Hippo Signaling Pathway

Current Biology 16, 2101–2110, November 7, 2006 ª2006 Elsevier Ltd All rights reserved

DOI 10.1016/j.cub.2006.09.045

Article Fat Cadherin Modulates Organ Size in Drosophila via the Salvador/Warts/Hippo Signaling Pathway F. Christian Bennett1 and Kieran F. Harvey1,* 1 Cell Growth and Proliferation Laboratory Peter MacCallum Cancer Centre 7 St Andrews Place East Melbourne, Victoria 3002 Australia

Summary Background: The atypical Fat cadherin has long been known to control cell proliferation and organ size in Drosophila, but the mechanism by which Fat controls these processes has remained elusive. A newly emerging signaling pathway that controls organ size during development is the Salvador/Warts/Hippo pathway. Results: Here we demonstrate that Fat limits organ size by modulating activity of the Salvador/Warts/Hippo pathway. ft interacts genetically with positive and negative regulators of this pathway, and tissue lacking fat closely phenocopies tissue deficient for genes that normally promote Salvador/Warts/Hippo pathway activity. Cells lacking fat grow and proliferate more quickly than their wild-type counterparts and exhibit delayed cell-cycle exit as a result of elevated expression of Cyclin E. fat mutant cells display partial insensitivity to normal developmental apoptosis cues and express increased levels of the anti-apoptotic DIAP1 protein. Collectively, these defects lead to increased organ size and organism lethality in fat mutant animals. Fat modulates Salvador/Warts/Hippo pathway activity by promoting abundance and localization of Expanded protein at the apical membrane of epithelial tissues. Conclusions: Fat restricts organ size during Drosophila development via the Salvador/Warts/Hippo pathway. These studies aid our understanding of developmental organ size control and have implications for human hyperproliferative disorders, such as cancers. Introduction To ensure organs of appropriate size are produced during development, rates of cell proliferation, cell growth and apoptosis must be tightly controlled. Many signaling pathways control organ size during development; these include the Insulin/PI3-kinase, Tor, and Ras/ MAP kinase pathways as well as the recently characterized Salvador/Warts/Hippo (Sav/Wts/Hpo) pathway [1]. Abnormal activation of these pathways can result in increased rates of cell growth and proliferation and abnormally large organs. Dysregulation of these signaling pathways also underlies many human hyperproliferative diseases, such as cancers. The Sav/Wts/Hpo pathway controls organ size by restricting cell growth and proliferation and by promoting

*Correspondence: [email protected]

apoptosis [2–11]. This pathway limits cell proliferation by controlling the abundance of Cyclin E, a protein critical for driving progression from G1 to S phase, and promotes apoptosis by downregulating the Drosophila inhibitor of apoptosis protein, DIAP1. Recently, the Sav/Wts/Hpo pathway has been reported to modulate organ size in part by regulating expression of the bantam microRNA [12]. The Sav/Wts/Hpo pathway includes the FERM domain proteins, Expanded (Ex) and Merlin (Mer), the serine-threonine kinases, Warts (Wts) and Hippo (Hpo), the scaffolding protein, Salvador (Sav), the Wts-activating protein, Mats (Mob as tumor suppressor), and the transcriptional enhancer Yesassociated protein (YAP), also known as Yorkie (Yki) [2–11]. This pathway modulates expression of cyclin E and DIAP1 at least in part by controlling abundance of mRNA of both of these genes, likely by modulating activity of Yki. All identified Sav/Wts/Hpo pathway components are conserved in mammals, and several, including sav (hWW45), mats (MATS1), merlin [Neurofibramatosis type 2 (NF2)], and yki (yap), have been implicated in the genesis of human cancers [2, 6, 10, 13–15]. It is unclear whether additional components of the Sav/Wts/Hpo signaling pathway exist, and the identities of the developmental cues that modulate pathway activity are also unknown. fat (ft) encodes an atypical cadherin [16], a transmembrane protein that localizes to apical membranes of epithelial cells in Drosophila imaginal discs [17, 18]. fat has long been known to control cell proliferation and organ size in Drosophila in an autonomous fashion [19]. Although proteins such as Dachs and Dachsous have been implicated in ft-dependent control of organ size [20, 21], the precise mechanism by which ft controls organ size has remained elusive. Ft also controls planar cell polarity (the organization of groups of cells into defined patterns) of several tissues, including the eyes, wings, and abdomen [17, 18, 22–24]. ft mutant tissue shares several phenotypic traits with tissue lacking genes that promote Sav/Wts/Hpo pathway activity; such traits include enlarged, hyperplastic imaginal discs and homozygous mutant clones that are round in shape when they are juxtaposed to wildtype tissue. Given these phenotypic similarities, we sought to determine whether ft restricts organ size via the Sav/Wts/Hpo pathway. We demonstrate that ft mutant cells grow more quickly than wild-type tissue and that ft is required for timely cell-cycle exit. We show that ft-deficient cells accumulate DIAP1 protein and mRNA and that ft is required for a portion of developmental apoptosis in the pupal retina. Significantly, modulating Sav/Wts/Hpo pathway activity by halving the dosage of yki suppresses overgrowth induced by loss of ft and rescues ft mutant animals from early pupal lethality. Additionally, we show that Ft promotes expression of Ex protein at the apical membrane of epithelial cells, suggesting a mechanism by which Ft modulates Sav/Wts/Hpo pathway activity.

