# The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth

Ditte S. Andersen<sup>1,2,3,4*</sup>, Julien Colombani<sup>1,2,3,4*</sup>, Valentina Palmerini<sup>5</sup>, Krittalak Chakrabandhu<sup>1,2,3,6</sup>, Emilie Boone<sup>1,2,3,4</sup>, Michael Röthlisberger<sup>7</sup>, Janine Toggweiler<sup>7</sup>, Konrad Basler<sup>7</sup>, Marina Mapelli<sup>5</sup>, Anne-Odile Hueber<sup>1,2,3,6</sup> & Pierre Léopold<sup>1,2,3,4</sup>

Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a *Drosophila* genome-wide genetic screen designed to identify molecules implicated in neoplastic growth¹, we identified grindelwald (grnd), a gene encoding a trans-membrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. Here we show that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique *Drosophila* TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling *in vivo*. Grnd also promotes the invasiveness of Ras<sup>v12</sup>/scrib<sup>-/-</sup> tumours through Egr-dependent Matrix metallo-protease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth.

We recently carried out a genome-wide screen to identify molecules that are required for neoplastic growth¹. The condition used for this screen was the disc-specific knockdown of *avalanche* (*rotund* (*rn*)>avl-RNAi; *avalanche* also known as *syntaxin 7*), a gene encoding a syntaxin that functions in the early step of endocytosis². *rn>avl*-RNAi results in ectopic Wingless (Wg) expression, neoplastic disc overgrowth² (Fig. 1a, b), and a 2-day delay in larva-to-pupa transition¹. We screened a collection of 10,100 transgenic RNA interference (RNAi) lines for their ability to rescue the pupariation delay and identified 121 candidate genes¹. Interestingly, only eight candidate genes also rescued ectopic Wg expression and neoplastic overgrowth (Extended Data Fig. 1a). These included five lines targeting core components of the JNK pathway (Bendless, Tab2, Tak1, Hemipterous and Basket; Extended Data Fig. 1b). Using a *puckered* enhancer trap (*puc-lacZ*) as a readout for JNK activity, we confirmed that JNK signalling is highly upregulated in *rn>avl*-RNAi discs (Fig. 1a, b, right). One of the remaining lines targets *CG10176*, a gene encoding a transmembrane protein. Reducing expression of *CG10176* by using two different RNAi lines was as efficient as *tak1* silencing to restore normal Wg pattern and suppresses JNK signalling and neoplastic growth in the *rn>avl*-RNAi background (Fig. 1c, d and Extended Data Fig. 1c-e). Sequence analysis of *GC10176* identified a cysteine-rich domain (CRD) in the extracellular part with homology to vertebrate TNFRs (Fig. 1e and Extended Data Fig. 2) harbouring a glycosphingolipid-binding motif (GBM) characteristic of many TNFRs including Fas³

(Fig. 1e and Extended Data Fig. 2). We named *CG10176 grindelwald (grnd)*, after a village at the foot of Eiger, a Swiss mountain that lent its name to the unique *Drosophila* TNF, Egr. Immunostaining and subcellular fractionation of disc extracts confirmed that Grnd localizes to the membrane (Extended Data Fig. 1f-h). Moreover, co-immunoprecipitation experiments showed that both Grnd full-length and Grnd-intra, a form lacking its extracellular domain, directly associate with Traf2, the most upstream component of the JNK pathway (Fig. 1f-h). This interaction is disrupted by a single amino acid substitution within a conserved Traf6-binding motif⁴ (human TRAF6 is the closest homologue to Traf2; Fig. 1f-h and Extended Data Fig. 3). Overexpression of Grnd-intra, but not full-length Grnd, is sufficient to induce JNK signalling, ectopic Wg expression and apoptosis (Fig. 1i-j and Extended Data Fig. 4a-c), and Grnd-intra-induced apoptosis is efficiently suppressed in a *hep*⁷⁵ (JNKK) mutant background (Extended Data Fig. 4d, e), confirming that Grnd acts upstream of the JNK signalling cascade.

The *Drosophila* TNF Egr activates JNK signalling and triggers cell death or proliferation, depending on the cellular context⁵. We therefore tested whether Grnd is required for the small-eye phenotype generated by Egr-induced apoptosis in the retinal epithelium⁶,⁷ (*GMR>egr*; Fig. 2a, b). As previously shown, inhibition of JNK signalling by reducing *tak1* (ref. 6) or *traf2* (ref. 8) expression, or by overexpressing *puckered*, blocks Egr-induced apoptosis and rescues the small-eye phenotype (Extended Data Fig. 5a-d). In contrast to a previous report⁹, RNAi silencing of *wengen* (*wgn*), a gene encoding a presumptive receptor for Egr, does not rescue the small-eye phenotype (Extended Data Fig. 5e). Furthermore, the small-eye phenotype is not modified in a *wgn*-null mutant background (Fig. 2c and Extended Data Fig. 5h, m, n), confirming that Wgn is not required for Egr-induced apoptosis in the eye. By contrast, reducing *grnd* levels partially rescues the Egr-induced small-eye phenotype, producing a 'hanging-eye' phenotype (Fig. 2d) that is not further rescued in a *wgn*-knockout (*wgn*<sup>KO</sup>) mutant background (Extended Data Fig. 5e-i). A similar phenotype was previously reported as a result of non-autonomous cell death induced by a diffusible form of Egr¹⁰ (Extended Data Fig. 5k, l). This suggests that Grnd prevents Egr from diffusing outside of its expression domain. Co-immunoprecipitation experiments show that both full-length Grnd and Grnd-extra, a truncated form of Grnd lacking the cytoplasmic domain, associate with Egr through its TNF-homology domain (Fig. 2j, k). Although Grnd-extra can bind Egr, it cannot activate JNK signalling. Therefore, we reasoned that Grnd-extra expression might prevent both cell-autonomous and non-autonomous apoptosis by trapping Egr and preventing its diffusion and binding to endogenous Grnd. Indeed, *GMR-Gal4*-mediated expression of *grnd-extra* fully rescues the Egr small-eye phenotype (Fig. 2e and Extended Data Fig. 5j). To confirm that the removal of Grnd induces Egr-mediated non-autonomous cell death, we generated wing disc

¹University of Nice-Sophia Antipolis, Institute of Biology Valrose, Parc Valrose, 06108 Nice, France. ²CNRS, Institute of Biology Valrose, Parc Valrose, 06108 Nice, France. ³INSERM, Institute of Biology Valrose, Parc Valrose, 06108 Nice, France. ⁴Genetics and Physiology of Growth laboratory, Institute of Biology Valrose, Parc Valrose, 06108 Nice, France. ⁵Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy. ⁶Death receptors Signalling and Cancer Therapy laboratory, Institute of Biology Valrose, Parc Valrose, 06108 Nice, France. ⁷Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

*These authors contributed equally to this work.
<figure><img src="image_1.png" /><figcaption>**Figure 1** | Grnd is a novel member of the TNFR superfamily. a–d, Wing discs stained for Wg (left) or LacZ (right). e, Sequence alignment of Grnd and human TNFRs. Blue, identity; yellow, strong similarity to Grnd; grey, weak similarity to Grnd. f, g, Co-immunoprecipitation experiments from S2R⁺ cells co-expressing haemagglutinin (HA)-Traf2 or Ihog-HA (control), and either Grnd-Flag, Grnd-intra-Flag or Grnd(E202A)-Flag (f), or HA-Egr and either Grnd-Flag, Grnd(E202A)-Flag or Ihog-Flag (control) (g). Input loading is quantified using β-tubulin. h, Pull-down experiments of Traf2 with various truncated forms of glutathione S-transferase (GST)-Grnd. Commassie blue staining is used to visualize the bound species. WT, wild type. i–j, Wing discs stained for Wg (left), LacZ (middle) and Grnd (right).</figcaption></figure>

clones expressing *egr* alone, *egr* + *tak1* RNAi, or *egr* + *grnd* RNAi (Fig. 2f–i). As expected, reducing *tak1* levels in *egr*-expressing clones prevents their elimination by apoptosis (Fig. 2f, g). Similarly, reducing *grnd* levels prevents autonomous cell death, but also induces non-autonomous apoptosis (Fig. 2h, i). This suggests that Egr, like its mammalian counterpart TNF-α, can be processed into a diffusible form *in vivo* whose interaction with Grnd limits the potential to act

<figure><img src="image_2.png" /><figcaption>**Figure 2** | Grnd is required for Egr-induced apoptosis. a–e, Light micrographs of *Drosophila* adult eyes of the indicated genotypes (all animals carry the *GMR-Gal4* driver). Original magnification, ×30. f–i, Wing discs carrying green fluorescent protein (GFP)-labelled clones (green) expressing *egr* (f), *egr* + *tak1* RNAi (*g*) or *egr* + *grnd* RNAi (*h*, *i*) and stained for Caspase 3 (Cas3; red). A close-up shows non-autonomous cell death around the *egr* + *grnd* RNAi clones. *j*, *k*, Co-immunoprecipitation (IP) experiments from S2R⁺ cells expressing HA-Egr or HA-EgrΔTNF (Egr lacking its TNF-homology domain; amino acids 278–415) and either Grnd-Flag, Grnd-extra-Flag or Ihog-Flag (control). Input loading is quantified using β-tubulin.</figcaption></figure>

at a distance. Flies carrying homozygous (*grnd*<sup>Minos/Minos</sup>) or transheterozygous (*grnd*<sup>Minos/Df</sup>) combinations of a transposon inserted in the *grnd* locus express no detectable levels of Grnd protein (Extended Data Fig. 6a, b) and are equally resistant to Egr-induced cell death (Extended Data Fig. 6e–j). In addition, *grnd*<sup>Minos/Minos</sup> mutant flies are viable and display no obvious phenotype (Extended Data Fig. 6c, d), suggesting that Grnd, like Egr, participates in a stress response to limit organismal damage. Collectively, our data demonstrate that Grnd is a new *Drosophila* TNF receptor that mediates most, if not all, Egr-induced apoptosis.

