TECHNIQUES AND RESOURCES RESEARCH ARTICLE

Validating upstream regulators of Yorkie activity in Hippo signalingthrough scalloped-based genetic epistasisJianzhong Yu1,2 and Duojia Pan1,*

ABSTRACTGenetic studies in Drosophila have been instrumental incharacterizing the Hippo pathway, which converges on the co-activator Yorkie to regulate target gene transcription. A routinely usedstrategy to interrogate upstream regulators of Yorkie involves theexamination of selected Hippo target genes upon loss or gain offunction of a suspected pathway regulator. A caveat with this strategyis that aberrant expression of a given Hippo target per se does notdistinguish whether it is caused by changes in Yorkie or Yorkie-independent inputs converging on the same target gene. Building onprevious findings that the DNA-binding transcription factor Scallopedmediates both Yorkie overexpression and loss-of-functionphenotypes yet is itself dispensable for normal eye development,we describe a simple strategy to distinguish these possibilities byanalyzing double-mutant clones of scalloped and a suspected Yorkieregulator. We provide proof of principle that this strategy can be usedeffectively to validate canonical Yorkie regulators and to excludeproteins that impact target expression independent of Yorkie. Thedescribed methodology and reagents should facilitate efforts toassess the expanding repertoire of proteins implicated in regulation ofYorkie activity.

KEY WORDS: Hippo signaling, Sd, Yki, Bantam, Expanded,Interommatidial cells

INTRODUCTIONThe Hippo signaling pathway is an evolutionarily conservedmechanism that regulates diverse physiological processes such asorgan size control, cell fate determination, tissue regeneration andstem cell renewal (Harvey and Tapon, 2007; Johnson and Halder,2014; Pan, 2010). This pathway comprises a core kinase cascadeinvolving Hippo (Hpo; Mst1/2, also known as Stk4/3 in mammals),Salvador (Sav; Sav1 in mammals), Warts (Wts; Lats1/2 inmammals) and Mob as tumor suppressor (Mats; Mob1A/B inmammals) that converges on the transcriptional co-activator Yorkie(Yki; YAP/TAZ in mammals). Phosphorylation of Yki/YAP/TAZexcludes it from the nucleus, where it normally functions as a co-activator for the transcription of growth-promoting genes.Consistent with the requirement of Hippo signaling for normaltissue homeostasis, YAP is a bona fide oncogene and is activated/overexpressed in a wide range of human cancers (Pan, 2010).

The TEF/TEAD family transcription factors, Sd in Drosophilaand TEAD1/2/3/4 in mammals, are the primary DNA-bindingpartners for the Yki/YAP/TAZ co-activators. Not only do they bindto Hippo target genes, such as Diap1 in Drosophila and Ctgf inmammals (Wu et al., 2008; Zhang et al., 2008; Zhao et al., 2008),the TEF/TEAD transcription factors have been identified as Yki/YAP-binding proteins in multiple unbiased protein-proteininteraction screens in both Drosophila and mammals (Giot et al.,2003; Vassilev et al., 2001; Wu et al., 2008). The physiologicalimportance of TEF/TEAD-Yki/YAP interactions is furthersupported by the discovery of a disease-causing point mutation inhuman TEAD1 (TEAD1Y421H underlying Sveinsson’s chorioretinalatrophy) (Kitagawa, 2007) and the unbiased recovery of a missensemutant allele in Drosophila Yki (YkiP88L) that specifically disruptsthis interaction (Wu et al., 2008), as well as structural studies ofTEAD-YAP co-crystals that independently pinpoint these residuesin the protein-binding interface (Chen et al., 2010; Li et al., 2010;Tian et al., 2010). Accentuating the physiological importance of thisinteraction, there is great interest in developing small moleculeinhibitors of TEAD-YAP interactions as potential therapeuticsagainst the YAP oncogene in human cancers (Liu-Chittenden et al.,2012).

Given its crucial role in normal development and tumorigenesis,there has been much interest in understanding the regulation of Yki/YAP/TAZ activity in Hippo signaling. In contrast to the relativelysimple molecular organization of the core kinase cascade leadingfrom Hpo/Mst to phosphorylation of Yki/YAP/TAZ, studies inDrosophila and mammalian cells have reported a complex array ofupstream inputs converging on Yki/YAP/TAZ, such as cell polarity,adhesion, mechanical forces and secreted ligands (Boggiano andFehon, 2012; Enderle and McNeill, 2013; Yu and Guan, 2013). Achallenge for the field is to understand how these diverse upstreaminputs intersect the Hippo pathway at a molecular level, and todefine the exact physiological contexts in which these inputsimpinge on Hippo signaling in vivo. Indeed, among the ever-expanding list of proteins implicated in regulating Yki/YAP/TAZactivity, few have been genetically validated in vivo. Thus, there is aneed for the development of simple and robust assays for validatingthese upstream regulators in vivo.

