Zyxin Links Fat Signaling to the Hippo Pathway

Using genetic and molecular analyses, the authors identify Zyx as a positive regulator of Hippo signaling and characterize its role within the pathway.


Introduction
The Hippo pathway has emerged as an important regulator of growth during metazoan development, and its dysregulation is implicated in diverse cancers [1][2][3]. Hippo signaling is effected by transcriptional co-activator proteins, Yorkie (Yki) in Drosophila and YAP and TAZ in mammals [4]. Three interconnected, upstream branches of Hippo signaling have been characterized in Drosophila: Fat-dependent, Expanded-dependent, and Merlin-dependent [1][2][3]. These upstream branches converge on the kinase Warts (Wts), which can phosphorylate Yki. Phosphorylated Yki is retained in the cytoplasm, whereas unphosphorylated Yki can enter the nucleus and, in conjunction with DNA-binding partners, promote the transcription of downstream genes. Upstream branches of Hippo signaling regulate both the activity of Wts and its abundance. Our understanding of many steps in Hippo signaling remains fragmentary, in part due to incomplete identification of pathway components. Here, we describe the identification of Zyx102 (Zyx, FBgn0011642) as a novel component of Hippo signaling and characterize its role in the pathway.
Fat is large cadherin that acts as a transmembrane receptor for one branch of Hippo signaling [1][2][3]5]. Fat-Hippo signaling influences the levels of Wts protein [6]. The molecular mechanism by which this is achieved is not understood, but dachs is genetically required for the influence of Fat on Wts levels, downstream gene expression, and organ growth [6][7][8]. Fat regulates the localization of Dachs to the sub-apical membrane: when fat is mutant, Dachs accumulates on the membrane around the entire circumference of the cell, and when Fat is over-expressed, Dachs is mostly cytoplasmic [7]. In imaginal discs and optic neuroepithelia, Dachs membrane localization is polarized within the plane of the tissue; this polarization reflects the graded expression of the Fat ligand Dachsous and the Fat pathway modulator Four-jointed [7,9,10]. The correlation of Dachs localization with Fat activity implicates Dachs regulation as a key step in Fat signaling, but how Dachs localization influences downstream events is unknown.
Zyx is a Drosophila homologue of the vertebrate Zyxin, Lipoma preferred partner (LPP), and Thyroid-receptor interacting protein 6 (TRIP6) proteins [11,12]. These proteins have three conserved LIM domains at their C-terminus, and they have been implicated in both cytoskeletal and transcriptional regulation [13][14][15]. Genetargeted mutations in murine Zyxin or Lpp have no significant effect on mouse development, presumably due to redundancy among family members [16,17]. Translocations involving LPP identified it as an oncogene involved in lipomas and other cancers [13]. In cultured cell assays, Zyxin and its paralogues can affect cell motility and actin polymerization and can localize to focal adhesions and adherens junctions [13,15,18]. Notably, Zyxin has been implicated as playing a key role in mechanotransduction, as its localization to focal adhesions can be influenced by the application of mechanical tension to cells in culture [18].
We report here that Zyx is an essential component of the Fat-Hippo signaling pathway, required for normal Yki activity and growth in Drosophila. Using genetic epistasis tests, we position the requirement for Zyx in between fat and wts. Binding studies show that Zyx protein binds to Dachs and binds to Wts in a Dachsregulated manner. Our observations suggest a model in which the regulated localization of Dachs to the membrane regulates Zyx-Wts binding, which then promotes Wts degradation. Dachs is a myosin protein, and its myosin motor domain contributes to interactions with Zyx and Wts, which raises the possibility that additional myosins might regulate Zyx-Wts interactions in other contexts.