TNFs probably represent a danger signal produced in response to tissue damage to rid the organism of premalignant tissue or to facilitate wound healing. Disc clones mutant for the polarity gene *scribbled* (*scrib*) induce an Egr-dependent response resulting in the elimination of *scrib* mutant cells by JNK-mediated apoptosis<sup>11,12</sup>. To test the requirement for Grnd in this process, we compared *scrib*-RNAi and *scrib*-RNAi + *grnd*-RNAi clones obtained 72 h after heat shock induction. As expected, *scrib*-RNAi cells undergo apoptosis and detach from
<figure><img src="image_3.png" /></figure>

eyFlp; UAS-Ras<sup>V12</sup>; scrib<sup>-/-</sup>

<figure><img src="image_4.png" /><figcaption>**Figure 3** | Grnd is required for the Egr-induced invasiveness of *Ras*<sup>V12</sup>/scrib<sup>-/-</sup> tumours. **a–f**, Wing discs dissected 5 days after egg deposition (AED) stained for Cas3 (red) and carrying GFP-labelled clones (green) of the indicated genotypes induced 2 days AED. **a, b, e**, Wild-type clones (**a**) and clones expressing the control *Rh3* RNAi (**b**) to exclude a Gal4 titration effect (**e**). **g–i**, Eye–brain complexes dissected 7 days AED carrying *Ras*<sup>V12</sup>/scrib<sup>-/-</sup> (**g**) or *Ras*<sup>V12</sup>/scrib<sup>-/-</sup> + grnd RNAi (**h**) clones (labelled by GFP), or *Ras*<sup>V12</sup>/scrib<sup>-/-</sup> GFP-labelled clones in an *egr* mutant background (**i**) stained for Mmp1 (**g–i**, middle, red) and 4',6-diamidino-2-phenylindole (DAPI; blue). *Ras*<sup>V12</sup>/scrib<sup>-/-</sup> clones invade the ventral nerve cord (**g**, white arrow). c.c., cephalic complex.</figcaption></figure>

the epithelium (Fig. 3a–d). By contrast, scrib-RNAi clones with reduced grnd expression survive (Fig. 3e, f), indicating that Grnd is required for Egr-dependent elimination of scrib-RNAi cells. Similar results were obtained by generating scrib<sup>-/-</sup> mutant clones in the eye disc (Extended Data Fig. 7a–d).

In both mammals and flies, TNFs are double-edged swords that also have the capacity to promote tumorigenesis in specific cellular contexts⁵. Indeed, scrib<sup>-/-</sup> eye disc cells expressing an activated form of *Ras* (*Ras*<sup>V12</sup>) exhibit a dramatic tumour-like overgrowth and metastatic behaviour, a process that critically relies on Egr¹³. *Ras*<sup>V12</sup>/scrib<sup>-/-</sup> metastatic cells show a strong accumulation of Grnd and Mmp1, and invade the ventral nerve cord¹¹,¹⁴,¹⁵ (Fig. 3g and Extended Data Figs 7e, 8a). Primary tumour cells reach peripheral tissues such as the fat body and the gut, where they form micrometastases expressing high levels of Grnd (Extended Data Fig. 7f, g). Reducing grnd levels in *Ras*<sup>V12</sup>/scrib<sup>-/-</sup> clones is sufficient to restore normal levels of Mmp1 and abolish invasiveness in a way similar to that observed in an *egr<sup>-/-</sup>* background¹³ (Fig. 3h, i and Extended Data Fig. 8a–c). Therefore, Grnd is required for the Egr-induced metastatic behaviour of *Ras*<sup>V12</sup>/scrib<sup>-/-</sup> tumorous cells. Similarly, reducing grnd, but not *wgn* levels, strongly suppresses Mmp1 expression in *Ras*<sup>V12</sup>/dlg-RNAi cells and limits tumour invasion (Extended Data Fig. 8d–g), indicating that Wgn does not have a major role in the progression of these tumours.

Perturbation of cell polarity is an early hallmark of tumour progression in epithelial cells. In contrast to small patches of polarity-deficient cells, for example, scrib<sup>-/-</sup> clones, organ compartments or animals fully composed of polarity-deficient cells become refractory to Egr-induced cell death and develop epithelial tumours. The formation of these tumours requires JNK/MAPK signalling, but not Egr<sup>13,16</sup>, suggesting Egr-independent coupling between loss of polarity and JNK/MAPK-dependent tumour growth. In line with these observations, we noticed that, in contrast to Grnd, Egr is not required to drive neoplastic growth in *rn>avl*-RNAi conditions (Extended Data Fig. 9a, b). This suggests that, in addition to its role in promoting Egr-dependent functions, Grnd couples loss of polarity with JNK-dependent growth independently of Egr. Disc immunostainings revealed that Grnd co-localizes with the apical determinant Crb in the marginal zone, apical to the adherens junction protein E-cadherin (E-cad) and the atypical protein kinase C (aPKC; Fig. 4a and Extended Data Fig. 9c, d). In *avl*-RNAi discs, Grnd and Crb accumulate in a wider apical domain² (Extended Data Fig. 9e, g). Apical accumulation of Crb is proposed to be partly responsible for the neoplastic growth induced by *avl* knockdown, since overexpression of Crb or a membrane-bound cytoplasmic tail of Crb (Crb-intra) mimics the *avl*-RNAi phenotype² (Extended Data Fig. 9f). We therefore examined whether Grnd might couple the activity of the Crb complex with JNK-mediated neoplastic growth. Indeed, reducing grnd levels, but not *wgn*, in *rn>crb-intra* discs suppresses neoplastic growth as efficiently as inhibiting the activity of the JNK pathway (Fig. 4c–f and Extended Data Fig. 9h, i). Notably, Yki activation is not rescued in these conditions (Extended Data Fig. 9j–l), illustrating the ability of Crb-intra to promote growth independently of Grnd by inhibiting Hippo signalling through its FERM-binding motif (FBM)¹⁷,¹⁸. Indeed, neoplastic growth and polarity defects induced by a form of Crb-intra lacking its FBM (CrbΔFBM-intra) are both rescued by Grnd silencing (Fig. 4i, j). As expected, the size of *rn>crbΔFBM-intra*;grnd-RNAi discs is reduced compared to the size of *rn>crb-intra*;grnd-RNAi discs (compare Fig. 4e, f with Fig. 4j).

Crb, Stardust (Sdt; PALS1 in humans), and Pals1-associated tight junction protein (Patj) make up the core Crb complex¹⁹, which recruits the adaptor protein Veli (MALS1–3 in humans)²⁰⁻²². In agreement with previous yeast two-hybrid data²³, we find that Grnd binds directly and specifically to the PDZ domain of Veli through a membrane-proximal stretch of 28 amino acids in its intracellular domain (Extended Data Fig. 10a–e). Grnd localization is unaffected in
<figure><img src="image_5.png" /><figcaption>**Figure 4** | *grnd* is required downstream of the apical Crb polarity complex for JNK-dependent neoplastic growth. a, b, Transverse sections of wild-type discs stained for Grnd (red), Crb (a, green) or Veli (b, green). c–h, Discs expressing *crb-intra* alone (c), or combined with the indicated genotypes (*d–h*), dissected 6 days AED, stained for Wg (green). i, j, Discs expressing</figcaption></figure>

*crb*⁻/⁻ and *veli* RNAi mutant clones (Extended Data Fig. 10f–h). However, reducing *veli* expression rescues the patterning defects and disc morphology of *rn>* *crb-intra* mutant cells (Fig. 4g, h), suggesting that Grnd couples Crb activity with JNK signalling through its interaction with Veli. Interestingly, aPKC-dependent activation of JNK signalling¹⁶ also depends on Grnd (Fig. 4k, l). aPKC is capable of directly binding and phosphorylating Crb, which is important for Crb function²⁴. This suggests that aPKC, either directly or through Crb phosphorylation, activates Grnd-dependent JNK signalling in response to perturbation of apico-basal polarity.