As Yki/YAP/TAZ represents the ultimate convergence of Hipposignaling, characterization of upstream regulators of Hipposignaling often involves examining the subcellular localization ofYki/YAP/TAZ, and, more sensitively/reliably in vivo, theexpression of selected target genes such as Diap1 in Drosophilaor Ctgf in mammals. However, it is important to bear in mind thatchanges in the expression of a given Hippo target gene per se do notnecessarily indicate changes in Yki/YAP/TAZ activity, because anyHippo target gene is likely to be regulated by a myriad oftranscriptional regulators in parallel with Yki/YAP/TAZ. Forexample, Diap1, one of the most commonly analyzed Hippotarget genes in Drosophila, is also regulated by parallel inputs, suchReceived 23 July 2017; Accepted 19 January 2018

1Department of Physiology, Howard Hughes Medical Institute, University of TexasSouthwestern Medical Center, Dallas, TX 75390-9040, USA. 2Department ofAnatomy & Physiology, Kansas State University College of Veterinary Medicine,Manhattan, KS 66506, USA.

*Author for correspondence (duojia.pan@utsouthwestern.edu)

D.P., 0000-0003-2890-4645

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as the JAK-STAT signaling component Stat92E (Betz et al., 2008).Similarly, Ctgf, one of the best-characterized Hippo targets inmammals, is regulated by other pathways such as TGFβ/Smad, Ras/MEK/ERK and JNK (Leask et al., 2003). Thus, it is important todistinguish whether any observed changes in the expression of aHippo target gene are actually due to modulation of Yki/YAP/TAZactivity. In Drosophila, this can be interrogated through geneticepistasis analysis in vivo by combining loss of function of a tumorsuppressor (or gain of function of an oncogene) that is suspected toconverge on Yki with loss of function of yki. In such analysis, onewould place a suspected gene upstream of yki if the resulting doublemutants display identical phenotypes to that induced by loss offunction of yki alone. On the other hand, if a gene impacts growth orHippo target gene expression independently of changes in Ykiactivity, the double mutants should present an intermediatephenotype. Indeed, epistasis analysis of this nature was used tosupport the suggestion that yki is genetically epistatic to the coreHippo pathway components hpo, sav or wts, by analyzing double-mutant clones of each tumor suppressor gene with yki (Huang et al.,2005). A potential complication with this approach is the prominentrequirement of yki for cell viability and basal-level Hippo targetgene expression (Huang et al., 2005), which could make it difficultto distinguish whether a double-mutant combination displays anintermediate phenotype or yki mutant phenotype. This point isespecially relevant given that many of the reported upstreamregulators of Yki display relatively subtle mutant phenotypes.Another limitation is that epistasis analysis cannot be conductedbetween loss of function of yki and a suspected regulator of Yki thatfunctions in the same direction as yki, as classical epistasis testrequires combining two mutations with opposite phenotypes.Lastly, from a technical standpoint, generating double-mutantclones is time-consuming because it requires complicated geneticcrosses and a yki rescue construct on the same FRT chromosome asthe tumor suppressor gene being tested (Huang et al., 2005). Thus, adefinitive, efficient and generally applicable strategy for epistasisanalysis would greatly facilitate the genetic characterization of theexpanding list of proteins that are thought to converge on theregulation of Yki activity.

RESULTS AND DISCUSSIONAs an alternative to yki, we explored the possibility of using loss-of-function mutations in Sd, the DNA-binding partner of Yki, ingenetic epistasis analysis. As expected of a critical Yki partner inHippo signaling, loss of sd not only fully rescues tissueovergrowth and elevated target gene transcription induced byYki overexpression (Wu et al., 2008), it also fully rescues tissueundergrowth and decreased Hippo target gene transcription in ykiloss-of-function mutations (Koontz et al., 2013). However, unlikeyki, which is required for normal growth and Hippo target geneexpression in Drosophila, sd is genetically dispensable for normalgrowth and basal-level expression of Hippo target genes inmost imaginal discs as a result of its default repressor activity(Koontz et al., 2013). These unique properties make sd a betterchoice than yki for genetic epistasis analysis, as the former is notcomplicated by genetic requirement in normal tissue growth orHippo target gene expression. Thus, it provides a more robustassay to distinguish whether changes in the expression of a Hippotarget gene are due to modulation of Yki activity or Yki-independent inputs into the same target gene; only the former isexpected to be rescued by loss of sd. Furthermore, because loss ofsd rescues both gain- and loss-of-yki phenotypes, it provides amore generally applicable assay than yki-based epistasis,

irrespective of whether a gene of interest functions in the sameor opposite direction as yki.