Results
In a screen for additional components of the Fat and Hippo pathways, we examined a collection of transgenic flies expressing UAS-hairpin constructs, which mediate RNAi. We focused on the X and 4 th chromosomes, which are under-represented in traditional genetic screens, and looked for phenotypes when these RNAi lines were expressed in the notum under pnr-Gal4 control, and in the wing under vg-Gal4 control. To enhance the strength of RNAi, the screening was done in flies expressing Dicer2 from a UAS-dcr2 transgene [19]. One hundred and forty-eight lines exhibiting either altered tissue growth or lethality were then rescreened for possible effects on Fat-Hippo signaling by assaying the expression of downstream targets of the pathway, Wingless (Wg) and thread (th, more commonly referred to as Diap1) [20,21], in wing discs in which RNAi lines were expressed in anterior cells under ci-Gal4 control (Table S1). The most promising candidates were then taken through four additional tests, involving confirmation of effects on additional downstream target genes, characterization of phenotypes when expressed under additional Gal4 drivers, confirmation of phenotypes with additional, independent UAS-RNAi lines, and characterization of genetic interactions with known pathway components. Based on these experiments, a single gene, Zyx102 (Zyx) [11,12], which is located at 102F7 near the tip of the fourth chromosome, was identified as a novel component of the Fat-Hippo signaling pathway.

Zyx Is Required for Hippo Signaling
Reduction of Zyx in the developing wing disc, under nub-Gal4 control ( Figure S1A), results in adult flies with small wings ( Figure 1A-C,S). Similar phenotypes were observed using two different RNAi lines, although NIG-32018R3 (RNAi-Zyx 32018 ), the line identified in our original screen, has slightly stronger phenotypes. Hippo signaling also regulates leg growth, and depletion of Zyx in developing legs results in shorter legs with fewer tarsal segments ( Figure S1I,J). In addition to observing similar phenotypes with two independent RNAi lines, confirmation that the phenotypes observed result specifically from reduction of Zyx was provided by the observation that overexpression of Zyx from a UAS transgene rescued the RNAi phenotypes ( Figure 1D,S). We also confirmed by Western blotting that that Zyx RNAi reduced Zyx protein levels ( Figure S1K).
Many different genes and pathways affect organ growth. To investigate the potential connection between Zyx and the Hippo pathway, we examined the expression of downstream target genes in wing discs in which Zyx was depleted by RNAi. As downstream targets we employed reporters of expanded (ex) expression (ex-lacZ) and th expression (th-lacZ, Diap1). When Zyx was depleted from posterior cells using en-Gal4, ex-lacZ, th-lacZ, and Diap1were all reduced (Figures 2A,B, S2A). Hippo signaling regulates transcription by controlling the sub-cellular localization of Yki: activation of Hippo signaling promotes cytoplasmic localization of Yki, whereas inactivation of Hippo signaling allows nuclear localization of Yki, which corresponds to Yki activation [22,23]. Zyx RNAi reduced nuclear Yki. This effect was subtle at late third instar, when levels of Yki in the nucleus are already low, but was evident in younger wing discs, which have higher levels of nuclear Yki ( Figure 2C,D). The decreased expression of Hippo pathway target genes, together with the reduction in nuclear Yki, identifies Zyx as a regulator or component of the Hippo pathway. The Hippo pathway is generally thought of as a negative regulator of growth and gene expression, because most genes in the pathway act as tumor suppressors and negatively regulate the activity of Yki. Zyx, by contrast, is positively required for Yki activity and organ growth.

Zyx Acts Genetically Within the Fat-Hippo Pathway
To position the genetic requirement for Zyx within the Hippo pathway, we performed a series of epistasis tests. RNAi lines targeted against several different tumor suppressor genes within the pathway (fat, ds, ex, wts, hpo, and mats), each of which phenocopy their respective mutants, were examined in combination with Zyx RNAi lines. The immediate upstream regulator of Yki is wts. Expression of a wts RNAi line under nub-Gal4 or en-Gal4 control is lethal at late third instar, but imaginal discs can be recovered and analyzed before lethality. Consistent with the expected de-repression of Yki, expression of wts RNAi resulted in upregulation of ex and Diap1 expression ( Figure 3A). This upregulation of ex and Diap1 was not suppressed by Zyx RNAi ( Figure 3B); hence, wts is epistatic to Zyx. Wts activity is directly regulated by a kinase, Hippo (Hpo), and a co-factor, Mats, and hpo and mats were also epistatic to Zyx ( Figure S3A-D). These observations imply that Zyx acts upstream of Wts.