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Figure 1. ft Interacts Genetically with Positive and Negative Regulators of the Sav/ Wts/Hpo Pathway Clones of wild-type FRT40A parental chromosome (A), ftfd (B), exe1 (C), or wtsLatsX1 (D) tissue generated in developing wing imaginal discs. Tissue derived from wild-type FRT40A parental chromosome or ex, wts, and ft mutant tissue fails to express green fluorescent protein (GFP; black clones indicated by arrows), whereas homozygous wild-type twinspots (white), and heterozygous tissue (gray) express GFP. (E–P) Genetic interactions between ft and Sav/Wts/Hpo pathway genes. Representative wing (E–H) and eye (I–L) imaginal discs from third-instar larvae of the following genotypes: ftfd/cyo (E and I); ftfd/ ft422 (F and J); ftfd/ft422;wtsLatsX1/+ (G and K); and ftfd ykiB5/ft422 (H and L). Pupa of genotype ftfd/ft422 (early pupal lethal) (M), and an adult ft transheterozygous fly rescued to viability by removal of one copy of yki (ftfd ykiB5/ft422) (N). Adult eyes from flies of genotype ftfd/ cyo (O), and ftfd ykiB5/ft422 (P). Scale bars represent 50 mM (A–L).

Results ft Interacts Genetically with the Sav/Wts/Hpo Pathway ft mutant tissue shares several phenotypic traits with tissue lacking genes that activate the Sav/Wts/Hpo pathway. ft mutant imaginal discs are significantly larger than wild-type discs ([19] and Figure 1F and 1J, compared with Figure 1E and 1I). In addition, clones of ft mutant tissue generated in Drosophila imaginal discs via the Flp/FRT technique outgrow their wild-type twinspot clones [25] and exhibit a rounded shape, properties shared with Sav/Wts/Hpo pathway genes, such as wts and ex (Figures 1B–1D). By contrast, clones of wildtype imaginal-disc tissue are generally jagged in appearance and are roughly equivalent in size to twinspot clones (Figure 1A). In addition, adult eyes harboring clones of ft mutant tissue are similar in appearance to eyes harboring clones of tissue derived from hypomorphic alleles of hpo [4]. ft mutant tissue (white) is overrepresented in comparison to wild-type tissue (red), and eyes are slightly rough in appearance (Figure S1 in the Supplemental Data available online). Based on these phenotypic similarities, we hypothesized that ft promotes activity of the Sav/Wts/Hpo pathway. To test this hypothesis, we examined whether the severity of phenotypes resulting from ft deficiency were modified in the context of heterozygosity for positive (wts), and negative (yki), components of the Sav/

Wts/Hpo pathway. The size and morphology of imaginal discs from identically aged third-instar larvae [6 days after egg deposition (AED) at 25 C] were analyzed from larvae of four different genotypes: (1) ftfd/cyo; (2) ftfd/ft422; (3) ftfd/ft422;wtslatsX1/+; and (4) ftfd ykiB5/ft422. ftfd (also known as ft8) [19], wtslatsX1 [26], and ykiB5 [11] have been reported as null alleles, whereas ft422 appears to be a strong hypomorphic allele [23]. Imaginal discs heterozygous for ftfd or ft422 were normal in size and shape and gave rise to normally sized and patterned adult organs (Figures 1E and 1I). Eye and wing imaginal discs mutant for ft (ftfd/ft422) exhibited substantial increases in size and displayed a ‘‘rippled’’ morphology when they were compared to wild-type imaginal discs, which were mostly flat (Figures 1F and 1J, compared with Figures 1E and 1I). Discs mutant for ft and heterozygous for wts (ftfd/ft422;wts latsX1/+) were even greater in size and more rippled than ftfd/ft422 discs (Figures 1G and 1K, compared with Figures 1F and 1J). In contrast, the size and morphological appearance of imaginal discs mutant for ft and heterozygous for yki (ftfd ykiB5/ ft422) were significantly suppressed and returned almost to those of wild-type discs (Figures 1H and 1L, compared with Figures 1F and 1J). Eye and wing imaginal discs heterozygous for wts or yki alone were normal in size and morphology and indistinguishable from wildtype discs (data not shown). The result of modifying components of the Sav/Wts/ Hpo pathway was also studied with respect to the