To facilitate double-mutant analysis with sd, we developed aclonal marking strategy based on double FRT chromosomescarrying a different fluorescent protein (GFP or RFP) on eachFRT chromosome, together with an eye-specific FLP source. Wehave previously used this strategy to show that the defects in growthand Diap1 expression in ykimutant clones were completely rescuedin sd; yki double-mutant clones (Koontz et al., 2013). Here, weextend this strategy to the analysis of negative regulators of Yki(potential tumor suppressors), loss-of-function mutations of whichpresumably lead to gain-of-Yki activity. As a proof-of-concept, wefirst tested whether the elevated expression of Diap1 in mutants ofthe core kinase cassette of the Hippo pathway, hpo, sav andwts, wasrescued by loss of sd. As illustrated schematically in Fig. 1 using sd;hpo double-mutant analysis as an example, sd mutant clones andhpomutant clones can be generated independently and labeled withdifferent markers: loss of GFP for sd clones and loss of RFP for hpoclones. In the merged channel, one should unambiguouslydistinguish four different genotypes in the same eye disc based onfluorescent markers: (1) double heterozygous genetic background(essentially a wild-type control): marked as GFP and RFP positive(appearing yellow); (2) sd single mutant clones: marked as GFPnegative and RFP positive (appearing red); (3) hpo single mutantclones: marked asRFP negative andGFP positive (appearing green);(4) sd; hpo double-mutant clones: marked as both GFP and RFPnegative (appearing black). This marking strategy therefore allowsone to reliably compare the expression of any Hippo pathway targetgene (such as Diap1) in hpo single mutant, sd; hpo double mutant,and wild-type cells right next to each other in the same eye disc.

Consistent with its established role in inhibiting Yki activity, lossof hpo caused a robust increase in Diap1 expression in the eye disc

Fig. 1. Schematic of sd-based genetic epistasis test for validating Hippopathway components and regulators, using sd; hpo double-mutantclones as an example. Double-mutant clones are generated in fliescontaining double FRT chromosomes carrying sd and hpomutations in trans todouble FRT chromosomes carrying GFP and RFP markers, together with aneye-specific flippase (FLP) source. This allows unambiguous marking of thedouble heterozygous background (yellow), sd single mutant clones (red), hposingle mutant clones (green), and sd; hpo double-mutant clones (black) in thesame eye disc. By determining whether aberrant expression of a Hippo targetgene (either upregulation or downregulation) in a givenmutant is genetically sddependent (such as hpo) or sd independent (such as Stat92E), one can inferwhether the mutation impacts target gene expression through Yki or Yki-independent inputs converging on the same Hippo target gene.

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(Fig. 2A-A‴). When sd was simultaneously removed, the elevatedDiap1 expression was completely rescued in sd; hpo double-mutantclones, as the double-mutant clones showed a similar level ofDiap1expression as the neighboring wild-type cells (Fig. 2A-A‴).Similarly, loss of sd completely rescued the elevated Diap1expression in sav and wts mutant clones, as evidenced by thesimilar level of Diap1 protein and Diap1-lacZ in sd; sav or sd; wtsdouble-mutant clones compared with the neighboring wild-typecells (Fig. 2B-C‴, Fig. S1A-B‴). We also applied this strategy totwo reported upstream regulators of the Hippo pathway, ex and ft.Consistent with these proteins being bona fide upstream regulatorsof Yki, loss of sd completely rescued the elevatedDiap1 expressionin ex and ft mutant clones (Fig. 3). Besides Diap1 and Diap1-lacZ,we also tested ex-lacZ, another widely used reporter of Yki activity.Similarly, loss of sd completely rescued the elevated ex-lacZexpression in hpo mutant clones (Fig. 2D-D‴).After demonstrating the efficacy of our double-mutant strategy in

validating known Yki regulators, we wished to test whether thisstrategy can be used to exclude genes that influence Diap1expression independently of changes in Yki activity. For this

purpose, we tested the JAK-STAT signaling component Stat92E.Stat92E mutant clones have been reported to show a modest cell-autonomous decrease in Diap1 expression in the wing discs (Betzet al., 2008; Recasens-Alvarez et al., 2017). After confirming thatStat92E mutant clones in the eye discs showed a similar decrease inDiap1 expression (Fig. 4A-A‴), we used our double-mutantstrategy to analyze sd; Stat92E double-mutant clones. In contrastto the complete rescue of decreased Diap1 expression in yki mutantclones by simultaneous loss of sd (Koontz et al., 2013), loss of sddid not rescue the decreased Diap1 expression in Stat92E mutantclones (Fig. 4B-B‴). Mechanistically, such findings are consistentwith the distinct location of the STAT-responsive element (withinthe promoter region) and Hippo/Yki-responsive element (within thefirst intron) in theDiap1 genomic locus (Betz et al., 2008; Wu et al.,2008; Zhang et al., 2008). Taken together, these results demonstratethat the sd-based epistasis test can be used not only to validateknown Yki regulators but also to exclude proteins that impact Hippotarget gene expression independently of Yki activity. To facilitatethe sd-based double-mutant analysis, we have developed a set of flystocks that can be used to test mutations on every autosomal arm,

