Tumor suppression by cell competition through regulation of the Hippo pathway

Homeostatic mechanisms can eliminate abnormal cells to prevent diseases such as cancer. However, the underlying mechanisms of this surveillance are poorly understood. This study investigated how clones of cells mutant for the neoplastic tumor suppressor gene scribble (scrib) are eliminated from Drosophila imaginal discs. When all cells in imaginal discs are mutant for scrib, they hyperactivate the Hippo pathway effector Yorkie (Yki), which drives growth of the discs into large neoplastic masses. Strikingly, when discs also contain normal cells, the scrib- cells do not overproliferate and eventually undergo apoptosis through JNK-dependent mechanisms. However, induction of apoptosis does not explain how scrib- cells are prevented from overproliferating. This study reports that cell competition between scrib- and wild-type cells prevents hyperproliferation by suppressing Yki activity in scrib- cells. Suppressing Yki activation is critical for scrib- clone elimination by cell competition, and experimental elevation of Yki activity in scrib- cells is sufficient to fuel their neoplastic growth. Thus, cell competition acts as a tumor-suppressing mechanism by regulating the Hippo pathway in scrib- cells (C. Chen, 2012).

This study shows that tumorigenic scrib- cells are removed from Drosophila imaginal discs by a cell-cell signaling event that suppresses elevated Yki activity in scrib- cells. Previous reports implicated JNK as a mediator of cell competition of scrib- clones, where it induces apoptosis and suppresses proliferation. However, it was not known how JNK prevents scrib- clones from hyperproliferating. This study now provides evidence that JNK prevents scrib- clones from hyperproliferating by regulating the activity of the Hippo pathway effector Yki. First, scrib- clones that do not face cell competition up-regulate Yki activity, which drives their hyperproliferation. Second, when scrib- clones do face cell competition, then JNK signaling prevents the upregulation of Yki activity. Third, experimental up-regulation of Yki activity is sufficient to rescue scrib- clones from being eliminated by cell competition. Fourth, experimental suppression of Yki activity in scrib- clones not subjected to cell competition is sufficient to suppress their hyperproliferation. Therefore, cell competition suppresses up-regulation of Yki activity in scrib- cells, and this suppression is important for the elimination of scrib- clones by cell competition. Previous reports showed that Hippo pathway reporters can be up-regulated in scrib- and lgl− mutant discs and clones and that Yki is required for the overgrowth of scrib-+BskDN cells not subjected to cell competition. However, these studies did not analyze the effects of cell competition on Yki activity in scrib- cells. This analysis now shows that scrib- cells facing cell competition do not up-regulate Yki activity and thereby identifies a mechanism that is critical for the elimination of scrib- cells. Although it was reported that scrib- and lgl clones can upregulate ex-lacZ expression and Yki activity (C. Chen, 2012).

However, upon quantification it was found that the majority of scrib- clones have normal or reduced levels of ex-lacZ expression, and only a small percentage of scrib- clones have elevated levels of ex-lacZ expression. Clones with elevated ex-lacZ expression were observed mainly in the hinge region of wing discs, which may provide an environment of reduced cell competition. Thus, outcompeted scrib- clones do not have elevated levels of Yki activity. In contrast, when scrib- clones are rescued from cell competition, they show highly elevated levels of ex-lacZ expression. Similarly, discs that are entirely mutant for scrib, thereby creating an environment that does not have competing normal cells, show hyperactivation of Yki. Cell competition thus prevents the hyperactivation of Yki in scrib- clones and turns a potential high-Yki 'supercompeting' scrib- cell into a cell of lower fitness and less resistance to apoptosis. Importantly, scrib- wts- and scrib-+Yki clones show greatly increased growth and survival compared with scrib- clones. These results show that elevated levels of Yki are sufficient to protect scrib- cells from being outcompeted. Thus, if Yki activity already was high in scrib- cells facing cell competition, those cells would not be outcompeted, and overexpression of Yki or loss of wts would not cause such dramatic effects on the survival and growth of scrib- clones. Apparently, Yki levels in scrib- cells facing cell competition are not high enough for these cells to evade cell competition. Thus, the amount of Yki activity in scrib- cells is a critical determinant of whether scrib- clones are eliminated or form tumorous tissue, and the suppression of Yki activity in scrib- clones is important for the elimination of scrib- clones by cell competition (C. Chen, 2012).

These studies show that JNK activity is required in scrib- cells for the suppression of Yki activity by cell competition. In contrast, JNK signaling can induce Yki activity during regeneration and compensatory proliferation in imaginal discs. Therefore, the effects of JNK signaling on Yki activity in scrib- cells are different from those in normal cells: JNK signaling activates Yki in normal cells promoted to regenerate but suppresses Yki in scrib- cells induced to be eliminated. Interestingly, both these effects are observed in discs with scrib- clones. In scrib- cells, JNK activity suppresses the hyperactivation of Yki, but in neighboring cells that are stimulated to proliferate and compensate for the loss of scrib- cells, the activities of both JNK and Yki are elevated. However, non-cell-autonomous effects on Yki reporters were still observed in egr−/− animals and in discs that ubiquitously inhibited JNK signaling by BskDN. Therefore, JNK-independent signals contribute to the non-cell-autonomous induction of Yki activity around scrib- clones. The regulation of Yki by JNK signaling thus is complex and context dependent and may involve several mechanisms (C. Chen, 2012).

The observation that wts- scrib- clones overgrow indicates that JNK and Wts function in parallel to regulate Yki or that JNK regulates the Hippo pathway upstream of Wts. JNK can phosphorylate and activate Yap1 to regulate apoptosis in mammalian cells. Notably, the JNK phosphorylation sites of Yap1 are different from the Lats phosphorylation sites, supporting a model in which JNK functions in parallel with Wts to regulate Yki activity. However, it is not known whether the same sites also act to suppress the activity of Yki in other contexts (C. Chen, 2012).

Although several models have been proposed to explain how cell-cell interactions between scrib- and normal cells lead to the elimination of scrib- clones from epithelia, it was not clear what properties normal cells must possess to perform this tumorsuppressive role. The data demonstrate that for scrib- cells to be eliminated they must be juxtaposed with cells that have higher levels of competitive fitness, not just proper cellular architecture. Overexpression of the Myc or RasV12 oncogenes in scrib- clones increases their fitness. As a result, in scrib- clones cell competition does not suppress Yki activity, which protects these clones from being eliminated. Interestingly, Myc expression also synergizes with loss of scrib to form tumors in mammals, and the data offer a model to explain this phenomenon. In addition to the cell-autonomous hyperproliferation, scrib- cells that are not removed from imaginal discs have profound non-cell-autonomous effects on the Hippo pathway. This non-cell-autonomous Hippo pathway-regulating signal may serve normally as a regenerative growth signal that facilitates the replacement of eliminated or dying cells, such as outcompeted scrib- cells. If scrib- clones are not eliminated efficiently, however, this signal may persist longer than required to restore the tissue, thereby causing overgrowth and deformation of neighboring tissue. Thus, continued residence of tumorigenic cells can stimulate growth beyond that needed for compensation, essentially hijacking the proliferation and regeneration programs of their normal neighbors. Therefore, the non-cell-autonomous activation of Yki by scrib- cells may have important implications for tumor-stromal interactions in human cancers (C. Chen, 2012).

In summary, it is concluded that cell competition is crucial in suppressing the tumorigenic capacity of scrib- cells and does so by regulating their Yki activity. Loss of this regulation results in overproliferation of both tumorigenic cells and neighboring wild-type cells. Efficient elimination of scrib- clones by cell competition prevents Yki-fueled overgrowth of mutant cells and prevents them from disrupting proliferation control of their normal neighbors. Thus, this study identified a tumor-suppression mechanism that depends on signaling between normal and tumorigenic cells. These data identify evasion of cell competition as a critical step toward malignancy and illustrate a role for wild-type tissue in preventing the formation of cancers (C. Chen, 2012).

Regulation of Hippo signaling by Jun kinase signaling during compensatory cell proliferation and regeneration, and in neoplastic tumors

When cells undergo apoptosis, they can stimulate the proliferation of nearby cells, a process referred to as compensatory cell proliferation. The stimulation of proliferation in response to tissue damage or removal is also central to epimorphic regeneration. The Hippo signaling pathway has emerged as an important regulator of growth during normal development and oncogenesis from Drosophila to humans. This study shows that induction of apoptosis in the Drosophila wing imaginal disc stimulates activation of the Hippo pathway transcription factor Yorkie in surviving and nearby cells, and that Yorkie is required for the ability of the wing to regenerate after genetic ablation of the wing primordia. Induction of apoptosis activates Yorkie through the Jun kinase pathway, and direct activation of Jun kinase signaling also promotes Yorkie activation in the wing disc. It was also shown that depletion of neoplastic tumor suppressor genes, including lethal giant larvae and discs large, or activation of aPKC, activates Yorkie through Jun kinase signaling, and that Jun kinase activation is necessary, but not sufficient, for the disruption of apical-basal polarity associated with loss of lethal giant larvae. These observations identify Jnk signaling as a modulator of Hippo pathway activity in wing imaginal discs, and implicate Yorkie activation in compensatory cell proliferation and disc regeneration (Sun, 2011).

Many tissues have the capacity respond to the removal or death of cells by increasing proliferation of the remaining cells. In Drosophila, this phenomenon has been characterized both in the context of imaginal disc regeneration and compensatory cell proliferation. These studies implicate the Hippo signaling pathway as a key player in these proliferative responses to tissue damage. After genetically ablating the wing primordia by inducing apoptosis, it was observed that Yki becomes activated to high levels in surrounding cells, based on its nuclear abundance and induction of a downstream target of Yki transcriptional activity. Moreover, high level Yki activation is crucial for wing disc regeneration, as even modest reduction of Yki levels, to a degree that has only minor effects on normal wing development, severely impaired wing disc regeneration. While it was known that Yki is required for wing growth during development, the current observations establish that Yki is also required for wing growth during regeneration, and moreover that regeneration requires higher levels of Yki activation than during normal development (Sun, 2011).

These studies identify Jnk activation as a promoter of Yki activity in the wing disc. Most aspects of imaginal disc development, including imaginal disc growth, normally do not require Jnk signaling. By contrast, Jnk signaling is both necessary and sufficient for Yki activation in response to wing damage. Jnk signaling has previously been linked to compensatory cell proliferation and regeneration in imaginal discs, and it is now possible to ascribe at least part of that requirement to activation of Yki. However, Jnk signaling also promotes the expression of other mitogens, including Wg, which were linked to regeneration and proliferative responses to apoptosis. Wg and Yki are not required for each other's expression, suggesting that they are regulated and act in parallel to influence cell proliferation after tissue damage. The mechanism by which Jnk activation induces Yki activation is not yet known. The observation that it could be suppressed by over-expression of Wts or Hpo suggests that it might impinge on Hippo signaling at or upstream of Hpo and Wts, but the possibility that Jnk-dependent Yki regulation occurs in parallel to these Hippo pathway components cannot be excluded. The high level of nuclear Yki localization is striking by contrast with the more modest effects of upstream tumor suppressors in the Hippo pathway, which suggests that Jnk might regulate Yki through a distinct mechanism, or simultaneously affect multiple upstream regulators (Sun, 2011).

Strong Yki activation was detected within the wing and haltere discs in response to Jnk activation, but weaker or non-existent effects in leg or eye discs. Jnk activation has previously been linked to oncogenic effects of neoplastic tumor suppressors in eye discs, and it is possible that Yki activation might be induced in eye discs if a distinct Jnk activation regime were employed. Nonetheless, since identical conditions were employed in both wing and eye discs, isolating them from the same animals, these studies emphasize the importance of context-dependence for Yki activation by Jnk. A link between Jnk activation and Yki activation is not limited to the wing however, as a connection between these pathways was recently discovered in the adult intestine, where damage to intestinal epithelial cells, and activation of Jnk, can activate also Yki (Sun, 2011).

There was a general correspondence between activation of Jnk and activation of Yki under multiple experimental conditions, including expression of Rpr, direct activation of Jnk signaling by Egr or Hep.CA (an activated form of the Jnk kinase Hemipterous), and depletion of lgl. Some experiments, most notably direct activation of Jnk by Hep.CA, revealed a non-autonomous effect on Yki, which could imply that the influence of Jnk on Yki activity is indirect. Although the basis for this non-autonomous effect is not yet known, the hypothesis that it is actually also mediated through Jnk signaling is favored, since it has been reported that Jnk activation can propagate from cell to cell in the wing disc. Consistent with this possibility, a non-autonomous activation of Jnk adjacent to lgl depleted cells was seen to be blocked by depletion of bsk solely within the lgl RNAi cells. Conversely, alternative signals previously implicated in compensatory cell proliferation do not appear to be good candidates for mediating Yki activation, since it was found that Wg is not required for Yki activation in regenerating discs, and prior studies did not detect a direct influence of Dpp pathway activity on Yki activation (Sun, 2011).

Activation of Yki adjacent to Egr- or Rpr-expressing cells was also reduced by over-expression of Wts. This might reflect an influence of Yki on signaling from these cells, but because expression of Wts inhibits Yki activity, and activated Yki promotes expression of an inhibitor of apoptosis (Diap1), it is also possible that this effect could be explained simply by Wts over-expression resulting in reduction or more rapid elimination of Egr- or Rpr-expressing cells; the reduced survival of these cells would then limit their ability to signal to neighbors (Sun, 2011).

Although Jnk has been implicated in compensatory cell proliferation and regeneration, it is better known for its ability to promote apoptosis. The dual, opposing roles of Jnk signaling as a promoter of apoptosis and a promoter of cell proliferation raise the question of how one of these distinct downstream outcomes becomes favored in cells with Jnk activation (see Diverse inputs and outputs of Jnk signaling). Given the links between Jnk activation and human diseases, including cancer, defining mechanisms that influence this is an important question, and the identification of the role of Yki activation in Jnk-mediated proliferation and wing regeneration should facilitate future investigations into how the balance between proliferation or apoptosis downstream of Jnk is regulated (Sun, 2011).

Hippo signaling is regulated by proteins that exhibit discrete localization at the subapical membrane, e.g., Fat, Ex, and Merlin. The observation that disruption of apical-basal polarity is associated with disruption of Hippo signaling underscores the importance of this localization to normal pathway regulation. These observations establish that Hippo signaling is inhibited by neoplastic tumor suppressor mutations, resulting in Yki activation, and that this activation of Yki is required for the tumorous overgrowths associated with these mutations (Sun, 2011).

Although these results agree with these recent studies in linking lgl to Hippo signaling (Grzeschik, 2010), there are some notable differences. A previous study examined lgl mutant clones in the eye imaginal disc, under conditions where cells retained apical-basal polarity, whereas this study examined wing imaginal discs, where apical-basal polarity was lost. Intriguingly this study found that conditions associated with activation of Yki by Jnk in the wing disc were not sufficient to activate Yki in the eye disc. This observation, together with the discovery that loss of polarity in lgl depleted wing cells requires Jnk activation, suggests as a possible explanation for why lgl null mutant clones retain apical-basal polarity in eye discs, that eye disc cells have a distinct, and apparently reduced, sensitivity to Jnk activation as compared to wing disc cells (Sun, 2011).

This study also identified distinct processes linked to Yki activation in the absence of lgl. A previous study reported an effect of lgl on Hpo protein localization (Grzeschik, 2010). In wing discs, the discrete apical localization of Hpo was observed in studies of eye discs. Thus, the proposed mechanism, involving activation of Yki via mis-localization of Hpo and dRassf, might not be relevant to the wing. By contrast, this study identified an essential role for Jnk signaling in regulating Yki activation in lgl-depleted cells in the wing. Because this study did not detect an effect of direct Jnk activation on Yki in eye discs, it is possible that Lgl can act through multiple pathways to influence Yki, including a Jnk-dependent pathway that is crucial in the wing disc, and a Jnk-independent pathway that is crucial in the eye disc. Grzeschik (2010) also linked the influence of lgl in the eye disc to its antagonistic relationship with aPKC. The observation that the influence of aPKC in the wing depends on Jnk activation is consistent with an Lgl-aPKC link, and identifies a role for Jnk activation in the oncogenic effects of aPKC (Sun, 2011).

The observation that the loss of polarity in lgl RNAi discs is dependent upon Jnk signaling was unexpected, but a related observation was recently reported by Zhu; 2010). These results suggest that the established role of the Lgl-Dlg-Scrib complex in maintaining epithelial polarity depends in part on repressing Jnk activity. However, since Jnk activation on its own was not sufficient to disrupt polarity, multiple polarity complexes might need to be disturbed in order for wing cells to lose apical-basal polarity, including both Lgl and additional, Jnk-regulated polarity complexes (Sun, 2011).

The discovery of the role of Jnk signaling in Yki activation provides a common molecular mechanism for the overgrowths observed in conjunction with mutations of neoplastic tumor suppressors, and those associated with compensatory cell proliferation, because in both cases a proliferative response is mediated through Jnk-dependent activation of Yki. Although the molecular basis for the linkage of these two pathways is not understood yet, it operates in multiple Drosophila organs, and thus appears to establish a novel regulatory input into Hippo signaling that is of particular importance in abnormal or damaged tissues. Moreover, Jnk activation has also been observed in conjunction with regeneration of disc fragments after surgical wounding, and thus its participation in regeneration is not limited to paradigms involving induction of apoptosis. It is also noteworthy that under conditions of widespread lgl depletion (i.e., lgl mutant or lgl RNAi), and consequent Jnk activation, the balance between induction of apoptosis and induction of cell proliferation is shifted towards a proliferative response. By contrast, in the wing disc clones of cells mutant for lgl fail to survive, unless oncogenic co-factors are co-expressed. The loss of lgl mutant clones in wing discs was recently attributed to cell competition. Together, these observations suggest that the choice between proliferative versus apoptotic responses to Jnk activation can be influenced by the Jnk activation status of neighboring cells (Sun, 2011).

The Salvador/Warts/Hippo pathway controls regenerative tissue growth in Drosophila melanogaster

During tissue regeneration, cell proliferation replaces missing structures to restore organ function. Regenerative potential differs greatly between organs and organisms; for example some amphibians can regrow entire limbs whereas mammals cannot. The process of regeneration relies on several signaling pathways that control developmental tissue growth, and implies the existence of organ size-control checkpoints that regulate both developmental, and regenerative, growth. This study explored the role of one such checkpoint, the Salvador-Warts-Hippo (SWH) pathway, in tissue regeneration. The Salvador-Warts-Hippo pathway limits tissue growth by repressing the Yorkie transcriptional co-activator. Several proteins serve as upstream modulators of this pathway including the atypical cadherins, Dachsous and Fat, while the atypical myosin, Dachs, functions downstream of Fat to activate Yorkie. Using Drosophila imaginal discs this study showed that Salvador-Warts-Hippo pathway activity is repressed in regenerating tissue and that Yorkie is rate-limiting for regeneration of the developing wing. Regeneration is compromised in dachs mutant wing discs, but proteins in addition to Fat and Dachs are likely to modulate Yorkie activity in regenerating cells. In conclusion these data reveal the importance of Yorkie hyperactivation for tissue regeneration and suggest that multiple upstream inputs, including Fat-Dachsous signaling, sense tissue damage and regulate Yorkie activity during regeneration of epithelial tissues (Grusche, 2011).

This study has shown that the SWH pathway regulates regenerative growth of Drosophila imaginal discs. Hyperactivation of Yki activity was observed in regenerating cells following tissue disruption using a range of genetic and surgical assays, and full Yki activity was essential for efficient wing regeneration. These findings imply that the important role that the SWH pathway has in specification of organ size during development involves an ability to control proliferation in response to tissue damage. These results extend recent data describing a role for the SWH pathway in regeneration of the cricket leg (Bando, 2009). Interestingly, while this manuscript was under review, the SWH pathway was found to modulate regeneration of other epithelial tissues such as the adult Drosophila gut and the murine small intestine in response to chemical and bacterial insults (Cai, 2010; Karpowicz, 2010; Shaw, 2010; Staley, 2010). Coupled with the data in this study, these findings suggest that the SWH pathway is a regulator of 'epithelial fitness', i.e. it is primed to sense damage in epithelial tissues, and to coordinate a robust repair mechanism in response to damage stimuli (Grusche, 2011).

Other growth-regulatory proteins such as Wg and Myc are important for D. melanogaster imaginal disc regeneration, as is the JNK pathway. Interestingly however, regeneration of wing imaginal discs does not appear to be modulated by all growth pathways. For example, another study provided evidence that the target of rapamycin pathway does not modulate regenerative growth of the wing imaginal disc. This lends weight to the hypothesis that the SWH pathway has a specific role in regulating regeneration, rather than regenerative tissue growth being controlled by all growth-regulatory pathways (Grusche, 2011).