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requirement of ft for organism viability. When reared at 18 C, ftfd/ft422 animals were lethal at the early pupal stages of development (Figure 1M). Halving the dosage of yki in ftfd/ft422 animals allowed the majority of flies to progress to the late pupal stage of development and, strikingly, rescued some flies to adulthood (Figure 1N). ftfd ykiB5/ft422 flies displayed defects in the size and morphology of eyes, wings and some bristles (Figures 1O and 1P). The genetic interactions between ft and members of the Sav/Wts/Hpo pathway described above (Figures 1E–1P) are consistent with the hypothesis that ft controls organ size by modulating the activity of this pathway. ft Mutant Cells Accumulate Cyclin E and Proliferate Ectopically Eye imaginal-disc cells lacking genes that normally promote Sav/Wts/Hpo pathway activity fail to exit the cell cycle posterior to the morphogenetic furrow (MF) [2– 10], which prompted us to examine whether ft mutant cells exhibit similar cell-cycle defects. Wild-type thirdinstar larval eye imaginal discs display a patterned array of cell-cycle events: Cells anterior to the MF cycle asynchronously; cells within the MF arrest in G1; cells immediately posterior to the MF re-enter the cell cycle in a synchronous fashion; and posterior to the MF almost all cells terminally exit the cell cycle and differentiate (Figures 2A and 2E). ft-deficient cells (which fail to express green fluorescent protein [GFP] in this and other figures] re-entered the cell cycle in an ectopic fashion several ommatidial rows posterior to the MF, where wild-type counterpart cells were quiescent, as measured by BrdU incorporation (Figures 2B–2D). An increased number of cells in S phase were also observed in clones of ft mutant tissue in anterior portions of eye imaginal discs, suggesting that cells lacking ft proliferate more quickly than wild-type cells in populations of cells that are proliferating asynchronously (Figures 2F–2H). Sav/Wts/Hpo-pathway mutant cells most likely reenter the cell cycle ectopically as a result of accumulation of Cyclin E, a critical regulator of the transition from G1 to S phase [2–10]. Cyclin E was also found to be elevated in clones of eye imaginal-disc tissue deficient for ft, as determined with an antibody to Cyclin E protein (Figures 2I–2M). Accumulation of Cyclin E was obvious in ft clones posterior to the MF but also occurred subtly in clones anterior to the MF. This is consistent with our finding that increased numbers of cells are evident in S phase in both anterior and posterior ft mutant eye tissue (Figures 2A–2H). These findings suggest that, akin to members of the Sav/Wts/Hpo pathway, ft normally limits cell proliferation by restricting expression of Cyclin E. ft Mutant Tissue Exhibits Commensurate Increases in Rates of Cellular Growth and Proliferation Clones of ft mutant tissue possess more cells than wildtype twinspot clones, demonstrating that ft mutant cells proliferate more rapidly than do wild-type cells [25]. To determine whether ft mutant cells also display elevated growth rates, we measured the size of cells from ft mosaic imaginal discs by using flow cytometry. ft mutant clones were induced by heat-shocking larvae 48 hr AED. Imaginal discs were then dissected

120 hr AED, dissociated with trypsin, and analyzed by flow cytometry. The size of ft mutant eye imaginal-disc cells, as determined by flow-cytometric analysis of forward scatter, was found to be marginally larger (by 5%) than that of wild-type cells (Figure 2O). Wing imaginal-disc cells were indistinguishable in size from wildtype cells (Figure S2B), consistent with a previous report by Garoia et al. (2005) [27]. To determine whether ft mutant cells displayed defects in their cell-cycle profile, we used flow cytometry to measure the DNA content of populations of cells from ft mosaic imaginal discs. We found that cells lacking ft spend roughly equal proportions of time in the G1, S, and G2 phases of the cell cycle as wild-type cells (Figure 2N; also Figure S2A). In order for cell size to remain consistent in the context of elevated rates of proliferation, cellular growth rates must also increase. Therefore, the observation that ft cells proliferate more quickly than wild-type cells [25] but that the size of ft cells is not significantly altered (Figure 2O; also Figure S2B) suggests that ft cells also grow more quickly than wild-type cells. These findings are consistent with those of cells mutant for positive regulators of the Sav/Wts/Hpo pathway, such as sav and hpo [2, 4, 5, 7, 8]. ft Restricts DIAP1 Expression but Is Largely Dispensable for Developmental Apoptosis The Sav/Wts/Hpo pathway promotes apoptosis of supernumerary cells in the developing Drosophila eye, as evidenced by the findings that eye tissue lacking positive regulators of the Sav/Wts/Hpo pathway contains many extra interommatidial cells and displays reduced apoptosis [2–10]. To analyze whether ft mutant eye tissue also possesses increased numbers of cells after normal developmental apoptosis has ceased, we stained ft mosaic eye discs 46 hr after puparium formation (APF) with an antibody specific for Discs-large, which highlights cell borders. In wild-type pupal eye discs a single layer of interommatidial cells was observed between cone-cell and photoreceptor clusters. In ft tissue, however, moderate increases in the number of interommatidial cells were observed (Figure 3A). The subtle increase in interommatidial cell number in ft tissue is reminiscent of that observed in ex mutant tissue [6], as opposed to the dramatic increase in interommatidial cell number observed in sav, wts, or hpo tissue and in ex,mer double-mutant tissue [2, 4–8]. To determine whether ft is required for apoptosis of retinal cells, we performed TUNEL analysis on ft mosaic eye discs at the mid-pupal phase of eye development (28 hr APF), when excess interommatidial cells are normally removed by apoptosis. Consistent with our finding that ft mutant eye tissue exhibits only a small increase in interommatidial cells, we observed significant apoptosis in both wild-type and ft mutant tissue (Figures 3B and 3C). If anything, an increase in apoptosis was observed in ft mutant clones; such an increase might be due to apoptosis of supernumerary cells generated by ectopic cell proliferation in ft tissue (Figure 2). The subtle increase in interommatidial cells in ft mutant tissue suggests that, unlike sav, wts, and hpo, ft is only required for apoptosis of a subset of retinal cells. The diminished apoptosis observed in tissue mutant for genes such as sav, wts, and hpo is likely a result of