Upstream branches of Hippo signaling have been characterized in Drosophila as Fat-dependent, Ex-dependent, or Mer-dependent. In the developing wing, fat and ex make substantial contributions to Yki regulation, whereas Mer has a lesser role [6,[24][25][26][27]. Thus, we investigated the relationship between the requirement for Zyx

Author Summary
Processes that control cell numbers are essential during normal development, when they are required to generate organs of the correct size, and during cancinogenesis, when they influence tumor growth. The Hippo pathway is an intercellular signaling pathway that relays information about cell-cell contact and cell polarity to a signal transduction pathway that regulates the transcription of genes controlling cell numbers. The role of Hippo signaling in controlling growth is conserved from fruit flies to humans, but many aspects of the Hippo signal transduction pathway remain poorly understood. In this article, we identify Zyx as a previously unknown component of the Hippo pathway in Drosophila, and characterize its role within the pathway. We show that Zyx plays an essential role in a branch of Hippo signaling that involves the transmembrane receptor protein Fat and its target Dachs, which is a myosin family protein. Our results suggest a model in which Fat regulates the localization of Dachs, Dachs subsequently binds Zyx, stimulating its binding with the kinase Warts/Lats, and thereby regulates downstream signaling events. Zyx is conserved in vertebrates and we suggest that vertebrate Zyx proteins might also be involved in the regulation of Hippo signaling and, thereby, organ growth. and those for fat and ex. Expression of fat or ex RNAi throughout the wing, under nub-Gal4 control, results in overgrown wings ( Figure 1E,I,S). Strikingly, the wing overgrowth phenotype associated with depletion of fat was suppressed by Zyx RNAi, resulting in adult wings of similar size to those of animals that only expressed Zyx RNAi ( Figure 1B,F,S). This epistasis of Zyx to fat was also visible at the level of target gene expression ( Figure 3D,E) and the subcellular localization of Yki ( Figure 4G,H). Zyx is also epistatic to the Fat ligand ds ( Figures 1S, S1C,D). These observations imply that Zyx acts downstream of fat. The ex RNAi phenotype, by contrast, was only slightly affected by Zyx RNAi, as the wings of Zyx ex double RNAi animals remained overgrown ( Figure 1J,S). Moreover, ex was epistatic to Zyx for effects on downstream target gene expression and Yki localization ( Figure 4A-D,J,K). Together, these observations indicate that Zyx specifically affects Fat-Hippo signaling and has little effect on Ex-Hippo signaling.
To refine our placement of Zyx within Fat-Hippo signaling, we examined requirements for Zyx relative to additional pathway components. dco encodes a kinase that phosphorylates the Fat cytoplasmic domain and participates in Fat-Hippo signaling [6,28,29]. The requirement for Dco within Fat signaling is uncovered by expression of an antimorphic isoform, Dco 3 . Expression of Dco 3 induces wing overgrowth ( Figure 1L) [29]. This overgrowth is suppressed by Zyx RNAi, suggesting that Zyx acts downstream of dco ( Figure 1P,S).
Like Zyx, dachs is required for normal wing and leg growth and acts genetically downstream of fat and dco but upstream of warts [6][7][8]. To examine the genetic relationship between Zyx and dachs, we took advantage of the observation that over-expression of Dachs can promote wing overgrowth ( Figure 1Q) [7]. This overgrowth was completely suppressed by Zyx RNAi (Figures 1S, S1G), as was the influence of Dachs over-expression on ex-lacZ expression ( Figure S4A,B). Thus, Zyx is required for Dachs-promoted activation of Yki. Over-expression of Zyx resulted in a mild wing overgrowth on its own (9% increase in wing area, Figure 1H,S), and synergized with Dachs over-expression, resulting in enhanced wing overgrowth ( Figure 1R,S). Together, these observations suggest that the functions of Zyx and Dachs in regulating growth are closely linked. However, the observation that Zyx depletion could enhance the small wing phenotype of a putative null allele of dachs ( Figures 1S, S1E,F) [7] implies that Zyx also has some Dachsindependent influence on growth.
Fat exerts a post-transcriptional influence on the levels of Wts protein [6]. The genetic placement of Zyx upstream of wts and within the Fat branch of the pathway suggested that Zyx might also affect Wts levels. Indeed, Zyx RNAi completely suppressed the reduction in Wts levels associated with fat RNAi (Figures 5A,B, S2B). Thus, Zyx is genetically required for the mechanism that links Fat activity to the regulation of Wts protein levels. The influence of fat on Warts levels also requires dachs [6]. Zyx RNAi did not detectably affect Dachs localization ( Figure S4D,E), nor did Zyx RNAi affect Fat localization ( Figure S5E,F). In addition to its effects on Wts, fat mutation also decreases the levels of Ex at the sub-apical membrane [30][31][32][33]. Zyx RNAi was not able to reverse this effect of fat on Ex levels ( Figure S5G-N). Depletion of Zyx in the wing disc also did not have visible effects on F-actin ( Figure  S5O,P).