What then are the upstream signaling mechanisms that, in response to tissue damage, permit Yki hyperactivation in regenerating cells? Given that a reduction in Wts-dependent phosphorylation of Yki was observed in regenerating tissue, obvious candidate signaling inputs are the three major classes of upstream regulatory proteins of the SWH pathway; the Ft–Ds branch, the KEM complex and the apicobasal polarity proteins, Lgl, Crb and aPKC. The Ft and Ds cadherins limit cell proliferation by engaging in physical interactions with each other on neighbouring cells and repressing the Yki activator, Dachs. Ft and Ds are also required for oriented mitoses of cells neighbouring apoptotic cells. Given this, it is hypothesized that in a damaged tissue, cell–cell contacts would be broken, causing Ft–Ds signaling to be abrogated and a resultant elevation in Dachs, and hence Yki, activity. dachs mutant wing imaginal discs displayed impaired regenerative capacity following γ-irradiation-induced apoptosis, thus implicating the Ft–Ds signaling module in the control of Yki-dependent regeneration. However, genetic wounding and tissue ablation assays of this study have suggested that Yki hyperactivation in regenerating cells involves signaling inputs in addition to the Ft–Ds branch of the SWH pathway, since Yki activation was still observed in regenerative tissue in the absence of dachs or ft (Grusche, 2011).

What are possible reasons for this apparent contradiction? Firstly, there could be subtle differences in Yki activation in wild type versus dachs cells that were not observed in this system, since lacZ enhancer trap lines offer poorly quantitative data, especially when comparing expression levels between independent tissues. Secondly, there is evidence that Ds controls SWH pathway activity by functioning not only as a ligand for Ft but also as a receptor. Whether Ds signaling to downstream SWH pathway proteins requires Dachs, has not yet been determined. Thirdly, Ft and Ds signaling might influence SWH pathway activity independent of Dachs in regenerating cells. For example Ft has been proposed to control Yki activity by influencing the subcellular localization and levels of the Ex protein. However this is is thought to be unlikely given that Yki hyperactivation in regenerative tissue was maintained in ex and ft mutant backgrounds. A more likely scenario is that upstream SWH pathway proteins in addition to Ft, Ds and Dachs participate in control of Yki activity in regenerative tissue growth. Candidates include the KEM complex components, Mer and Kibra, as well as the apicobasal polarity proteins, Crumbs and atypical Protein Kinase C. Another possibility is that the altered physical status of cells that surround tissue wounds might regulate SWH pathway activity via a mechanism such as tension, which has been hypothesized to influence the specification of Drosophila wing size. It is also plausible that signaling pathways that are known to control regeneration, such as the JNK pathway, regulate the SWH pathway during tissue regeneration (Grusche, 2011).

Regenerating cells have been postulated to revert to a more primitive differentiation state. Recently the murine Yki orthologue, YAP, was found to promote stem cell pluripotency along with other transcriptional regulatory proteins including Myc. Significantly, Myc has previously been shown to be induced in regenerating Drosophila tissues, and to be a potent driver of the regenerative response. Therefore a plausible hypothesis is that Yki hyperactivation promotes regeneration by altering the cell's transcriptional program, and thus cellular plasticity, in conjunction with Myc (Grusche, 2011).

Regeneration has often been used as a paradigm to describe the idea of an organ size-checkpoint that limits the size of tissues during both development and adult homeostasis in metazoans. The current findings suggest that the SWH pathway, which is a crucial regulator of organ size, also controls regeneration, providing evidence for a molecular link between the two processes. In the future, it will be interesting to determine whether the SWH pathway controls regeneration in animals with robust regenerative capacity such as axolotls, hydra and planaria. It will also be important to determine whether the SWH pathway controls tissue regeneration in mammals; if this proves to be the case, modulation of SWH pathway activity might be a powerful approach to modulate tissue regeneration post trauma or surgery. Finally, aberrant tissue regeneration upon chronic injury or inflammation has been proposed to contribute to tumour formation. Since increasing evidence points to a role of aberrant SWH pathway signaling in cancer, the current findings provide a potential molecular link between regeneration and tumorigenesis (Grusche, 2011).

The tumour suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements

In Drosophila, defects in asymmetric cell division often result in the formation of stem cell derived tumors. This study shows that very similar terminal brain tumor phenotypes arise through a fundamentally different mechanism. Brain tumors in l(3)mbt mutants originate from overproliferation of neuroepithelial cells in the optic lobes caused by de-repression of target genes in the Salvador-Warts-Hippo (SWH) pathway. ChIP-seq was used to identify L(3)mbt binding sites, and it was shown that L(3)mbt binds to chromatin insulator elements. Mutating l(3)mbt or inhibiting expression of the insulator protein gene mod(mdg4) results in upregulation of SWH-pathway reporters. As l(3)mbt tumors are rescued by mutations in bantam or yorkie or by overexpression of expanded the deregulation of SWH pathway target genes is an essential step in brain tumor formation. Therefore, very different primary defects result in the formation of brain tumors, which behave quite similarly in their advanced stages (Richter, 2011).

Drosophila nervous system recapitulates many steps in mammalian neurogenesis. Neurons in the adult fly brain arise from stem cells called neuroblasts which undergo repeated rounds of asymmetric cell division during larval stages. After division, one daughter cell remains a neuroblast while the other is called the ganglion mother cell (GMC) and divides just once more into two differentiating neurons. Most larval neuroblasts are inherited from the embryo but the so-called optic lobe neuroblasts (NB) located laterally on each brain lobe pass through a neuroepithelial (NE) stage and are therefore a particularly suitable model for mammalian neurogenesis. During early larval stages, the NE cells of the optic lobes (OL) proliferate and separate into the inner (IOA) and outer (OOA) optic anlagen. During late larval stages, NE cells switch to a neurogenic mode. On the medial side, they generate optic lobe neuroblasts (OL NBs), which generate the neurons of the medulla, the second optic ganglion. OL neurogenesis is controlled by a wave of lethal of scute (l(1)sc) expression passing through the neuroepithelium from medial to lateral. The activity of the Jak/STAT pathway inhibits neural wave progression and thereby controls neuroblast number. Differentiation of neuroepithelial cells also involves the Notch, Epidermal Growth Factor (EGF) and Salvador-Warts-Hippo (SWH) pathways (Richter, 2011).

Characterization of Drosophila genes identified in brain tumor suppressor screens has demonstrated that defects in neuroblast asymmetric cell division result in the formation of stem cell derived tumors that metastasize and become aneuploid upon transplantation. These screens also identified lethal (3) malignant brain tumor (l(3)mbt), a conserved transcriptional regulator that is also required for germ-cell formation in Drosophila. L(3)mbt binds to the cell cycle regulators E2F and Rb but the relevance of these interactions is unclear. This study shows that in Drosophila, L(3)mbt regulates target genes of the Salvador-Warts-Hippo (SWH) pathway that are important in proliferation and organ size control. The SWH-pathway is regulated by the membrane proteins Expanded (Ex) and Fat, which activate a protein complex containing the kinases Hippo and Warts to phosphorylate the transcriptional co-activator Yorkie. Yorkie acts together with the transcription factors Scalloped and Homothorax to activate proliferative genes like Cyclin E and the microRNA bantam (ban) and Drosophila inhibitor of apoptosis 1 (diap1: thread). Upon phosphorylation, Yorkie is retained in the cytoplasm and its target genes are not activated. In Drosophila the main role of the SWH-pathway is to limit proliferation in imaginal discs and its absence leads to tumorous overgrowth. In vertebrates, many homologs of key pathway members are tumor suppressors indicating that this function is conserved (Richter, 2011).

L(3)mbt contains three MBT domains which bind mono- or dimethylated histone tails. Biochemical experiments in vertebrates have suggested a role in chromatin compaction but whether this role is conserved is not known. Results published while this paper was under review have shown that germline genes are upregulated in l(3)mbt mutant brains and are necessary for tumor formation. The current data indicate that L(3)mbt is bound to insulator sequences, which affect promoter-enhancer interactions and influence transcription. In Drosophila, the proteins CTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4) and GAF are found at insulator sequences but how these factors act is largely unknown (Richter, 2011).

The data presented in this study show that tumor formation in l(3)mbt mutants is initiated by the uncontrolled overproliferation of neuroepithelial cells in the optic lobes due to the upregulation of proliferation control genes normally repressed by the SWH-pathway. L(3)mbt is located at DNA sequences bound by chromatin insulators and we propose that the function of L(3)mbt as a chromatin insulator is essential for repressing SWH target genes and preventing brain tumor formation (Richter, 2011).

brat, lgl and dlg were previously identified as Drosophila brain tumor suppressors. In all cases, defects in asymmetric cell division cause a huge expansion of the neuroblast pool. In l(3)mbt mutants, however, the neuroblast pool is expanded because an upregulation of SWH target genes results in a massive expansion of neuroepithelial tissue. Why those neuroblasts proliferate indefinitely upon transplantation is currently not understood for any of those mutants (Richter, 2011).

While the SWH-pathway is essential for tumorigenesis in l(3)mbt mutants, its overactivation can not recapitulate the neuroblast tumor phenotype seen in l(3)mbt mutants (this study and Reddy, 2010). Similar to the multifactorial origin of mammalian tumors, therefore, the combined deregulation of several signaling pathways could be required. The Notch pathway could be involved as it regulates the formation of OL neuroblasts from neuroepithelia and Notch pathway gene insulator sequences are bound by L(3)mbt. Increased activity of the Jak/STAT pathway, a major regulator of OL development, was also observed. Finally, the deregulation of germline genes in l(3)mbt mutants that has been described while this manuscript was under review (Janic, 2010) could provide another exciting explanation (Richter, 2011).

The results indicate that L(3)mbt acts on insulator elements, which isolate promoters from the activity of nearby enhancers acting on other genes. the analysis showed that L(3)mbt binding sites overlap with CP190, CTCF and BEAF-32, placing the protein into what has been called the class I of chromatin insulators (Negre, 2010; Richter, 2011 and references therein).

The identification of a DNA consensus motif for a histone binding protein like L(3)mbt is highly unexpected as insulators are typically nucleosome free. Currently, the activity of these important transcriptional regulators could be explained in several ways. Either, they form physical barriers blocking the interaction between enhancers and promoters. Alternatively, they mimic promoters and compete with endogenous promoters for enhancer interaction. Finally, they could interact with each other or nuclear structures to form loop domains that regulate transcriptional activity. The data suggests another model in which insulators interact with histones on nearby nucleosomes and influence the structure of higher order chromatin. Importantly, in the regions flanking CTCF binding sites nucleosomes are enriched for histones that are mono- and di-methylated on H3K4 or mono-methylated on H3K9 or H4K20, the variants to which MBT domains can bind in vitro. As the human L(3)mbt homolog L3MBTL1 was shown to compact nucleosome arrays in vitro, a model becomes feasible in which simultaneous binding to insulators and the surrounding nucleosomes reduces flexibility and thereby restricts the ability of nearby enhancers to interact with promoters on the other side of the insulator. However, the data could equally well be worked into the other prevalent models for insulator activity. Since L(3)mbt is currently the only chromatin insulator besides CTCF that is conserved in vertebrates, analysis of its homologs will certainly allow to distinguish between those possibilities (Richter, 2011).

OL development resembles vertebrate neurogenesis. Both processes consist of an initial epithelial expansion phase followed by neurogenesis through a series of asymmetric divisions. Together with previous findings, these data demonstrate that l(3)mbt and the SWH-pathway are crucial regulators of the initial neuroepithelial proliferation phase. Interestingly, the SWH-pathway has been implicated in regulating neural progenitors in the chicken embryo and it will be exciting to test the role of mammalian L(3)mbt in this process. It is remarkable that YAP is upregulated and L3MBTL3 is deleted in a subset of human medulloblastomas. Medulloblastoma is the leading cause of childhood cancer death and investigating the role of the SWH-pathway might contribute to the progress in fighting this disastrous disease (Richter, 2011).

The sterile 20-like kinase tao controls tissue homeostasis by regulating the hippo pathway in Drosophila adult midgut

The proliferation and differentiation of adult stem cells must be tightly controlled in order to maintain resident tissue homeostasis. Dysfunction of stem cells is implicated in many human diseases, including cancer. However, the regulation of stem cell proliferation and differentiation is not fully understood. This study shows that the sterile-like 20 kinase, Tao, controls tissue homeostasis by regulating the Hippo pathway in the Drosophila adult midgut. Depletion of Tao in the progenitors leads to rapid intestinal stem cell (ISC) proliferation and midgut homeostasis loss. Meanwhile, it was find that the STAT signaling activity and cytokine production are significantly increased, resulting in stimulated ISC proliferation. Furthermore, expression of the Hippo pathway downstream targets, Diap1 and bantam, is dramatically increased in Tao knockdown intestines. Consistently, it was shown that the Yorkie (Yki) acts downstream of Tao to regulate ISC proliferation. Together, these results provide insights into understanding of the mechanisms of stem cell proliferation and tissue homeostasis control (X. Huang, 2014).

Targets of Activity

Activation of yki leads to increased transcription of diap1 and CycE. The increased cell proliferation and decreased apoptosis resulting from yki overexpression are strikingly similar to those caused by loss of hpo, sav, or wts, suggesting that Yki functions in the Hpo pathway. To further explore this possibility, the transcription of cell-death inhibitor diap1 and cell-cycle regulator cycE, known targets of the Hpo pathway (Wu, 2003) were examined. Elevated DIAP1 protein is detected in yki-overexpressing clones in the eye discs. This regulation is largely mediated at the level of diap1 transcription since the expression of thj5c8, a P[lacZ] enhancer trap reporter inserted into the diap1 locus, is similarly elevated in yki-overexpressing clones in a cell-autonomous manner. A cycE-lacZ reporter containing 16.4 kb of the 5′ regulatory sequence of cycE is also increased in yki-overexpressing clones, especially those close to the MF, although the effect is less profound than that observed with the diap1 reporter. Thus, like loss of hpo, sav, or wts, overexpression of yki results in increased transcription of diap1 and cycE. It is worth noting that previous analyses of hpo mutant clones also revealed a 'tighter' regulation of diap1: while diap1 transcription is elevated in all hpo mutant cells irrespective of their relative position to the MF, cycE transcription is only elevated in hpo mutant cells close to the MF (Wu, 2003). These observations suggest that diap1 might represent a more direct transcriptional target of the Hpo pathway (Huang, 2005).

The transcriptional coactivator activity of Yki is negatively regulated by the Hpo pathway

The results are consistent with a model wherein Yki acts antagonistically to Hpo, Sav, and Wts in a common signaling pathway that coordinately controls cell proliferation and apoptosis. Based on the physical interactions between Yki and Wts, and given that YAP, the mammalian homolog of Yki, is known to function as a transcriptional coactivator (Yagi, 1999; Strano, 2001; Vassilev, 2001), it was further hypothesized that Yki functions downstream of Wts to regulate transcription of genes such as diap1 and that the Hpo pathway negatively regulates the coactivator activity of Yki (Huang, 2005).

To test the hypothesis that the coactivator activity of Yki is negatively regulated by the Hpo pathway, a transcription assay was established for Yki activity in Drosophila S2 cells. Since the cognate transcription factor(s) that partner with Yki are not yet identified, Yki was fused to the DNA binding domain (DB) of the yeast Gal4 transcription factor. The activity of this fusion construct was then assayed using a Gal4-responsive reporter. Consistent with previous reports of YAP as a transcriptional coactivator in mammalian cells, the Gal4DB-Yki fusion protein exhibited potent transcriptional activation. Strikingly, transcriptional activity of the Gal4DB-Yki fusion was abolished when Hpo, Sav, and Wts plasmids were coexpressed. This effect is specific to Yki since activity of the full-length Gal4 (with its own activation domain) was unaffected by the coexpression of Hpo, Sav, and Wts. These results suggest that the Hpo pathway negatively regulates the coactivator activity of Yki (Huang, 2005).

To further probe a functional link between yki and the Hpo pathway, their genetic interactions were investigated. While expression of hpo or wts directly from the GMR promoter results in viable flies with rough or slightly rough eyes, respectively, cointroduction of GMR-hpo and GMR-wts into the same animals results in 100% lethality at early pupal stage (Wu, 2003). Strikingly, such lethality is completely rescued by coexpression of yki from a GMR-yki transgene. Interestingly, this lethality is also completely rescued by coexpression of the human YAP gene. In another line of experiments, advantage was taken of the complete pupal lethality caused by the overexpression of UAS-hpo driven by the GMR-Gal4 driver. Interestingly, this lethality is also rescued by the coexpression of yki (100% rescue) or YAP (21% rescue). Taken together, these genetic interactions further support the model that Yki acts antagonistically to Hpo, Sav, and Wts in a common signaling pathway. The ability of a human YAP transgene to rescue the lethality of flies caused by Hpo pathway hyperactivation reveals a functional conservation between Yki and YAP, suggesting that YAP might play a similar role in mammalian growth control (Huang, 2005).

bantam is a target of the hippo tumor-suppressor pathway

The Hippo tumor-suppressor pathway has emerged as a key signaling pathway that controls tissue size in Drosophila. Hippo signaling restricts tissue size by promoting apoptosis and cell-cycle arrest, and animals carrying clones of cells mutant for hippo develop severely overgrown adult structures. The Hippo pathway is thought to exert its effects by modulating gene expression through the phosphorylation of the transcriptional coactivator Yorkie. However, how Yorkie regulates growth, and thus the identities of downstream target genes that mediate the effects of Hippo signaling, are largely unknown. This study reports that the bantam microRNA is a downstream target of the Hippo signaling pathway. In common with Hippo signaling, the bantam microRNA controls tissue size by regulating cell proliferation and apoptosis. hippo mutant cells had elevated levels of bantam activity; and bantam is required for Yorkie-driven overgrowth. Additionally, overexpression of bantam is sufficient to rescue growth defects of yorkie mutant cells and to suppress the cell death induced by Hippo hyperactivation. Hippo regulates bantam independently of cyclin E and diap1, two other Hippo targets, and overexpression of bantam mimics overgrowth phenotypes of hippo mutant cells. These data indicate that bantam is an essential target of the Hippo signaling pathway to regulate cell proliferation, cell death, and thus tissue size (Nolo, 2006).

To test whether the activity of the bantam miRNA is regulated by Hpo signaling, use was made of a GFP bantam sensor that reports the spatial activity of bantam. This bantam sensor expresses GFP under the control of a ubiquitously active tubulin promoter and has two perfect bantam target sites in its 3′ UTR. When present, the bantam miRNA reduces GFP expression through its RNAi effect. The expression pattern of GFP is thus a negative image of the activity pattern of the bantam miRNA. In third-instar wing imaginal discs, the bantam sensor is expressed in a complex pattern with higher levels along the presumptive wing margin, in the anterior compartment along the anteroposterior compartment boundary, and in several patches in the thorax region. Overexpression of the bantam miRNA in the developing wing eliminated the GFP expression of the bantam sensor in the corresponding region, demonstrating that the expression of GFP is indeed under the control of bantam. In developing eye discs, the bantam sensor is also broadly expressed, with higher levels in differentiating photoreceptor cells. As in wing discs, overexpression of bantam downregulated GFP expression in eye discs. The bantam sensor thus reflects the activity of the bantam miRNA in eye and wing discs (Nolo, 2006).

To address whether Hpo signaling regulates the activity of the bantam miRNA, GFP expression of the bantam sensor was monitored in imaginal discs that had defects in Hpo signaling. It was found that hpo or wts mutant cells had lower levels of bantam-sensor-driven GFP expression throughout the mutant clones. Significantly, hpo and wts mutant clones showed lower levels of GFP in multiple tissues, including the wing, antenna, and eye imaginal discs. In eye imaginal discs, wts clones affected the bantam sensor anterior to the morphogenetic furrow, where cells are still uncommitted as well as posterior to the furrow in differentiating photoreceptor cells. In all cases, the regulation of the bantam sensor was cell autonomous. In addition, wing imaginal discs that overexpressed Yki had lower levels of bantam sensor expression in the entire region of Yki overexpression. In summary, it is concluded that Hpo signaling generally regulates bantam expression in multiple imaginal discs and cell types (Nolo, 2006).

A model is postulated in which bantam is an essential target of the Hpo signaling pathway to regulate cell proliferation, cell death, and thus tissue size. This model is based on several observations. First, it was found that bantam is regulated by Hpo signaling broadly and in various tissues. This regulation is a specific downstream effect of Hpo signaling and is not simply the consequence of the cell proliferation induced in hpo mutant cells. Second, bantam is required for Yki to drive tissue overgrowth, because removal of bantam suppresses the overgrowth phenotypes caused by overexpression of Yki in the retina. Third, overexpression of bantam rescues the cell death induced by overexpressed Hpo and significantly rescues growth defects of yki mutant cells. And fourth, bantam overexpression mimics the phenotypes of hypomorphic hpo mutations. Taken together, these data support a model in which bantam is an important downstream target of the Hpo pathway (Nolo, 2006).