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Figure 2. Cells Lacking ft Exhibit Delayed Cell-Cycle Exit and Accumulate Cyclin E but Are Normal in Size (A–M) Third-instar larval eye imaginal discs. Anterior is to the right. Wild-type tissue expresses GFP (green), whereas ft mutant tissue does not. Arrowheads denote the MF. (A–H) Cells entering S phase, as visualized by BrdU-incorporation in red, in wild-type eye imaginal discs (A and E) or ftfd mosaic discs (B–D and F–H). Arrows indicate ft mutant cells re-entering the cell cycle posterior to the MF in (B) and (D) and anterior to the MF in (F) and (H). (I–M) Cyclin E protein expression (red) in ftfd mosaic eye imaginal discs; images merged with the GFP clonal marker are shown in (K) and (M). Arrows indicate examples of ft clones that have accumulated Cyclin E. Scale bars represent 50 mM (A–K) and 10 mM (L and M). (N) The cell-cycle profile of wild-type (red) and ftfd (blue) third-instar larval eye imaginal-disc cells as determined by flow-cytometric analysis of DNA content. Inset numbers indicate the percentage of cells in different phases of the cell cycle (G1, S, or G2). (O) The size of wild-type (red) and ftfd (blue) third-instar larval eye imaginal-disc cells as measured by forward scatter. The inset number indicates the relative size of ft cells compared to wild-type cells.

increased expression of the Drosophila inhibitor of apoptosis protein 1 (DIAP1) [2–5, 8]. We therefore stained ft chimaeric larval eye discs with an antibody directed against DIAP1 and found that ft mutant eye tissue displayed significant increases in expression of DIAP1 (Figures 3D–3H). As described previously, the Sav/ Wts/Hpo pathway controls abundance of DIAP1 transcriptionally and posttranscriptionally [2–5, 8]. Therefore, we used a DIAP1 enhancer trap to measure the abundance of DIAP1 mRNA in larval eye discs chimaeric for ft. As predicted, we observed significantly increased

activity of the DIAP1 enhancer trap in ft mutant clones, suggesting that ft, like other members of the Sav/Wts/ Hpo pathway, modulates DIAP1 expression at least in part by restricting abundance of DIAP1 mRNA (Figures 3I–3M). ft and ex Mutant Tissue Closely Phenocopy One Another ft and ex mutant tissue share several phenotypic characteristics, including a proliferative advantage over wildtype tissue, a subtle increase in interommatidial cells,

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and planar cell-polarity defects [6, 17–19, 21–25, 28, 29], making it likely that these genes function in the same signaling pathway. To further explore this possibility, we analyzed several phenotypic aspects of ftfd,exe1 double-mutant tissue. Like ft or ex mutant tissue alone, ft,ex double-mutant tissue displayed only a subtle increase in interommatidial cell number, implying that both ft and ex are dispensable for the majority of pupal retinal apoptosis (Figure 3N). This hypothesis was confirmed by the finding that significant apoptosis, as measured by TUNEL, was observed in both ex (Figures S3G–S3I) and ft,ex mutant tissue (Figures 3O and 3P), 28 hr APF. Like ft tissue, ft,ex double-mutant tissue accumulated Cyclin E protein (Figures S3A–S3C) and underwent apoptosis despite expressing high levels of DIAP1 protein (Figures S3D–S3F). Collectively, these data suggest that ex and ft modulate organ size by functioning in the same signaling pathway.