Our data are consistent with a model whereby Grnd integrates
signals from Egr, the unique fly TNF, and apical polarity determi-
nants to induce JNK-dependent neoplastic growth or apoptosis in a
context-dependent manner (Fig. 4m). Recent work reveals a correla-
tion between mammalian Crb3 expression and tumorigenic potential
in mouse kidney epithelial cells²⁵. The conserved nature of the Grnd
receptor suggests that specific TNFRs might carry out similar func-
tions in vertebrates, in which the link between apical cell polarity and
tumour progression remains elusive.

Crb-intra lacking its FBM (*crbΔFBM-intra*) alone (i) or with grnd RNAi (j) stained for Wg (in green). k, l, Discs from *puc-LacZ* animals expressing activated aPKC (*aPKCΔN*)²⁶ in the posterior domain alone (k) or together with grnd RNAi (l). m, A model for Grnd function.

**Online Content** Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper.

Received 25 September 2013; accepted 9 February 2015.

Published online 15 April 2015.

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**Acknowledgements** We thank G. Jarretou for technical assistance, the Vienna *Drosophila* RNAi Centers, the *Drosophila* Genetics Resource Center, the Bloomington Stock Center, N. Caridi and S. Pasqualato for technical assistance and P.L. laboratory members for comments on the manuscript. This work was supported by the CNRS, INSERM, Agence Nationale de la Recherche, Fondation pour la Recherche Médicale, Association pour la Recherche contre le Cancer (grant no. PJA20131200042 to J.C.), European Research Council (Advanced grant no. 268813 to P.L.), Marie Curie Life Long Training (grant no. 252373 to D.S.A.), the Labex Signalife program (grant ANR-11-LABX-0028-01 to P.L.), the Italian Association for Cancer Research (AIRC IG-12877) and the Italian Ministry of Health (GR-2008-1134103) to M.M.

**Author Contributions** D.S.A., J.C., K.C., A.-O.H., K.B., M.M. and P.L. designed the research; D.S.A., J.C., K.C., V.P., E.B., M.R. and J.T. performed experiments; D.S.A., J.C., K.C., E.B., A.-O.H., K.B., V.P., M.M. and P.L. analysed the data; D.S.A., J.C. and P.L. wrote the manuscript.
METHODS

Fly strains and food. Animals were reared at 25 °C on fly food containing, per litre: 17 g inactivated yeast powder, 83 g corn flour, 10 g agar, 60 g white sugar and 4.6 g Nipagin M (in ethanol). The *rn*Gal4, *UAS-egr*, *tub*Gal80<sup>ts</sup> animals were raised at 18 °C, shifted to 30 °C for 40 h during early third instar larval development (7 days AEL), and subjected to dissection immediately after (Extended Data Fig. 6).

Cell culture. *Drosophila* S2 and S2R⁺ cells were grown in complete Schneiders medium (CSM; Schneiders medium (Invitrogen) supplemented with 10% heat-inactivated FBS (BioWhittaker), 50 U ml⁻¹ penicillin and 50 µg ml⁻¹ streptomycin (Invitrogen)) at 25 °C. Transfections were done using Effectene reagent (Qiagen).

Plasmids. *grnd*, *grnd-extra* and *grnd-intra* coding sequences were PCR amplified from BDGP EST complementary DNA clones RE28509, and cloned into the pENTR/D-TOPO vector using the following gene-specific primers: sense primers CACCATGTCGGTCAGGAAGTTGAG (*grnd* and *grnd-extra*) and CACCA TGCTGATCCTCTTGCTGCTGACTATCC (*grnd-intra*); and antisense primers GAAAGCGACGGGAATCGTCGC (*grnd* and *grnd-intra*) and CCATCGAAGG AACTGAAACGCG (*grnd-extra*). The mutations giving rise to the single E202A and double P200A/E202A amino acid changes in Grnd and Grnd¹²⁵⁻²⁴¹ were introduced using the QuickChange site-directed mutagenesis kit (Stratagene), *grnd*-pENTR or *grnd*¹²⁵⁻²⁴¹-*pGEX-6PI* templates, and the following primers: sense, CGGTGAGCACTCGCTATCCGGCGGCAAACAGTACCACGCCCAC; and antisense, GTGGGCGTGGTACTGTTTGCCGCCGGATAGCGAGTGCTCAC CG for *grnd*E202A; sense, CGGTGAGCACTCGCTATGCGGCGGCAAACAGT ACCACGCCCAC; and antisense, GTGGGCGTGGTACTGTTTGCCGCCGCA TAGCGAGTGCTCACCG for *grnd*P200A/E202A. To generate *grnd-Flag*, *grnd*E202A- *Flag*, *grnd-extra-Flag* and *grnd-intra-Flag*, *grnd*E202A, *grnd-extra* and *grnd-intra* coding sequences were cloned into the Gateway Destination vector pActin-3×Flag (*Drosophila* Gateway Vector Collection) for expression in S2 and S2R⁺ lines. In addition, *grnd*, *grnd-intra* and *grnd-extra* coding sequences were cloned into pUASg.ATTB for generation of UAS-*grnd*, UAS-*grnd-intra* and UAS-*grnd-extra* transgenic lines. The *HA-traf2* plasmid was from K.B.²⁷. The *HA-egr* and *HA-egrΔTNF* plasmids were a gift from M. Miura. pActin-iHog-HA and pActin-iHog-Flag were gifts from L. Ruel.

Transgenic flies. To generate the *UAS-grnd* *UAS-grnd-intra* and *UAS-grnd-extra* lines, the *pUASattB-grnd*, *pUASattB-grnd-intra* and *pUASattB-grnd-extra* constructs were introduced into the germ line by injections in the presence of the PhiC31 integrase and inserted in the 86F8-landing site on the 3R chromosome (Bloomington *Drosophila* Stock Center, BL24749, BestGene). The following RNAi lines were from the GD or KK collections of the Vienna *Drosophila* RNAi Center (VDRC): *grnd* RNAi (GD43454, sense primer CGCGAATTCCTCCTTGCTG CTGACTATCCTTCTCGT, and antisense primer GCGTCTAGAGTTGTGG GCGTGGTACTGTTTCC; and KK104538, sense primer TGGCGCCCCTAG ATGAACGGTTATGACGGAGATCG, and antisense primer CGCATGTAGC CTGCCGTTTGTGGTGATTGCGAATG, *tak1* RNAi (KK101357, sense primer TGGCGCCCCTAGATGGCTTGCCTTGAAGTCCTGC, and antisense primer CGACGCCCGCTGATAGCATGAGCAGGAGATTGTCA), *bendless* RNAi (KK109638, sense primer TGGCGCCCCTAGATGCGCATCATCAAGGAGA CTCA, and antisense primer CGCATGTAGCCTGCCTTCTGGAAAAACGCC GATAC), *tab2* RNAi (KK100326, sense primer TGGCGCCCCTAGATGGCA CTTTCAGCGGAAGAAAC, and antisense primer TAGCCTCCCTAGCG CAGCTCTGCACTGGGATAGGA), *traf2* RNAi (GD16125, sense primer CGC GAATTCAGCACCCACTCCGCCCAAGA, and antisense primer GCGTCTAG ACCGAACAGCCCAAGCGAGGA), *hep* RNAi (KK109277, sense primer TGG CGCCCCTAGATGGGCTTCTTGGACAGCTTGAG, and antisense primer CG CATGTAGCCTGCCAGGAGAACAAACGCATCCTG), *bsk* RNAi (KK104569, sense primer TGGCGCCCCTAGATGAGCATAGCCTTCCTCAGCAT, and antisense primer TAGGTCTAGCCCGCGGTACACCATCACCCTCGTT), *scrib* RNAi (GD 45556, sense primer CGCGAATTCCGAAGAGGACAACG CACAGCA, and antisense primer GCGTCTAGACTGGGAGCGGGCAAAGT GTAAG), *avl* (GD5413, sense primer CGCGAATTCCAACAGTAGCCGC AGCAGCA, and antisense primer CGCTCTAGACCGTGCCCTGCGAGACA AAA), *dilp8* (KK102604, sense primer TGGCGCCCCTAGATGACTCGATG GACCTTCTGTCG, and antisense primer CGCATGTAGCCTGCCGTCATCG GAGTCTGTTGCCT), and *rh3* RNAi (KK100853, sense primer TGGCGCCCC TAGATGATCTGGGATGACTTATGGCG, and antisense primer TAGCCTCC CTAGCGCTCGATACGCACTCTTTGTG). The *veli*-RNAi lines were from U. Thomas. The *wengen*-RNAi line (*wengen-IR*) was provided by M. Miura. The *GMR*-*Gal4* (on chromosome II), *UAS-egr* (on chromosome III), *GMR*-*Gal4*, *UAS-ecto-egr60* (on chromosome II), *GMR*-*Gal4*, *UAS-egr* (on chromosome II), *UAS-Crb4*ΔFBM-*intra*, *DIAP*-*GFP*, aPKCΔN, *FRT*, *Crb4*¹¹A²² (null) and *UAS-wgn* transgenic lines were previously published⁷,¹⁰,²⁷⁻²⁹, the *UAS-puc*²A line

was described previously³⁰, the *UAS-crb-intra* transgenic lines and *veli*⁶⁶ mutant
flies were from E. Knust, the *egr*¹ and *egr*³ mutant lines and the UAS-ras¹²,
FRT82B, scrib¹/TM6b and UAS-ras¹², *egr*³; FRT82B, scrib¹/TM6b and yw,
eyfFlp1; act>y+>Gal4, UAS-GFP; FRT82B, tubgal80 and yw, eyfFlp1;
act>y+>Gal4, UAS-GFP, *egr*¹; FRT82B, tubGal80 lines were from M. Vidal,
the w¹¹¹⁸; +; *rn*Gal4, UAS-eiger, *tub*Gal80⁵/TM6B line was provided by I.
Hariharan, the yw, *hep*75/FM7, GFP; Sal⁵PE/cyo line was provided by F. Serras.
The *rn*-*Gal4*, *nub*-*Gal4*, *puc*-LacZ, *grnd*⁴Minos (Mi{MIC}CG10176M¹⁰⁵²⁹²), and
*grnd*⁵(DF(BSC149) lines were provided by the Bloomington *Drosophila* Stock Center.