Fig. 2. Validation of Hippo pathway core components by sd-based double-mutant analysis. In all panels, mutant clones of sdand the tumor suppressor gene being tested were marked by loss ofGFP and RFP, respectively. (A-B‴) Eye discs containing mutantclones of the indicated genotypes were stained for Diap1 protein.Note the increased Diap1 staining in hpo or wts single mutant clones(arrows, green areas in the merged channel), but not thecorresponding double-mutant clones with sd (arrowheads, blackareas in the merged channel). (C-C‴) An eye disc containing mutantclones of sd (GFP negative) and wts (RFP negative) was stained forDiap1-lacZ. Note the increased Diap1-lacZ staining in wts singlemutant clones (arrows, green areas in the merged channel), but notsd; wts double-mutant clones (arrowheads, black areas in themerged channel). (D-D‴) An eye disc containing mutant clones of sd(GFP negative) and hpo (RFP negative) was stained for ex-lacZ.Note the increased ex-lacZ staining in hpo single mutant clones(arrows, green areas in the merged channel), but not sd; hpo double-mutant clones (arrowheads, black areas in the merged channel).

Fig. 3. Validation of Hippo pathway upstream regulators bysd-based double-mutant analysis. (A-B‴) In all panels, mutantclones of sd and the tumor suppressor gene being tested weremarked by loss of GFP and RFP, respectively. Eye discscontaining mutant clones of the indicated genotypes were stainedfor Diap1 protein. Note the increased Diap1 staining in ex or ftsinglemutant clones (arrows, green areas in themerged channel),but not the corresponding double-mutant clones with sd(arrowheads, black areas in the merged channel).

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allowing one to obtain definitive epistasis information in twogenerations (see Materials and Methods for details).In principle, one could apply the sd-based double-mutant

analysis to the expression of any Yki target genes besides Diap1and ex, as long as target gene expression can be followed in adifferent detection channel as the GFP and RFP markers in confocalmicroscopy. We tested this possibility by examining the expression

of the miRNA bantam (Nolo et al., 2006; Thompson and Cohen,2006). bantam presents a particularly interesting Yki target gene asprevious studies suggested that, unlike Diap1, transcriptionalregulation of bantam by Yki is mediated by other DNA-bindingproteins, including Mad, Tsh and Hth (Oh and Irvine, 2011; Penget al., 2009). We note, however, that these non-Sd transcriptionfactors acting as mediators of Yki’s regulation of bantam expression

Fig. 4. Loss of sd did not rescue the decreased Diap1expression in Stat92E mutant clones. (A-A‴) An eye disccontaining Stat92E mutant clones (RFP negative) was stained forDiap1 protein. Note the modest decrease of Diap1 protein level inStat92E mutant clones (arrows). (B) An eye disc containing sd;Stat92Emutant clones was stained for Diap1. Mutant clones of sdand Stat92E were marked by loss of GFP and RFP, respectively.Note the decreased Diap1 protein level in both Stat92E singlemutant clones (arrows, green areas in the merged channel) andsd; Stat92E double-mutant clones (arrowheads, black areas in themerged channel).

Fig. 5. Loss of sd completely rescues aberrant bantamexpression resulting from defective Hippo signaling. (A-A‴) Aneye disc containing mutant clones of sd (GFP negative) and hpo(RFP negative) was stained for bantam-lacZ. Note the increasedbantam-lacZ expression in hpo single mutant clones (arrows, greenareas in the merged channel), but not sd; hpo double-mutant clones(arrowheads, black areas in the merged channel). (B-B‴) An eyedisc containing mutant clones of sd (GFP negative) and hpo (RFPnegative) was stained for brC12-lacZ. Note the increased brC12-lacZ expression in hpo single mutant clones (arrows, green areas inthe merged channel), but not sd; hpo double-mutant clones(arrowheads, black areas in the merged channel). (C-C‴) An eyedisc containing yki mutant clones (RFP negative) was stained forbantam-lacZ. Note the modest decrease of bantam-lacZ staining inthe yki mutant clones (arrow). (D-D‴) An eye disc containing sd; ykidouble-mutant clones was stained for bantam-lacZ. Note the similarbantam-lacZ level in the double-mutant clone (arrowheads) as in theneighboring wild-type cells. (E-E″) An eye disc containing yki-overexpressing clones (GFP positive) and stained for bantam-lacZ.Note the dramatic increase of bantam-lacZ staining in the Yki-overexpressing clones (arrows). (F-F″) An eye disc containing sdmutant clones with yki overexpression (GFP positive), showingsimilar bantam-lacZ staining in the clones (arrows) as theneighboring wild-type cells.