The finding that Hpo signaling regulates the expression of bantam raises the question of how important this effect is for Hpo signaling to control tissue size. Removal of bantam suppresses the induction of extra interommatidial cells in the retina by Yki overexpression but does not cause a general elimination of retinal cells in a wild-type background. These data indicate that the regulation of bantam is an essential downstream effect of Hpo signaling to regulate tissue size. However, loss of bantam only partially suppresses the effects of Yki overexpression, indicating that Yki regulates other targets in addition to bantam. Hpo was found to regulate bantam independently of cyclin E and diap1, two other genes known to be regulated by Hpo signaling. bantam is thus not a component of the Hpo signal transduction pathway itself, but is one of several downstream target genes. Yki must have targets in addition to bantam, cyclin E, and diap1, because overexpression of bantam, Cyclin E, and DIAP1 together did not induce the amount of overgrowth caused by Yki overexpression in wing discs. Nevertheless, overexpression of bantam alone caused phenotypes resembling hypomorphic situations for Hpo signaling, indicating that bantam is a critical mediator of Hpo function. Whether the regulation of bantam by a Yki-containing transcription factor complex is direct remains to be determined. However, the fact that Hpo regulates bantam cell autonomously and in multiple tissues is consistent with such a model (Nolo, 2006).

bantam expression is spatially modulated, and patterning signals such as Wg and Dpp also regulate the expression of bantam to generate its expression pattern. These patterning signals regulate specific aspects of the bantam expression pattern, and they have different effects on cell proliferation as well as bantam activity in different regions in various imaginal discs. In contrast, hpo mutant cells upregulate bantam activity independently of cell type and in multiple imaginal discs, indicating an intimate relationship. Hpo is thus a more general and ubiquitous regulator of bantam expression in imaginal discs. An important question that remains to be answered is how these patterning signals regulate tissue growth and bantam expression and whether they regulate bantam expression directly and independently of Hpo signaling or through the regulation of Hpo activity (Nolo, 2006).

Surprisingly, just the opposite of hpo mutant cells, TSC1 mutant cells had lower levels of bantam activity although these cells overgrow, indicating that TSC1 mutant cells induce growth independently of bantam. Neither Myc, Ras, nor Cyclin D-Cdk4 expression induced bantam, although they induce cell growth and proliferation. bantam is thus not simply a part of the cell-intrinsic machinery that executes cell growth and division but rather acts as an upstream component to instruct cells to proliferate. In summary, although Hpo is a key regulator of bantam expression, bantam is also regulated by other pathways potentially integrating the effects of several growth-regulatory and patterning pathways (Nolo, 2006).

miRNAs and their target genes often show mutually exclusive expression patterns, and miRNAs induced during differentiation tend to target messages that were abundant in the previous developmental stage. miRNAs may thus provide a rapid and effective means to suppress expression of residual, unwanted mRNAs while the transcriptional program in a cell is changing. Hpo signaling is involved in regulating cell proliferation and apoptosis in developing imaginal discs. Cell lineages and cell proliferation show significant plasticity in growing imaginal discs, which can rapidly respond to surgical ablation or genetic insults by regenerating missing (eliminated) cells or by ablating unwanted (extra) cells. This adjustment of cell proliferation and apoptosis requires a mechanism that can rapidly change the growth properties of a cell. Yki appears to regulate cell number on the one hand by inducing the expression of positive regulators of cell proliferation and cell survival and on the other hand by inducing the expression of bantam, which posttranscriptionally suppresses the expression of proteins that inhibit cell proliferation and induce apoptosis. An example of such cooperative action of Yki and bantam is the regulation of Hid: Yki suppresses the expression of hid, but also induces bantam, which then suppresses the translation of hid mRNAs that may still be present in a cell. The induction of bantam by Yki may also accelerate the repression of negative growth regulators, thereby enabling a cell to more quickly and robustly adjust its rate of cell proliferation. It will be interesting to elucidate how bantam regulates growth and how its growth targets are integrated with other targets of Hpo signaling (Nolo, 2006).

The Hippo pathway regulates the bantam microRNA to control cell proliferation and apoptosis in Drosophila

The Hippo signaling pathway acts upon the Yorkie transcriptional activator to control tissue growth in Drosophila. Activated Yorkie drives growth by stimulating cell proliferation and inhibiting apoptosis, but how it achieves this is not understood. Yorkie is known to activate Cyclin E (CycE) and the apoptosis inhibitor DIAP1. However, overexpression of these targets is not sufficient to cause tissue overgrowth. This study shows that Yorkie also activates expression of the bantam microRNA, a known regulator of both proliferation and apoptosis. bantam overexpression mimics Yorkie activation while loss of bantam function slows the rate of cell proliferation. bantam is necessary for Yorkie-induced overproliferation and bantam overexpression is sufficient to rescue survival and proliferation of yorkie mutant cells. Finally, bantam levels are shown to be regulated during both developmentally programmed proliferation arrest and apoptosis. In summary, the results show that the Hippo pathway regulates expression of bantam to control tissue growth in Drosophila (Thompson, 2006).

The Hippo pathway is unique in its direct and dedicated role in the intrinsic program of growth in proliferating tissues. The potency of the Hippo pathway in driving tissue growth appears to reside in its ability to coordinately stimulate cell proliferation and suppress apoptosis. A key goal is to understand how this coordinate control is achieved. The results show that the bantam microRNA, a known regulator of both cell proliferation and apoptosis, is a critical target of the Hippo pathway. Activated Yki is necessary and sufficient to induce bantam expression and to stimulate cell survival and proliferation. bantam appears to be a key target of Yki because loss of Yki can be rescued by overexpression of bantam. Finally, bantam clearly has an important role in both normal growth and Yki-driven overgrowth because loss of bantam strongly reduces the rate of cell proliferation in either case. Although the bantam microRNA appears not to be conserved in vertebrates, it is possible that other microRNAs play a functionally equivalent role as effectors of the Hippo pathway. Recent work has identified human microRNAs involved in this pathway (Thompson, 2006).

Two lines of evidence indicate that bantam is not the only relevant target of the Hippo pathway. Firstly, loss of bantam does not completely mimic loss of Yki in every respect, because bantam mutant cells do not undergo apoptosis. This difference is likely to reflect the contribution of the Yki target DIAP1, whose absence is known to trigger apoptosis. Secondly, Yki retains some ability to stimulate cell proliferation even in the absence of bantam. Again, this activity may reflect the role of other Yki targets, including CycE, in driving cell proliferation. Thus, the results favor the view that bantam acts in a highly cooperative way with other Yki target genes to mediate the effects of the Hippo pathway on cell proliferation and apoptosis (Thompson, 2006).

The expression of bantam during normal development shows a striking pattern of regulation; it is expressed in proliferating cells but not in quiescent cells or, as has been shown in this work, in certain cells destined for apoptosis. These findings indicate that regulation of bantam is a key feature of the normal program of tissue growth. Previous work has shown that high levels of the Wingless (Wnt) morphogen represses bantam as cells arrest proliferation at the presumptive wing margin. Since the results show that the Hippo pathway regulates bantam, the pattern of bantam expression may reflect regulation of Hippo pathway activity by positional signals. Thus, positional signals could determine the behavior of cells along the spectrum from rapid proliferation to apoptosis simply by controlling the Hippo pathway. Alternatively, positional signals and the Hippo pathway may act independently, with the bantam locus being a regulatory nexus that integrates information from a number of different signaling pathways (Thompson, 2006).

The final finding is that the Hippo pathway also influences epithelial morphogenesis. This function appears to be independent of its role in controlling cell survival and proliferation and does not involve bantam. Interestingly, expression of Yki and Hippo have reciprocal effects on the epithelium, with overexpressed Yki driving apical bulging and overexpressed Hippo causing basal outfolding. In both cases, the cells remain epithelial, indicating that the Hippo pathway controls cell shape without affecting epithelial polarity or integrity. These observations are consistent with previous reports that clones of cells with elevated pathway activity (i.e. mutant for Hippo or other negatively acting components) have a rounded appearance, indicating altered cell affinities, and that mutation of warts also causes apical bulging of epithelia that is attributed to an expanded apical membrane domain. Why cells use the same pathway to control survival, proliferation, and shape remains an intriguing question (Thompson, 2006).

A fuller understanding of the network connecting the Hippo pathway with the basic machinery controlling survival, proliferation, and morphology will be needed to understand how size regulation is connected to pattern formation during normal development. This work allows sketching of the outline of one facet of this network, with the Yki targets bantam, CycE, and DIAP1 cooperating to control survival and proliferation (Thompson, 2006).

Transcription factor choice in the Hippo signaling pathway: homothorax and yorkie regulation of the microRNA bantam in the progenitor domain of the Drosophila eye imaginal disc

The accurate control of cell proliferation and survival is critical for animal development. The Hippo tumor suppressor pathway regulates both of these parameters by controlling the nuclear availability of the transcriptional coactivator Yorkie (Yki), which regulates downstream target genes together with Scalloped (Sd), a DNA-binding protein. This study provides evidence that Yki can also regulate target genes in conjunction with Homothorax (Hth) and Teashirt (Tsh), two DNA-binding transcription factors expressed in the uncommitted progenitor cells of the Drosophila eye imaginal disc. Clonal analyses demonstrate that Hth and Tsh promote cell proliferation and protect eye progenitor cells from apoptosis. Genetic epistasis experiments suggest that Hth and Tsh execute these functions with Yki, in part by up-regulating the microRNA bantam. A physical interaction between Hth and Yki can be detected in cell culture, and this study shows that Hth and Yki are bound to a DNA sequence approximately 14 kb upstream of the bantam hairpin in eye imaginal disc cells, arguing that this regulation is direct. These data suggest that the Hippo pathway uses different DNA-binding transcription factors depending on the cellular context. In the eye disc, Hth and Tsh provide spatial information to this pathway, promoting cell proliferation and survival in the progenitor domain (Peng, 2009).

The evidence suggests that Hth and Tsh function as partners to carry out two main functions in anterior eye disc cells: They repress the expression of the later-acting retinal determination factors, and they promote cell proliferation. That these functions require hth is supported by both loss-of-function studies as well as gain-of-function studies. For example, hthP2 clones fail to survive anterior to the MF, and Tsh's ability to induce overgrowths when ectopically expressed is abolished in the absence of hth. The involvement of Tsh is supported by gain-of-function experiments and the finding that Hth and Tsh directly interact with each other in vivo. Carrying out loss-of-function genetics for tsh is difficult because this gene is located proximal to the standard Flp recombination targets (FRTs) used to generate mitotic recombination. In addition, the highly related gene tio, which is closely linked to tsh, functions redundantly with tsh in several instances, including some aspects of eye development. Nevertheless, knocking down tsh using RNAi in a tio-null background results in poor survival in the progenitor domain. Taken together, these data provide a compelling argument for Hth + Tsh functioning together to promote cell survival in the anterior eye disc (Peng, 2009).

A functional relationship between Hth and Tsh also exists in other tissues in Drosophila, most notably in both wing and leg imaginal discs, where they are coexpressed in cells that will give rise to the proximal domains of these appendages. In both wings and legs, Tsh has the capacity to regulate hth when expressed in clones, and both tsh and hth have the ability to suppress distal appendage development when misexpressed. However, in these tissues, and unlike the eye disc, Hth + Tsh expression is not correlated with proliferation, which occurs uniformly throughout these discs. Consistently, the expression pattern exhibited by the bantam sensor does not correlate with Hth or Tsh in the leg or wing. The special relationship between proliferation and Hth + Tsh in the eye may be due in part to the Drosophila Pax6 homolog Eyeless (Ey), which is critical for eye identity. Moreover, Ey is found in a complex with Hth in vivo and participates with Hth and Tsh in the repression of retinal determination genes. Thus, it may also be the case that Ey directly participates in the regulation of bantam together with Hth and Yki (Peng, 2009).

Although hthP2 clones fail to survive in the eye progenitor domain, the data demonstrate that hth is not absolutely required for cells in this domain to proliferate. The effects observed on the bantam sensor are consistent with the idea that hth promotes, but is not essential for, cells to proliferate in the eye progenitor domain. In hthP2 clones, bantam sensor levels increased above those normally observed in progenitor cells, but not as high as the levels observed in differentiated photoreceptors. Thus, if the level in photoreceptors represents the complete absence of bantam, these data imply that hth only up-regulates bantam over a basal hth-independent level. Moreover, the levels of the bantam sensor in other tissues, such as the wing disc, rarely approach those observed in photoreceptors, suggesting that most cells have some bantam expression, and that bantam regulators, such as hth, only serve to modulate bantam levels (Peng, 2009).

If eye progenitor cells have the capacity to proliferate in the absence of hth, how important is the proliferation-promoting function of hth? Although normal eyes can develop in animals in which hthP2 clones are generated, this is likely due to the ability of neighboring wild-type cells to compensate in this mosaic situation. In contrast, when wild-type and heterozygous cells are killed (using the EGUF method [ey-Gal4/UAS-flp/GMF-hidwe find that the remaining hthP2 tissue is unable to produce normal-sized eyes. This experiment suggests that the proliferation-promoting functions of hth in the eye progenitor domain are critical for normal eye development, likely by providing a sufficient pool of progenitor cells prior to differentiation (Peng, 2009).

The Hippo pathway has emerged recently as an important regulator of cell proliferation and survival in both vertebrates and invertebrates. In Drosophila, this pathway appears to regulate proliferation in nearly all tissues. For example, wts- clones or Yki+ clones have the capacity to induce overgrowths throughout the body. As Yki and its mammalian ortholog Yap are transcriptional coactivators that do not have their own DNA-binding domain, they are thought to partner with DNA-binding transcription factors to regulate gene expression. Prior to this work, the only transcription factor proposed to work directly with Yki was Sd, a member of the TEAD/TEF family of DNA-binding proteins. However, unlike other components of the Hippo pathway, the available data suggest that Sd plays a more limited role in cell proliferation and survival in Drosophila. In contrast to its essential role in the wing pouch, sd- clones survive well in other tissues, including the region of the wing disc that will give rise to the notum of the fly. Similarly, sd-null clones grow well in the eye progenitor domain. Thus, unlike in the wing pouch, sd is not required for cell survival and proliferation in the eye progenitor domain (Peng, 2009).

In contrast to the survival of sd clones in this domain, hthP2 clones fail to survive in the eye progenitor domain. Thus, analogous to sd in the wing pouch, hth is required for cells to survive and proliferate in the anterior eye imaginal disc. This observation suggests that hth could play an analogous role to sd in this progenitor domain, a view that is supported by the results. This evidence includes (1) Hth can interact with Yki when coexpressed in S2 cells, (2) Hth + Tsh regulate the Yki target bantam, and (3) Hth and Yki are both bound to the same region of the bantam locus in eye discs. Genetically, it was shown that the Hippo pathway is unable to induce overgrowths in the eye progenitor domain without hth, and that Hth + Tsh cannot induce overgrowths in the absence of Yki. These results suggest that Hth + Tsh comprise the DNA-binding transcription factors that function with Yki to regulate proliferation and survival genes, such as bantam. Thus, analogous to Sd in the wing pouch, Hth + Tsh are transcription factors used by the Hippo signaling pathway in eye progenitor cells (Peng, 2009).

The finding that Hth + Tsh play an analogous role in the eye progenitor domain as Sd does in the wing pouch has several implications for how the Hippo pathway is regulated in vivo. For one, the use of different DNA-binding transcription factors to regulate Hippo target genes suggests a previously unknown degree of specificity available to this pathway. Hth, a TALE family homeodomain protein, and Tsh, a Zn finger protein, are likely to bind very different target DNA sequences than Sd, a TEAD/TEF domain DNA-binding factor. Accordingly, it was found that ectopic Hth + Tsh clones in the eye disc do not consistently up-regulate diap1 or expanded, known Sd-Yki targets in the wing disc (Peng, 2009).

These results also imply that the transcriptional regulation of hth, tsh, and sd has the potential to change the output of the Hippo pathway. Because hth and tsh are transcriptionally repressed by signals coming from the MF, these factors are not available to work with the Hippo pathway posterior to the MF. However, loss of Hippo kinase activity can lead to proliferation of differentiated cells posterior to the MF. In these cells, sd is expressed, suggesting that Yki may use this transcription factor in this context. Analogously, loss of Hippo kinase activity can cause overgrowths in the notum as well as in the wing pouch. As sd- clones grow well in the notum, but not in the wing pouch, these data suggest that the notum overgrowths may be mediated by a transcription factor other than Sd. hth clones also survive well in the notum, implying that yet another transcription factor or factors may work with Yki in this tissue. In sum, it is suggested that Yki, and thus the Hippo pathway, may be able to work with multiple transcription factors to regulate target genes. In principle, the use of several transcription factors that are themselves developmentally regulated allows the Hippo pathway to be interpreted in different ways in different contexts (Peng, 2009).

Although the data suggest that the Hippo pathway uses Hth + Tsh to up-regulate bantam, they also suggest that both Hth + Tsh and Yki have additional, independent targets. For example, the loss of Hippo kinase activity leads to the up-regulation of diap1 throughout the eye disc. Because diap1 is not affected when Hth + Tsh are coexpressed, the Hippo pathway has the capacity to regulate some genes independently of Hth + Tsh, even in the eye progenitor domain. Moreover, at least when Yki is ectopically expressed, sd appears to be required in all regions of the eye disc for diap1 activation. Thus, although it has not been shown that sd is required for endogenous diap1 expression in this tissue, these data suggest that Yki may use both Sd and Hth + Tsh to regulate gene expression in the eye disc. In fact, it has been suggested that sd is also a modifier of bantam expression in the eye disc and that sd is required for normal-sized eyes. However, these clones, which used RNAi to knock down Sd, grew well in the eye progenitor domain. In addition, the smaller eyes observed when sd was knocked down could be due to the earlier embryonic expression of the Gal4 driver used in these experiments. In contrast, when generated during larval stages, hth- clones, but not sd- clones, fail to survive in the eye progenitor domain, arguing that, at least post-embryonically, gene regulation by Hth + Tsh, not Sd, is critical for cell survival in this tissue. This conclusion is also supported by the finding that Hth + Tsh can induce proliferation in the absence of sd (Peng, 2009).

As shown previously, Hth + Tsh play a key role in blocking eye differentiation by repressing the retinal determination genes eya and so. The available data do not yet resolve whether this repression works independently of the Hippo pathway. In contrast, the loss of Hippo kinase activity leads to overgrowths without blocking differentiation, arguing that nuclear Yki promotes proliferation without changing cell fate. Consistently, it was found that wts- or Yki+ clones do not alter Elav expression in differentiated photoreceptors. Curiously, however, ectopic expression of Hth + Tsh did not block differentiation in the absence of Yki. Although these data could be interpreted to suggest that Yki is directly required for repressing differentiation, they could alternatively suggest that repression requires cell proliferation. Consistently, Hth + Tsh were also unable to block differentiation in the absence of bantam. These observations raise the possibility that the absence of bantam or yki indirectly inhibits Hth + Tsh's ability to repress differentiation by compromising the proliferation of these cells, although other indirect affects are also possible (Peng, 2009).

Hth + Tsh are also likely to regulate genes in addition to bantam to promote proliferation and survival in the eye progenitor domain. This is most strongly supported by the observation that ectopic expression of bantam only partially rescues the survival of hthP2 clones. In addition, it was found that the overgrowths generated by ectopic expression of Hth + Tsh are only partially suppressed by the coexpression of Hpo, whose overexpression removes Yki from the nucleus. These data suggest that some of the Hth + Tsh targets that mediate growth and survival in the eye progenitor domain are regulated independently of Yki (Peng, 2009). In summary, these results suggest that the transcriptional regulation of hth and tsh along the anterior-posterior axis of the eye disc changes the output of the Hippo pathway. In the eye progenitor domain, where Hth and Tsh are both present, the pathway uses these transcription factors to promote proliferation and cell survival, at least in part by up-regulating bantam. Once hth and tsh are repressed by signals coming from the MF, the Hippo pathway may use other transcription factors, such as Sd, to regulate a different set of target genes. Thus, together with other functions carried out by these transcription factors, their regulation across the anterior-posterior axis coordinates the complex switch from proliferation to differentiation during eye development (Peng, 2009).

A screen for conditional growth suppressor genes identifies the Drosophila homolog of HD-PTP as a regulator of the oncoprotein Yorkie

Mammalian cancers depend on 'multiple hits,' some of which promote growth and some of which block apoptosis. A screened was performed for mutations that require a synergistic block in apoptosis to promote tissue overgrowth and myopic (mop), the Drosophila homolog of the candidate tumor-suppressor and endosomal regulator His-domain protein tyrosine phosphatase (HD-PTP), was identified. Myopic was found to regulate the Salvador/Warts/Hippo (SWH) tumor suppressor pathway: Myopic PPxY motifs bind conserved residues in the WW domains of the transcriptional coactivator Yorkie, and Myopic colocalizes with Yorkie at endosomes. Myopic controls Yorkie endosomal association and protein levels, ultimately influencing expression of some Yorkie target genes. However, the antiapoptotic gene diap1 is not affected, which may explain the conditional nature of the myopic growth phenotype. These data establish Myopic as a Yorkie regulator and implicate Myopic-dependent association of Yorkie with endosomal compartments as a regulatory step in nuclear outputs of the SWH pathway (Gilbert, 2011).

This study describes a screening strategy to identify mutations in Drosophila that require a synergistic block in cell death in order to promote tissue overgrowth. Using this approach, the endosomal protein Myopic, which is the Drosophila homolog of the candidate mammalian tumor suppressor HD-PTP, was identified as a regulator of the SWH growth inhibitory pathway. Through multiple approaches, this study demonstrates that Mop regulates Yki activity via a mechanism involving direct binding and modulation of Yki endosomal association (Gilbert, 2011).