Flies' eye pictures are acquired on a Leica Fluorescence Stereomicroscope MZ16 FA with a Leica digital camera DFC 490.

**Generation of wgn<sup>KO/KO</sup> mutant flies.** The *wgn* knockout allele was generated by ends-in gene targeting as described previously³¹. The pTV2 targeting construct carried frameshift mutations in exon 1 and 2, an I-SceI site between these two exons, a *white*⁻ marker gene, an I-CreI site, up- and downstream homology sequences and two flanking FRT sites. The frameshift mutations were introduced by cutting the unique intrinsic restriction sites AclI and NotI followed by T4 DNA polymerase-mediated fill-in. F3 Larvae carrying the targeting construct together with an *hsp70-flp* and a *hsp70-I-SceI* transgene were heat shocked (1 h at 38 °C on day 2 and 3 after egg laying) to generate a linearized targeting construct that aligns and recombines with the endogenous wild-type locus. After crossing the F3 flies to eye-FLP-expressing flies, the F4 was screened for non-mosaic *white*⁺ eyes carrying an insertion of a processed targeting construct. After processing with FLP and I-SceI, only one FRT site remained next to the *white*⁺ marker gene in these flies. Flies still carrying the original donor insertion lost the *white*⁺ marker gene, since it was still flanked by two FRT sites. Female F4 *w*⁺ *wgn*<sup>KO</sup>, flies, carrying two *wgn* copies on the X chromosome were crossed with males of the genotype *v*/Y; +/+; [*v*+*, 70I-CreI] 1A/TM6*. The F5 offspring were heat shocked (1 h at 38 °C on day 2 after egg laying). F5 offspring males were selected against TM6 (*w*⁺/−74/Y; +/+; [*v*+*, 70I-CreI] 1A/+*) and typically displayed *white*⁺/⁻ mosaics. When they were individually crossed to the X-chromosomal balancer stock 'bin/"852", more than 90% of the males were sterile. The F6 flies that hatched were either *white*⁺ or *white*⁻. The *white*⁻ flies were tested for the presence of the mutations. The stocks *y* w (v); P[ry+, 70FLP]4 P[v+, 70I-SceI]2B Sco/S2 Cyo' and *v*; +/+; [*v*+*, 70I-CreI] 1A/ TM6, Ubx, e' were provided by K. Golic and Y. Rong.

Genome-wide screen. As an unbiased approach to identify molecules that are required for neoplastic growth, we used the *elav*Gal80; *rn>avl*-RNAi/ *TM6b*, *tub>Gal80* testerline, which is characterized by neoplastic growth in the discs and a delay of about 2 days in pupariation. We crossed this line to the Vienna *Drosophila* RNAi Center (VDRC) collection of *phiC31* (KK) RNAi lines (10,100 lines) and screened for candidates that were able to rescue the delay at pupariation as described previously¹. One-hundred and twenty-one candidates significantly rescued the pupariation delay in *rn>avl*-RNAi larvae. Of these, only 8 lines rescued the neoplastic disc growth (Extended Data Fig. 1).

Immunostainings of larval tissues. Tissues dissected from larvae in 1× PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, pH 8) at the indicated hour AED, were fixed in 4% formaldehyde (Sigma) in PBS for 20 min at room temperature washed in PBS containing 0.1% Triton X-100 (PBT), blocked for 2 h in PBT containing 10% FBS (PBS-TF), and incubated overnight with primary antibodies at 4 °C. The next day, cells and tissues were washed, blocked in PBS-TF, and incubated with secondary antibodies at 1/500 dilution (Rhodamine Red-X-conjugated donkey anti-rat, Cy3 conjugated donkey anti-rabbit, Cy3 conjugated donkey anti-chicken, Cy3 conjugated donkey anti-mouse and Cy3 conjugated mouse anti-guinea pig, Cy5 conjugated donkey anti-rat, and Cy5 conjugated donkey anti-guinea pig from Jackson ImmunoResearch, goat anti-mouse Alexa Fluor 488, donkey anti-rat Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 488 from Invitrogen) for 2 h at room temperature. After washing, tissues were mounted in Vectashield containing DAPI for staining of DNA (Vector Labs). Fluorescence images were acquired using a Leica SP5 DS (×20 and ×40 objectives) and processed using Adobe Photoshop CS5.

**Lysis and immunoprecipitations.** Cells were lysed in 100 µl buffer A (50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% NP-50, 1 mM EGTA, 0.5 M NaF, phosphatase inhibitor cocktail 2 (Sigma), complete protease inhibitor cocktail (Roche)). Immunoprecipitations were performed from about 1 × 10⁷ S2 cells. Cells were lysed in 200 µl buffer A, and cell extracts were cleared of membranous material by centrifugation at 10,000 r.p.m. for 15 min. The cleared extracts were incubated with protein G-Sepharose beads (Sigma) for 1 h to reduce unspecific binding of proteins to the beads in the subsequent purifications. Next, the precleared extracts were incubated with 80 µl of protein G-Sepharose beads and 1 µl of the relevant antibody for 3 h. Subsequently, beads were washed three times in buffer A, boiled in sample and reducing buffers (Invitrogen).
**Protein preparation.** *Drosophila* Veli full length (Veli¹⁻¹⁹⁵) and PDZ domain (Veli⁸⁵⁻¹⁹⁵) were expressed with an amino-terminal His₆-tag from pET43 (Novagen) in *Escherichia coli* strain BL21 Rosetta, and purified by affinity on Ni-NTA beads (Qiagen). The Veli L27 domain (Veli¹⁻⁸¹) was cloned in a modified pET43 plasmid as a His₆-MBP-tagged construct. The fusion protein was expressed in bacteria similarly to the other constructs. After a first step of affinity purification on amylose beads (NEB), the protein was incubated with TEV protease (Invitrogen Life Technologies) to cleave the MBP tag, and loaded on a Resource-Q anion-exchange column (GE-Healthcare). The TRAF domain of *Drosophila* Traf2 (*Traf2*³¹⁹⁻⁴⁷⁵) was expressed similarly, and purified by affinity followed by ion-exchange chromatography on a Resource-S column (GE-Healthcare). All Grnd constructs were expressed as GST-fusion proteins using a modified version of the pGEX-6P1 vector (GE-Healthcare), and purified by affinity on glutathione sepharose (GSH) beads. The purifications were carried out at 4 °C. The PDZ domains of human Par3 (Par3-PDZ2 and Par3-PDZ3 encompassing residues 465–548 and 582–685, respectively) and *Drosophila* Bazooka (Baz-PDZ1 corresponding to residues 331–419) were expressed as GST-fusion proteins. After the first step of affinity purification on GSH-beads, the GST tag was removed from the constructs by cleavage with PreScission protease, and the PDZ domains were further purified by ion-exchange chromatography.

**In vitro binding assay.** To test the interaction properties of intracellular Grnd constructs, GST-Grnd fusion proteins (5 µM) adsorbed on GSH-beads were incubated for 40 min at 4 °C with 10 µM Veli<sup>FL</sup>, Veli<sup>L127</sup> or Veli<sup>PDZ</sup> in a buffer containing 40 mM HEPES pH 7.4, 30 mM NaCl, 5% glycerol, 0.5 mM EDTA and 1 mM dithiothreitol (DTT). After two washing steps, the bound species were resolved on SDS-PAGE and visualized by Coomassie staining. To test the specificity of binding, analogous pull-down assays were performed with the PDZ domains of Par3 and Bazooka under the same conditions. To monitor the ability of Traf2 to bind Grnd, 2 µM GST-Grnd¹²⁵⁻²⁴¹ wild type or mutated on P200 and/or E202 were absorbed on GSH beads and incubated with 10 µM Traf2³¹⁹⁻⁴⁷⁵ in a buffer consisting of 10 mM HEPES pH 7.4, 0.15 M NaCl, 5% glycerol, 0.5 mM EDTA, 1 mM DTT and 0.05% Tween. After washing, complexes immobilized on beads were analysed by SDS-PAGE.