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cannot be readily reconciled with the observation that loss of sdcompletely rescues the growth defects of ykimutant clones (Koontzet al., 2013), as the latter would support Sd as a physiologicalpartner of Yki in the expression of all growth-relevant genes,including bantam. It is also hard to imagine how Yki could switchbetween different DNA-binding partners to regulate different targetgenes as proposed by these studies (Oh and Irvine, 2011; Peng et al.,2009). Thus, the most parsimonious model is that, like the other Ykitargets, any changes in bantam expression resulting from loss orgain of Yki activity should be dependent on sd.To test this model, we applied the double-mutant labeling

strategy described above to examine whether aberrant bantamexpression resulting from defective Hippo signaling is dependent onsd function. As expected of a Yki target, bantam expression wasstrongly upregulated in loss-of-function mutant clones of hpo orsav, as revealed by a bantam-lacZ reporter inserted into theendogenous locus (Herranz et al., 2012) (Fig. 5A-A‴, Fig. S1C-C‴).Strikingly, when sd was simultaneously removed from hpo or savmutant clones, the elevated bantam expression was completelyrescued, as sd; hpo or sd; sav double-mutant clones showed a similarlevel of bantam expression as the neighboring wild-type cells(Fig. 5A-A‴, Fig. S1C-C‴). Conversely, loss of yki resulted indecreased bantam expression (Fig. 5C-C‴), and the decreasedbantam expression in yki mutant clones was completely rescued insd; yki double-mutant clones (Fig. 5D-D‴). As an additional,independent test for the genetic requirement of sd in Yki-mediatedbantam expression, we used the MARCM (mosaic analysis with arepressible cell marker) technique to generate sdmutant clones withYki overexpression. As expected, Yki overexpression inducedstrong upregulation of bantam expression (Fig. 5E-E″), and suchupregulation was completely rescued by simultaneous loss of sd(Fig. 5F-F″). We applied the same double-mutant labeling strategyto examine the regulation of brC12-lacZ, a Yki-dependent bantamreporter that was reported to be regulated in a Mad-dependent butSd-independent manner (Oh and Irvine, 2011). Contrary to theprevious report, we found that when sdwas simultaneously removedfrom hpo mutant clones, the elevated brC12-lacZ expression was

completely rescued to a similar level to the neighboring wild-typecells (Fig. 5B-B‴). We conclude that, like Diap1 and ex, aberrantbantam expression resulting from both gain and loss of Yki activityis completely dependent on Sd function. We further infer from theseresults that Sd functions as a default repressor for the transcription ofbantam, similar to its role in Diap1 and ex transcription (Koontzet al., 2013). Taken together, these findings support the central roleof Sd in Yki-mediated transcriptional regulation. We emphasize thatthis conclusion is not intended to imply that Yki target genes cannotbe regulated by transcription factors other than Sd. Like Diap1,which contains distinct STAT- and Yki-responsive enhancers (Betzet al., 2008;Wu et al., 2008; Zhang et al., 2008), any Yki target geneis likely to be regulated by several DNA-binding transcriptionfactors that bind to distinct enhancers, and these enhancer-bindingtranscription factors can still interact with each other on chromatin toinfluence transcriptional output. The essence of our conclusion isthat any changes of Yki target genes upon loss or gain of Ykiactivity cannot be realized without Sd binding to these target loci.