In addition to regulating transcription, Fat also regulates planar cell polarity (PCP) (reviewed in [1,5]). PCP in the adult wing is manifest in the orientation of wing hairs, which point distally. The anterior, proximal wing is particularly sensitive to Fat-PCP signaling, and fat RNAi results in strong PCP phenotypes in this region, including reversals of hair polarity ( Figure S1M). PCP phenotypes have also been described in this region of dachs mutant wings [34]. Zyx RNAi, by contrast, had no detectable effect on wing PCP ( Figure S1N), and a PCP phenotype was also still detected in fat Zyx double RNAi wings ( Figure S1O). Genes previously identified as influencing Fat-PCP signaling (i.e., fat, ds, fj, app, dachs, lft) also influence cross-vein spacing. Zyx RNAi wings sometimes have extra cross-veins, but by contrast to dachs mutants, the anterior and posterior cross-veins remain well-separated in Zyx RNAi flies ( Figure 1B,C), and the influence of fat on cross-vein spacing is not suppressed by Zyx ( Figure 1F). Our observations suggest that Zyx is specifically required for Fat-Hippo signaling, and not for Fat-PCP signaling, although because Zyx RNAi might not completely eliminate Zyx, we cannot exclude the possibility that low levels of Zyx are sufficient for PCP, but not for Hippo signaling.

Localization of Zyx to the Sub-Apical Membrane
As our anti-Zyx sera did not work for immunostaining, we made use of a V5-tagged UAS transgene that rescues the Zyx RNAi phenotype ( Figure 1) to investigate the subcellular localization of Zyx in imaginal discs. We also examined a UAS-Ypet:Zyx transgene [35]. Although our localization studies are subject to the caveat that Zyx protein was over-expressed, the two different tagged Zyx proteins have similar localization profiles, and similar localization profiles were observed using different Gal4 drivers. Zyx was preferentially localized to the sub-apical membrane of disc cells ( Figure 6). This sub-apical membrane staining was at the same apical-basal position as E-cadherin (E-cad), and just basal to Fat ( Figure 6A-D). This is similar to the membrane localization of Dachs [7]. Indeed, when we compared Zyx and Dachs localization, using epitope-tagged constructs, we observed that the membrane staining is at the same apical-basal position and that they partially co-localize ( Figure 6G,H). A distinguishing feature of Dachs localization is its polarization within the plane of the epithelium, which occurs in response to the Fj and Ds gradients ( Figure 6J) [7,9]. Zyx, by contrast, is not planarpolarized ( Figure 6I); hence, Zyx and Dachs are expected to overlap on only one side of wing disc cells. A distinguishing feature of Zyx staining is that it often displays puncta of larger, more intense staining at the vertices where three cells meet ( Figure 6G). Intriguingly, Ex protein also displays uneven staining, but Ex puncta are partially complementary to Zyx puncta ( Figure 6E,F). These observations suggest that even though Ex and Zyx localize to a similar apical-basal position, they assemble into distinct protein complexes. Dachs localization was not visibly affected by RNAi of Zyx ( Figure S4E), nor was Zyx localization affected by mutation of dachs ( Figure S5B), which indicates that neither protein depends upon the other for its localization. Zyx localization was also not visibly affected by mutation or RNAi of fat, ex, or wts ( Figure S5 and unpublished data).

Dachs Promotes Zyx-Wts Binding
The similar genetic requirements for Zyx and dachs in Fat-Hippo signaling, together with their partial co-localization in imaginal discs, raised the possibility that Zyx and Dachs might interact. This was investigated by expressing tagged isoforms in cultured Drosophila S2 cells and assaying for physical interactions through co-immunoprecipitation. Indeed, Zyx and Dachs could be specifically co-precipitated from S2 cells ( Figure 7B). This observation suggests that Dachs and Zyx can interact directly, although it is also possible that they interact indirectly through a larger complex including endogenously expressed proteins within S2 cells.