This study defines a pool of cytoplasmic Yki that binds Mop and colocalizes with it on endosomes. Data from discs and cultured cells indicate Mop controls endosomal association of this pool of Yki and that a positive correlation exists between Yki colocalization with EEA1-positive early endosomes, and Yki levels and activity. A growing body of genetic and molecular data support a role for endosomes as key signaling centers for signal transduction pathways that influence the nuclear translocation of latent cytoplasmic transcription factors. For example, the activated c-Met receptor associates with the STAT3 transcription factor on EEA1-positive endosomes prior to STAT3 nuclear accumulation, and c-Met delivery to a perinuclear endosomal compartment is necessary to sustain nuclear STAT3. The enrichment of Yki on EEA1 endosomes and activation of a subset of Yki nuclear targets in mop mutant cells suggests that Yki, perhaps in association with receptor complexes, may take a similar route to the nucleus. Intriguingly, microtubule-regulated perinuclear transport of Merlin (Mer) controls nucleocytoplasmic shuttling of Yki). The direct link between Mer transport and Yki shuttling is not clear. However, as Mer can control internalization of transmembrane receptors, perinuclear transport of Mer might in turn modulate endosomal internalization and transit of Yki:receptor complexes en route to the nucleus (Gilbert, 2011).

Genetic data show that exogenous Mop is sufficient to restrict ectopic expression of the Yki-target ex but not diap1 and that loss of endogenous Mop upregulates a set of Yki targets that do not include diap1. Mop thus appears to define a regulatory step in determining outputs of the SWH pathway, perhaps as part of the endosomal sorting process. Trafficking of transmembrane proteins down alternate endosomal routes contributes to the activation of different nuclear programs in the Notch, Jak/STAT and Akt pathways. Similarly, association of Yki-containing complexes with different endosomal compartments may shift Yki nuclear output, perhaps by bringing Yki into contact with post-translational modifiers or binding partners that affect its ability to activate its suite of target promoters. Further studies will be required to establish whether loss of Mop indeed alters Yki post-translational modification or the assembly of Yki transcriptional complexes (Gilbert, 2011).

In the context of SWH signaling, the differential effect of mop loss on ex and diap1 expression place Mop within the growth-regulatory arm of the SWH network. Differential effects on the growth and apoptotic outputs of the SWH pathway is also a feature of mutations in ex and mer, which preferentially drive Yki-dependent clonal growth or anti-apoptotic signals respectively and whose combined mutant phenotypes are more severe than those of single mutants. The synergy between ex and mop alleles on IOC number extends this model and supports the hypothesis that Ex is downstream of wts in growth control but upstream of wts in apoptotic control (Gilbert, 2011).

mop mutant cells undergo high rates of caspase-dependent apoptosis in developing eye and wing imaginal discs. It is probable that this apoptosis is not caused by an effect on diap1 expression but rather a requirement for Mop in additional pro-survival mechanisms. Knockdown of vertebrate HD-PTP/PTPN23 elevates levels of tyrosine phosphorylated focal adhesion kinase (FAK), which is implicated in cell migration and integrin-mediated survival signals. Mop facilitates trafficking of the EGFR receptor into late endosomal compartments and promotes Ras/MAPK signaling downstream of EGFR in the developing retina. Because the Ras/MAPK module is required to restrain cell death pathways, reduced EGFR-dependent signaling seems likely to contribute to a subset of the apoptotic phenotype of mop mutant cells (Gilbert, 2011).

The Mop:Yki interaction involves a WW:PPxY interaction mechanism shared by the SWH proteins Ex, Wts, and Hpo that can bind Yki directly and regulate its activity independent of S168 phosphorylation status. Mop represses growth driven by the YkiS3A mutant, indicating that its repressive mechanism is not dependent on Wts kinase activity. As Mop controls the distribution of Yki across endosomal compartments, the paired Bro1 and PPxY domains in Mop could function as a bridge between Yki-containing SWH signaling complexes in the cytoplasm and complexes on the outer membrane of endosomes such as ESCRTs. These complexes could be fairly static or they could assemble and disassemble in response to specific signals. The fact that Mop, Ex, Hpo and Wts share a WW:PPxY binding mechanism suggests these proteins might compete for Yki binding in the cytosol, or that Mop acts as an endocytic scaffolding factor in a 'hand-off' mechanism from the upstream components Ex, Hpo, and Wts. Indeed understanding the dynamics and composition of the Mop:Yki complex is a significant question going forward. Intriguingly loss of the Lgl kinase, which regulates cell polarity and membrane compartmentalization, elevates Yki activity by mislocalizing Hpo and the SWH component RASSF in the cytoplasm of disc cells, suggesting that Hpo and RASSF proteins participate in dynamic and localized interactions in the cytoplasm that are important for their Yki-regulatory function (Gilbert, 2011).

The human HD-PTP/PTPN23 gene resides in a region of the genome (3p21.3) associated with loss-of-heterozygosity (LOH) in greater than 90% of small cell (SCLC) and non-small cell (NSCLC) lung cancers. Yap protein is predominantly nuclear in a subset of primary NSCLC samples, promotes cell proliferation and invasion in NSCLC cell lines, and its expression correlates with poor prognosis in NSCLC patients. Thus mutations that deregulate Yap levels and activity are predicted to promote the inappropriate growth and invasiveness of lung epithelial cells. The mechanism of growth suppression by HD-PTP is not known, but its ability to suppress colony formation of human renal cancer cells is independent of catalytic PTPase activity in much the same way regulation of Yki by Mop does not require PTPase activity. Although HD-PTP lacks a canonical PPxY motif, genetic data indicate that Mop retains the ability to inhibit Yap activity in the Drosophila eye. The extent to which HD-PTP binds Yap or Taz has yet to be examined, but if the relationship between the orthologous Drosophila proteins is conserved in vertebrates, this link to Yki/Yap may contribute to growth regulatory roles of vertebrate HD-PTP proteins in development and disease (Gilbert, 2011).

Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila

The conserved Hippo tumor suppressor pathway is a key kinase cascade that controls tissue growth by regulating the nuclear import and activity of the transcription co-activator Yorkie. This study reports that the actin-Capping Protein αβ heterodimer, which regulates actin polymerization, also functions to suppress inappropriate tissue growth by inhibiting Yorkie activity. Loss of Capping Protein activity results in abnormal accumulation of apical F-actin, reduced Hippo pathway activity and the ectopic expression of several Yorkie target genes that promote cell survival and proliferation. Reduction of two other actin-regulatory proteins, Cofilin and the cyclase-associated protein Capulet, cause abnormal F-actin accumulation, but only the loss of Capulet, like that of Capping Protein, induces ectopic Yorkie activity. Interestingly, F-actin also accumulates abnormally when Hippo pathway activity is reduced or abolished, independently of Yorkie activity, whereas overexpression of the Hippo pathway component expanded can partially reverse the abnormal accumulation of F-actin in cells depleted for Capping Protein. Taken together, these findings indicate a novel interplay between Hippo pathway activity and actin filament dynamics that is essential for normal growth control (Fernández, 2011).

The Hippo pathway has emerged as a crucial regulator of tissue size in both Drosophila and mammals. In Drosophila, the Hpo pathway comprises a kinase cascade in which the Hpo kinase binds the WW domain adaptor protein Salvador (Sav) to phosphorylate and activate the Warts (Wts) kinase. Wts, in turn, facilitated by Mats, phosphorylates and prevents nuclear translocation of the transcriptional co-activator Yorkie (Yki). This leads to transcriptional downregulation of target genes that positively regulate cell growth, survival and proliferation, including the Drosophila inhibitor of apoptosis protein 1 (Diap1; thread - FlyBase), expanded (ex), Merlin (Mer) and wingless (wg) in the inner distal ring, within the proximal wing imaginal disc. The upstream components Ex, Hpo and Wts are also thought to regulate Yki through a phosphorylation-independent mechanism, by directly binding to Yki, sequestering it in the cytosol and thereby abrogating its nuclear activity (Fernández, 2011).

Multiple upstream inputs are known to regulate the core Hpo kinase cassette at various levels. Thus, the atypical cadherin Fat was identified as an upstream component of the Hpo pathway and was proposed to transduce signals from the atypical cadherin Dachsous (Ds) and Four-jointed (Fj), a Golgi-resident kinase that phosphorylates Fat and Ds. Moreover, the two Ezrin-Radixin-Moesin (ERM) family members, Ex and Mer have been reported to lie upstream of the Hpo pathway. Mer and Ex can heterodimerize and are believed to exert their growth suppression activity by activating the Hpo kinase. However, how the different inputs that feed into the core kinase cassette are coordinated to regulate Yki activity is unknown (Fernández, 2011).

ERM proteins form a structural linkage between transmembrane components and actin filaments (F-actin). For instance, mammalian Mer binds numerous cytoskeletal factors, including actin, and appears to act as an inhibitor of actin polymerization. Interestingly, the Merlin-actin cytoskeleton association is required for growth suppression and inhibition of epidermal growth factor (EGFR) signaling. Moreover, F-actin depolymerization promotes activation of the Hpo orthologs MST1 and MST2 in mouse fibroblasts (Densham, 2009). These observations suggest a role for F-actin dynamics in modulating Hpo pathway activity (Fernández, 2011).

Actin filament growth, stability and disassembly are controlled by a plethora of actin-binding proteins. Among these, the Capping Protein (CP) heterodimer, composed of α (Cpa) and β (Cpb) subunits, acts as a functional heterodimer to restrict the accessibility of the filament barbed end, inhibiting addition or loss of actin monomers (Cooper, 2008). In Drosophila, mutations in either cpb, lead to accumulation of F-actin within the cell and give rise to identical developmental phenotypes that are tissue specific. In the wing blade (BL), the most distal domain of the imaginal disc, cpa and cpb prevent cell extrusion and death, but they are not required for this function in the most proximal domain, the prospective body wall and the hinge wing imaginal disc (Janody, 2006). The Cofilin homolog Twinstar (Tsr) and the Cyclase-associated protein Capulet (Capt) also restrict actin polymerization: Tsr severs filaments and enhances dissociation of actin monomers from the pointed end, whereas Capt sequesters actin monomers, preventing their incorporation into filaments (Fernández, 2011).

This study investigated the relationship between the control of the actin cytoskeleton and Hpo pathway activity. Actin-binding proteins CP and Capt, but not Tsr, were shown to enhance Hpo signaling activity. Moreover, a new relationship was uncovered between the Hpo pathway and the machinery that regulates F-actin, and it was revealed that Hpo signaling activity, like CP, limits F-actin accumulation at apical sites independently of Yki. Finally, it is proposed that regulation of an apical F-actin network by Hpo signaling activity and CP sustains Hpo pathway activity, thereby limiting Yki nuclear import and the activation of proliferation and survival genes (Fernández, 2011).

This report shows an interdependency between Hpo signaling activity and F-actin dynamics in which CP and Hpo pathway activities inhibit F-actin accumulation, and the reduction in F-actin in turn sustains Hpo pathway activity, preventing Yki nuclear translocation and upregulation of proliferation and survival genes (Fernández, 2011).

It is suggested that Ex prevents apical F-actin accumulation through Hpo signaling activity but independently of Yki. ERM proteins can form a structural linkage between transmembrane components and the actin cytoskeleton. Mammalian Mer appears to act as an inhibitor of actin polymerization. Moreover, the Mer-actin cytoskeleton association has a crucial role for growth suppression and inhibition of EGFR signaling. In Drosophila, Mer and Ex are structurally related and appear to have partially redundant functions but vary in their requirement depending on the tissue or developmental stage. In imaginal discs, loss of ex shows stronger phenotypes when compared with those of Mer. Ex might also have a stronger requirement on F-actin dynamics, as loss of ex, but not that of Mer, triggered F-actin accumulation. Surprisingly, loss of hpo, sav, mats or wts also triggered apical F-actin accumulation. Ex is likely to affect F-actin through activation of the Hpo kinase cassette because in most ex mutant clones, overexpressing hpo suppressed F-actin accumulation. Some clones seemed to contain increased F-actin. However, these clones also constricted apically, suggesting that the effect on F-actin levels results from a reduction of the apical cell surface and that in the absence of ex, differential activity of overexpressed hpo triggers cell shape changes. Together, these observations argue that Ex prevents apical F-actin accumulation through Hpo signaling activity but independently of Yki (Fernández, 2011).

Loss of Hpo pathway activity or CP triggerw apical F-actin accumulation. Ex localizes to the sub-apical region of epithelial cells, and colocalizes with an HA-tagged form of Cpa, Ex, Hpo, Sav and Wts all interact with each other through WW and PPXY motifs (Oh, 2009; Reddy, 2008). Therefore, a pool of Hpo, Sav and Wts, localized at apical sites, could directly regulate an actin-regulatory protein. Hpo pathway activity might act downstream of CP on F-actin. In agreement with this, ex overexpression significantly suppresses F-actin accumulation in cells with reduced CP levels. The role of Hpo signaling activity might be to inhibit an actin-nucleating factor, which adds new actin monomers to filament barbed ends free of the capping activity of CP. However, it cannot be excluded that ex overexpression enhances the activity of residual Cpa in cells knocked down using RNAi, nor that Hpo pathway activity acts in parallel to CP on F-actin. Interestingly, although endogenous Ex is upregulated in cells lacking CP, mutant cells still accumulated F-actin. wts mutant clones also upregulated Ex, which, when overexpressed, suppresses growth of wts mutant clones. Therefore, the increased levels of endogenous Ex in cells lacking either CP or wts appears to be insufficient to fully suppress the effects of loss of CP or wts on F-actin and growth, respectively (Fernández, 2011).

The data indicate that CP inhibits Yki nuclear accumulation, activation of Yki target genes, and consequently overgrowth of the proximal wing epithelium. Interestingly, Yki was also found to accumulate in nuclei of wild-type cells adjacent to the clone border. Consistent with a non-autonomous effect of CP loss on Hpo pathway activity, ex-lacZ and diap1-lacZ were upregulated in wild-type cells adjacent to CP mutant clones. However, Ex levels were reduced in wild-type neighboring cells. Cells expressing different amounts of ds and fj also upregulate ex-lacZ, but show reduced levels of Ex. Therefore, loss of CP might affect Fj or Ds levels, creating a sharp boundary of their expression. However, in contrast to clones overexpressing ds or mutant for fj, cell lacking CP also upregulated Ex and Mer inside the mutant clones, indicating that CP also acts cell-autonomously to promote Hpo signaling activity. CP might facilitate Yki phosphorylation by the Hpo kinase cassette as cpa-depleted tissues contain decreased phospho-Yki levels. But, the possibility cannot be excluded that CP also favors the direct binding of non-phosphorylated Yki to Ex, Hpo or Wts (Oh, 2009). Further analysis will be required to elucidate the mechanisms by which CP restricts Yki activity cell autonomously and in wild-type neighboring cells (Fernández, 2011).

The results argue for a constitutive role of CP in Hpo pathway activity, since Yki target genes are upregulated in the whole wing and eye imaginal discs. However, loss of CP did not fully recapitulate the phenotype for core components of the hpo pathway. Despite that, on average, cpb mutant clones located in the proximal wing disc domain were 25% larger than wild-type twin spots; 60% of mutant clones failed to grow. Moreover, in the distal wing epithelium, reducing CP levels induces mislocalization of the adherens junction components Armadillo and DE-Cadherin, extrude and death. Furthermore, in Drosophila, CP also prevents retinal degeneration (Delalle, 2005; Johnson, 2008). This indicates that although loss of CP can, under certain conditions, result in tissue overgrowth due to inhibition of Hpo pathway activity, other factors such as the polarity status, also determine the survival and growth of the mutant tissue. Therefore, in addition to promoting Hpo pathway activity, CP has additional developmental functions in epithelia. However, the possibility cannot be excluded that, like most upstream inputs that feed into the Hpo pathway, CP has a tissue-specific requirement in Hpo pathway activity (Fernández, 2011).

CP, Capt and Tsr all restrict F-actin assembly directly. CP and Capt control F-actin formation near the apical surface and inhibit ectopic expression of Yki target genes, whereas Tsr acts around the entire cell cortex and has no effect on Yki target genes. This argues that Hpo signaling activity is not affected by the excess of F-actin per se but provides significant support to the view that stabilization of an apical F-actin network by CP, Capt and Hpo signaling activity feeds back on the Hpo pathway to sustain its activity (Fernández, 2011).

These findings do not lead to an understanding of where F-actin accumulation intersects Hpo signaling activity because both Hpo signaling activity and F-actin dynamics feedback to each other. For instance, hpo or ex overexpression suppressed growth of CP-depleted cells. But, overexpressed ex and possibly hpo also suppress F-actin accumulation of Cpa-depleted cells. The control of F-actin by Hpo signaling activity and CP might constitute a parallel input, which sustains Hpo pathway activity. Alternatively, F-actin could act upstream of the core kinase cascade, which in turn feeds back to F-actin, to maintain its activity. The identification of additional actin cytoskeletal components that either promote Hpo pathway activity or act downstream of Hpo pathway activity on F-actin would help to discriminate between these possibilities (Fernández, 2011).

How F-actin influences Hpo signaling activity remains to be determined. The apical F-actin network, which regulates the formation and movement of endocytic vesicles from the plasma membrane, might promote the recycling or degradation of Hpo pathway components. Increased F-actin at apical sites would, therefore, affect protein turnover. Alternatively, apical F-actin might act as a scaffold to tether Hpo pathway components apically. In support of this, Ex, Hpo, Sav, Wts and Yki could all interact between each other through WW and PPXY motifs at apical sites (Oh, 2009; Reddy, 2008). Moreover, expression of a membrane-targeted form of Mats enhances Hpo signaling (Ho, 2010). Although Ex and Mer are properly localized in CP mutant cells, other members of the pathway might be mislocalized in the presence of excess F-actin. Interestingly, in mouse fibroblasts, the Hpo orthologs MST1 and MST2 colocalize with F-actin structures and are activated upon F-actin depolymerization (Densham, 2009), suggesting that by tethering Hpo pathway components, F-actin dynamics modulates their activity. Finally, the F-actin network might act as a mechanical transducer. Most of the mechanosensitive responses require tethering to force-bearing actin filaments. Tissue surface tension has been proposed to be a stimulus for a feedback mechanism that could regulate tissue growth. The tension exerted by neighboring cells might be sensed at the cell membrane by the actin cytoskeleton and translated to the regulation of cell proliferation through the Hpo signaling pathway (Fernández, 2011).

Akt is negatively regulated by Hippo signaling for growth inhibition in Drosophila

Tissue growth is achieved through coordinated cellular growth, cell division and apoptosis. Hippo signaling is critical for monitoring tissue growth during animal development. Loss of Hippo signaling leads to tissue overgrowth due to continuous cell proliferation and block of apoptosis. As cells lacking Hippo signaling are similar in size compared to normal cells, cellular growth must be properly maintained in Hippo signaling-deficient cells. However, it is not clear how Hippo signaling might regulate cellular growth. This study shows that loss of Hippo signaling increases Akt (also called Protein Kinase B, PKB) expression and activity, whereas activation of Hippo signaling reduces Akt expression in developing tissues in Drosophila. While yorkie is sufficient to increase Akt expression, Akt up-regulation caused by the loss of Hippo signaling is strongly dependent on yki, indicating that Hippo signaling negatively regulates Akt expression through Yki inhibition. Consistently, genetic analysis reveals that Akt plays a critical role in facilitating growth of Hippo signaling-defective tissues. Thus, Hippo signaling not only blocks cell division and promotes apoptosis, but also regulates cellular growth by inhibiting the Akt pathway activity (Ye, 2012).

Growth inhibition mediated by Hippo signaling is essential for tissue growth and organ size control. Loss of Hippo signaling generates extra cells with their size similar to normal cells, suggesting that both cell division and cellular growth are promoted in cells lacking Hippo signaling activity. If Hippo signaling is only involved in inhibiting cell division, loss of Hippo signaling would result in extra cells that are smaller than normal cells. By altering activities of some cell-cycle regulators, it has been previously shown that an increase of cell division rate is insufficient to drive cellular growth, and therefore, cell division and cellular growth can be separately regulated. In the case of Hippo signaling, this growth-inhibitory pathway appears to play an active role to negatively control both cell division and cellular growth. This study found that akt expression is negatively regulated by Hippo signaling as a way to reduce the Akt pathway activity. Moreover, growth-promoting factor Yki is required for activating akt expression in developing tissues. The genetic evidence is also consistent with a role of akt as a critical downstream target of Hippo signaling. Thus, these results support a model in which Hippo signaling negatively regulates akt expression through Yki inhibition to coordinate cellular growth and cell division and ultimately control tissue and organ size during Drosophila development (Ye, 2012).