Ft Functions at the Apical Point of the Sav/Wts/Hpo Pathway Misexpression of different Sav/Wts/Hpo pathway components induces ectopic pathway activation, resulting in reduced cell proliferation and increased apoptosis, culminating in a reduction of organ size [2–6, 8]. Creating tissue lacking ft, in the context of misexpression of other Sav/Wts/Hpo pathway components, allowed us to perform genetic epistasis experiments to determine at which point Ft functions in this pathway. We performed epistasis experiments in the developing Drosophila eye by misexpressing genes under control of the GMR promoter and creating eyes mosaic for ft by using eyFlpdirected mitotic recombination. GMR-driven expression of ex at 25 C elicited a severe reduction in eye size, whereas expression of sav and wts together at 28 C induced a more mild reduction in eye size and roughness (Figures 4A–4C). To determine whether ft is required for ex alone or for sav and wts together to induce a small rough eye, we created clones of tissue lacking ft in eyes misexpressing either ex at 25 C or sav and wts at 28 C (Figures 4D–4F). In each case the small-eye phenotype induced by misexpression of either ex or both sav and wts was epistatic to the eye-size increase resulting from loss of ft. These results suggest that ft is not required for either ex or both sav and wts to induce a reduction in eye size when these genes are misexpressed under control of the GMR promoter. We performed TUNEL analysis to examine the requirement of ft for apoptosis induced by misexpression of hpo (Figures 4G–4I) or ex (Figures 4J–4L) by the GMR promoter in third-instar larval eye imaginal discs. GMRhpo (Figure 4G) and GMR-ex (Figure 4J) both induced apoptosis in the GMR expression domain, posterior to the MF. In eye discs mosaic for ft, GMR-hpo- (Figures 4H and 4I) and GMR-ex- induced apoptosis (Figures 4K and 4L) proceeded unabated in tissue lacking ft. These data are consistent with our findings that ft is dispensable for the ability of ex to induce a reduction in adult eye size. Genetic epistasis experiments performed here, in conjunction with phenotypic and genetic interaction data described above, suggest that ft modulates Sav/Wts/Hpo-pathway activity by functioning upstream of sav, wts, hpo, and ex.

Ft Is Required for Ex Protein Abundance and Localization Ex mRNA and protein are dramatically elevated in tissue lacking positive regulators of the Sav/Wts/Hpo pathway [6], which is likely to constitute a regulatory feedback mechanism used to maintain appropriate signaling output of this pathway. Given our above results suggesting that ft is a positive-regulatory component of the Sav/ Wts/Hpo pathway, we predicted that Ex would also accumulate in ft mutant tissue. Using quantitative PCR analysis, we found ex mRNA to be elevated 3.3-fold in wing imaginal discs deficient for ft (ftfd/ft422) in comparison to ex mRNA in wild-type control tissue (Figure 5A). This result is consistent with activation of ex transcription, or stabilization of ex mRNA, when the Sav/Wts/Hpo pathway is inhibited. We also analyzed the expression of Ex protein in clones of imaginal-disc tissue lacking either ft or wts (Figures 5D–5I). Ex protein is predominantly localized at the apical membrane of Drosophila imaginal-disc epithelial cells, the same subcellular localization shown by Ft protein (Figure S4) [6, 17, 18, 29, 30]. Consistent with previous findings, we found Ex protein to be elevated in tissue deficient for genes, including wts, that promote Sav/Wts/Hpo pathway activity (Figures 5D–5F). Contrary to our prediction that Ex protein would be elevated in ft tissue, we observed that, strikingly, Ex protein was undetectable at the apical membranes of clones of wing imaginal-disc ft tissue, whereas Ex expression was normal in neighboring wild-type tissue (Figures 5G–5I). We also observed decreases in Ex protein expression in ft clones in other imaginal discs, including eye-antennal, haltere, and leg discs (Figure S5, and data not shown). Interestingly, clones of ft eye imaginal-disc tissue exhibited only partial reduction of Ex expression at the apical membrane (Figure S5). To determine whether Ex protein is absent from ft imaginal-disc tissue altogether, we used immunoblot analysis to analyze total Ex protein expression in wildtype or ft mutant (ftfd/ft422) eye (Figure 5B) or wing (Figure 5C) discs. Consistent with immunofluorescence results, expression of Ex protein was significantly elevated in wtsLatsX1 mutant eye discs (Figure 5B). Total Ex protein expression appeared unchanged in ft eye imaginal-disc tissue (Figure 5B) and was reduced by approximately 60% in ft wing imaginal-disc tissue (Figure 5C). Together with our finding that ex mRNA is elevated in ft mutant tissue, immunoblot and immunofluorescence studies suggest that Ft is normally required for the translation, correct localization, and/or stability of Ex protein (Figure 5). Discussion Ft Modulates Organ Size via the Sav/Wts/Hpo Pathway The Sav/Wts/Hpo signaling pathway controls organ size in Drosophila by restricting cell growth and proliferation and by promoting apoptosis. Tissue in which the Sav/ Wts/Hpo pathway is repressed exhibits unique phenotypic signatures, distinct from those of tissue hyperactivated for growth-stimulatory signaling pathways such as the Tor, Insulin, and Ras/Map kinase pathways. Tissue deficient for ft shares many of these phenotypic

