has important functions in specifying somatic gonadal cell types and labels the ICs (Li et al. 2003), Vasa and α-Spectrin. We observed that, like their wild-type counterparts, l (3)

L(3)mbt mutant somatic larval cells ectopically express the germline marker vasa
Previous studies suggested that L(3)mbt loss results in derepression of germline genes in the larval brain and cultured somatic cells (Georlette et al., 2007;Janic et al., 2010;Meier et al., 2012;Sumiyoshi et al., 2016). Thus, we asked whether germline genes were ectopically expressed in mutant larval somatic cells. Indeed, we observed faint Vasa antibody signal in the somatic tissues of l(3)mbt mutant ovaries, especially in the apical compartment ( Fig. 3B and S3).
To further demomstrate these initial observations, we induced l(3)mbt mutant clones using the c587-Gal4 driver to express FLP recombinase specifically in the somatic tissues of the larval ovary (Zhu and Xie, 2003). Induction of wild-type control clones (labeled by the absence of GFP) in somatic cells of the basal or apical compartment did not cause Vasa expression (Fig.   3C). In contrast, l(3)mbt GM76 homozygous clones of somatic cells exhibited Vasa staining (Fig.   3D). From this, we conclude that L(3)mbt represses the germ cell marker vasa in the somatic cells of the larval ovary.

L(3)mbt mutant somatic ovarian cells simultaneously express somatic gonad and germlinespecific genes
To gain a genome-wide view of gene expression changes induced by loss of L(3)mbt in adult somatic ovarian cells in vivo, we performed RNA-sequencing (RNA-seq) analysis. To distinguish between germline and somatic ovarian tissues, we took advantage of tud maternal mutations (tud M ), which give rise to progeny that lack germ cells and develop into adults devoid of germline (Arkov et al., 2006;Smendziuk et al., 2015; see File S1 and Materials and  (Table   S1). 44 upregulated genes were shared with the 101 MBTS genes (Janic et al., 2010), and 115 out of the 681 genes found upregulated in Δ−l(3)mbt Ovarian Somatic Cells (OSC; Sumiyoshi et al., 2016). These derepressed genes include piRNA pathway components and germline-specific genes such as nos, Pxt, vas, aub, tej, krimp, AGO3, and CG9925 (Fig. 4A, Fig. S4A). The effect of l(3)mbt mutation on gene expression was more pronounced for repressed genes, while genes normally expressed in the soma showed only low fold-changes in the mutant (124/141 had a Log 2 fold-change between -0.3 and -1). To investigate whether mutant somatic cells retained their somatic identity, we performed immuno-stainings against the key transcription factors Traffic Jam (TJ) and Zfh1, which are essential for gonad development and exclusively expressed in somatic cells (Leatherman and Dinardo, 2008;Li et al., 2003). Despite the gross morphological abnormalities, we observed TJ or Zfh1 expressing cells surrounding germ cells in mutant ovaries (

Expression of Nos is necessary and sufficient to cause ovarian defects
Aberrant growth of l(3)mbt brain tumors was shown to rely on the ectopic expression of nos, aub, and vasa (Janic et al., 2010). We find that each of these genes is indeed upregulated in compromised somatic ovarian cells lacking L(3)mbt ( Fig. 4A and S4A). We therefore asked whether their misexpression contributed to the ovarian defects observed of l(3)mbt mutants by generating double mutant animals. Due to lethality, we were unable to assess ovarian phenotypes in vas, l(3)mbt mutants. However we found that aub, l(3)mbt double mutant ovaries were phenotypically similar to l(3)mbt single mutant, containing many apoptotic cells and aberrant egg chambers with more than 16 germ cells (Fig. S5A). In contrast, nos BN /nos L7 mutations dramatically suppressed the l(3)mbt ovarian defects: nos, l(3)mbt double mutant ovaries contained late stages egg chambers with ovarioles phenotypically indistinguishable from wild type and 85% of double mutant egg chambers contained 16 germ cells and only one oocyte ( Fig.   5C-E, n=88). Consistently, depletion of Nanos' co-factor Pumilio in l(3)mbt mutant ovaries also suppressed the mutant phenotypes, with 81% of pum 680 , l(3)mbt double mutant ovarioles resembling wild-type morphology ( Fig. 5F-H, n=181). These data suggest that Nanos is a critical factor leading to l(3)mbt mutant ovarian phenotypes. To test whether nos misexpression in somatic ovarian cells is sufficient to cause l(3)mbt-like ovarian defects, we ectopically expressed nos in the somatic cells of the ovary using the tj-Gal4 driver. Nos misexpression during larval stages caused lethality, we therefore restricted nos expression in the soma to adult stages using the Gal80 ts system (McGuire et al., 2004). Nos somatic expression perturbed ovarian morphology resulting in defects of the follicle epithelium and poorly individualized egg chambers (Fig. S5B).
While this phenotype resembles the l(3)mbt phenotype, it does not fully recapitulate it, possibly because nanos was only misexpressed in adult tissues likely at higher levels compared to l(3)mbt mutants. In contrast, ectopic expression of aub or vas in somatic ovarian cells did not yield a morphologically significant phenotype (Fig. S5C, D). Together, these results suggest that ectopic expression of nos, but not aub or vas, is necessary and sufficient to cause aberrant somatic ovarian development.