Because the DNA-binding protein Scalloped (Sd) interacts with Yki to regulate transcription of downstream target and three putative Sd-binding sites were found in the downstream intergenic region within a 30-kb akt genomic region, the potential enhancer activity of these elements in mediating the transcription activation property of Yki was tested. Two genomic fragments that contain these sites were tested for their potential enhancer activity to drive a GFP reporter gene expression. Neither fragment was able to respond to Yki overexpression to activate gene transcription in cultured Drosophila S2 cells, whereas a previously identified diap1 enhancer that contains Sd-binding sites was able to activate gene expression in responding to Yki. Furthermore, from a dataset generated by the Drosophila Regulatory Elements modENCODE Project, a genome-wide ChIP analysis did not detect the akt locus as an obvious target of Yki. Therefore, how Yki functions to directly or indirectly to control akt expression will need to be further investigated (Ye, 2012).

Since bantam (ban) miRNA has been shown to act autonomously to increase Akt expression in epithelial cells and non-autonomously to decrease Akt expression in neighboring neuronal cells, whether ban miRNA is involved in regulating the level of akt expression was tested by expressing ban in Drosophila S2 cells as well as larval wing discs. Preliminary data showed that ban overexpression can slightly increase the level of Akt protein. Thus, while ban is a critical downstream target of yki, ban might contribute to the upregulation of Akt expression in tissues such as larval wing discs during development (Ye, 2012).

As the Hippo pathway is highly conserved in evolution, it is possible that Akt regulation by Hippo signaling also occurs in mammalian cells. Indeed, knockdown of LATS1 in human MCF10A breast epithelial cells activated the AKT pathway as shown by the increased levels of activated AKT kinase protein, although the total Akt protein level was not. Interestingly, AKT upregulation caused by reduction of LATS1 function is critically dependent on YAP activity. However, this regulation of AKT activity is likely mediated at the post-translational level because the total AKT protein level was not changed by LATS1 knockdown or LATS1/YAP double knockdown. Although the mechanism of this AKT inhibition by Hippo signaling in mammalian cells is currently uncharacterized, clarification of how Hippo signaling can negatively regulate the AKT pathway activity would reveal mechanisms by which these two important cellular signaling pathways cross-talk for a proper developmental control of cell growth, cell division and cell death. More excitingly, this study implicates that Hippo signaling might influencing the AKT pathway activity for its nutrient control of growth, homeostasis, and longevity in animals (Ye, 2012).

The cell adhesion molecule echinoid functions as a tumor suppressor and upstream regulator of the Hippo signaling pathway

The Hippo (Hpo) signaling pathway controls tissue growth and organ size in species ranging from Drosophila to mammals and is deregulated in a wide range of human cancers. The core pathway consists of the Hpo/Warts (Wts) kinase cassette that phosphorylates and inactivates the transcriptional coactivator Yorkie (Yki). This study reports that Echinoid (Ed), an immunoglobulin domain-containing cell adhesion molecule, acts as an upstream regulator of the Hpo pathway (see Ed regulates organ size through the Hpo-Yki pathway). Loss of Ed compromises Yki phosphorylation, resulting in elevated Yki activity that increases Hpo target gene expression and drives tissue overgrowth. Ed physically interacts with and stabilizes the Hpo-binding partner Salvador (Sav) at adherens junctions. Ed/Sav interaction is promoted by cell-cell contact and requires dimerization of Ed cytoplasmic domain. Overexpression of Sav or dimerized Ed cytoplasmic domain suppressed loss-of-Ed phenotypes. It is proposed that Ed may link cell-cell contact to Hpo signaling through binding and stabilizing Sav, thus modulating the Hpo kinase activity (Yue, 2012).

The Hpo signaling pathway has emerged as a conserved regulatory pathway that controls tissue growth and organ size. Although the core pathway components (i.e., the Hpo/Sav/ Wts/Mats kinase cassette and its effector Yki/Yap), have been well defined, the upstream regulators, especially the membrane receptors that link cell-cell communication to Hpo signaling, remain poorly defined. This study provides both genetic and biochemical evidence that the transmembrane cell adhesion molecule Ed functions as a upstream regulator of the Hpo pathway. Evidence is provided that Ed physically interacts with Sav/Hpo and regulates the abundance of Sav at adherens junctions. Loss of Ed compromises the ability of Hpo/Wts kinase cassette to phosphorylate Yki, leading to elevated levels of nuclear Yki that drive tissue overgrowth. Ed/Sav association is facilitated by cell-cell contact, raising an interesting possibility that Ed may serve as a mechanism that links Hpo signaling to cell contact inhibition (Yue, 2012).

The atypical cadherin Ft functions as a receptor for the Hpo pathway; however, Ft mainly acts through Dachs to control the stability of Wts. Genetic study indicated that Ed does not act through Ft-Dachs to regulate Yki activity because inactivation of Dachs did not block Yki activation induced by loss of Ed. Furthermore, loss of Ed synergized with loss of Ds to induce the expression of Hpo-responsive genes, supporting a model in which Ed acts in parallel with Ds/Ft in the Hpo pathway. Several lines of evidence suggest that Ed regulates Hpo signaling, at least in part, through Sav. (1) Using coimmunoprecipitation, colocalization, and FRET assays, it was demonstrated that Ed physically interacts with Sav. (2) Deleting the Sav-interacting domain in Ed compromises its in vivo activity. (3) Ed regulates the abundance and subcellular localization of Sav both in vitro and in vivo. (4) Overexpression of Sav suppresses tissue overgrowth induced by loss of Ed. Sav is a binding partner and activator of Hpo. Hence, Ed could influence the Hpo kinase activity by regulating the abundance and subcellular location of the Sav/Hpo complex. How Ed regulates Sav stability is currently unknown; however, it was found that Sav is degraded in a proteasome-dependent manner. It is possible that binding of Ed to Sav leads to some modifications of Sav and prevents it from ubiquitin/proteasome-mediated degradation (Yue, 2012).

Sav binds Ed and Hpo through its N- and C-terminal regions, respectively, and thus may function as a bridge to bring Hpo to Ed. Indeed, enhanced Ed/Hpo association was observed in the presence of cotransfected Sav. It has been suggested that apical membrane recruitment of Hpo promotes phosphorylation of Wts. Thus, it is conceivable that Ed may regulate the Hpo kinase by recruiting Sav/Hpo complex to the apical membrane. It was found that Ed/Sav interaction requires membrane association and dimerization/oligomerization of Ed intracellular domain. As Sav also forms a dimer/oligomer, dimerization/oligomerization of Ed intracellular domain may enhance binding to Sav through multimeric interactions. It is also possible that membrane association and dimerization/oligomerization could lead to a modification of Ed intracellular domain, resulting in increased binding affinity toward Sav (Yue, 2012).

It has been shown that the Hpo pathway can mediate cell contact inhibition in mammalian cultured cells, although the underlying mechanism has remained poorly defined. Interestingly, this study found that cell-cell contact can facilitate the recruitment of Sav to Ed at the contact site. Cell-cell contact may facilitate homophilic interaction in trans and clustering of Ed intracellular domain or induce posttranslational modification of Ed C-tail at the contact site, leading to enhanced Sav association. It is proposed that regulation of Ed/Sav association may provide a mechanism for cell-cell contact to modulate Hpo signaling and tissue growth (Yue, 2012).

The mechanism by which Ed regulates Hpo signaling is likely to be more complex than simply regulating Sav/Hpo. For example, it was also observed that Ed interacts with Ex/Mer/Kibra as well as Yki. It has been proposed that enrichment of Hpo pathway components to the apical membrane domain may facilitate the activation of the kinase cassette and increase the accessibility of Yki to its kinase (Genevet, 2011). The finding that Ed facilitates the apical localization of Sav lends further support to this notion. Through interacting with multiple components of the Hpo pathway, Ed could function as a molecular scaffold to facilitate Hpo activation and Yki phosphorylation. Loss of Ed did not alter the apical membrane localization of Ex and Mer in wing discs even though overexpression of Ed in S2 cells facilitates membrane recruitment of Ex. The apical localization of Ex and Mer is likely to be mediated by other upstream components such as Ft and Crb in the absence of Ed. Indeed, Crb physically interacts with Ex, and both loss and gain of function of Crb caused mislocalization of a fraction of Ex to the basal region. It has been shown that Ex physically interacts with Yki, which may sequester Yki in the cytoplasm independent of Yki phosphorylation. The finding that Ed interacts with Yki through a domain distinct from those mediating its binding to the upstream Hpo pathway components raises a possibility that Ed may also directly sequester Yki in the cytoplasm in addition to regulating its subcellular localization through phosphorylation (Yue, 2012).

It is interesting to note that Ed is related to TSLC1, a tumor suppressor implicated in human non-small-cell lung cancer and other cancers including liver, pancreatic, and prostate cancers. Like Ed, TSLC1 also mediates cell-cell adhesion through homophilic interactions. TSLC1 interacts with MPP3, a human homolog of Drosophila tumor suppressor Discs large (Dlg) that has been implicated in the Hpo pathway, as well as DAL-1, a FERM-domain containing tumor suppressor related to Ex/Mer. Therefore, it would be interesting to determine whether TSLC1 inhibits tumor formation through the Hpo pathway (Yue, 2012).

Mutual repression by bantam miRNA and Capicua links the EGFR/MAPK and Hippo pathways in growth control

The epidermal growth factor receptor (EGFR) and Hippo signaling pathways control cell proliferation and apoptosis to promote tissue growth during development. Misregulation of these pathways is implicated in cancer. Understanding of the mechanisms that integrate the activity of these pathways remains fragmentary. This study identifies bantam microRNA as a common target of these pathways and suggests a mechanistic link between them. The EGFR pathway acts through bantam to control tissue growth. bantam expression is regulated by the EGFR pathway, acting via repression of the transcriptional repressor Capicua. Thus EGFR signaling induces bantam expression by alleviating the effects of a repressor. bantam in turn acts in a negative feedback loop to limit Capicua expression. bantam appears to be a transcriptional target of both the EGFR and Hippo growth control pathways. Feedback regulation by bantam on Capicua provides a means to link signal propagation by the EGFR pathway to activity of the Hippo pathway and may play an important role in integration of these two pathways in growth control (Herranz, 2012).

The ability of the EGFR pathway to drive tissue growth resides in its ability to coordinately stimulate cell proliferation and suppress apoptosis. Understanding how coordinated control is achieved depends on identification of the effector mechanisms that mediate these outputs along with the connections to other growth regulatory pathways. The results show that the bantam miRNA is a critical target of the EGFR pathway. Further, a mechanism is outlined by which bantam serves as a link between the EGFR and Hippo pathways (Herranz, 2012).

In Drosophila, EGFR pathway effectors include the transcription factors Pointed, Tramtrack, and Yan, and the HMG-box repressor Capicua. Capicua has an important role in early embryonic patterning and as a negative growth regulator. Although several Capicua targets involved in embryonic patterning have been identified, how Capicua regulates tissue growth was unknown. These results identify bantam as an important target of Capicua required to mediate EGFR-dependent tissue growth (Herranz, 2012).

A key finding of this study is the regulatory feedback relationship between bantam and Capicua. Each represses the activity of the other. Viewed from the perspective of the EGFR/MAPK pathway alone, the outcome of this relationship would be signal amplification, with downregulation of Capicua levels by bantam reinforcing direct MAPK-induced turnover of Capicua protein. This adds a new mechanism to the repertoire of positive and negative feedback loops affecting EGFR pathway activity. These feedback mechanisms are thought to be important in disease, and their regulation is complex. Relatively little is known about Capicua in cancer, although one recent study reports mutants in the human Cic protein in oligodendroglioma (Bettegowda, 2012; Herranz, 2012 and references therein).

An alternative logic for the relationship between bantam and Capicua may be seen in the fact that it links the output of the EGFR pathway to the output of the Hippo pathway, mediated through transcriptional regulation of bantam by Yorkie. EGFR signaling via MAPK and bantam cooperate to downregulate Capicua protein levels. Thus the transcriptional output of the Hippo pathway via Yorkie can be seen as potentiating EGFR signaling by 'lowering' the effective threshold of MAPK activity needed to reduce Capicua to a given level. Alternatively, the lack of sufficient Yorkie activity would lower bantam activity and thereby raise the threshold of EGFR activity required to reach an effective level of Capicua downregulation. This provides a mechanism to ensure coordination of the growth regulatory pathways. Signaling via the Hippo pathway has also been shown to induce the EGFR ligand amphiregulin to promote tissue growth in a nonautonomous manner. Thus, there appear to be multiple levels of crosstalk between these pathways (Herranz, 2012).

Considerable evidence is emerging linking miRNAs to robustness of regulatory feedback networks. It is intriguing that miRNAs are now implicated in regulation of all three of the known transcriptional effectors of EGFR signaling. miR-7 acts in two feed-forward loops downstream of EGFR to control photoreceptor specification and differentiation in the Drosophila eye. EGFR acts via the transcription factors Yan and Pointed. Yan is a direct target of miR-7. Yan also represses miR-7 transcription directly as well as indirectly. In the same cells, the ETS-1 factor Pointed-P1 activates miR-7 to repress Yan as well as acting directly to repress Yan. Use of interlinked motifs is thought to provide stability to the cell differentiation program controlled by EGFR. The current findings link bantam to regulation of a third EGFR transcriptional effector, Capicua, in addition to its regulation by the Hippo pathway. Coordination of diverse growth control inputs by miRNAs might contribute to robustness (Herranz, 2012).

Notch signaling activates Yorkie non-cell autonomously in Drosophila

In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. This study shows that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. These data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth (Graves, 2012).

This study identifies a novel role of Notch signaling for non-cell autonomous control of apoptosis via induction of Yki activity in neighboring cells. It has previously been shown that Notch signaling controls cell proliferation both autonomously and non-cell autonomously in the developing eye. The non-cell autonomous component of proliferation control was attributed to Notch-dependent activation of Jak/Stat signaling although that recently came into question. Nevertheless, Jak/Stat activation is not sufficient to mediate the effect of Notch on non-cell autonomous control of apoptosis. This study identified the Hpo/Wts/Yki pathway as a target of Notch signaling for the non-cell autonomous control of apoptosis both in eye and wing imaginal discs. Because the Hpo/Wts/Yki pathway also controls proliferation, it is likely that Notch promotes non-cell autonomous proliferation through both Jak/Stat and Hpo/Wts/Yki activities (Graves, 2012).

It is also interesting to note that this non-autonomous control of the Hpo/Wts/Yki pathway by Notch occurs in a position-dependent manner. For example, vps25 mutant clones or NICD-expressing clones located in the hinge and notum of wing discs triggered non-cell autonomous up-regulation of ex-lacZ, while clones in the wing pouch did not. Additionally, vps25 mutant clones located anterior to the morphogenetic furrow triggered non-cell autonomous up-regulation of ex-lacZ, while clones in the posterior of the eye disc did not. The reason for this position-dependence is unknown. However, the regions which do not induce Hpo/Wts/Yki signaling non-autonomously correspond to the zone of non-proliferating (ZNP) cells in the wing disc and post-mitotic, differentiating cells in the eye disc. Therefore, one potential reason for the position-dependence may be that the post-mitotic nature of the cells in the ZNP of the wing pouch and in the posterior of the eye disc render them inert to growth-promoting signals that trigger the Hpo/Wts/Yki pathway. However, while this is one possibility, there may also be additional mechanisms that influence the response to growth-promoting signals (Graves, 2012).

How Notch exerts this non-autonomous effect is an important and interesting question. Based on its function as a transcriptional regulator, it is possible that increased Notch signaling in vps25 mutant cells could lead to transcription of a secreted or transmembrane protein that communicates to surrounding tissue and induces Yki activity. Expression of proteins known to non-cell autonomously activate Yki signaling such as Fat, Dachsous (Ds) and Four-Jointed as well as ds-lacZ, however, are not altered in vps25 mosaic discs. Identification of this non-cell autonomous signaling mechanism may also be critical for understanding tumorigenesis, as mutations in the Notch pathway, the Hippo pathway and in ESCRT components have been implicated in many different types of human cancer. In conclusion, this study provides a mechanism by which neoplastic cells influence the behavior of neighboring wild-type cells, which may be critical for generating a supportive microenvironment for tumor growth by preventing cell death and promoting the proliferation of wild-type cells (Graves, 2012).

Insulin/IGF signaling drives cell proliferation in part via Yorkie/YAP

The insulin/IGF signaling (IIS) pathway is a potent inducer of cell proliferation in normal development and in cancer. The mechanism by which this occurs, however, is not completely understood. The Hippo signaling pathway regulates cell proliferation via the transcriptional co-activator Yorkie/YAP, however the signaling inputs regulating Hippo activity are not fully elucidated. This study presents evidence linking these two conserved, oncogenic pathways in Drosophila and in mammalian cells. It was found that activation of IIS and of Yorkie signaling correlate positively in hepatocellular carcinoma. IIS activates Yorkie in vivo, and that Yorkie plays an important role in the ability of IIS to drive cell proliferation. Interestingly, the converse was also found -- that Yorkie signaling activates components of the insulin/TOR pathway. In sum, this crosstalk between IIS and Yorkie leads to coordinated regulation of these two oncogenic pathways (Strassburger, 2012).

This study provides evidence that the insulin/IGF signaling (IIS) pathway and the Hpo/Yki signaling pathway are intricately interlinked in normal development and in cancer. In addition to observing a correlation in activation of the two pathways in hepatocellular carcinoma, mechanistic studies suggest that IIS activates Yki signaling and vice-versa. IIS appears to activate Yki via two mechanisms, a minor one involving Akt and a second major one via another target of PDK1. Both appear to function via Hpo repression. A previous report found that Akt can phosphorylate and inactivate MST1 in HeLa cells in the context of pro-apoptotic signaling. Whether a direct phosphorylation of Hpo by Akt might explain the growth effects observed was tested in this this study. However, to Akt-mediated phosphorylation of Hpo was observed either by in vitro kinase assay or by isoelectric focusing of cell extracts treated in the presence or absence of insulin. Furthermore, by expressing phospho-mimetic or alanine mutant versions of Hpo in vivo in the fly, no effect on Hpo activity was observed. In sum, no data was found supporting the idea that the growth-promoting effect of IIS occurs via direct phosphorylation of Hpo by Akt, in agreement with the finding that the major mechanism by which IIS affects Yki is Akt-independent . In addition, a previous report found that Akt might also directly phosphorylate and inhibit YAP, which is surprising given that both Akt and YAP have similar - and not antagonistic - effects on cell growth and proliferation. Another study was not able to confirm this finding leaving this an open issue. Likewise, the connections from Yki to IIS appear to be multiple, as transcriptional regulation of a large number of components of the IIS pathway was seen upon Yki activation. These interconnections lead to coordinated activation of the two pathways (Strassburger, 2012).

The main finding of this study is that insulin/IGF signaling drives proliferation partly via Hpo/Yki signaling. It was found that IIS regulates Yki activity in vivo, both at the biochemical level, and in terms of Yki transcriptional activity. Although analysis of Yki phosphorylation and of four Yki targets (DIAP1, merlin, expanded and cyclin E) consistently indicate that IIS activates Yki, surprisingly no increase was seen in expression of another yorkie target, bantam, upon expression of Dp110-CAAX, consistent with previous findings. Since bantam is an outlier compared to all other yorkie targets tested, this likely suggests bantam has other transcriptional regulatory inputs. Since the growth caused by Dp110-CAAX expression is abrogated in the absence of Hpo and highly sensitive to Yki gene dosage, this indicates that Yki is an important and limiting effector mechanism by which IIS drives tissue growth. Since the regulation of Yki/YAP by IIS is conserved from flies to humans, this mechanism likely represents an ancestral mechanism by which IIS drives tissue growth (Strassburger, 2012).

Hpo appears to be a central hub for integrating numerous inputs that affect tissue growth. The overgrowth caused by removal of upstream regulators of Hpo, such as Merlin, Expanded, Fat, Dachsous, Dco, Kibra and Crumbs, are mild in comparison to the overgrowth caused by loss of Hpo, suggesting these upstream components function in a combinatorial and additive manner to regulate Hpo. The work described in this study provides an additional input into Hpo signaling. Since IIS is responsive to nutrient conditions in addition to growth factor signaling, this links nutrient status to Hpo/Yki signaling and animal growth. It is tempting to speculate that nutrient overload and hyperactivation of the IIS pathway over an extended time in an adult organism might, via this mechanism, potentially contribute towards hyperplasia (Strassburger, 2012).

Recently, the YAP signaling pathway was found to be linked to several other oncogenic pathways including TGF-beta, beta-catenin and EGF signaling. In each case, however, the link is that YAP affects signaling through the other pathway. For instance, YAP interacts with β-catenin to regulate a subset of Wnt target genes, and YAP upregulates expression of the EGFR ligand amphiregulin to activate EGF signaling. The findings presented in this study are the first in which the link also goes in the other direction, with IIS regulating Hpo and Yki signaling, thereby adding growth factor signaling as an input into Hpo/Yki (Strassburger, 2012).

Thus far, Yki has been found to regulate expression of genes affecting mainly cell proliferation (e.g. CycE) and apoptosis (e.g. diap1). The data presented in this study suggest a mechanism by which Yki can induce tissue growth by also affecting TOR activity, thus activating both cell proliferation and cell growth in Drosophila. This further extends and is in line with recently published data that show that YAP overexpression can drive IIS in mouse cardiomyocytes (Strassburger, 2012).