As Dachs can also associate with Warts in co-immunoprecipitation assays [6], and both Zyx and dachs are required for the fatdependent regulation of Wts levels, we also investigated binding between Zyx and Wts. When tagged full-length proteins were coexpressed in S2 cells, little or no Zyx-Wts co-precipitation was detected ( Figure 7C,H). However, in addition to their role in Hippo signaling, functions for LATS proteins have also been identified in mitosis, and LATS1 has been localized to the mitotic apparatus [36,37]. In the context of a study of mitotic functions of LATS1, it was reported that the C-terminus of human Zyxin, including the LIM domains, could bind to human LATS1, even though full-length Zyxin did not bind [36]. When we expressed a C-terminal polypeptide comprising the LIM domains of Zyx (Zyx-LD) in S2 cells, only very low levels of protein could be detected ( Figure 7B-D). Nonetheless, this C-terminal polypeptide bound efficiently to Wts ( Figure 7C). Thus, the LIM domains of Zyx can associate with Wts, but this association is normally inhibited within full-length Zyx.
The discovery of this latent ability of Zyx to bind Wts, together with our discovery of Zyx-Dachs binding, and previous identification of Dachs-Wts binding [6], indicates that Dachs, Zyx, and Wts each have the ability to bind to one another. To gain further insight into complex formation among these proteins, we mapped their interaction domains. Wts bound to the LIM domains of Zyx. Dachs, by contrast, bound most strongly to the C-terminal LIM domains but also bound to the N-terminal half of Zyx ( Figure 7B). Dachs contains a large central myosin motor domain and could bind to both Zyx and Wts through this motor domain ( Figure 7D,G and unpublished data). Zyx-LD bound to Wts   Figure 7E). Dachs bound both to this region and also to the Wts kinase domain ( Figure 7F). Thus, Zyx, Dachs, and Wts interact with each other through partially overlapping domains.
To assay for potential sequential, cooperative, or competitive interactions amongst Zyx, Dachs, and Wts, we examined binding interactions when all three proteins were co-expressed together in S2 cells. A key feature of Zyx's interactions with Wts is that fulllength Zyx does not bind efficiently to Wts, but the LIM domains do. However, we found that Dachs enhanced the co-precipitation of full-length Zyx with Wts ( Figure 7H). Two basic models for this stimulation of Zyx-Wts association by Dachs can be envisioned: (a) Dachs might bridge Wts and Zyx within a Wts-Dachs-Zyx complex, or (b) Dachs might trigger a conformational change in Zyx that reveals the latent Wts-binding activity of the Zyx LIM domains ( Figure 8A,B). By employing V5 epitope tags on both Zyx and Dachs, and assaying their co-precipitation with FLAG-tagged Wts, we could directly compare their association with Wts. A simple trimeric complex model (e.g., one subunit each of Zyx, Wts, and Dachs) would predict that Zyx and Dachs should be present within the Wts trimeric complex at equal levels. However, we found instead that Zyx could be much more abundant in Wts complexes than Dachs ( Figure 7H). This suggests that rather than remaining stably associated with Zyx and Wts in a trimeric complex, Dachs is able to stimulate a conformational change in Zyx that exposes the LIM domains and enables them to bind Wts. Consistent with this model, Dachs stimulated Zyx binding to Wts but did not stimulate the binding of Zyx-LD to Wts ( Figure S6A).

The Requirement for Jub in Hippo Signaling Is Distinct from that of Zyx
Zyx is a Drosophila member of a group of cytoskeletal-associated proteins with three C-terminal LIM domains [38]. These comprise two families: the Zyxin family, which in vertebrates includes Zyxin, Lipoma preferred partner (LPP), and Thyroid-receptor interacting protein 6 (TRIP6), and the Ajuba family, which in vertebrates includes Ajuba, LIM domain containing 1 (LIMD1), and Wilms tumor protein 1-interacting protein (WTIP). Drosophila have a single member of each family; Zyx is a member of the Zyxin family, and Ajuba LIM protein (Jub) is a member of the Ajuba family. Ajuba has been reported to interact with a human homologue of Warts, LATS2 [39], and Das Thakur et al. (2010) recently reported that mutation or RNAi-mediated depletion of Jub reduces growth through interactions with the Hippo pathway, and through genetic and protein interaction experiments positioned Jub as a regulator of Wts [40]. In agreement with this, we found that RNAi-mediated depletion of Jub reduces wing growth ( Figure 1M,N,S), expression of Hippo pathway target genes, and nuclear Yki ( Figure S7), and that wts is epistatic to Jub ( Figure 3C). As for Zyx, depletion of Jub did not detectably influence wing hair PCP ( Figure S1P,K).