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Figure 3. ft Restricts DIAP1 Expression and Is Partially Required for Apoptosis in the Developing Drosophila Eye ft mutant tissue is marked by absence of GFP, whereas wild-type tissue is GFP positive. (A) Cell architecture of a ftfd mosaic pupal eye disc 46 hr APF, as visualized with an antibody directed against Discs-large protein (red). Extra interommatidial cells are observed in ft mutant portions of the eye disc (examples indicated by arrows). (B and C) Apoptosis in ftfd mosaic pupal eye discs 28 hr APF, as detected by TUNEL (red), is observable in both wild-type tissue (green in C) and ft mutant tissue. (D–M) Third-instar larval eye imaginal discs. Arrowheads denote the MF. (D–H) DIAP1 protein (red) is elevated in ftfd eye imaginal-disc clones (examples are indicated by arrows). (I–M) Expression of a DIAP1-lacZ enhancer trap, which assays DIAP1 mRNA expression (magenta), is elevated in ftfd eye imaginal-disc clones. Arrows indicate examples of ft clones with

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components of the Sav/Wts/Hpo pathway are intracellular proteins. Genetic epistasis data performed here suggest that Ft functions near the apical point of the Sav/ Wts/Hpo pathway, upstream of Ex, Hpo, Sav, and Wts (Figure 4). Ex and Mer are cytoplasmic proteins that localize predominantly to the apical epithelial membrane, raising the possibility that Ft, Ex, and Mer form a signaling complex that relays an as-yet-unidentified extracellular signal from Fat to downstream components of the Sav/Wts/Hpo pathway to ultimately repress Yki-dependent gene transcription (Figure 6). Ft might also function together with Ex and Mer to control endocytosis of membrane proteins important for proliferation control and cell adhesion [31].

Figure 4. ft Functions at the Apical Point of the Sav/Wts/Hpo Pathway (A–F) Scanning-electron-microscopy images of adult eyes. (A) Wildtype, (B) GMR-sav/wts, (C) GMR-ex, (D) ftfd mosaic, (E) ftfd mosaic; GMR-sav/wts, (F) ftfd mosaic; GMR-ex. (G–L) TUNEL analysis of eye imaginal discs. The MF is indicated by arrowheads. (G) GMRhpo, (H and I) ftfd mosaic; GMR-hpo, (J) GMR-ex, (K and L) ftfd mosaic; GMR-ex. Dying cells (TUNEL positive) are red in (G)–(L). ft mutant tissue fails to express GFP (green in [I] and [L]). Anterior is to the right in all images. Scale bars represent 50 mM (A–L).

signatures, e.g.: (1) ft mutant and chimaeric organs are oversized; (2) clones of imaginal-disc ft tissue are round in morphology; (3) ft mutant cells proliferate more quickly than wild-type cells; (4) ft mutant cells display increased growth rates; (5) ft eye tissue accumulates Cyclin E; (6) ft tissue expresses increased levels of DIAP1; (7) ft eye tissue possesses supernumerary interommatidial cells; and (8) ft tissue expresses elevated amounts of ex mRNA. In addition, halving the dosage of yki suppresses imaginal-disc overgrowth and lethality in flies lacking ft (Figure 1), providing strong evidence that ft restricts organ size by modulating activity of the Sav/Wts/Hpo pathway. Ft Functions at the Apical Point of the Sav/Wts/Hpo Pathway Ft is a transmembrane protein, localized at the apical membrane of Drosophila epithelia, whereas most known