Although loss-of-function mutations are preferred in epistasisanalysis, there might be situations in which mutant alleles areunavailable for a suspected Yki regulator, or when mutant alleles areunsuitable for clonal analysis for reasons such as cell lethality,protein endurance and genetic redundancy. Indeed, many studies ofHippo signaling have relied on the use of the UAS-Gal4 system toknockdown or overexpress a gene of interest, often in a specificregion of the wing or eye imaginal disc. In order to extend sd-basedepistasis analysis to such genes, we devised a way to combine UAS-Gal4-mediated transgene expression with sd mutant clones in theeye. For this purpose, we took advantage of a Gal4 driver (DE-Gal4)that is specifically expressed in the dorsal half of the eye discs(Morrison and Halder, 2010). The expectation is that if aberrantDiap1 expression (or any other Hippo targets) upon perturbation ofa given gene is truly due to abnormal Yki activity, such aberrationshould be sd dependent. We first tested Ex, a known regulator of Ykiactivity, as a proof of concept. As expected, knockdown of ex byDE-Gal4 caused increased Diap1 expression specifically in thedorsal half of the eye disc (Fig. 6A,A′), and the upregulation of

Fig. 6. Application of sd-based epistasis analysis to genetic background created by UAS-Gal4-mediated transgene expression. The eye disc is orientedwith dorsal side up in all panels. (A,A′) An eye disc expressing UAS-exRNAi and UAS-GFP under the control of DE-Gal4 was stained for Diap1 protein. Note theenhanced Diap1 staining in the dorsal half of the eye disc. (B,B′) An eye disc containing sdmutant clones (GFP negative) and expressing UAS-exRNAi under thecontrol of DE-Gal4 was stained for Diap1 protein. The increased Diap1 staining in the dorsal half of the eye disc was specifically reduced in sd mutant clones(arrowheads) to a level comparable to that in the ventral half of the eye. (C,C′) An eye disc expressing UAS-DmIKKɛRNAi and UAS-GFP under the control of DE-Gal4 was stained for Diap1 protein. Note the enhanced Diap1 staining in the dorsal half of the eye disc. (D,D′) An eye disc containing sd mutant clones (GFPnegative) and expressingUAS-DmIKKɛRNAi under the control ofDE-Gal4was stained for Diap1 protein. The increased Diap1 staining in the dorsal half of the eyedisc was not affected in sd mutant clones (arrowheads).

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Diap1 expression was completely rescued by loss of sd (Fig. 6B,B′).As a negative control, we tested the Drosophila IKK-related kinaseDmIKKɛ (IKKε). RNAi knockdown of DmIKKɛ by the en-Gal4driver was previously reported to result in amild upregulation of Diap1protein level in the posterior compartment of the wing discs (Kuranagaet al., 2006). We confirmed that knockdown of DmIKKɛ in the eyediscs resulted in a similar increase of Diap1 level (Fig. 6C,C′). Incontrast to the knockdown of ex, however, DmIKKɛ RNAi-inducedupregulation of Diap1 expression was not rescued by loss of sd(Fig. 6D,D′). Indeed, whereas Hippo signaling controls Diap1transcription, DmIKKɛ is known to impact Diap1 expression post-transcriptionally by regulating Diap1 protein stability (Kuranaga et al.,2006). Together with our analysis of Stat92E, these results demonstratethe efficacy of sd-based epistasis analysis in identifying proteins thatimpinge on Hippo target expression independently of Yki activity.Besides aberrant target gene expression, another routinely

assayed phenotype suggestive of elevated Yki activity is increasedinterommatidial cell number in pupal retina. As with our concernsabout target gene expression, it is crucial to distinguish whether anobserved alteration in interommatidial cell number is due to changesin Yki activity or other modalities impacting interommatidial cellnumber. We reasoned that sd-based epistasis analysis can besimilarly applied in this context, as any alteration in interommatidialcell number caused by changes in Yki activity should be completelyrescued by loss of sd. We tested this idea by comparing two differentgenetic backgrounds resulting in increased interommatidial cellnumber in pupal retina: overexpression of Yki or its downstreamtarget bantam. Our prediction is that only the former can be rescuedby loss of sd. Indeed, Yki overexpression induced amassive increasein interommatidial cells [average >40 extra cells per cluster (ECPC),n=11; Fig. 7A,A′], and this increase was completely rescued bysimultaneous loss of sd (0 ECPC, n=16; Fig. 7B,B′). In contrast,even though bantam overexpression caused a milder increase ofinterommatidial cells (average 16.4 ECPC, n=15; Fig. 7C,C′)compared with Yki overexpression, the bantam-induced increase ininterommatidial cell number was not rescued at all by loss of sd(average 16.1 ECPC, n=13; Fig. 7D,D′). Thus, besides Hippo targetgenes, sd-based epistasis analysis can be applied as a specific anddefinitive way to determine the causality of the interommatidial cellphenotype with respect to changes in Yki activity.