The determination that Zyx and Jub are each genetically required for Hippo signaling suggests that they have distinct functional roles, and consistent with this, we observed that overexpression of Zyx could not rescue Jub RNAi phenotype ( Figure  S1H) and that Zyx Jub double RNAi induced an even greater reduction of wing size than when they were expressed individually ( Figure 1O,S). Das Thakur et al. (2010) did not address the relationship of Jub to upstream regulators of Hippo signaling. Intriguingly, we found that depletion of Jub suppressed both fat and ex phenotypes. This suppression was evident upon examination of adult wings ( Figure 1G,K,S), expression of downstream target genes in wing discs ( Figures 3F, 4E,F), and the sub-cellular localization of Yki ( Figure 4I,L). Thus, by contrast to Zyx, which functions specifically within Fat-Hippo signaling, Jub is required for both Ex-Hippo and Fat-Hippo signaling. This observation confirms that these two LIM-domain proteins have functionally distinct roles within the Hippo pathway.
The distinct genetic role of Jub in Hippo signaling is also reflected in distinct binding interactions. By contrast to the crucial role of Dachs in stimulating binding between full-length Zyx and Wts, full-length Jub binds efficiently to Wts, and full-length vertebrate homologues of Jub bind to LATS proteins [39,40]. Moreover, Jub bound only very weakly Dachs ( Figure S6B). Thus, although Zyx and Jub share the ability to associate with Wts through their LIM domains, both genetic and biochemical studies indicate that the regulation and consequences of these LIMdomain-Wts interactions are distinct.

Discussion
Our characterization of Zyx identifies a role for it as a novel and integral component of the Hippo pathway, which is required for the Fat branch, but not the Ex branch, of Hippo signaling. Unlike most previously identified components, loss of Zyx reduces the activity of the key transcriptional effector of the pathway, Yki, and consequently its loss reduces organ growth. Genetic epistasis experiments position the requirement for Zyx in between fat and wts, and concordant protein binding experiments identify a Dachsstimulated ability of Zyx to bind Wts protein. We infer that this association of Zyx with Wts then downregulates Wts, at least in part, by targeting it for degradation.  Zyx localizes to the sub-apical membrane independently of Fat or Dachs. Since Fat regulates the localization of Dachs [7], this regulated localization provides a mechanism by which Fat could modulate the interaction of Dachs with Zyx (although we note that Fat might affect the activity of Dachs in addition to affecting its localization). Since Dachs stimulates Zyx-Wts binding, this regulated localization provides a means for Fat signaling to modulate Zyx-Wts binding. We infer that Dachs effects a conformational change in Zyx, as in the absence of Dachs a Zyx LIM-domains polypeptide binds efficiently to Wts, whereas fulllength Zyx binds poorly. Intriguingly, the association of vertebrate homologues of Zyx and Warts can also be post-translationally regulated, as the ability of the LIM domains of human LATS1 to bind Zyxin is masked within full-length Zyxin, but uncovered by Cdc2-mediated phosphorylation, presumably due to conformational change [36]. We hypothesize that the ability of Dachs to bind to both the N-terminus and the LIM domains of Zyx enables it to effect a conformational change in Zyx, resulting in an open configuration that can bind to Wts ( Figure 8B). It is also possible that Dachs binding stimulates a post-translational modification of Zyx to induce a conformational change.
Prior studies identified two mechanisms by which Fat signaling could influence Yki activity, as fat mutation reduces both the levels of Wts protein [6] and the amount of Ex at the sub-apical membrane [31][32][33]. It has not been possible to completely uncouple these two pathways for Fat-Hippo signaling, although the observation that over-expression of Wts can efficiently suppress fat overgrowth phenotypes, but only partially suppresses ex overgrowth phenotypes [30], suggested that the influence of Fat on Wts levels might be more critical. Analysis of the influence of Zyx on Ex is complicated by its influence on ex transcription, but our observation that reduction of Zyx does not appear to suppress the influence of fat on Ex staining, even though it does suppress the influence of fat on Wts levels, also suggests that the influence of Fat on Wts levels might be more critical than its effects on Ex. Intriguingly, mutation of dachs did suppress the influence of fat on Ex levels [30]. Although it is possible that this difference between dachs and Zyx results from technical differences in the experimental paradigms (e.g., mutant clones versus RNAi), it is also possible that dachs can influence Ex levels independently from its association with Zyx.