**Subcellular fractioning.** Subcellular fractioning of 300 wing imaginal discs dissected from wild-type animals was performed using the Qproteome Cell Compartment Kit (Qiagen) according to the manufacturer's protocol.

**Western blotting.** Proteins were resolved by SDS-PAGE using 4–12% gradient gels (NuPAGE Novex gel, Invitrogen) and transferred electrophoretically to polyvinylidene difluoride membranes (Milipore). The membranes were incubated for 1 h in blocking buffer (PBS, 5% milk) and incubated overnight at 4 °C in the same buffer containing primary antibodies at 1:1,000 dilutions: rabbit anti-Smoothened³², mouse anti-Lamin (Developmental Studies Hybridoma Bank (DSHB)), mouse anti-Dlg (4F3) from DSHB, mouse anti-Myc 9E10 (sc-40, Santa Cruz Biotechnology), rabbit anti-Myc A-14 (sc-789, Santa Cruz Biotechnology), mouse anti-Flag F3165 (Sigma), rat anti-HA 3F10 (Roche), guinea pig anti-Grnd at 1:1,000, anti-tubulin (Sigma) at 1:2,000. The anti-Wgn antibody was used as described previously²⁷. Membranes were washed three times in PBS-T, blocked for 1 h, and probed with secondary antibodies in blocking buffer for 1 h at room temperature. After three washes in PBS-T, chemiluminescence was observed using the ECL-Plus western blotting detection system (Amersham Biosciences). Images were generated using the Fujifilm Multi Gauge software.

**Antibodies.** To generate purified antisera specific for the Grnd protein, two peptides, CAYPHPATANGKDLNA and NAINHPGSDLRAQSQ, corresponding to amino acids 140–154 and 161–176, respectively, were used as immunogens in guinea pigs (Eurogentec). The following primary antibodies were used for immunofluorescence in this study: mouse anti-Wg (4D4) from DSHB 1/50, mouse anti-Dlg (4F3) from DSHB 1/500, rat anti-E-Cad (DCAD2) from DSHB 1/500, mouse anti-MMP1 (3A6B4/5H7B11/3B8D12; the three different antibodies were mixed in amounts (1:1) from DSHB (1:25)), chicken anti-β-galactoside from GeneTek 1/1,000, rabbit anti-cleaved caspase-3 (Asp 175) from Cell Signaling Technology 1/500, rabbit anti-aPKC from Santa Cruz Biotechnology 1/1,000, guinea pig anti-Grnd 1/100, rabbit anti-Veli 1/1,000 (gift from G. Pietrinari), and rat anti-Crb 1/1,000 (gift from E. Knust).

**Sequence alignment and homology modelling.** Multiple sequence alignment, per cent identity matrix, and phylogenetic data were generated using Clustal Omega (http://www.ebi.ac.uk/Tools/services/web/toolform.ebi?tool=clustalo). Data of five human death receptors of the TNFR superfamily with the highest identity with Grnd are shown. Slight manual adjustment of the alignments was performed. Amino acids are highlighted where Grnd shares identity or similarity with death receptor(s) (Fig. 1) or Grnd-like proteins from other insect species (Extended Data Fig. 2d), or where Fas shares identity or similarity with Grnd and/or Wgn (Extended Data Fig. 2b). Amino acid similarity is presented based on scores in

Gonnet PAM 250 matrix (>0.5, highly similar; ≤0.5, weakly similar). Amino acid positions are indicated in parentheses or at the end of the shown sequences. The phylogram was drawn using interactive Tree of Life³³ (http://itol.embl.de/). Number on each branch indicates branch length (Extended Data Fig. 2a). UniProt protein identifiers are: Fas (P25445), DR4 (O00220), DR5 (O14763), DcR1 (O14798), DcR2 (Q9UBN6), and Wengen (Q9VWS4). Information of protein domains in TNFR superfamily members used to construct protein domain diagram (Extended Data Fig. 2d) was obtained from UniProt (http://www.uniprot.org/).

**Genotypes.** Fig. 1a: *w*⁻; +; *rn*‐Gal4/*puc‐lacZ*. Fig. 1b: *w*⁻; *elav*‐Gal80/+; *rn*‐Gal4, *UAS* *avl* RNAi GD/*puc‐lacZ*. Fig. 1c: *w*⁻; *elav*‐Gal80/*UAS* grnd RNAi KK; *rn*‐Gal4, *UAS* *avl* RNAi GD/*puc‐lacZ*. Fig. 1d: *w*⁻; *elav*‐Gal80/*UAS* tak1 RNAi KK; *rn*‐Gal4, *UAS* *avl* RNAi GD/*puc‐lacZ*. Fig. 1i: *w*⁻; *nub*‐Gal4/+; *UAS* grnd / *puc‐lacZ*. Fig. 1j: *w*⁻; *nub*‐Gal4/+; *UAS* grnd‐intra / *puc‐lacZ*.

Fig. 2a: *w*⁻; GMR‐Gal4/+; +. Fig. 2b: *w*⁻; GMR‐Gal4, *UAS* egr+/+; MKRS+/+. Fig. 2c: wgn<sup>KO</sup>; GMR‐Gal4, *UAS* egr+/+; MKRS+/+. Fig. 2d: *w*⁻; GMR‐Gal4, *UAS* egr/ *UAS* grnd RNAi KK; MKRS+/+. Fig. 2e: *w*⁻; GMR‐Gal4, *UAS* egr+/+; MKRS/ *UAS* grnd‐extra. Fig. 2f: *yw*, *hs‐FLP*; *Act*>CD2>Gal4, *UAS* GFP+/+; *UAS* egr+/+. Fig. 2g: *yw*, *hs‐FLP*; *Act*>CD2>Gal4, *UAS* GFP/ *UAS* tak1 RNAi KK; *UAS* egr+/+. Fig. 2h‐i: *yw*, *hs‐FLP*; *Act*>CD2>Gal4, *UAS* GFP/ *UAS* grnd RNAi KK; *UAS* egr+/+.

Fig. 3a: *yw*, *hs‐FLP*; *Act*>CD2>Gal4, *UAS* GFP+/+; +. Fig. 3b: *yw*, *hs‐FLP*; *Act*>CD2>Gal4, *UAS* GFP/ *UAS* rh3 RNAi KK; +. Fig. 3c: *yw*, *hs‐FLP*; *Act*>CD2>Gal4, *UAS* GFP/ *UAS* grnd RNAi KK; +. Fig. 3d: *yw*, *hs‐FLP*; *Act*>CD2>Gal4, *UAS* GFP+/+; *UAS* scrib RNAi GD+/+. Fig. 3e: *yw*, *hs‐FLP*; *Act*>CD2>Gal4, *UAS* GFP/ *UAS* rh3 RNAi KK; *UAS* scrib RNAi GD+/+. Fig. 3f: *yw*, *hs‐FLP*; *Act*>CD2>Gal4, *UAS* GFP/ *UAS* grnd RNAi KK; *UAS* scrib RNAi GD+/+. Fig. 3g: *yw*, *ey‐FLP*; *Act>*y⁺>Gal4, *UAS* GFP/ *UAS* ras<sup>V12</sup>/+; *FRT82B*, scrib<sup>1</sup>/ FRT82B, tubGal80. Fig. 3h: *yw*, *ey‐FLP*; *Act>*y⁺>Gal4, *UAS* GFP/ *UAS* ras<sup>V12</sup>, *UAS* grnd RNAi KK / +; *FRT82B*, scrib<sup>1</sup>/ FRT82B, tubGal80. Fig. 3i: *yw*, *ey‐FLP*; *Act>*y⁺>Gal4, *UAS* GFP; egr<sup>1</sup> / *UAS* ras<sup>V12</sup>, egr<sup>3</sup>; *FRT82B*, tubGal80/ *FRT82B*, scrib<sup>1</sup>.

Fig. 4a, b: *w*⁻. Fig. 4c: *w*⁻; *UAS* crb‐intra+/+; *rn*‐Gal4 / +. Fig. 4d: *w*⁻; *UAS* crb‐intra/ *UAS* puc<sup>2A</sup>; *rn*‐Gal4 / +. Fig. 4e: *w*⁻; *UAS* crb‐intra/ *UAS* grnd RNAi KK; *rn*‐Gal4 / +. Fig. 4f: *w*⁻; grnd<sup>Minos</sup>/ grnd<sup>Minos</sup>; *rn*‐Gal4 / *UAS* crb‐intra. Fig. 4g: *w*⁻; *UAS* crb‐intra/ *UAS* veli RNAi; *rn*‐Gal4/ *UAS* veli RNAi. Fig. 4h: *w*⁻; *UAS* crb‐intra/ *nub*‐Gal4; veli<sup>66</sup>/ veli<sup>66</sup>. Fig. 4i: *w*⁻; +; *UAS* crbΔFBM‐intra/ *rn*‐Gal4. Fig. 4j: *w*⁻; *UAS* grnd RNAi KK/+; *UAS* crbΔFBM‐intra/ *rn*‐Gal4. Fig. 4k: *w*⁻; *en*‐Gal4, *UAS* GFP+/+; *UAS* aPKCΔN/ *puc‐lacZ*. Fig. 4l: *w*⁻; *UAS* grnd RNAi KK/ *en*‐Gal4, *UAS* GFP; *UAS* aPKCΔN/ *puc‐lacZ*.