In sum, the data presented in this study reveal one molecular mechanism by which IIS drives proliferation, they identify a novel input into Hpo signaling, and they elucidate cross-talk between two established oncogenic pathways of relevance for cancer development (Strassburger, 2012).

Opposite Feedbacks in the Hippo Pathway for Growth Control and Neural Fate

Signaling pathways are reused for multiple purposes in plant and animal development. The Hippo pathway in mammals and Drosophila coordinates proliferation and apoptosis via the coactivator and oncogene, YAP/Yorkie (Yki), which is homeostatically regulated through negative feedback. In the Drosophila eye, cross-repression between the Hippo pathway kinase, LATS/Warts (Wts), and growth regulator, Melted, generates mutually exclusive photoreceptor subtypes. This study shows that this all-or-nothing neuronal differentiation results from Hippo pathway positive feedback: Yki both represses its negative regulator, warts, and promotes its positive regulator, melted. This postmitotic Hippo network behavior relies on a tissue-restricted transcription factor network - including a conserved Otx/Orthodenticle-Nrl/Traffic Jam feedforward module - that allows Warts-Yki-Melted to operate as a bistable switch. Altering feedback architecture provides an efficient mechanism to co-opt conserved signaling networks for diverse purposes in development and evolution (Jukam, 2013).

A fundamental strategy in animal development is to re-purpose the same signaling pathways for a diversity of functions. This study identified a tissue-specific transcription factor network that enables the otherwise homeostatic Hippo growth control pathway to act as a bistable switch for terminal cell fate. This alteration in network level properties—such as positive versus negative feedback—within biochemically conserved pathways is an efficient means to re-use a signaling network in contexts as distinct as proliferation and terminal differentiation (Jukam, 2013).

How is the R8-specific Hippo regulatory circuit achieved? The two interlinked positive feedback loops (one with wts, one with melt) provide the R8 Hippo pathway with multiple points of potential regulation. Context-specific expression of wts and melt is defined by overlapping expression of four transcription factors: Otd, Tj, Pph13, and Sens. Otd and Pph13 are expressed in all photoreceptors and generate a permissive context that endows the initially equipotent R8s with the competence to become either subtype: Otd promotes melt/Rh5 whereas Pph13 promotes wts/Rh6 expression. This competence is further restricted by expression of Tj in R7 and R8, and Sens in R8s, which ensures that melt and wts cross-regulation is restricted to R8s. Importantly, it is the status of Yki activity and resulting feedback that assures the outcome of pR8/Rh5 vs. yR8/Rh6 (p vs. y) fate: in pR8s, Yki functions with Otd and Tj to promote melt and Rh5; in yR8s, wts inhibits Yki, preventing melt and Rh5 expression and allowing 'default' wts and Rh6 expression by Pph13 and Sens. Each of these four transcription factors regulates a partially overlapping subset of R8 subtype fate genes, and together, the network cooperates at multiple regulatory nodes to provide the specific context for repurposing the Hippo pathway (Jukam, 2013).

While other instances of pathways with both positive and negative feedback exist, these are conceptually different from R8 Hippo regulation. For example, in Sprouty (hSpry) regulation of Ras/MAPK-mediated EGFR signaling, EGFR induces hSpry2 expression but hSpry2 inhibits EGFR function (negative feedback); however, hSpry2 also promotes EGFR activity by preventing Cbl-dependent EGFR inhibition (positive feedback). hSpry2 positive feedback is likely coupled to its negative feedback to fine-tune the length and amplitude of receptor activation. In contrast, the opposite Hippo pathway feedbacks occur in vastly different cell types (mitotic epithelial cells versus post-mitotic neurons), and both forms of feedback cannot co-exist in R8 since Yki’s repression of wts expression (positive feedback) would make Yki up-regulation of Hippo regulators (negative feedback) inconsequential (Jukam, 2013).

Gaining positive feedback or losing negative feedback within Hippo signaling could permit oncogenesis. Indeed, the Yki ortholog, YAP, is an oncogene and is amplified in multiple tumors, and LATS1/2 (Wts) down-regulation is associated with non-small cell lung carcinomas, soft tissue sarcoma, metastatic prostate cancers, retinoblastoma, and acute lymphoblastic leukemia. Otx and MAF factors are also oncogenic in a number of tissues. Thus, understanding the regulatory networks identified here in other contexts will be crucial for deciphering how normal signaling pathways can go awry (Jukam, 2013).

The current findings also reveal that a Crx/Otd-Nrl/Tj feedforward module plays a conserved role in post-mitotic photoreceptor fate specification in both flies and mammals. Both induce one photoreceptor fate at the expense of another, and both regulate opsins with a feedforward loop wherein Crx/Otd activates Nrl/Tj expression and Crx-Nrl or Otd-Tj synergistically activate downstream targets (Hao, 2012). Given such deep evolutionary conservation, this module may be critical for generating photoreceptor diversity in other complex visual systems (Jukam, 2013).

This work has two main implications. First, although positive feedback is well documented in other switch-like, irreversible cell fate decisions such as in Xenopus oocyte maturation or cell cycle entry, this work suggests that positive feedback could have a broad role in terminal neuronal differentiation, which often requires permanent fate decisions to maintain a neuron’s functional identity. Second, the changes in network topology in R8 photoreceptors allows a finely tuned growth control pathway to be used as a switch in a permanent binary cell fate decision. Context-specific regulation allows the feedback architecture to change in an otherwise conserved signaling module. This may be a general mechanism to endow signaling networks with new systems properties and diversify cell fates in development and evolution (Jukam, 2013).

Divergent transcriptional regulatory logic at the intersection of tissue growth and developmental patterning

The Yorkie/Yap transcriptional coactivator is a well-known regulator of cellular proliferation in both invertebrates and mammals. As a coactivator, Yorkie (Yki) lacks a DNA binding domain and must partner with sequence-specific DNA binding proteins in the nucleus to regulate gene expression; in Drosophila, the developmental regulators Scalloped (Sd) and Homothorax (Hth) are two such partners. To determine the range of target genes regulated by these three transcription factors, genome-wide chromatin immunoprecipitation experiments for each factor was performed in both the wing and eye-antenna imaginal discs. Strong, tissue-specific binding patterns are observed for Sd and Hth, while Yki binding is remarkably similar across both tissues. Binding events common to the eye and wing are also present for Sd and Hth; these are associated with genes regulating cell proliferation and 'housekeeping' functions. In contrast, tissue-specific binding events for Sd and Hth significantly overlap enhancers that are active in the given tissue, are enriched in Sd and Hth DNA binding sites, respectively, and are associated with genes that are consistent with each factor's tissue-specific functions. Overall these results suggest that both Sd and Hth use distinct strategies to regulate distinct gene sets during development: one strategy is shared between tissues and associated with Yki, while the other is tissue-specific, generally Yki-independent and associated with developmental patterning (Slattery, 2013).

The control of gene expression in multicellular eukaryotes depends on a limited set of transcription factors that are reused in different contexts and combinations to execute a diverse array of cellular functions. To gain insight into this process this study used tissue-specific, genome-wide ChIP to explore the global DNA targeting properties of three transcriptional regulators – Yki, Sd, and Hth. Yki is a transcriptional coactivator that regulates tissue growth in all tissues, and it does so in part through interactions with the DNA binding TFs Sd and Hth. However, in addition to their Yki-dependent roles in promoting tissue growth, Sd and Hth also have highly tissue-specific developmental roles. Thus, this group of regulators provides an ideal starting point for addressing the logic by which TFs execute both tissue-specific and -nonspecific gene regulatory functions in vivo. The implications of the differences this study uncovered between these modes of binding for Hth and Sd are discussed, as well as the unexpectedly large number of shared binding sites for Yki (Slattery, 2013)

Drosophila Yki was initially identified as an essential transcriptional coactivator in the Hippo tumor suppressor pathway. Loss of function clones of yki grow very poorly, while gain of function Yki clones result in tissue overgrowths that are similar to those generated when the upstream kinases (Hippo and Warts) are compromised. These observations suggested that Yki, with the help of DNA binding proteins, would target genes required for cell proliferation and survival, including the known Hippo pathway targets cycE and diap1. Consistent with this expectation, this study observed Yki binding to these and other genes that are regulated by the Hippo pathway. Unexpectedly, however, in addition to known Hippo pathway genes Yki binding to several thousands of genes was observed in both the eye-antenna and wing imaginal discs, implying that Yki targets many more genes than those regulated by the Hippo pathway, or that the Hippo pathway targets many more genes than previously thought. Consistent with the latter possibility, over 1000 of the genes identified as tissue-shared Yki targets in this study are upregulated >2-fold in wts- wing discs relative to wild-type based on recently published RNA-seq data. In addition, Yki was recently shown to bind and activate several genes required for mitochondrial fusion. Moreover, the mammalian homologs of Yki, Yes-associated protein (YAP) and TAZ (transcriptional coactivator with PDZ-binding motif) are thought to regulate many genes in a wide variety of contexts, including human embryonic stem cells and several adult human tissues. Taken together, these results suggest that Yki may be a widely used transcriptional coactivator in Drosophila and vertebrates. The severe cell proliferation defects associated with yki mutant clones may have obscured its other functions in other pathways. These results are consistent with the idea that Yki and its vertebrate orthologs interact with a wide variety of transcription factor. Together, the data imply that DNA binding proteins in addition to Sd and Hth may recruit Yki to a large number of broadly active CRMs (Slattery, 2013)

The view that Yki is recruited to DNA by factors other than Sd was recently questioned by experiments suggesting that, in the eye imaginal disc, sd yki double mutant clones proliferate better than yki single mutant clones. These observations were interpreted to suggest that Sd is a default repressor of proliferation and survival-promoting genes. However, this conclusion is complicated by the observation that both Sd and Yki are also important for specifying non-retinal (peripodial epithelium) fates in the eye imaginal disc: thus, the partially rescued growth of sd yki clones could in part be due to a fate transformation. Further, this study found that the activity of the ban-eye enhancer is not affected in sd clones, but is lost in hth clones, arguing that at least for this direct Hippo pathway target Hth, not Sd, is the primary activator. It is noteworthy that although their activities can be separated, the ban wing and eye enhancers identified in this study are adjacent to each other in the native ban locus. It is plausible that Sd+Yki input provides a basal level of activity in both tissues and that Hth and Sd boost this level in the eye and wing, respectively. Regardless, the improved growth of sd yki clones does not argue against the idea that Yki is recruited to survival genes by Hth in wild type eye discs. Taken together with genome-wide binding and ban enhancer studies, it is suggested that the absence of Sd results in both a fate change and some derepression of survival genes, but that wild type proliferation and gene regulation in the eye disc requires the recruitment of Yki to the DNA by Hth (Slattery, 2013)

In contrast to the widespread and largely tissue-nonspecific binding observe for Yki, Sd and Hth exhibit both tissue-specific and tissue-shared binding events. Multiple characteristics distinguish these types of binding. First, tissue-shared binding by both Sd and Hth is frequently associated with Yki binding and often close to cell cycle and housekeeping genes, while tissue-specific binding is not. These observations are consistent with previous studies showing that Yki controls cell survival and proliferation in all imaginal discs, an activity that is regulated by the Hippo pathway. Second, compared to tissue-shared binding, DNA sequences bound by Sd and Hth in a tissue-specific manner are more conserved, more likely to contain the TF's consensus binding site, less likely to be promoter proximal, and more likely to be associated with key developmental regulatory loci. Third, tissue-specific Sd and Hth binding events are more likely to overlap with enhancers active in the corresponding tissue. To illustrate this point, the newly identified tissue-specific TF-CRM interactions at wg match the known roles for Sd and Hth. Taken together, these results suggest that regulation at the level of TF-DNA binding is a significant mechanism by which Sd and Hth regulate tissue-specific gene expression. Tissue-specific binding could be regulated through direct or indirect interactions with additional transcription factors, through tissue-specific differences in DNA accessibility, or through a combination of these factors (Slattery, 2013)

This study also found that distinct chromatin types are differentially correlated with tissue-specific and -nonspecific binding, even though these chromatin categories were defined in Kc cells. All tissue-shared binding events have a strong tendency to occur in actively transcribed chromatin states. Tissue-specific (W>EA) Sd and Hth binding is also enriched in RED chromatin (see Filion, 2010) but is uniquely enriched in BLUE chromatin. BLUE chromatin is associated with Polycomb-mediated repression. The W>EA Sd and Hth binding in Polycomb-associated chromatin indicate that these factors target tissue-specific enhancers that are also regulated by PcG proteins during development (Slattery, 2013)

Despite the importance of tissue-specific binding as a regulatory mechanism for Sd and Hth activity, both factors also displayed a significant amount of tissue-shared binding. These tissue-shared binding events can be broken down into distinct groups based on the local chromatin environment. The majority of tissue-shared binding occurs in YELLOW chromatin and is associated with ubiquitously expressed housekeeping genes. However, binding that occurs in BLUE chromatin, and to a lesser extent in RED chromatin, is more conserved and more likely to be associated with a TF's motif, both characteristics of tissue-specific binding. In the case of the bantam eye and wing enhancers, Sd and Hth binding in BLUE chromatin is direct and apparently able to drive tissue-specific, rather than ubiquitous, expression patterns. Other examples of enhancers in RED or BLUE chromatin that drive patterned expression and have tissue shared binding are shown in this study. These observations suggest that gene regulation by Sd and Hth may also be controlled at a step beyond DNA binding, perhaps via interactions with additional transcription factors at a given enhancer. Alternatively, some of the binding events called as tissue-shared may turn out to be specific binding events in distinct cell types within each imaginal disc (e.g. hinge, notum, and pouch in the wing disc and antenna, eye progenitor domain, and photoreceptors in the eye-antenna disc). Regardless, the hundreds of Sd- and Hth-CRM interactions identified in this study provide a tremendous resource for further dissecting the mechanisms by which Sd and Hth regulate patterned gene expression (Slattery, 2013)

Notably, few of the above conclusions would have been clear had genome-wide binding been measured in only one of the two tissues. Tissue-specific binding is not the most highly enriched (that is, the signal is generally weaker compared to tissue-shared events) and might have been overlooked had just one tissue been characterized, where the strongest peaks are generally the focal point. The tissue-specific binding events detected in this study may also occur in subsets of cells in the wing or eye-antennal discs, which are also heterogeneous in cell type. This would explain why tissue-specific binding signals may be weaker, because the ChIP data represent an average of all cell types in a single imaginal disc type. If correct, it would be an error to focus on only the strongest peaks when analyzing in vivo TF binding, particularly in heterogeneous tissues. It is possible that ChIP signal is more biologically meaningful in highly homogenous tissues like the blastoderm Drosophila embryo, or in cell culture. Still, distinct TF-DNA binding mechanisms (long residence time versus rapid binding turnover) with different functional outcomes can lead to indistinguishable, strong ChIP peaks, making it difficult to interpret ChIP data on strength of signal alone. Despite their lower intensity, many biologically relevant binding events, such as those identified in this study, may only stand out when looking at the influence of tissue context on binding (Slattery, 2013).

Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling

Chromatin remodeling processes are among the most important regulatory mechanisms in controlling cell proliferation and regeneration. Drosophila intestinal stem cells (ISCs) exhibit self-renewal potentials, maintain tissue homeostasis, and serve as an excellent model for studying cell growth and regeneration. This study shows that Brahma (Brm) chromatin-remodeling complex is required for ISC proliferation and damage-induced midgut regeneration in a lineage-specific manner. ISCs and enteroblasts exhibit high levels of Brm proteins; and without Brm, ISC proliferation and differentiation are impaired. Importantly, the Brm complex participates in ISC proliferation induced by the Scalloped-Yorkie transcriptional complex, and the Hippo (Hpo) signaling pathway directly restricts ISC proliferation by regulating Brm protein levels by inducing caspase-dependent cleavage of Brm. The cleavage resistant form of Brm protein promotes ISC proliferation. These findings highlighted the importance of Hpo signaling in regulating epigenetic components such as Brm to control downstream transcription and hence ISC proliferation (Jin, 2013).

SWI/SNF complex subunits regulate the chromatin structure by shutting off or turning on the gene expression during differentiation. Recently, the findings from several research reports based on the stem cell system reveal important roles of chromatin remodeling complex in stem cell state maintenance. The current study suggests that the chromatin remodeling activity of Brm complex is required for the proliferation and differentiation of Drosophila ISCs. Based on these findings, it is proposed that Brm is critical for maintaining Drosophila intestinal homeostasis. High levels of Brm in the ISC nucleus represent high proliferative ability and are essential for EC differentiation; low levels of Brm in the EC nucleus may be a response for homeostasis. Changes in Brm protein levels result in the disruption of differentiation and deregulation of cell proliferation. In line with previous findings in human, the cell-type-specific expression of Drosophila homologs BRG1 and BRM are also detected in adult tissues. BRG1 is mainly expressed in cell types that constantly undergo proliferation or self-renewal, whereas BRM is expressed in other cell types. These observations indicate that Brm may act similarly to BRG1 and BRM in controlling proliferation and differentiation (Jin, 2013).

The Hpo pathway restricts cell proliferation and promotes cell death at least in two ways: inhibiting the transcriptional co-activator Yki and inducing activation of pro-apoptotic genes such as caspases directly. This study has identified a novel regulatory mechanism of the Hpo pathway in maintaining intestinal homeostasis. In this scenario, Brm activity is regulated by the Hpo pathway. In normal physiological conditions, under the control of Hpo signaling, the function of Yki–Sd to promote ISC proliferation is restricted and the pro-proliferation of target genes such as diap1 that inhibits Hpo-induced caspase activity cannot be further activated. Therefore, Hpo signaling normally functions to restrict cell numbers in the midgut by keeping ISC proliferation at low levels. Yki is enriched in ISCs, but predominantly inactivated in cytoplasm by the Hpo pathway. The knockdown of Yki in ISCs did not cause any phenotype in the midgut, suggesting that Yki is inactivated in ISCs under normal homeostasis. During an injury, Hpo signaling is suppressed or disrupted, Yki translocates into the nuclei to form a complex with Sd, which may allow Yki–Sd to interact with Brm complex in the nucleus to activate transcriptional targets. Of note, the loss-of-function of Brm resulted in growth defect of ISCs, suggesting that Brm is required for ISC homeostasis and possessing a different role of Brm from Yki in the regulation of ISCs. It is possible that the function of Brm on ISC homeostasis is regulated via other signaling pathways by recruiting other factors. Therefore, different phenotypes induced by the loss-of-function of Brm and Yki in midgut might be due to different regulatory mechanisms. Despite its unique function cooperating with Yki in midgut, that Brm complex is essential for Yki-mediated transcription might be a general requirement for cell proliferation. While this manuscript was under preparation, Irvine lab reported a genome-wide association of Yki with chromatin and chromatin-remodeling complexes (Oh, 2013). These results support the model developed in this paper (Jin, 2013).

The current results also suggest that the interaction between Brm and Yki–Sd transcriptional complex is under tight regulation. The loss of Hpo signaling stabilizes Brm protein, whereas the active Hpo pathway restricts Brm levels by activating Drosophila caspases to cleave Brm at the D718 site and inhibiting downstream target gene diap1 transcription simultaneously. In addition, overexpression of Brm complex components induces only a mild enhancement on midgut proliferation. One possibility is that overexpressing only one of the Brm complex components does not provide full activation of the whole complex; the other possibility is that due to the restriction of the Hpo signaling, as overexpressing BrmD718A mutant protein in ISCs/EBs exhibits a stronger phenotype than expressing the wild-type Brm and coexpression of BrmD718A completely rescues the impairment of Hpo-induced ISC proliferation. D718A mutation blocks the caspase-dependent Brm cleavage and exhibits high activity in promoting ISC proliferation. This study has defined a previously unknown, yet essential epigenetic mechanism underlying the role of the Hpo pathway in regulating Brm activity (Jin, 2013).

It is a novel finding that Brm protein level is regulated by the caspase-dependent cleavage. To focus on the function of Brm cleavage in the presence of cell death signals, attempts were made to examine the activities of the cleaved Brm fragments. Although in vivo experiments did not show strong activity of Brm N- and C-cleavage products in promoting proliferation of ISCs, the C-terminal fragment of Brm that contains the ATPase domain exhibits a relative higher activity than the N-terminal fragment in ISCs. The cleavage might induce faster degradation of Brm N- and C-terminus, since it was difficult to detect N- or C-fragments of Brm by Western blot analysis without MG132 treatment. It reveals that the degradation events of Brm including both ubiquitination and cleavage at D718 site can be important for Brm functional regulation under different conditions. To this end, the intrinsic signaling(s) may balance the activity of Brm complex through degradation of some important components, such as Brm, to maintain tissue homeostasis. Of note, the cleavage of Brm at D718 is occurred at a novel DATD sequence that is not conserved in human Brm. It has been reported that Cathepsin G, not caspase, cut hBrm during apoptosis, suggesting that the cleavage regulatory mechanism of Brm is relatively conserved between Drosophila and mammals (Jin, 2013).