Ex Expression Levels Are Critical for Modulation of Sav/Wts/Hpo Pathway Activity Several points of evidence indicate that expression levels of Ex are critical for regulation of the Sav/Wts/ Hpo pathway: (1) Loss of ex leads to inhibition of pathway activity [6]; (2) overexpression of ex can stimulate pathway activity and lead to ectopic apoptosis and reduced organ size [6, 28]; (3) ex mRNA expression is induced in an apparent regulatory feedback loop when the Sav/Wts/Hpo pathway is repressed ([6] and Figure 5A); and (4) total Ex protein is significantly reduced in ft-deficient wing imaginal-disc cells and is absent from the apical membrane of these cells (Figure 5). The observation that Ex protein is reduced in ft-deficient cells, despite the fact that ex mRNA is increased, suggests that Ft is required for translation or stability of Ex protein and/or for recruitment or retention of Ex at the apical membrane of epithelial cells. The precise mechanism by which Ft controls expression and/or localization of Ex protein is currently unclear but could be a critical event in modulation of organ size by the Sav/Wts/Hpo pathway. Divergent Roles for Components of the Sav/Wts/Hpo Pathway Certain phenotypes of ft and ex mutants resemble each other more closely than they resemble other components of the Sav/Wts/Hpo pathway. First, both ft and ex exhibit defects in planar cell polarity [17, 18, 22–24, 28], whereas in sav, wts, hpo, and mer tissue such defects have not been reported. A possible explanation for this phenotypic difference is that ft and ex function together to restrict organ size via the Sav/Wts/Hpo pathway but that other processes, such as planar cell polarity, employ a divergent signaling pathway (Figure 6). Secondly, ft and ex appear to be dispensable for most developmental apoptosis in the pupal retina, whereas sav, wts, and hpo are required (Figure 3; Figure S3; [2– 5, 8]). This is especially surprising given that DIAP1 protein and mRNA accumulate in tissue deficient for ft, ex and mer, sav, wts, or hpo (Figure 3; [2–6, 8]). One possibility is that Sav, Wts, and Hpo mediate apoptosis by modulating expression or activity of a protein(s) other than DIAP1 and that this protein(s) is not subject to regulation by Ex and Ft. Alternatively, a secondary

high DIAP1 mRNA. (N) Extra interommatidial cells (indicated by arrows) are observed in a ftfd,exe1 mosaic pupal eye disc 46 hr APF. (O and P) Apoptosis in ftfd,exe1 mosaic pupal eye discs 28 hr APF, as detected by TUNEL (red), is observable in both wild-type tissue (green in P) and ftfd,exe1 mutant tissue. Scale bars represent 10 mM (A, G, H, and L–N) and 50 mM (B–F, I–K, O, and P).

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Figure 5. Ex mRNA, but not Protein, Is Elevated in ft Tissue (A) ex mRNA expression in wild-type or ft mutant (ftfd/ft422) wing imaginal discs, as determined by quantitative PCR. ex expression was normalized to a wild-type expression level of 1. Results depict the mean expression levels 6 SEM of three experiments, where p < 0.005. (B and C) Total Ex protein expression in wild-type, ft mutant (ftfd/ft422), or wtsLatsX1 mutant eye imaginal discs (B) or wing imaginal discs from wild-type or ft mutant (ftfd/ ft422) animals (C). Molecular mass markers (in kD) and relative Ex protein expression levels are indicated. Actin protein expression was determined to ensure even protein loading and allow relative quantification of Ex protein (bottom immunoblots in [B] and [C]). (D–I) Expression of Ex protein (red) in wtsLatsX1 (D– F) and ftfd (G–I) mosaic wing imaginal discs. Mutant tissue lacks GFP expression (green) in (E), (F), (H), and (I). Expanded protein (red) is evident in (D) and (G) and in the merged images, (F) and (I). Please note that confocal images of Ex protein in wts chimaeric discs were captured with a lower laser power than in ft mosaic discs to avoid overexposure of significantly increased levels of Ex protein observed in wts clones. The scale bar represents 50 mM (D–I).

phenotype of ft and ex mutant cells (e.g., planar cellpolarity defects), might render them sensitive to developmental apoptosis cues, despite the fact that they accumulate DIAP1. Interestingly, despite accumulating DIAP1, eye tissue lacking mats also undergoes developmental apoptosis, probably because of differentiation defects of mats tissue, which frequently cause cells to apoptose [10]. Further experimentation is required for investigating the roles of Ft, Ex, MATS, and DIAP1 in apoptosis regulated by the Sav/Wts/Hpo pathway. Concluding Remarks Recessive clonal screens in Drosophila have very effectively isolated organ-size-controlling genes, including sav, wts, and hpo. All known components of the Sav/ Wts/Hpo pathway are conserved in mammals, and several of their orthologs have been implicated in mammalian tumorigenesis [2, 6, 10, 14, 15, 32]. Currently, however, the only human Sav/Wts/Hpo pathway ortholog that has been defined as a bona fide tumor suppressor gene is the mer ortholog, NF2 (reviewed in [13]). In future studies, it will be important to determine whether components of the Sav/Wts/Hpo pathway other than NF2 are mutated in neurofibromatosis and other human hyperplastic disorders. Experimental Procedures Drosophila Stocks Loss-of-function tissue harboring mutations in different genes was generated by creation of transheterozygous animals, or in a clonal fashion by the use of the Flp/FRT technique, and the following mutant alleles: ftfd [19], ft422 [23], exe1 [28], ftfd exe1 (generated by meiotic recombination), wtslatsX1 [26] and hpoMGH3 [4]. Misexpression