Extended Data Fig. 1c: *w*⁻; *elav*‐Gal80/+; *rn*‐Gal4, *UAS* *avl* RNAi GD / +. Extended Data Fig. 1d: *w*⁻; *elav*‐Gal80/ *UAS* grnd RNAi GD; *rn*‐Gal4, *UAS* *avl* RNAi GD / +. Extended Data Fig. 1e: *w*⁻; *elav*‐Gal80/ *Rh3* RNAi KK; *rn*‐Gal4, *UAS* *avl* RNAi GD / +. Extended Data Fig. 1f, g: *w*⁻; *ptc*‐Gal4, *UAS*‐GFP/ *UAS* grnd RNAi KK; +.

Extended Data Fig. 4a: *w*⁻; +; *rn*‐Gal4 / +. Extended Data Fig. 4b: *w*⁻; +; *rn*‐Gal4 / *UAS* grnd. Extended Data Fig. 4c, d: *w*⁻; +; *rn*‐Gal4 / *UAS* grnd‐intra. Extended Data Fig. 4e: *yw*⁻; sal‐Gal4 / +; *UAS* grnd‐intra / +. Extended Data Fig. 4f: *yw*⁻, hep<sup>75</sup>; sal‐Gal4 / +; *UAS* grnd‐intra / +.

Extended Data Fig. 5a: *w*⁻; *GMR*‐Gal4, *UAS* egr+/+; MKRS+/+. Extended Data Fig. 5b: *w*⁻; *GMR*‐Gal4, *UAS* egr/ *UAS*‐traf2 RNAi GD; MKRS+/+. Extended Data Fig. 5c: *w*⁻; *GMR*‐Gal4, *UAS* egr/ *UAS* puc<sup>2A</sup>; MKRS+/+. Extended Data Fig. 5d: *w*⁻; *GMR*‐Gal4, *UAS* egr/ *UAS* tak1 RNAi KK; MKRS+/+. Extended Data Fig. 5e: *w*⁻; *GMR*‐Gal4, *UAS* egr/ *UAS*‐wengen RNAi IR; MKRS+/+. Extended Data Fig. 5f: *w*⁻; *GMR*‐Gal4, *UAS* egr/ *UAS*‐grnd RNAi GD; MKRS+/+. Extended Data Fig. 5g: wgn<sup>KO</sup>; *GMR*‐Gal4, *UAS* egr/ *UAS*‐grnd RNAi GD; MKRS+/+. Extended Data Fig. 5h: wgn<sup>KO</sup>; +; +. Extended Data Fig. 5i: *w*⁻; *GMR*‐Gal4/ *UAS*‐grnd RNAi KK; +. Extended Data Fig. 5j: *w*⁻; *GMR*‐Gal4/+; *UAS*‐grnd‐extra/+.

Extended Data Fig. 5k: *w*⁻; *GMR*‐Gal4, *UAS*‐ecto‐egr60/+; MKRS+/+. Extended Data Fig. 5l: *w*⁻; *GMR*‐Gal4, *UAS*‐ecto‐egr60/ *UAS* puc<sup>2A</sup>; MKRS+/.

Extended Data Fig. 6c: *w*⁻. Extended Data Fig. 6d: *w*⁻; grnd<sup>Minos</sup>/ grnd<sup>Minos</sup>; +. Extended Data Fig. 6e: *w*⁻; +; *rn*‐Gal4, *UAS* egr, tubGal80<sup>1s</sup> / +. Extended Data Fig. 6f: *w*⁻; *UAS*‐grnd RNAi KK/+; *rn*‐Gal4, *UAS* egr, tubGal80<sup>1s</sup> / +. Extended Data Fig. 6g: *w*⁻; *UAS*‐traf2 RNAi GD / +; *rn*‐Gal4, *UAS* egr, tubGal80<sup>1s</sup> / +. Extended Data Fig. 6h: *w*⁻; *UAS*‐tak1 RNAi KK/+; *rn*‐Gal4, *UAS* egr, tubGal80<sup>1s</sup> / +. Extended Data Fig. 6i: *w*⁻; grnd<sup>Minos</sup>/ grnd<sup>Minos</sup>; *rn*‐Gal4, *UAS* egr, tubGal80<sup>1s</sup> / +. Extended Data Fig. 6j: *w*⁻; grnd<sup>Minos</sup>/ grnd<sup>Df</sup>; *rn*‐Gal4, *UAS* egr, tubGal80<sup>1s</sup> / +.

Extended Data Fig. 7a: *yw*, *ey‐FLP*; *Act>*y⁺>Gal4, *UAS* GFP; +; *FRT82B/ FRT82B*, tubGal80. Extended Data Fig. 7b: *yw*, *ey‐FLP*; *Act>*y⁺>Gal4, *UAS* GFP; +; *FRT82B*, scrib<sup>1</sup> FRT82B, tubGal80. Extended Data Fig. 7c: *yw*, *ey‐FLP*; *Act>*y⁺>Gal4, *UAS* GFP; *UAS* grnd RNAi KK / +; *FRT82B*, scrib<sup>1</sup> FRT82B,
tubGal80. Extended Data Fig. 7d: ey-FLP; Act>CD2>Gal4, UAS GFP/ UAS grnd RNAi KK +. Extended Data Fig. 7e-g: yw, ey-FLP, Act>y⁺>Gal4, UAS GFP; UAS ras<sup>V12</sup>+/; FRT82B, scrib<sup>1</sup>/ FRT82B, tubGal80.

Extended Data Fig. 8a: yw, ey-FLP; Act>y⁺>Gal4, UAS GFP; UAS ras<sup>V12</sup>+/; FRT82B, scrib<sup>1</sup>/ FRT82B, tubGal80. Extended Data Fig. 8b: yw, ey-FLP; Act>y⁺>Gal4, UAS GFP; UAS ras<sup>V12</sup>, UAS grnd RNAi KK +/; FRT82B, scrib<sup>1</sup>/ FRT82B, tubGal80. Extended Data Fig. 8c: yw, ey-FLP; Act>y⁺>Gal4, UAS GFP; UAS ras<sup>V12</sup>, UAS dilp8 RNAi KK +/; FRT82B, scrib<sup>1</sup>/ FRT82B, tubGal80. Extended Data Fig. 8d: w; UAS ras<sup>V12</sup>, UAS-dlg RNAi GD +/; ey-FLP; Act>►Gal4, UAS GFP +/. Extended Data Fig. 8e: w; UAS ras<sup>V12</sup>, UAS-dlg RNAi GD / UAS-grnd RNAi GD ; ey-FLP; Act>►Gal4, UAS GFP +/. Extended Data Fig. 8f: w; UAS ras<sup>V12</sup>, UAS-dlg RNAi GD / UAS-grnd RNAi KK ; ey-FLP; Act>►Gal4, UAS GFP +/. Extended Data Fig. 8g: wgn<sup>KO</sup>; UAS ras<sup>V12</sup>, UAS-dlg RNAi GD /+; ey-FLP; Act>►Gal4, UAS GFP /+.

Extended Data Fig. 9a: w⁻; +; *rn*-Gal4, UAS *avl* RNAi GD/+.
Extended Data Fig. 9b: w⁻; *egr*¹ / *egr*³; *rn*-Gal4, UAS *avl* RNAi GD/+.
Extended Data Fig. 9c, d: w⁻; +; +.
Extended Data Fig. 9e: w⁻; *elav*-Gal80/+; *rn*-Gal4, UAS *avl* RNAi GD/+.
Extended Data Fig. 9f: w⁻; UAS *crb*-intra/+; *rn*-Gal4 / +.
Extended Data Fig. 9g: w⁻; *elav*-Gal80/+; *rn*-Gal4, UAS *avl* RNAi GD/+.
Extended Data Fig. 9h: w⁻; UAS *crb*-intra/+; *rn*-Gal4 / +.
Extended Data Fig. 9i: wgn<sup>KO</sup>; UAS *crb*-intra/+; *rn*-Gal4 / +.
Extended Data Fig. 9j: w⁻; +; *rn*-Gal4 / diap-GFP.
Extended Data Fig. 9k:

w⁻; UAS crb-intra/+; *rn*-Gal4 / diap-GFP. Extended Data Fig. 9l: w⁻; UAS crb-intra/ UAS-grnd RNAi KK; *rn*-Gal4 / diap-GFP.

Extended Data Fig. 10f, g: yw, hsFLP; +; FRT82B, Ubi-GFP/ FRT82B, crb¹¹A²².
Extended Data Fig. 10h: yw, hs-FLP; Act>CD2>Gal4, UAS GFP/ UAS veli RNAi;
UAS veli RNAi / +.