This study provides evidence that the Brm complex plays an important role in Drosophila ISC proliferation and differentiation and is regulated by multi-levels of Hpo signaling. The findings indicate that Hpo signaling not only exhibits regulatory roles in organ size control during development but also directly regulates epigenetics through a control of the protein level of epigenetic regulatory component Brm. In mammals, it is known that Hpo signaling and SWI/SNF complex-mediated chromatin remodeling processes play critical roles in tissue development. Malfunction of the Hpo signaling pathway and aberrant expressions of SWI/SNF chromatin-remodeling proteins BRM and BRG1 have been documented in a wide variety of human cancers including colorectal carcinoma. Thus, this study that has implicated a functional link between Hpo signaling pathway and SWI/SNF activity may provide new strategies to develop biomarkers or therapeutic targets (Jin, 2013).

Crumbs promotes expanded recognition and degradation by the SCFSlimb/beta-TrCP ubiquitin ligase

In epithelial tissues, growth control depends on the maintenance of proper architecture through apicobasal polarity and cell-cell contacts. The Hippo signaling pathway has been proposed to sense tissue architecture and cell density via an intimate coupling with the polarity and cell contact machineries. The apical polarity protein Crumbs (Crb) controls the activity of Yorkie (Yki)/Yes-activated protein, the progrowth target of the Hippo pathway core kinase cassette, both in flies and mammals. The apically localized Four-point-one, Ezrin, Radixin, Moesin domain protein Expanded (Ex) regulates Yki by promoting activation of the kinase cascade and by directly tethering Yki to the plasma membrane. Crb interacts with Ex and promotes its apical localization, thereby linking cell polarity with Hippo signaling. This study shows that, as well as repressing Yki by recruiting Ex to the apical membrane, Crb promotes phosphorylation-dependent ubiquitin-mediated degradation of Ex. Skp/Cullin/F-boxSlimb/beta-transducin repeats-containing protein (SCFSlimb/beta-TrCP) was identifed as the E3 ubiquitin ligase complex responsible for Ex degradation. Thus, Crb is part of a homeostatic mechanism that promotes Ex inhibition of Yki, but also limits Ex activity by inducing its degradation, allowing precise tuning of Yki function (Ribeiro, 2014).

Recent work in flies and mammals has implicated the apical polarity determinant Crb as a transmembrane receptor for Hpo signaling. Accordingly, clonal loss of crb function leads to Yki derepression and increased growth. However, the observation that Crb is required for Ex membrane localization apparently conflicts with the finding that Crbintra overexpression reduces Ex levels and, like crb loss of function, leads to increased Yki activity. This study reconciles these findings by showing that Crb is not only required for Ex tethering at the apical membrane but also for promoting its degradation via the SCFSlimb/β-TrCP E3 ubiquitin ligase. Indeed, immediately downstream of its FERM domain, Ex contains a sequence that conforms to the D/S/TSGφXS consensus sequence for canonical Slmb targets, which is conserved in Ex orthologs from arthropod species but absent from related FERM domain proteins such as Moe and Mer. In addition, loss of Slmb increases Ex levels in vivo, whereas Slmb depletion prevents Crbintra-induced Ex degradation in cell culture. Thus, in crb mutants, Ex no longer reaches the apical membrane and is protected from degradation in the cytoplasm, where it accumulates but is presumably unable to repress Yki. When Crbintra is overexpressed, Ex turnover at the membrane (or in an endocytic compartment if Ex degradation occurs after Crb internalization) is accelerated, leading to its depletion and consequent Yki activation. Therefore, in both cases, the outcome is Yki derepression, albeit for different reasons (Ribeiro, 2014).

How is the Slmb:Ex association regulated by Crb? Previous observations point to the involvement of a phosphorylation-dependent degradation mechanism, because Crb induces Ex phosphorylation. Accordingly, mutation of the phosphodegron (Ser453) or the putative priming site (Ser462) leads to loss of Slmb:Ex association and Ex stabilization. The most obvious candidate Ex kinase in this context is aPKC, which is known to interact with the Crb polarity complex and to phosphorylate Crb at its FBM. However, aPKC is thought to be recruited to the Crb complex via Par-6, which interacts with Crb through the PBM at the C terminus of the Crb intracellular domain. This is inconsistent with the fact that the PBM is dispensable for Crb to promote both Ex phosphorylation and degradation. Moreover, a dominant-negative version of aPKC is unable to rescue the overgrowth phenotype of Crbintra overexpression in the wing. The Hpo downstream kinase Wts is another attractive candidate, because mammalian LATS1/2 promotes YAP phosphorylation on Ser381, providing priming for phosphorylation by CK1δ/ε and triggering degradation by SCFβ-TrCP. However, neither Ser462 nor Ser453 conform to the Wts/LATS consensus. The identity of the kinases therefore remains open (Ribeiro, 2014).

Regulation of tissue growth during development and adult life depends on the maintenance of tissue architecture, which, in turn, relies on cell-cell and cell-matrix interactions. Due to its intimate coupling to polarity and cytoskeletal regulators, the Hpo signaling pathway is thought to sense epithelial integrity and couple tissue architecture to growth control. In particular, YAP has been shown to sense contact inhibition in cell culture, such that its progrowth activity is silenced as cultured epithelial cells reach confluence. This is thought to depend on the assembly of tight junctions, leading to repression of YAP by the mammalian Crb complex. Recent work in flies and zebrafish has suggested that Crb can form homodimers in trans. This suggests that Crb mediates local cell-cell communication in epithelial tissues. Indeed, clonal loss of Crb leads to Crb depletion in the junctions of wild-type cells abutting the crb mutant tissue. This loss of Crb at the clone boundary is mirrored by loss of Ex, which is dependent on Crb for its apical localization. Thus, loss of Crb caused by loss of polarity or cell death can be transmitted to neighboring cells. This has been proposed as a means of cell- cell communication to induce regenerative proliferation through Yki activation. Indeed, genetic induction of epithelial wounds in imaginal discs has been shown to up-regulate Yki activity in cells neighboring the wound. In addition, Yki/ YAP is activated and promotes tissue regeneration upon injury in the vertebrate and fly intestine, as well as in the mouse liver (Ribeiro, 2014).

These findings suggest that Crb (and perhaps other polarity proteins) functions as a sensor of cell density and tissue integrity during development. In this model, disruption of Crb function would lead to Yki/YAP derepression, which, upon tissue injury, would allow regenerative growth to ensue. Another interesting question is whether liganded Crb behaves differently to unliganded Crb with respect to regulation of Ex stability. For example, it is possible that unliganded Crb promotes Ex turnover faster than its liganded counterpart, which might provide a sensitive means of responding to the status of neighboring cells. Further work will be needed to resolve this issue. The present work indicates that Crb fulfills a dual function in Hpo signaling, both recruiting Ex apically to repress Yki activity and promoting Ex turnover through phosphorylation and Slmb-dependent degradation. This mechanism could ensure constant turnover of Ex at the apical membrane, allowing Yki activity to rapidly respond to changing environmental conditions. This dynamic equilibrium could be particularly important to promote fast tissue regeneration upon injury (Ribeiro, 2014).

Coupling of Hedgehog and Hippo pathways promotes follicle stem cell maintenance by stimulating proliferation

It is essential to define the mechanisms by which external signals regulate adult stem cell numbers, stem cell maintenance, and stem cell proliferation to guide regenerative stem cell therapies and to understand better how cancers originate in stem cells. This paper shows that Hedgehog (Hh) signaling in Drosophila melanogaster ovarian follicle stem cells (FSCs) induces the activity of Yorkie (Yki), the transcriptional coactivator of the Hippo pathway, by inducing yki transcription. Moreover, both Hh signaling and Yki positively regulate the rate of FSC proliferation, both are essential for FSC maintenance, and both promote increased FSC longevity and FSC duplication when in excess. It was also found that responses to activated Yki depend on Cyclin E induction while responses to excess Hh signaling depend on Yki induction, and excess Yki can compensate for defective Hh signaling. These causal connections provide the most rigorous evidence to date that a niche signal can promote stem cell maintenance principally by stimulating stem cell proliferation (J. Huang, 2014).

The Hippo pathway regulates hematopoiesis in Drosophila melanogaster

The Salvador-Warts-Hippo (Hippo) pathway is an evolutionarily conserved regulator of organ growth and cell fate. It performs these functions in epithelial and neural tissues of both insects and mammals, as well as in mammalian organs such as the liver and heart. Despite rapid advances in Hippo pathway research, a definitive role for this pathway in hematopoiesis has remained enigmatic. The hematopoietic compartments of Drosophila melanogaster and mammals possess several conserved features. D. melanogaster possess three types of hematopoietic cells that most closely resemble mammalian myeloid cells: plasmatocytes (macrophage-like cells), crystal cells (involved in wound healing), and lamellocytes (which encapsulate parasites). The proteins that control differentiation of these cells also control important blood lineage decisions in mammals. This study defines the Hippo pathway as a key mediator of hematopoiesis by showing that it controls differentiation and proliferation of the two major types of D. melanogaster blood cells, plasmatocytes and crystal cells. In animals lacking the downstream Hippo pathway kinase Warts, lymph gland cells overproliferated, differentiated prematurely, and often adopted a mixed lineage fate. The Hippo pathway regulated crystal cell numbers by both cell-autonomous and non-cell-autonomous mechanisms. Yorkie and its partner transcription factor Scalloped were found to regulate transcription of the Runx family transcription factor Lozenge, which is a key regulator of crystal cell fate. Further, Yorkie or Scalloped hyperactivation induced ectopic crystal cells in a non-cell-autonomous and Notch-pathway-dependent fashion (Milton, 2014).

Impaired Hippo signaling promotes Rho1-JNK-dependent growth

The Hippo and c-Jun N-terminal kinase (JNK) pathway both regulate growth and contribute to tumorigenesis when dysregulated. Whereas the Hippo pathway acts via the transcription coactivator Yki/YAP to regulate target gene expression, JNK signaling, triggered by various modulators including Rho GTPases, activates the transcription factors Jun and Fos. This study shows that impaired Hippo signaling induces JNK activation through Rho1. Blocking Rho1-JNK signaling suppressed Yki-induced overgrowth in the wing disk, whereas ectopic Rho1 expression promoted tissue growth when apoptosis was prohibited. Furthermore, Yki directly regulates Rho1 transcription via the transcription factor Sd. These results identify a novel molecular link between the Hippo and JNK pathways and implicate the essential role of the JNK pathway in Hippo signaling-related tumorigenesis (Ma, 2005).

Recent studies have revealed a complex interaction network between Hippo and other key signaling pathways, including TGF- β /SMAD and Wnt/β-catenin pathways, whereas its communication with JNK signaling remains elusive. This study provides genetic evidences that impaired Hippo signaling promotes overgrowth through Rho1-JNK signaling in Drosophila. First, loss of Hippo signaling induces JNK activation and its target gene expression. Second, Yki-induced overgrowth is suppressed by blocking Rho1-JNK signaling. Third, ectopic Rho1 expression phenocopies Yki-triggered overgrowth and proliferation when cell death is compromised (Ma, 2005).

Yki/YAP's ability in promoting tissue growth depends on transcription factors, including Sd/TEADs and SMADs. Consistent with this notion, this study found Sd, but not Mad, is essential for Yki-induced JNK activation, whereas ectopic Sd expression is sufficient to activate JNK signaling by itself. The Rho1 GTPase was further implicated as the critical factor that bridges the interaction between Hippo and JNK signaling. Rho1 not only mediates Yki-induced JNK activation and overgrowth, but also serves as a direct transcriptional target of Yki/Sd complex. Intriguingly, Rho1 activation was also found to promote nuclear translocation of Yki in wing discs, and reducing Yki activity significantly impeded Rho1 induced growth, implying the existence of a potential positive feedback loop to amplify Yki-induced overgrowth and to help maintain signaling in a steady state. Consistent with thi observation, recent studies reported that GPCRs could activate YAP/TAZ through RhoA in mammals, whereas elevated JNK signaling in Drosophila could stimulate Yki nuclear translocation during regeneration and tissue growth. Thus, these results provide the other side of the story about a novel cross-talk between Hippo and JNK signaling (Ma, 2005).

Intriguingly, it was found that ectopic Yki expression driven by ptc-Gal4 induced MMP1 activation, puc-LacZ expression, rho1 transcription, and Yki target gene transcription predominantly in the proximal region of wing disk, but not that of the dorsal/ventral boundary. This is consistent with a recently published paper showing that tension in the center region of Drosophila wing disk is lower than that in the periphery, which correlates with lower Yki activity. It is also worth noting that despite the requirement of JNK signaling in Yki-induced wing overgrowth, JNK was not activated strictly in an autonomous manner upon Yki overexpression. This could be caused by supercompetitive activity of Yki expression clones, or, alternatively, through a propagation of JNK signal into neighboring cells, which would be very interesting to study further (Ma, 2005).

Apart from its role in growth control, the Hippo pathway also regulates tumor invasion and metastasis. Similarly, JNK signaling plays a major role in modulating metastasis in both flies and mammals. Rho1 was also reported to cooperate with oncogenic Ras to induce large invasive tumors. Hence, it is likely that Rho1 also acts as the molecular link between Yki and JNK signaling in modulating metastasis as well (Ma, 2005).

Control of mitochondrial structure and function by the Yorkie/YAP oncogenic pathway

Mitochondrial structure and function are highly dynamic, but the potential roles for cell signaling pathways in influencing these properties are not fully understood. Reduced mitochondrial function has been shown to cause cell cycle arrest, and a direct role of signaling pathways in controlling mitochondrial function during development and disease is an active area of investigation. This study shows that the conserved Yorkie/YAP signaling pathway implicated in the control of organ size also functions in the regulation of mitochondria in Drosophila as well as human cells. In Drosophila, activation of Yorkie causes direct transcriptional up-regulation of genes that regulate mitochondrial fusion, such as opa1-like (opa1) and mitochondria assembly regulatory factor (Marf), and results in fused mitochondria with dramatic reduction in reactive oxygen species (ROS) levels. When mitochondrial fusion is genetically attenuated, the Yorkie-induced cell proliferation and tissue overgrowth are significantly suppressed. The function of Yorkie is conserved across evolution, as activation of YAP2 in human cell lines causes increased mitochondrial fusion. Thus, mitochondrial fusion is an essential and direct target of the Yorkie/YAP pathway in the regulation of organ size control during development and could play a similar role in the genesis of cancer (Nagaraj, 2012).

Previous studies have shown that the Hippo pathway functions in flies as well as vertebrates to control organ size. Furthermore, mutations in components of this pathway have been implicated in multiple forms of cancer. This pathway has been shown to directly promote cell proliferation and repress apoptosis. This study shows that the mitochondrion is an important additional target of the Hippo pathway. An increase in Yki activity causes an increase in mitochondrial fusion due to direct transcriptional activation of major mitochondrial fusion genes. Increased mitochondrial fusion has been linked to regulation of the G1-S checkpoint and Cyclin E activity, and cells harboring fused mitochondria are resistant to stress-induced apoptosis. Interestingly, these are precisely the phenotypes seen upon activation of the Hpo/Yki pathway. It seems likely that this pathway independently activates the cell cycle, represses apoptosis, and promotes mitochondrial and metabolic changes described in this study that together cause tumor growth. However, since mitochondrial fusion plays a role in S-phase entry and stress resistance, it is attractive to speculate that the mitochondrial effects could indirectly affect Yki's up-regulation of Cyclin E and DIAP. This is supported by-observation that in hpo, opa1 double-mutant clones, Cyclin E expression is suppressed when compared with that seen in hpo mutant clones. The increase in fusion gene levels upon Yki activation is modest. This may be important in producing the observed phenotype of fused but functional mitochondria. Gross overexpression of fusion genes leads to abnormal and globular mitochondria. Moreover, the modification of mitochondrial structure by Yki is accompanied by an up-regulation of antioxidant enzymes and subunits of complex I of the electron transport chain and a dramatic reduction in intracellular ROS. It has recently been demonstrated that other oncogenes (Ras, Raf, and Myc) also reduce ROS, but in the current system, these oncogenes do not up-regulate mitochondrial fusion as seen with activation of the Yki pathway, suggesting that different oncogenic pathways could alter ROS by distinct mechanisms. The importance of mitochondrial fusion in Yki signaling is further highlighted by the observation that a reduction in mitochondrial fusion suppresses Yki-mediated growth phenotypes. This observed link between the Hpo/Yki pathway and mitochondrial fusion is of significance to both normal development and cancer biology. This study reveals that increased mitochondrial fusion and expansion by Yki is evolutionarily conserved and that this function of the Hippo pathway is relevant for both normal and patho-physiological situations (Nagaraj, 2012).

Drosophila C-terminal Src kinase regulates growth via the Hippo signaling pathway

The Hippo signaling pathway is involved in regulating tissue size by inhibiting cell proliferation and promoting apoptosis. Aberrant Hippo pathway function is often detected in human cancers and correlates with poor prognosis. The Drosophila C-terminal Src kinase (d-Csk) is a genetic modifier of warts (wts), a tumor-suppressor gene in the Hippo pathway, and interacts with the Src oncogene. Reduction in d-Csk expression and the consequent activation of Src are frequently seen in several cancers including hepatocellular and colorectal tumors. Previous studies show that d-Csk regulates cell proliferation and tissue size during development. Given the similarity in the loss-of-function phenotypes of d-Csk and wts, the interactions of d-Csk with the Hippo pathway were investigated. Multiple lines of evidence are presented suggesting that d-Csk regulates growth via the Hippo signaling pathway. Loss of dCsk caused increased Yki activity, and genetic epistasis places dCsk downstream of Dachs. Furthermore, dCsk requires Yki for its growth regulatory functions, suggesting that dCsk is another upstream member of the network of genes that interact to regulate Wts and its effector Yki in the Hippo signaling pathway (Kwon, 2014).

Brahma regulates the Hippo pathway activity through forming complex with Yki-Sd and regulating the transcription of Crumbs

The Hippo signaling pathway restricts organ size by inactivating the Yorkie (Yki)/Yes-associated protein (YAP) family proteins. The oncogenic Yki/YAP transcriptional coactivator family promotes tissue growth by activating target gene transcription, but the regulation of Yki/YAP activation remains elusive. In mammalian cells, Brg1, a major subunit of chromatin-remodeling SWI/SNF family proteins, was identified that interacts with YAP. This finding led the authors to investigate the in vivo functional interaction of Yki and Brahma (Brm), the Drosophila homolog of Brg1. Brm was found to function at the downstream of Hippo pathway and interacts with Yki and Scalloped (Sd) to promotes Yki-dependent transcription and tissue growth. Furthermore, it was demonstrated that Brm is required for the Crumbs (Crb) dysregulation-induced Yki activation. Interestingly, it was also found that crb is a downstream target of Yki-Brm complex. Brm physically binds to the promoter of crb and regulates its transcription through Yki. Together, this study has shown that Brm functions as a critical regulator of Hippo signaling during tissue growth and plays an important role in the feedback loop between Crb and Yki (Zhu, 2014).

The core signaling cascade of Hippo pathway has been extensively studied. However, the regulatory mechanism of Yki/YAP activation remains largely elusive. This study found that Brm, a component of SWI/SNF complex, interacts with Yki and regulates organ growth in Drosophila. The findings indicated that Brm is indispensable for Yki activation to drive the expression of target genes. Interestingly, it was also found that the expression of crb is regulated by Yki-Brm complex. The ChIP assay showed that Brm and Yki physically bound to the promoter region of crb, and knockdown of Yki reduced the binding of Brm to crb promoter. Taken together, this study presents a novel feedback loop between Crb and the Yki-Brm complex. Thus, the mutual regulation between apical polarity and Hippo pathway could be critical for tissue growth and homeostasis (Zhu, 2014).

Considering the transcriptional co-dependence with RNA Polymerase II and broad localization in actively transcribed regions, the Brm complex may be present on multiple promoters and is required by global gene transcription. However, the expression of only 872 genes has been observed significantly altered by Brm knockdown. This suggests that there is a selectivity of Brm-mediated transcriptional regulation. In addition to Hippo pathway, morphogens, such as Wingless (Wg), Hedgehog (Hh) and Decapentaplegic (Dpp), also play fundamental roles in organ patterning and growth. Therefore, the readouts of Wg, Hh and Dpp pathway in wing discs with brm knockdown were examined. Interestingly, only Wg secretion was found to be altered, which argued that there was gene specificity of Brm-mediated transcription. Many other factors including the interaction of transcription factors and co-factors, histone modifications and DNA methylation have been also shown essential in this process. Therefore, how Brm regulates the specific targets transcription needs to be further investigated (Zhu, 2014).

These data demonstrate that the enhancement of Crb levels caused by yki overexpression can be suppressed by brm knockdown. However, the overexpression of Brm alone cannot alter the Crb levels. These results suggest that the regulatory function of Brm complex is indispensable for crb but relies on Yki. Together with the fact that Brm physically associate with Yki, it is argued that Brm complex is recruited to target gene promoters through Brm-Yki interaction. Accordingly, another Drosophila transcription factor Zeste has been reported that recruits the Brm complex to chromatin to initiate transcription. Beside of Brm, Moira (Mor), another subunit of the complex, has also been reported to directly interact with Yki and occupy on the promoters of Hpo target genes, which support the working model that Yki facilitates the recruitment of Brm complex onto crb promoters. Taken together, this study draws a conclusion that the specificity of Brm for regulating Hippo targets is determined by Yki recruitment. Given that both the Brm complex and Hippo pathway are conserved in mammals, these results shed a light on a conserved interaction, which might be critical for mammalian growth and tissue homeostasis (Zhu, 2014).