The discovery of the Fat-specific effect on Wts levels, by contrast to the Hippo-pathway-mediated effect on Wts kinase activity, established the concept of distinct mechanisms for regulating Wts-one that affects Wts levels and another that affects Wts activity [6]. Our identification of distinct genetic requirements for Zyx and Jub provide further support for this concept. As Jub is equally required for both Fat-Hippo and Ex-Hippo signaling and acts genetically between hippo and wts [40], Jub appears to inhibit Wts activation. In our working model ( Figure 8C), the epistasis of Jub to fat could be explained by an increased activity of residual Wts, which then acts catalytically to repress Yki activity. Zyx is required for the influence of fat on Wts levels. We note that when measured within a whole tissue lysate, Wts levels are only reduced to approximately half their normal levels. However, as Wts appears to function within multi-protein complexes, including some components that can localize preferentially to the sub-apical membrane [41,42], we hypothesize that Fat signaling affects a discrete pool of Wts within a complex at the membrane that is crucial for Hippo signaling, whereas there might be additional pools of Wts within the cell that are unaffected. We also note that indicates that equal amounts of pUAS-Zyx:V5 and pUAS-dachs:V5 plasmids were used, and 3x and 6x indicate corresponding increases in amounts of pUAS-dachs:V5 plasmid transfected. Note that in the absence of Dachs, no binding between full-length Zyx and Wts was detected when proteins were precipitated using anti-V5 beads and GFP:V5 was used as a negative control (panel C), but weak binding was detected when proteins were precipitated using anti-FLAG beads and GFP:FLAG was used as a negative control (H). doi:10.1371/journal.pbio.1000624.g007 while we clearly see effects on Wts protein levels, our results do not exclude the possibility that Fat signaling also influences Wts activity.
Our characterization of Zyx and Jub also provides new tools for analyzing critical steps in Hippo signaling. For example, in addition to influencing Hpo and Wts kinase activity, it has been observed that Ex can bind directly to Yki and that when Ex is over-expressed it can repress Yki through a mechanism that involves direct sequestration of Yki, rather than regulation of Yki phosphorylation [43,44]. Because this direct repression mechanism was based on over-expression experiments, the extent to which it contributes to normal Yki regulation in vivo remained uncertain. The observations that Jub acts genetically upstream of wts, yet is required for ex phenotypes, suggests that Ex regulates Yki principally through its effects on Wts activity, rather than through direct interaction with Yki.
The ability of Zyx LIM domains to interact with Wts is conserved in their human homologues [36]. Although the functional significance of this interaction in vertebrates has not yet been established, our observations raise the possibility that the oncogenic effects of human LPP mutations [13] could be due to an ability of these aberrant LPP fusion proteins to negatively regulate LATS proteins, resulting in inappropriate activation of YAP or TAZ.
One of the most intriguing aspects of Zyxin family proteins is their role in mediating effects of mechanical force on cell behavior [18]. Zyxin family proteins can localize to focal adhesions of cultured fibroblasts, and this localization is modulated by mechanical tension [15,18,45]. The observation that increasing tension on stress fibers stimulates Zyxin accumulation at focal adhesions is intriguing in light of our observation that Zyx tends to accumulate at higher levels at intercellular vertices in imaginal discs, as these could be points of increased tension. As the association of unconventional myosins with F-actin can also be influenced by external force [46], our discovery of binding between a myosin protein (Dachs) and Zyx raises the possibility that other myosins might also interact with Zyxin family proteins, which could potentially influence either their tension-based recruitment or their activity.
Finally, we note that theoretical models of growth control in developing tissues have proposed that growth should be controlled by mechanical tension [47,48], and direct evidence for mechanical effects on growth has been obtained in cultured cell models [49]. However, a mechanism for how this might be achieved has been lacking. Our discovery that Zyx, a member of a family of proteins implicated in responding to and transducing the effects of mechanical tension, is also a component of the Hippo signaling pathway, a crucial regulator of growth from Drosophila to humans, raises the intriguing possibility that Zyxin family proteins might form part of a molecular link between mechanical tension and the control of growth.