27. Geuking, P., Narasimamurthy, R. & Basler, K. A genetic screen targeting the tumor necrosis factor/Eiger signaling pathway: identification of *Drosophila* TAB2 as a functionally conserved component. *Genetics* **171**, 1683–1694 (2005).

28. Izaddoost, S., Nam, S. C., Bhat, M. A., Bellen, H. J. & Choi, K. W. *Drosophila* Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres. *Nature* **416**, 178–183 (2002).

29. Zhang, L. et al. The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control. *Dev. Cell* **14**, 377–387 (2008).

30. Martín-Blanco, E. et al. puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in *Drosophila*. *Genes Dev.* **12**, 557–570 (1998).

31. Rong, Y. S. et al. Targeted mutagenesis by homologous recombination in *D. melanogaster*. *Genes Dev.* **16**, 1568–1581 (2002).

32. Ruel, L., Rodriguez, R., Gallet, A., Lavenant-Staccini, L. & Therond, P. P. Stability and association of Smoothened, Costal2 and Fused with Cubitus interruptus are regulated by Hedgehog. *Nature Cell Biol.* **5**, 907–913 (2003).

33. Ciccarelli, F. D. et al. Toward automatic reconstruction of a highly resolved tree of life. *Science* **311**, 1283–1287 (2006).
<figure><img src="image_6.png" /></figure>

**Extended Data Figure 1 |** Identification of Grnd in a genetic screen for molecules implicated in neoplastic growth.

a, Schematic of the genome-wide screen. Ten-thousand one-hundred RNAi lines were screened for their abilities to rescue the delay of the *rn>avl*-RNAi condition. Of the 121 candidates able to rescue the delay of the *rn>avl* condition, only 8 also rescued the neoplastic growth.

b, Schematic of the five components of the JNK pathway shown to be required for the neoplastic overgrowth in the *avl* loss of function condition. Although no RNAi line targeting Traf2 was included in the original screen, Traf2 was subsequently shown to be required for neoplastic overgrowth in the

*avl*-RNAi condition using an alternative RNAi line. c-e, Wing discs of the indicated genotypes dissected 5 days (left) or 6 days (right) AED and stained for Wg (green) and Caspase3 (red). f, g, Transverse (f) or xy (g) sections of dissected wing discs expressing *grnd* RNAi in the patch domain (GFP, green) stained for Grnd (red) and DAPI (g, right, blue) to visualize the nuclei.

h, Subcellular fractionation of cleared extract from dissected wing discs shows that Grnd, like the transmembrane receptor Smoothened, localizes to the membrane fraction (PM), whereas Disc-large remains in the cytoplasmic fraction (Cyto). Laminin serves as a control for nuclear fractions.
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**Extended Data Figure 2 | Sequence homology of the extracellular domain of Grnd to CRDs of human TNFRs.** a, Per cent identity matrix and phylogenetic data from multiple local sequence alignment of the entire extracellular domains of Grnd and Wgn with extracellular domains (corresponding to a part of CRD2 and all of CRD3) of various human TNFR superfamily members. The extracellular domain of Grnd shares the highest identity with the GBM-harbouring CRD of Fas (26%). A similar result is observed for Wgn, although it exhibits lower identity (22%) and a higher degree of divergence to Fas.

b, Sequence alignment of Fas, Grnd and Wgn demonstrates that Grnd shares a higher degree of similarity with the GBM-harbouring CRD of Fas than Wgn. This is particularly apparent in the immediate region encompassing the functionally critical F134 (marked with an asterisk) of the GBM of Fas. Blue indicates residue identity, yellow strong similarity, and grey weak similarity of Fas with Grnd and/or Wgn. c, Schematic representation of Grnd, Wgn, and

various members of the human TNFR superfamily showing a high level of diversity in the number and arrangement of CRDs. The colour codes used are: red for signal peptide, green for extracellular domains, orange for transmembrane domains, and blue for intracellular domain. Numbers in each box indicate corresponding amino acid positions. Pro, prodomain; T1-5, TAPE1-5 (threonine-, alanine-, proline- and glutamine-rich repeats).

d, Multiple sequence alignment showing the conservation of the extracellular domain of Grnd in other insect species. Whereas global similarity of the extracellular domain of Grnd degenerates outside the melanogaster group, the cysteine residues of the CRD (highlighted in red) and the aromatic residue (F/Y, marked with an asterisk) corresponding to F134 of Fas CRD3 remain highly conserved throughout the Hymenoptera order. Blue indicates residue identity; yellow, strong similarity; and grey, weak similarity to Grnd.

Accepted Manuscript
<figure><img src="image_8.png" /><figcaption>Extended Data Figure 3 | Identification of a conserved TRAF6-like binding motif in the intracellular domain of Grnd proteins. Multiple sequence alignment of the intracellular domain of Grnd and Grnd-like proteins in other species. A binding motif for TRAF6 (PxExx(aromatic/acidic residue)), the closest homologue of *Drosophila* Traf2, was previously identified in human TNFRs⁴. The position of a related putative Traf2-binding motif in the</figcaption></figure>

intracellular domain of Grnd is indicated. Note that, although sequence similarity of the intracellular domain of Grnd degenerates outside the family Drosophilidae, the core residues of this motif (highlighted in red) remain highly conserved throughout the order Hymenoptera. Blue indicates residue identity; yellow, strong similarity to Grnd.
<figure><img src="image_9.png" /><figcaption>**Extended Data Figure 4** | Grnd acts upstream of JNK signalling. a–d, Wild-type wing discs (a) or discs expressing *grnd* (b) or *grnd-intra* (c, d) in the *rn* domain were dissected 5 days AED and stained for Wg (left; green), *Cas3* (middle; red), and Grnd (right; grey). d, e, Wing discs expressing *grnd-intra* in</figcaption></figure>

the *sal* domain (marked by a truncated line) in a wild-type (d) or *hep*<sup>75</sup> mutant (e) background were dissected 5 days AED and stained for Wg (left; green), *Cas3* (middle; red), and Grnd (right; grey).
<table><thead><tr><th>GMR-Gal4</th><th>GMR-Gal4</th></tr></thead><tbody><tr><td><strong>a</strong><br>UAS-egr</td><td><strong>j</strong><br>UAS-grnd-extra</td></tr><tr><td><strong>b</strong><br>UAS-egr + traf2 RNAi</td><td><strong>k</strong><br>UAS-ecto-egr-60</td></tr><tr><td><strong>c</strong><br>UAS-egr + UAS-puc<sup>2A</sup></td><td><strong>l</strong><br>UAS-ecto-egr-60 + UAS-puc<sup>2A</sup></td></tr><tr><td><strong>d</strong><br>UAS-egr + tak1 RNAi</td><td><strong>m</strong><br>70<br>wgn WT: CACAA - CGTTTCA<br>wgn KO: CACAACGCGTTTCA<br><br>175<br>wgn WT: GG - - - CGGCCGC<br>wgn KO: GGCGGCCGCCGC</td></tr><tr><td><strong>e</strong><br>UAS-egr + wengen RNAi</td><td></td></tr><tr><td><strong>f</strong><br>UAS-egr + grnd RNAi (GD)</td><td></td></tr><tr><td><strong>g</strong><br>UAS-egr + grnd RNAi KK + wgn<sup>KO</sup></td><td></td></tr><tr><td><strong>h</strong><br>wgn<sup>KO</sup></td><td></td></tr><tr><td><strong>i</strong><br>UAS-grnd RNAi KK</td><td></td></tr></tbody></table>
**Extended Data Figure 5 | Characterization of the *wgn*<sup>KO</sup> mutant allele.**

a-e, Light micrographs of *Drosophila* adult eyes are shown. Original magnification, ×30. The small-eye phenotype caused by *GMR*-driven expression of *egr* (a) is rescued by co-expressing *puc* (c) or reducing the activity of *Traf2* (b), but not by reducing *Wgn* activity (d; also serves as a control to exclude a *Gal4* titration effect). f, g, Reducing *Grnd* activity partially rescues the small-eye phenotype, giving rise to the hanging-eye phenotype (f) and is not further rescued in a *wgn*<sup>KO</sup> mutant background (g). h, *wgn*<sup>KO</sup> mutant flies do not display any eye phenotype. i, j, Reducing *Grnd* levels or expressing *Grnd*-extra in the *GMR* domain does not affect eye morphology. k, *GMR*-Gal4-

induced expression of a soluble form of Egr lacking the transmembrane and cytoplasmic domain (Ecto-Egr-60) gives rise to a small-eye phenotype¹⁰. l, Co-expression of *puc*²A in a *GMR>ecto-egr-60* background partially rescues the small-eye phenotype, giving rise to a hanging-eye phenotype¹⁰. m, Sequence analysis of DNA extracted from flies homozygous for the *wgn*<sup>KO</sup> mutation showing the two frameshift mutations introduced by cutting the unique intrinsic restriction sites AclI (exon 1) and NotI (exon 2) followed by T4 DNA polymerase-mediated fill-in. n, Western blot of fly extracts from the indicated genotypes probed with an anti-Wgn antibody²⁷. KO, knockout; OE, overexpressing; WT, wild type.
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<figure><img src="image_12.png" /><figcaption>**Extended Data Figure 6** | Characterization of the *grnd*<sup>Minos</sup> mutant.</figcaption></figure>

a, Schematic representation of the Minos transposon insertion in the *grnd* gene region corresponding to the 5' untranslated region of the transcript. b, Western blot of wing disc extracts from the indicated genotypes probed with anti-Grnd (top) and anti-β-tubulin (bottom) antibodies. Grnd protein migrates at the predicted size of 27 kDa (lane 1, control). Grnd protein levels are decreased below detection level in both *grnd*<sup>Minos/Minos</sup> and *grnd*<sup>Minos/Df</sup> mutant flies, indicating that *grnd*<sup>Minos/Minos</sup> is indistinguishable from a null mutant.

c, d, Polarity is not affected in *grnd*<sup>Minos/Minos</sup> and *grnd*<sup>Minos/Df</sup> wing discs.