ConclusionsGenetic epistasis analyses are instrumental in dissectingdevelopmental pathways and determining the relationshipsbetween genes of interest (Huang and Sternberg, 2006). We havepresented a simple genetic epistasis test, as well as the necessary fly

reagents, to validate proteins implicated in regulating Yki activity,taking advantage of the unique genetic property of sd as an essentialmediator of both loss- and gain-of-Yki phenotypes. A key feature ofthis strategy is that it examines the ability of loss of sd, which byitself does not affect normal growth and basal-level Hippo targetgene expression in the eye, to revert/rescue the mutant phenotype ofanother gene potentially linked to the regulation of Yki activity,irrespective of whether the gene acts positively or negatively on yki.This method provides a specific, sensitive and versatile assay toascertain whether aberrant expression of a given Hippo target geneis due to changes in Yki activity, as we have shown for hpo, sav,wts,ex and ft, or Yki-independent inputs converging on the same targetgene, as we have shown for Stat92E and DmIKKɛ. Such sd-basedepistasis analysis is not limited to target gene expression, and can beextended to any Yki-dependent phenotypes, as we have shown forthe interommatidial cell phenotype. We suggest that sd-basedepistasis analysis should be broadly applied to assess the expandingrepertoire of proteins and inputs that have been suggested toconverge on the regulation of Yki activity.

MATERIALS AND METHODSDrosophila geneticsThe bantam-lacZ reporter P{lacW}banL1170a (stock ID 10154 fromBloomington Drosophila Stock Center) is a lacZ-containing P-elementenhancer trap line inserted in the promoter of bantam (Herranz et al., 2012;Dent et al., 2015). The UAS-DmIKKɛ RNAi line was also obtained fromBloomington Drosophila Stock Center (stock ID 35266). UAS-ex RNAiflies were obtained from the Vienna Drosophila Resource Center(transformant ID 22994). The following flies have been describedpreviously: Diap1-lacZ reporter thj5c8 (Wu et al., 2003), brC12-lacZ (Ohand Irvine, 2011), UAS-yki and ykiB5 (Huang et al., 2005), UAS-ban(Brennecke et al., 2003), sd47M (Srivastava et al., 2004), hpo42-47 (Wu et al.,2003), sav3 (Tapon et al., 2002), wtsX1 (Xu et al., 1995), ft8 (Bryant et al.,1988), exe1 (Hamaratoglu et al., 2006), Stat92EP1681 (Hou et al., 1996).

For MARCM, all clones were induced 68-72 h after egg deposition andheat-shocked at 38°C for 30 min. Double-mutant clones in the eye imaginaldiscs were generated using flies containing double FRT chromosomes withGFP and RFP markers together with an eye-specific FLP source asdescribed previously (Koontz et al., 2013).

The following genotypes were used:yki overexpression clones: tub-Gal80 FRT19A/FRT19A; UAS-GFP hs-

FLP/UAS-yki, bantam-lacZsd clones overexpressing yki: tub-Gal80 FRT19A/sd47M FRT19A; UAS-

GFP hs-FLP/UAS-yki, bantam-lacZsd clones overexpressing bantam: tub-Gal80 FRT19A/sd47M FRT19A;

UAS-GFP hs-FLP/UAS-bansd; hpo double-mutant clones: ey-FLP, Ubi-GFP FRT19A/sd47M

FRT19A; FRT42D hpo42-47/FRT42D Ubi-RFP

Fig. 7. Application of sd-based epistasis analysis to determine the causality of interommatidial cell phenotype. (A,A′) A mid-pupal retina containing yki-overexpressing clones (GFP positive) and stained for Discs large (Dlg). Note the dramatic increase of interommatidial cells (average >40 ECPC, n=11) in yki-overexpressing clones. (B,B′) A mid-pupal retina containing sdmutant clones with yki overexpression (GFP positive) and stained for Dlg. Note the similar numberof interommatidial cells (0 ECPC, n=16) in the clones as the neighboring wild-type cells. (C,C′) Amid-pupal retina containing bantam-overexpressing clones (GFPpositive) and stained for Dlg. Note the increase of interommatidial cells (average 16.4 ECPC, n=15) in bantam-overexpressing clones. (D,D′) A mid-pupal retinacontaining sd mutant clones with bantam overexpression (GFP positive) and stained for Dlg, showing an average of 16.1 ECPC (n=13).

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sd; sav double-mutant clones: ey-FLP, Ubi-GFP FRT19A/sd47M

FRT19A; FRT82B sav3/FRT82B Ubi-RFPsd; wts double-mutant clones: ey-FLP, Ubi-GFP FRT19A/sd47M