Drosophila Genetics
RNAi screening was conducted using lines from the NIG-Fly Stock Center (http://www.shigen.nig.ac.jp/fly/nigfly/index.jsp), which were crossed to vg-Gal4 UAS-dcr2 or pnr-Gal4 UAS-dcr2. Those with growth phenotypes were then re-screened for effects on Diap1 and Wg expression in imaginal discs by crossing to ci-Gal4 UAS-dcr2 or en-Gal4 UAS-dcr2. All crosses were carried out at 28.5 C to obtain stronger phenotypes. Approximately 1,200 lines were examined in the initial screen (Table S1) Figure  S3E,F. Both Zyx RNAi lines gave similar effects on growth and gene expression in combination with multiple Gal4 lines and also behaved similarly in epistasis tests. UAS lines employed include UAS-dco 3 [29,48] Adult wing phenotypes were scored by crossing UAS-dcr2; nub-Gal4 females to males of RNAi lines or Oregon-R males as a control. Wings of male progeny were photographed, all at the same magnification. For quantitation, between 9 and 12 wings per genotype were traced using NIH Image J, and wing areas were normalized to the average area in control males. Standard error of the mean (s.e.m.) and t tests were calculated using Graphpad Prism software.

Plasmid Constructs
Details of plasmid construction are in Text S1.
For analysis of Wts protein levels, tub-Gal4 UASdcr2/ TM6b females were crossed to white (control), RNAi-fat, RNAi-Zyx, RNAi-fat; RNAi-Zyx, or UAS-Zyx:V5 males, and wing discs were dissected from third instar larval progeny and lysed in RIPA buffer. Amounts loaded were adjusted to try to load equivalent amounts of total protein in each lane. Wts was detected using a published Wts anti-sera [6] at 1:4,000. Protein bands were detected using anti-mouse IRdye680 and goat anti-rabbit IRdye800 (1:10,000, LiCor) and scanning on a LiCor Odyssey. Bands were quantified using LiCor Odyssey software. Relative Wts levels were determined by comparison to bands detected by anti-Actin antibodies (mouse anti-Actin at 1:5,000, Calbiochem). To enable the relative levels of Wts to be averaged across different blots, we normalized the ratios on each blot to that detected for the control lane, which was set as 1.
For confirmation of the influence of Zyx RNAi on Zyx protein levels, tub-Gal4 UASdcr2/TM6b females were crossed to white (control), or RNAi-Zyx 32018 , and cultured at 29 C, and wing discs were dissected from third instar larval progeny and lysed in RIPA buffer. A rabbit anti-Zyx sera was used at a 1:2,000 dilution, and subsequently the blot was re-probed with rabbit anti-actin (1:10,000, Sigma). Fluorescent detection was performed as described above. Anti-Zyx sera was obtained by immunization of rabbits with a KLH conjugated peptide (KRRLDIPPKPPIKY), performed by Open Biosystems. Table S1 Primary screening of RNAi lines. All fly lines for the primary screening were obtained from the NIG collection. The first two columns identify the RNAi line and the gene (some genes are represented by two independent RNAi lines). The genes screened included all of the lines targeted against X chromosome genes that were available at the time the screen was initiated, plus a selection of lines for 4 th chromosome genes, kinases, phosphatases, and myosins. The third column indicates the phenotype when RNAi lines were crossed to a pnr-Gal4 UAS-dcr2 chromosome. Pnr is expressed in a broad stripe along the center of the notum. A blank entry means that no visible phenotype was detected. The fourth column indicates the phenotype when RNAi lines were crossed to a vg-Gal4 UAS-dcr2 chromosome. Vg is expressed in a broad stripe along the dorsal-ventral compartment boundary, mostly in the wing but also extending into the hinge and notum tissue. A blank entry means that no visible phenotype was detected. The fifth column indicates the phenotype when RNAi lines were crossed to a ci-Gal4 UAS-dcr2 chromosome. Ci is expressed in anterior cells. For this cross, we only examined third instar wing imaginal discs, which were stained with antibodies against Diap1 and Wg. For this column, a blank entry means that this genotype was not examined.