L(3)mbt functions through the LINT complex to secure ovarian development
L(3)mbt has been associated with two chromatin complexes that repress developmental and germline genes: the dREAM and LINT complexes ( Fig. 6 A-B; Georlette et al., 2007;Meier et al., 2012). To determine the function of these complexes in ovarian development, we depleted somatic cells from E2f2 and Mip120, two core repressors of the dREAM complex. Mutant somatic clones for E2f2 (Fig. 6C) or ovaries deficient for mip120 (Fig. 6E) did not result in phenotypic aberrations reminiscent of those observed in l(3)mbt mutants. We also asked whether mip120 mutation affected L(3)mbt nuclear localization in the ovary, as previously described in salivary glands (Blanchard et al., 2014). Using the TdTomato::L(3)mbt fusion protein we observed L(3)mbt nuclear localization comparable to wild type in mip120 67.9A.9 mutants (Fig.   S6B). Together, these results suggest that L(3)mbt critical function in somatic ovarian cells is independent of the dREAM complex.

L(3)mbt is autonomously required in the germline for egg chamber survival and represses neuronal and testis-specific genes in the female germline
In addition to its role in the development of the somatic cells of the Drosophila ovary, L(3)mbt also has a maternal, germline-autonomous role that supports nuclear divisions during early embryogenesis (Yohn et al., 2003). Similarly, we observed that embryos laid by mutant females, which somatically expressed the complementing l(3)mbt::myc transgene, failed to hatch. Further, while most egg chambers appeared morphologically normal in somatically-complemented l(3)mbt mutant ovaries, we noticed that 69% of ovarioles contained apoptotic egg chambers (Fig.   7A,B). This suggests that in addition to its previously identified maternal effect function for early embryonic development, L(3)mbt is autonomously required in the germline for egg chamber development.
Our data highlight a role for L(3)mbt in suppressing germline specific genes in somatic tissues of the ovary. However, our experiments also uncovered a germline autonomous requirement for egg chamber development. To gain a genome-wide view of the changes in gene expression specifically induced by loss of L(3)mbt in the germline, we performed RNA-seq analysis on embryos laid by l(3)mbt mutant or heterozygous mothers expressing the L(3)mbt::myc fusion in the somatic ovary. We used early embryos prior to activation of the zygotic genome as the early embryonic RNA pool is exclusively composed of maternally provided transcripts and thereby reflects the germline expression profile during oogenesis (Edgar and Schubiger, 1986). Our analysis identified 878 differentially expressed genes (adjusted p-value<0.05), of which 342 were upregulated and 536 downregulated in embryos laid by mutant females (Table S2). Most upregulated genes were uncharacterized and not enriched for specific Gene Ontology terms.
Thus, to better characterize the group of genes upregulated in the l(3)mbt mutant germline, we performed two-way hierarchical clustering to identify any tissue specific expression signatures.
Two major groups were readily identified: one composed of genes highly expressed in neuronal tissues (brain, thoracicoabdominal ganglion (tag), larval CNS, eye, and head; Fig. 7C) and another comprising of testis-specific genes. As in mutant somatic ovaries, two-thirds (365/536) of the downregulated genes had their expression level reduced by less than two-fold compared to the heterozygous controls (Fig. S7). We observed only a limited overlap between the genes derepressed in l(3)mbt mutant somatic and germline ovarian tissues, with two thirds of genes being specifically upregulated in one of the two tissues (Fig. 7D). Taken together, these results indicate that L(3)mbt function is not restricted to repressing the germline program in somatic tissues, but that L(3)mbt regulates distinct sets of genes, in a tissue-specific manner.