<figure><img src="image_13.png" /></figure>

Transverse sections of dissected control (c) or *grnd*<sup>Minos/Minos</sup> (d) wing discs stained for Grnd (top; red), Crb (middle top; blue) and Dlg (middle bottom; green). e–j, Wing discs dissected from third instar larvae after 40 h of Egr expression in the *rn* domain. Pouch ablation, ubiquitous Wg expression and apoptosis observed in *rn>*egr, *Tub*-Gal80<sup>ts</sup> discs (e) are suppressed to various extents upon co-expression of *grnd*-RNAi (f) or reduction of JNK activity (*g*, *h*) and fully suppressed in *grnd*<sup>Minos/Minos</sup> (i) and *grnd*<sup>Minos/Df</sup> (j) mutant backgrounds.
eyFlp; FRT control

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eyFlp; FRT scrib⁻/⁻

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eyFlp; grnd RNAi

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eyFlp; UAS-Ras<sup>V12</sup>; scrib⁻/⁻

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**Extended Data Figure 7** | Grnd is highly expressed in Ras<sup>V12/scrib<sup>-/-</sup></sub> micro-metastases. a–c, Reducing *grnd* levels in *scrib<sup>-/-</sup>* mutant clones prevent their elimination. Eye discs dissected 5 days AED carrying GFP-labelled MARCM clones of the indicated genotypes stained for Grnd (middle; red) and DAPI (right; grey). d, Reducing Grnd levels does not affect cell viability. Eye discs dissected 5 days AED carrying GFP-labelled *grnd* RNAi Flip-

out clones stained for Grnd (middle; red) and DAPI (right; grey). **e–g**, Invasive brain tumours and micro-metastases display high levels of Grnd protein. Dissected eye–brain complex (**e**), gut (**f**) and fat body (**g**) displaying GFP-labelled invasive Ras<sup>V12/scrib<sup>-/-</sup></sub> clones (**e**; green) or micro-metastases (**f**, **g**; green) stained for Grnd (red) and DAPI (blue).

<figure><img src="image_35.png" /></figure>
<figure><img src="image_36.png" /><figcaption>**Extended Data Figure 8** | Grnd, but not Wgn, is required for Mmp1 expression both in *Ras*<sup>V12</sup>/scrib<sup>1</sup> and *Ras*<sup>V12</sup>/Dlg RNAi tumours. a–c, Eye–brain complexes dissected 7 days AED carrying *Ras*<sup>V12</sup>/scrib<sup>−/−</sup> (*a*), *Ras*<sup>V12</sup>/scrib<sup>−/−</sup> + *grnd*–RNAi (*b*), or *Ras*<sup>V12</sup>/scrib<sup>−/−</sup> + *dilp8*–RNAi (*c*; control RNAi to exclude a Gal4 titration effect) clones labelled by GFP and stained for Mmp1 (middle; red). *Ras*<sup>V12</sup>/scrib<sup>−/−</sup> and *Ras*<sup>V12</sup>/scrib<sup>−/−</sup> + *dilp8*–RNAi clones</figcaption></figure>

invade the ventral nerve cord (a, c; see white arrow). **d–g**, Eye–brain complexes dissected 7 days AED carrying *Ras*<sup>V12</sup>/Dlg–RNAi (**d**) or *Ras*<sup>V12</sup>/Dlg–RNAi + *grnd*–RNAi (**e, f**) clones (labelled by GFP), or *Ras*<sup>V12</sup>/Dlg–RNAi GFP-labelled clones in a *wgn*-null mutant background (**g**) stained for Mmp1 (middle; red) and DAPI (right; blue). *Ras*<sup>V12</sup>/Dlg RNAi clones invade the ventral nerve cord in *wgn*-null mutant animals (**h**; see white arrow).
avl RNAi-induced neoplastic growth.".5,1,0,0,600pt,600pt,0pt,0pt >

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**Extended Data Figure 9** | Egr is not required for the *rn>avl* RNAi-induced neoplastic growth. a, b, Wing discs of the indicated genotypes dissected 6 days AED and stained for Wg (green). c, d, Transverse sections of dissected wild-type discs stained for Grnd (red) and either E-cad (c; in green), or aPKC (d; in green). e, g, Transverse sections of *rn>avl* RNAi wing discs dissected 5 days AED and stained for Grnd (top, bottom; red) and either E-cad (e, middle, bottom; green) or Crb (g, middle, bottom; green). The *rn* expression domain is indicated (e, top). f, Wing discs expressing *crb-intra* in the *rn* domain (*rn>crb-intra*) dissected 6 days AED and stained for Disc-large (Dlg, green) to visualize

neoplastic structures and Grnd (red). h, i, Wing discs expressing *crb-intra* in the *rn* domain of wild-type (h) or *wgn*-knockout mutant (i) animals dissected 6 days AED and stained for Wg (green). j–l, Reducing *grnd* levels does not affect *crb-intra*-mediated inhibition of Hippo signalling, which causes an upregulation of the Yki transcriptional target Diap1. Control discs (j) or wing discs expressing Crb-intra alone (k) or together with *grnd* RNAi (l) in the *rn* domain were dissected from animals carrying a reporter gene for Hippo pathway activity (*Diap-GFP*, green) and stained for Wg (red).
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<figure><img src="image_39.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_41.png" /><figcaption>e</figcaption></figure>

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**Extended Data Figure 10** | Grnd directly interacts with the PDZ domain of Veli and neither depends on Veli nor Crb for its proper localization. a, b, Schematic representation of Grnd and Veli (also known as Lin-7) domains and the various truncations used to map the interface of the Grnd–Veli interaction. L27, Lin-2/Lin-7 domain; PDZ, PSD-95/Dlg/ZO-1 domain; TM, transmembrane domain. c, GST–Grnd<sup>125–241</sup> immobilized on GSH beads was incubated with full-length Veli (Veli<sup>FL</sup>) or the complementary Veli fragments L27 (Veli<sup>L27</sup>) and PDZ domain (Veli<sup>PDZ</sup>). The PDZ1 domain of the polarity protein Bazooka was included in the pull-down as a specificity control (Baz<sup>PDZ1</sup>). Coomassie-blue stained SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was used to visualize species retained on beads. Veli<sup>FL</sup> and Veli<sup>PDZ</sup> bind to Grnd (lanes 10 and 12), but not Veli<sup>L27</sup> nor Baz<sup>PDZ1</sup> (lanes 11 and 9), indicating that Grnd specifically recognizes the PDZ domain of Veli. d, To map the interface between Grnd and Veli<sup>PDZ</sup>, the same binding assay was

performed using a battery of Grnd deletion mutants adsorbed on GSH beads. Veli<sup>PDZ</sup> forms a complex with Grnd<sup>125–241</sup> and Grnd<sup>125–168</sup> (lanes 1 and 3), but not with the carboxy-terminal region of Grnd (lanes 2 and 4) or GST alone (lane 5, control). e, To address further the specificity of the binding of Grnd<sup>125–241</sup> to the PDZ domain of Veli, we repeated the pull-down assay with the PDZ domains of Baz and its human orthologue PAR3. Veli<sup>PDZ</sup> is retained on GST–Grnd<sup>125–241</sup> beads (lane 9), while PAR3<sup>PDZ2</sup>, PAR3<sup>PDZ3</sup> and Baz<sup>PDZ1</sup> are not (lanes 10, 11 and 12). f–g, Reducing Crb levels affects the localization of Veli, but not Grnd. f–g, xy (f) or transverse (g) sections of dissected wing discs bearing crb<sup>-/-</sup> mutant clones (labelled by absence of GFP) stained for Grnd (f, bottom left, g, bottom; white) or Veli (f, bottom right; red). h, Reducing Veli levels does not affect Crb<sup>20</sup> or Grnd localization. Transverse sections of dissected wing discs bearing clones expressing veli RNAi (h, bottom; labelled by the presence of GFP) stained for Grnd (top; red) and Crb (middle; blue).