FRT19A; FRT82B wtsx1/FRT82B Ubi-RFPex RNAi clones: UAS-GFP/+; DE-Gal4/UAS-exRNAi

sd clones with ex RNAi: ey-FLP,Ubi-GFP FRT19A/sd47M FRT19A; DE-Gal4/UAS-exRNAi

DmIKKɛ RNAi clones: UAS-GFP/+; DE-Gal4/UAS-IKKɛRNAi

sd clones with DmIKKɛ RNAi: ey-FLP, Ubi-GFP FRT19A/sd47M

FRT19A; DE-Gal4/UAS-IKKɛRNAi

sd clones with yki overexpression: ey-FLP, Ubi-GFP FRT19A/sd47M

FRT19A; DE-Gal4/UAS-ykiStat92E mutant eyes: ey-FLP; FRT82B Stat92EP1681/FRT82B Ubi-GFPsd; Stat92E double-mutant clones: ey-FLP, Ubi-GFP FRT19A/sd47M

FRT19A; FRT82B Stat92EP1681/FRT82B Ubi-RFPyki mutant eyes: ey-FLP, Ubi-GFP FRT19A/+; FRT42D ykiB5/FRT42D

Ubi-RFPsd; yki double-mutant clones: ey-FLP,Ubi-GFP FRT19A/sd47M FRT19A;

FRT42D ykiB5/FRT42D Ubi-RFPsd; ft double-mutant clones: ey-FLP, Ubi-GFP FRT19A/sd47M FRT19A;

ft8FRT40A/Ubi-RFP FRT40Asd; ex double-mutant clones: ey-FLP, Ubi-GFP FRT19A/sd47M FRT19A;

exe1FRT40A/Ubi-RFP FRT40A.Tool flies developed for sd-based genetic epistasis test: ey-FLP, Ubi-GFP

FRT19A; Adv/T(2;3)SM6-TM6B, sd47M FRT19A/FM6; FRT40A Ubi-RFP/CyO (for candidate genes on 2L), sd47M FRT19A/FM6; FRT42D Ubi-RFP/CyO (for candidate genes on 2R), sd47M FRT19A/FM6; Ubi-RFP FRT80B/TM6B (for candidate genes on 3L), sd47M FRT19A/FM6; Ubi-RFP FRT80B/TM6B (for candidate genes on 3R), sd47M FRT19A/FM6; DE-Gal4/TM6B.

To generate sd; hpo double-mutant clones, male FRT42D hpo/CyO flieswere first crossed to ey-FLP, Ubi-GFP FRT19A; Adv/T(2;3)SM6-TM6Bvirgin females. The resulting F1 male progeny of the genotype ey-FLP,Ubi-GFP FRT19A/Y; FRT42D hpo/T(2;3)SM6-TM6B were crossed to sd47M

FRT19A/FM6; FRT42D Ubi-RFP/Cyo virgin females. Female third instarlarva without the T(2;3)SM6-TM6B balancer from the cross are predicted tobe ey-FLP, Ubi-GFP FRT19A/sd47M FRT19A; FRT42D hpo42-47/FRT42DUbi-RFP, the genotype that allows the generation of sd; hpo double-mutantclones. A similar mating scheme can be used to examine double mutants ofsd and candidate genes on other chromosome arms, by replacing the sd47M

FRT19A/FM6; FRT42D Ubi-RFP/Cyo flies with a fly stock for a candidategene’s chromosome arm (see the list of tool flies above).

To generate sd mutant clones in the genetic background of UAS-Gal4-mediated transgene expression (ex RNAi as an example) in the eye, maleUAS-exRNAi flies were first crossed to ey-FLP, Ubi-GFP FRT19A; Adv/T(2;3)SM6-TM6B virgin females. The resulting F1 male progeny of thegenotype ey-FLP, Ubi-GFP FRT19A/Y; UAS-exRNAi/T(2;3)SM6-TM6Bwere crossed to sd47M FRT19A/FM6; DE-Gal4/TM6B virgin females.Female third instar larva without the T(2;3)SM6-TM6B balancer from thecross are predicted to be ey-FLP, Ubi-GFP FRT19A/sd47M FRT19A; DE-Gal4/UAS-exRNAi, the genotype that allows the generation of sd mutantclones in the presence of ex RNAi.

Image analysisAll confocal images were acquired on a Carl Zeiss 700 microscope andanalyzed using ImageJ.

AcknowledgementsWe thank the Confocal Microscopy Core and the Molecular Biology Core funded byKSU-CVM.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: J.Y., D.P.; Methodology: J.Y.; Validation: J.Y.; Formal analysis:J.Y., D.P.; Investigation: J.Y., D.P.; Resources: J.Y.; Data curation: J.Y.; Writing -original draft: J.Y.; Writing - review & editing: J.Y., D.P.; Supervision: D.P.; Projectadministration: J.Y., D.P.; Funding acquisition: D.P.

FundingThis study was supported in part by grants from the National Institutes of Health(EY015708). D.P. is an investigator of the Howard Hughes Medical Institute.Deposited in PMC for release after 6 months.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.157545.supplemental

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