Discussion
By combining developmental and molecular analysis, we show that l (3) Our experiments demonstrate that ectopic expression of nanos is necessary and sufficient to induce aberrant development of l(3)mbt mutant ovaries. These defects are likely not due to a direct interference at the transcriptional level but are rather caused by Nos' function as a translational repressor. In support, we find that Pumilio, the sequence-specific translational repressor and co-factor of Nos, is also essential for the l(3)mbt ovarian phenotype. Nos was recently shown to modulate Pum RNA-binding and target-specificity in somatic S2 cells (Weidmann et al., 2016). Since Pum is ubiquitously expressed, we propose that ectopic Nos stabilizes Pum binding at target mRNAs essential for somatic functions. Interestingly, ectopic expression of NANOS1 was found to be required for growth of human pRb deficient tumor cells.
In this case, NANOS1 and PUM repress p53 translation allowing cells to bypass apoptosis (Miles et al., 2014). Thus, ectopic Nos-Pum complexes may alter tissue maintenance at the posttranscriptional level in other systems as well. As Nos has been found to repress somatic genes in germ cells of multiple organisms (Hayashi et al., 2004, Lee et al., 2017, we would expect to find key regulators of somatic fate among mRNAs aberrantly targeted by Nos-Pum in somatic Drosophila tissues. In contrast to the widespread effects of nos derepression observed in multiple somatic tissues upon loss of L(3)mbt, ectopic expression of additional genes may define the exact phenotypic consequences, which depend on tissue type. For example, piRNA pathway genes are ectopically expressed in l(3)mbt larval brain tumors and somatic ovarian cells, however, depleting them ameliorates the brain tumor but not the ovarian phenotype. This difference may be explained by the fact that the somatic ovary uses core components of the piRNA pathway to regulate transposable elements (Handler et al., 2013). Similarly, we did not observe derepression of Hippo target genes in ovarian tissues. Consistent with our finding that in l(3)mbt mutants new and original tissue identities are co-expressed, these results suggest that the phenotypic consequences of l(3)mbt mutation depend on the context of the original tissue identity.
Our results demonstrate that L(3)mbt function in the ovary is independent of the dREAM complex. The dREAM complex has a well-established role in cell-cycle regulation (Sadasivam and Decaprio 2013). Indeed, Mip120, a core dREAM component, was recently found to be required for decondensation of nurse cell nuclei (Cheng et al., 2017) and E2F2 is required for endo-replication of follicle cells (Cayirlioglu et al., 2001). We did not observe a role for l(3)mbt in the regulation of nurse and follicle cell endo-replication. Instead, our data support the hypothesis that in the ovary, L(3)mbt functions predominantly through the LINT complex and that this complex can be functionally separated from of the dREAM complex. Considering the moderate phenotype of lint1 1 mutants and that gene function is apparently dispensable at 25˚C, we speculate that L(3)mbt exerts most of the repressive activity of this new complex, possibly with additional, yet unidentified interactors and that Lint1 has an accessory role.
Loss of L(3)mbt causes the ectopic expression of a number of genes including cell identify regulators that interfere with original cellular function and affect tissue development. In contrast to a previously suggested soma-to-germline transformation, our results favor the hypothesis that l(3)mbt mutation imbalances tissue-homeostasis whereby normally mutually exclusive lineage determinants become simultaneously expressed. In support, L(3)mbt depletion in neuronal, somatic ovarian, and germ cells does not lead to loss of original tissue-specific markers, but genes characteristic of other lineages are derepressed (Richter et al. 2011, this study). Moreover, our results suggest that the role of L(3)mbt is not solely restricted to prevent ectopic expression of germline genes, but instead L(3)mbt represses distinct, broader sets of genes in a tissue-specific manner. Therefore, we propose that L(3)mbt secures tissue identity by stabilizing gene expression profiles established during differentiation.     mutant early embryos, as measured by RNA-seq (normalized counts, log 10 ). Up-regulated genes are shown in red, down-regulated genes are shown in blue.

Generation of transgenic lines
To generate UASp-l(3)mbt::myc transgenic flies, l(3)mbt coding sequence was amplified from LD05287 gold cDNA (Drosophila Gemonics Resource Center), cloned using the p-ENTR/D-TOPO system and recombined into the pPWM destination vector (Drosophila Gateway Vector Collection) using Gateway technology (Invitrogen). pPWM-l(3)mbt was randomly inserted on the 2 nd chromosome through P-transposition. To generate the lint1 1 mutation, the target sequence (chrX:11044844-11044866) was identified by the flyCRISPR Optimal Target Finder tool (Gratz et al., 2014), amplified from genomic DNA using lint-CRISPR oligos (Supplemental Oligos   Table) and ligated in the pU6-BbsI-gRNA plasmid (Gratz et al., 2013). The resulting construct was injected in FRT19A;; vas-Cas9 embryos and progeny was screened by PCR and sequencing.

RNA-seq data analysis
Sequencing results were demultiplexed and converted to FASTQ format using Illumina Bcl2FastQ software (Illumina). Reads were aligned to the fly genome (build dm3/BDGP5) using the splice-aware STAR aligner (Dobin et al., 2013). PCR duplicates were removed using the Picard toolkit (https://broadinstitute.github.io/picard/). HTSeq package was used to generate counts for each gene based on how many aligned reads overlap its exons (Anders et al., 2015).
These counts were then used to test for differential expression using negative binomial generalized linear models implemented by the DESeq2 R package.

Tissue expression clustering
Expression of deregulated genes was extracted from FlyAtlas (Chintapalli et al., 2007) using FlyBaseIDs, normalized to fly average and log2 transformed. Distance matrix was calculated using the "Manhattan" method and data clustered using "ward.D2". Heatmap was generated using the heatmap.2 function of the gplots R package.