experiments were conducted with the following stocks: UAS-ex [33], UAS-hpo [8], GMR-sav, GMR-wts [2, 4], and GMR-Gal4. Other stocks included thjc58 [34], ykiB5 [11], y w eyFlp;FRT40A P[W+ ubiGFP], y w hsFlp;FRT40A P[W+ ubi-GFP], y w eyFlp;FRT42D P[W+ ubi-GFP], y w eyFlp;FRT82B l(3)cl-R3 [1]/TM6B and y w hsFlp;FRT82B P[W+ ubi-GFP]. Information on genotypes used in each experiment can be found in the Supplemental Experimental Procedures. Immunohistochemistry Antibodies used were as follows: anti-Cyclin E (from H. Richardson) [35], anti-DIAP1 (from B. Hay) [36], anti-Expanded (from A. Laughon) [29], anti-Fat (from H. McNeill), anti-Discs large (DSHB), anti-BrdU (Becton Dickinson), anti-b-galactosidase (Sigma), and anti-mouse, anti-rat, and anti-rabbit secondary antibodies (Molecular probes). Tissues were fixed in 4% paraformaldehyde and incubated with antibodies overnight at 4 C in phosphate-buffered saline and 0.3% Triton X-100. Flow Cytometry Larvae were heat shocked 48 hr after egg deposition (AED), and imaginal discs were dissected 120 hr AED. Discs were dissociated in PBS Trypsin-EDTA and stained with 0.5 mg/ml Hoechst 33342. Sample data were acquired on a Becton Dickinson LSR II flow cytometer via a UV laser with a 450/65 nM BP collection filter and were analyzed with FCS-Express (DeNovo Software). Apoptosis and Cell-Proliferation Assays TUNEL assays were performed on pupal eye discs 28 hr APF with a kit from Roche. Discs were permeabilized overnight at 4 C in 0.3% Triton X-100 in PBS, then 30 min at 65 C in 0.1% sodium citrate, 0.1% Triton X-100 in PBS. Cell proliferation was analyzed by BrdU incorporation according to standard protocols, including a 90 min BrdU pulse. Quantitative PCR RNA was extracted from third-instar larval wing imaginal discs by the use of Trizol (Invitrogen). Equivalent amounts of total RNA

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Stock Centre for fly stocks and antibodies. We thank D. Chiarelli and S. Chakravarti for assistance with flow cytometry. We thank H. McNeill for discussion of results prior to publication. K.H is a Leukemia and Lymphoma Society Special Fellow and the recipient of a Career Development Award from the International Human Frontier Science Program. This work was supported by a Project Grant from the National Health and Medical Research Council of Australia. Received: July 11, 2006 Revised: September 4, 2006 Accepted: September 22, 2006 Published online: October 12, 2006 References

Figure 6. The Sav/Wts/Hpo Signaling Pathway We propose a model where the transmembrane protein Ft, together with Ex and Mer, transduce a signal to downstream components of the Sav/Wts/Hpo pathway, Sav, Wts, Hpo and Mats, which in turn inhibit the transcriptional coactivator protein, Yki. In combination with an as-yet-unidentified transcription factor(s) (X), Yki drives expression of genes regulated by the Sav/Wts/Hpo pathway; such genes include cyclin E, DIAP1, expanded, bantam, and other unidentified genes that promote cell growth. Ft and Ex also control planar cell polarity independently of Mer, Sav, Wts, Hpo, and Mats.

from imaginal discs of different genotypes were used to produce first-strand cDNA with a Superscript II kit (Invitrogen). Quantitative PCR reactions were analyzed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with SYBR green reagents and primers designed to detect ex and GAPDH messages with similar amplification curves. PCR reactions were visualized on an agarose gel to ensure that each reaction amplified a single product. Relative levels of ex mRNA were compared by the comparative CT method. Statistical significance was determined with an unpaired Student’s t test, which showed p < 0.005. Immunoblotting Third-instar larval wing imaginal discs were lysed in NETN and boiled in protein sample buffer. Samples were subjected to SDSPAGE, transferred to Immobilon-P membrane, and probed with anti-Ex [29] or anti-Actin (clone JLA20, DSHB). Secondary antibodies used were anti-rabbit-HRP and anti-mouse-HRP (Jackson Immunochemicals). Ex protein expression in each sample was quantified relative to Actin with Image J. Supplemental Data Supplemental data include five figures and fly genotypes and are available online at http://www.current-biology.com/cgi/content/ full/16/21/2101/DC1/. Acknowledgments We thank A. Brumby, I. Hariharan, N. Harvey, and H. Richardson for comments and G. Halder, I. Hariharan, B. Hay, A. Laughon, H. McNeill, M. Mlodzik, D. Pan, H. Richardson, P. Sinha, N. Tapon, the Developmental Studies Hybridoma Bank, and the Bloomington

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