The transcriptional response to tumorigenic polarity loss in Drosophila

Loss of polarity correlates with progression of epithelial cancers, but how plasma membrane misorganization drives oncogenic transcriptional events remains unclear. The polarity regulators of the Drosophila Scribble (Scrib) module are potent tumor suppressors and provide a model for mechanistic investigation. RNA profiling of Scrib mutant tumors revealed multiple signatures of neoplasia, including altered metabolism and dedifferentiation. Prominent among these was upregulation of cytokine-like Unpaired (Upd) ligands, which drive tumor overgrowth. This study identified a polarity-responsive enhancer in upd3, which was activated in a coincident manner by both JNK-dependent Fos and aPKC-mediated Yki transcription. This enhancer, and Scrib mutant overgrowth in general, were also sensitive to activity of the Polycomb Group (PcG), suggesting that PcG attenuation upon polarity loss potentiated select targets for activation by JNK and Yki. These results link epithelial organization to signaling and epigenetic regulators that control tissue repair programs, and provide insight into why epithelial polarity is tumor-suppressive (Bunker, 2015).

Transformed Drosophila cells evade diet-mediated insulin resistance through wingless signaling

Cancer cells demand excessive nutrients to support their proliferation but how cancer cells sense and promote growth in the nutrient favorable conditions remain incompletely understood. Epidemiological studies have indicated that obesity is a risk factor for various types of cancers. Feeding Drosophila a high dietary sugar was previously demonstrated to not only direct metabolic defects including obesity and organismal insulin resistance, but also transform Ras/Src-activated cells into aggressive tumors. This study demonstrates that Ras/Src-activated cells are sensitive to perturbations in the Hippo signaling pathway. Evidence that nutritional cues activate Salt-inducible kinase, leading to Hippo pathway downregulation in Ras/Src-activated cells. The result is Yorkie-dependent increase in Wingless signaling, a key mediator that promotes diet-enhanced Ras/Src-tumorigenesis in an otherwise insulin-resistant environment. Through this mechanism, Ras/Src-activated cells are positioned to efficiently respond to nutritional signals and ensure tumor growth upon nutrient rich condition including obesity (Hirabayashi, 2015).

The prevalence of obesity is increasing globally. Obesity impacts whole-body homeostasis and is a risk factor for severe health complications including type 2 diabetes and cardiovascular disease. Accumulating epidemiological evidence indicates that obesity also leads to elevated risk of developing several types of cancers. However, the mechanisms that link obesity and cancer remain incompletely understood. Using Drosophila, a whole-animal model system has been developed to study the link between diet-induced obesity and cancer: this model has provided a potential explanation for how obese and insulin resistant animals are at increased risk for tumor progression (Hirabayashi, 2015).

Drosophila fed a diet containing high levels of sucrose (high dietary sucrose or ‘HDS') developed sugar-dependent metabolic defects including accumulation of fat (obesity), organismal insulin resistance, hyperglycemia, hyperinsulinemia, heart defects and liver (fat body) dysfunctions. Inducing activation of oncogenic Ras and Src together in the Drosophila eye epithelia led to development of small benign tumors within the eye epithelia. Feeding animals HDS transformed Ras/Src-activated cells from benign tumor growths to aggressive tumor overgrowth with tumors spread into other regions of the body (Hirabayashi, 2013). While most tissues of animals fed HDS displayed insulin resistance, Ras/Src-activated tumors retained insulin pathway sensitivity and exhibited an increased ability to import glucose. This is reflected by increased expression of the Insulin Receptor (InR), which was activated through an increase in canonical Wingless (Wg)/dWnt signaling that resulted in evasion of diet-mediated insulin resistance in Ras/Src-activated cells. Conversely, expressing a constitutively active isoform of the Insulin Receptor in Ras/Src-activated cells (InR/Ras/Src) was sufficient to elevate Wg signaling, promoting tumor overgrowth in animals fed a control diet. These results revealed a circuit with a feed-forward mechanism that directs elevated Wg signaling and InR expression specifically in Ras/Src-activated cells. Through this circuit, mitogenic effects of insulin are not only preserved but are enhanced in Ras/Src-activated cells in the presence of organismal insulin resistance (Hirabayashi, 2015).

These studies provide an outline for a new mechanism by which tumors evade insulin resistance, but several questions remain: (1) how Ras/Src-activated cells sense the organism's increased insulin levels, (2) how nutrient availability is converted into growth signals, and (3) the trigger for increased Wg protein levels, a key mediator that promotes evasion of insulin resistance and enhanced Ras/Src-tumorigenesis consequent to HDS. This study identifies the Hippo pathway effector Yorkie (Yki) as a primary source of increased Wg expression in diet-enhanced Ras/Src-tumors. Ras/Src-activated cells are sensitized to Hippo signaling, and even a mild perturbation in upstream Hippo pathway is sufficient to dominantly promote Ras/Src-tumor growth. Functional evidence is provided that increased insulin signaling promotes Salt-inducible kinases (SIKs) activity in Ras/Src-activated cells, revealing a SIKs-Yki-Wg axis as a key mediator of diet-enhanced Ras/Src-tumorigenesis. Through this pathway, Hippo-sensitized Ras/Src-activated cells are positioned to efficiently respond to insulin signals and promote tumor overgrowth. These mechanisms act as a feed-forward cassette that promotes tumor progression in dietary rich conditions, evading an otherwise insulin resistant state (Hirabayashi, 2015).

Previously work has demonstrated that Ras/Src-activated cells preserve mitogenic effects of insulin under the systemic insulin resistance induced by HDS-feeding of Drosophila (Hirabayashi, 2013). Evasion of insulin resistance in Ras/Src-activated cells is a consequence of a Wg-dependent increase in InR gene expression (Hirabayashi, 2013). This study identified the Hippo pathway effector Yki as a primary source of the Wnt ortholog Wg in diet-enhanced Ras/Src-tumors. Mechanistically, functional evidence is provided that activation of SIKs promotes Yki-dependent Wg-activation and reveal a SIK-Yki-Wg-InR axis as a key feed-forward signaling pathway that underlies evasion of insulin resistance and promotion of tumor growth in diet-enhanced Ras/Src-tumors (Hirabayashi, 2015).

In animals fed a control diet, at most a mild increase was observed in Yki reporter activity within ras1G12V;csk-/- cells. A previous report indicates that activation of oncogenic Ras (ras1G12V) led to slight activation of Yki in eye tissue. Activation of Src through over-expression of the Drosophila Src ortholog Src64B has been shown to induce autonomous and non-autonomous activation of Yki. In contrast, inducing activation of Src through loss of csk (csk-/-) failed to elevate diap1 expression. The results indicate that activation of Yki is an emergent property of activating Ras plus Src (ras1G12V;csk-/-). However, this level of Yki-activation was not sufficient to promote stable tumor growth of Ras/Src-activated cells in the context of a control diet: Ras/Src-activated cells were progressively eliminated from the eye tissue (Hirabayashi, 2013). It was, however, sufficient to sensitize Ras/Src-activated cells to upstream Hippo pathway signals: loss of a genetic copy of ex-which was not sufficient to promote growth by itself-dominantly promoted tumor growth of Ras/Src-activated cells even in animals fed a control diet. These data provide compelling evidence that Ras/Src-transformed cells are sensitive to upstream Hippo signals (Hirabayashi, 2015).

SIK was recently demonstrated to phosphorylate Sav at Serine-413, resulting in dissociation of the Hippo complex and activation of Yki (Wehr, 2013). SIKs are required for diet-enhanced Ras/Src-tumor growth in HDS. Conversely, expression of a constitutively activated isoform of SIK was sufficient to promote Ras/Src-tumor overgrowth even in a control diet. Mammalian SIKs are regulated by glucose and by insulin signaling. However, a recent report indicated that glucagon but not insulin regulates SIK2 activity in the liver. The current data demonstrate that increased insulin signaling is sufficient to promote SIK activity through Akt in Ras/Src-activated cells. It is concluded that SIKs couple nutrient (insulin) availability to Yki-mediated evasion of insulin resistance and tumor growth, ensuring Ras/Src-tumor growth under nutrient favorable conditions (Hirabayashi, 2015).

The results place SIKs as key sensors of nutrient and energy availability in Ras/Src-tumors through increased insulin signaling and, hence, increased glucose availability. SIK activity promotes Ras/Src-activated cells to efficiently respond to upstream Hippo signals, ensuring tumor overgrowth in organisms that are otherwise insulin resistant. One interesting question is whether this mechanism is relevant beyond the context of an obesity-cancer connection: both Ras and Src have pleiotropic effects on developmental processes including survival, proliferation, morphogenesis, differentiation, and invasion, and these mechanisms may facilitate these processes under nutrient favorable conditions. From a treatment perspective the current data highlight SIKs as potential therapeutic targets. Limiting SIK activity through compounds such as HG-9-91-01 may break the connection between oncogenes and diet, targeting key aspects of tumor progression that are enhanced in obese individuals (Hirabayashi, 2015).

Scaling the Drosophila wing: TOR-dependent target gene access by the Hippo pathway transducer Yorkie

How cells integrate distinct inputs to generate organs of the appropriate size and shape is largely unknown. The transcriptional coactivator Yorkie (Yki, a YES-Associated Protein, or YAP) acts downstream of patterning morphogens and other tissue-intrinsic signals to promote organ growth. Yki activity is regulated primarily by the Warts/Hippo (Wts/Hpo) pathway, which impedes nuclear access of Yki by a cytoplasmic tethering mechanism. This study shows that the TOR pathway regulates Yki by a separate and novel mechanism in the Drosophila wing. Instead of controlling Yki nuclear access, TOR signaling governs Yki action after it reaches the nucleus by allowing it to gain access to its target genes. When TOR activity is inhibited, Yki accumulates in the nucleus but is sequestered from its normal growth-promoting target genes. TOR also promotes wing growth by liberating Yki from nuclear seclusion, a parallel pathway that is proposed to contribute to the scaling of wing size with nutrient availability (Parker, 2015).

The Hippo pathway effector Yki downregulates Wg signaling to promote retinal differentiation in the Drosophila eye

The evolutionarily conserved Hippo signaling pathway is known to regulate cell proliferation and maintain tissue homeostasis during development. This study found that activation of Yorkie (Yki), the effector of the Hippo signaling pathway, causes separable effects on growth and differentiation of the Drosophila eye. It presented evidence supporting a role for Yki in suppressing eye fate by downregulation of the core retinal determination genes. Other upstream regulators of the Hippo pathway mediated this effect of Yki on retinal differentiation. The study showed that in the developing eye, Yki could prevent retinal differentiation by blocking morphogenetic furrow (MF) progression and R8 specification. The inhibition of MF progression was due to ectopic induction of Wingless (Wg) signaling and Homothorax (Hth), the negative regulators of eye development. Modulating Wg signaling could modify Yki-mediated suppression of eye fate. Furthermore, ectopic Hth induction due to Yki activation in the eye was dependent on Wg. Last, using Cut (Ct), a marker for the antennal fate, it was shown that suppression of eye fate by hyperactivation of yki did not change the cell fate (from eye to antenna-specific fate). In summary, this study provides the genetic mechanism by which yki plays a role in cell fate specification and differentiation - a novel aspect of Yki function that is emerging from multiple model organisms (Wittkorn, 2015).

Protein Interactions

Studies of the Hpo signaling pathway placed Wts as the most downstream component among Hpo, Sav and Wts. In an effort to extend this pathway further downstream, a yeast two-hybrid screen was carried out for Wts binding proteins. Using the noncatalytic N-terminal portion of Wts (1-608) as bait and from 1 million cDNA clones, three independent clones were isolated representing partial sequences of a gene annotated as CG4005 by the Berkeley Drosophila Genome Project. This gene was named yorkie (yki) after Yorkshire Terriers, one of the world’s smallest breeds of pet dogs, according to its loss-of-function phenotype. Consistent with the yeast two-hybrid results, Wts and Yki coimmunoprecipitate with each other in Drosophila S2 cells (Huang, 2005).

The three independent Wts-interacting clones isolated from the yeast two-hybrid screen define the C-terminal half of Yki (residues 229-418) as a Wts binding region. This region contains the two predicted WW domains, suggesting that the WW domains are required for Yki-Wts binding. Consistent with this hypothesis, mutating two critical residues of the WW domains abolishes the binding between Yki and Wts. Likewise, the N-terminal half of the Yki protein, which does not contain the WW domains, did not bind to Wts in the same assay. Thus, the WW domains of Yki are required for its interaction with Wts (Huang, 2005).

Given the direct interaction between Yki and Wts and that Wts encodes a protein kinase, it was hypothesized that Yki is regulated by the Hpo pathway through Wts-mediated phosphorylation. To test this possibility, phosphorylation of Yki by the Hpo pathway was tested using an S2 cell-based assay. Coexpression of Wts and Yki results in a small mobility retardation of Yki. Coexpression of Hpo-Sav with Yki also results in a mobility shift of Yki, and coexpression of Hpo-Sav-Wts results in an even greater mobility shift of Yki. The mobility shift of Yki induced by Hpo-Sav-Wts expression was abrogated by phosphatase treatment, demonstrating that this shift is due to protein phosphorylation. It is worth noting that the increasing phosphosphorylation of Yki induced by Wts, Hpo-Sav, and Hpo-Sav-Wts in the S2 cell assay correlates with the severity of the overexpression phenotype caused by the respective transgenes in vivo: expression of Wts by the GMR promoter results in slightly rough eyes; expression of Hpo-Sav results in strong rough eyes with reduced size, and expression of Hpo-Sav-Wts results in complete animal lethality. These results suggest that Yki phosphorylation is a relevant output of the Hpo signaling pathway (Huang, 2005).

To determine whether Yki is a direct substrate of Wts, in vitro kinase assays were performed. When expressed alone, Wts shows little kinase activity on Yki. When coexpressed with Hpo-Sav, however, Wts displays specific kinase activity on Yki but not a control substrate. Moreover, a kinase-dead mutation of Wts completely abolishes the in vitro kinase activity of Wts toward Yki. These data confirm that Yki is a kinase substrate of Wts. Furthermore, the observation that Hpo-Sav coexpression stimulates the kinase activity of Wts on Yki is consistent with the activation of Wts by Hpo-Sav as measured by the phosphorylation status of Wts (Huang, 2005).

If Hpo-Sav activates Wts, which in turn phosphorylates Yki, one would predict that the mobility shift of Yki induced by transfected Hpo-Sav or Wts in the S2 cell assay might require the endogenous Wts or Hpo, respectively. Indeed, RNAi of wts completely reverses the mobility shift of Yki induced by Hpo-Sav expression, and RNAi of hpo completely reverses the mobility shift of Yki induced by Wts expression. These data further support the model that Yki is phosphorylated by Wts upon activation of the Hpo pathway (Huang, 2005).

yki is genetically epistatic to hpo, sav, and wts. The genetic evidence presented so far suggests that yki acts antagonistically to hpo, sav, and wts. Biochemical studies further refined this model and demonstrate that Yki is phosphorylated and inactivated by the Hpo pathway via Wts-mediated phosphorylation. A prediction of this model is that loss-of-function mutations of yki should be genetically epistatic to those of hpo, sav, or wts. To test this hypothesis, clones of cells were generated that were doubly mutant for hpo-yki, sav-yki, or wts-yki. While loss of hpo, sav, or wts results in increased diap1 transcription and overgrowth (Wu, 2003), hpo-yki, sav-yki, or wts-yki double mutant clones display phenotypes indistinguishable from those of yki mutant clones, including retarded growth, decreased DIAP1 protein levels, and decreased diap1 transcription. These genetic observations further strengthen the molecular model implicating Yki as a target of Wts in the Hpo pathway (Huang, 2005).

Dmp53 activates the Hippo pathway to promote cell death in response to DNA damage

Developmental and environmental signals control a precise program of growth, proliferation, and cell death. This program ensures that animals reach, but do not exceed, their typical size. Understanding how cells sense the limits of tissue size and respond accordingly by exiting the cell cycle or undergoing apoptosis has important implications for both developmental and cancer biology. The Hippo (Hpo) pathway comprises the kinases Hpo and Warts/Lats (Wts), the adaptors Salvador (Sav) and Mob1 as a tumor suppressor (Mats), the cytoskeletal proteins Expanded and Merlin, and the transcriptional cofactor Yorkie (Yki). This pathway has been shown to restrict cell division and promote apoptosis. The caspase repressor DIAP1 appears to be a primary target of the Hpo pathway in cell-death control. Firstly, Hpo promotes DIAP1 phosphorylation, likely decreasing its stability. Secondly, Wts phosphorylates and inactivates Yki, decreasing DIAP1 transcription. Although some of the events downstream of the Hpo kinase are understood, its mode of activation remains mysterious. This study shows that Hpo can be activated by Ionizing Radiations (IR) in a p53-dependent manner and that Hpo is required (though not absolutely) for the cell death response elicited by IR or p53 ectopic expression (Colombani, 2006).

Hpo is the ortholog of the Mammalian Sterile Twenty-like (MST) kinases, which belong to the Ste20 family of kinases. MSTs are highly similar to Hippo (Hpo) in their N-terminal serine/threonine kinase domains as well as in the C-terminal Salvador (Sav) binding region (or SARAH domain). MST1 functions both downstream and upstream of caspases to promote chromatin condensation and nuclear fragmentation, as well as activation of the JNK (Jun N-terminal kinase) and p38 pathways. Like most Ste20 family kinases, MST1/2 auto- or trans-phosphorylates at a number of residues. One of these, T183 in the activation loop, has been shown to be required for full kinase activity and has been used as a useful marker of MST1 activation in cultured cells. In order to study events upstream of Hpo, antibodies that have previously been shown to recognize MST1/2 phosphorylated on T183 were tested for their ability to cross-react with Hpo on the equivalent residue (T195). Interestingly, it was found antibodies that specifically recognized the phosphorylated form of Hpo upon treatment with staurosporine (sts), a known activator of MST1/2. This signal is abolished by RNAi-mediated Hpo depletion and disappears upon phosphatase treatment. Moreover, the antibodies recognize overexpressed tagged Hpo before immunoprecipitation. By contrast, the antibodies did not recognize a nonphosphorylable (T195A) Hpo mutant protein. Myc-tagged wild-type and T195A Hpo were immunoprecipitated and their auto-kinase activity and their activity on an exogenous substrate (Histone H2B, not shown) were measured in both the presence and absence of sts. As has been observed for MST1/2, overexpression of Hpo leads to its activation, presumably via trans-phosphorylation. Sts treatment potently stimulates Hpo kinase activity (5-fold). By contrast, the T195A mutant is severely compromised both in its unstimulated and stimulated activities, suggesting that T195 phosphorylation is crucial to normal Hpo kinase activity. Thus, these phospho-specific antibodies can be used as readouts of Hpo pathway activity (Colombani, 2006).

In the course of testing stimuli that would activate Hpo in tissue culture, it was observed that γ-irradiation potently and rapidly induced Hpo activation. The fly p53 ortholog has been shown to mediate cell death upon ionizing radiation (IR)-induced DNA damage. Although the pro-apoptotic genes reaper (rpr), hid, and sickle are p53 transcriptional targets, removal of these three proteins via chromosomal deficiencies only partially suppresses the cell-death effects of IR in embryos, suggesting that additional death signals act downstream of p53. This prompted an examination of whether the Hpo pathway could function downstream of Drosophila p53 in the response to IR (Colombani, 2006).

Initially, wing imaginal discs (the larval precursors of the adult wing) containing clones of hpo, wts, and sav mutant cells were treated with γ-rays and cell death was examined by staining for activated caspases. Interestingly, although caspase activation was efficiently induced in wild-type tissue or control discs, cell death was severely reduced in hpo, wts, and sav mutant clones and in p53 mutant discs. Quantification of the caspase staining indicated that apoptosis was reduced by 2- to 3-fold in hpo, wts, and sav clones compared to wild-type tissue. This was also true in eye imaginal discs (Colombani, 2006).

Overexpression of p53 in the posterior portion of late larval eye imaginal dics was sufficient to induce apoptosis. Loss of function of hpo, wts, and sav decreased cell death in this context, although the effect was less pronounced in sav clones, perhaps as a reflection of the weaker phenotype of the sav mutants. This suggests that the Hpo complex may function as an effector in the p53-mediated response to IR. To test this hypothesis, Hpo activation was measured in cultured cells treated with γ-rays in the presence or absence of dsRNAs directed against p53. Excitingly, depletion of Dmp53 markedly reduced Hpo phosphorylation by IR. The residual level of Hpo activation observed in p53-depleted cells can probably be explained by the fact that the dsRNA-mediated p53 depletion was never complete, as measured by RT-PCR. To check that the increased Hpo phosphorylation observed corresponded to increased activity, IP kinase assays were performed on cells expressing ectopic Hpo. It was observed that IR treatment potently induced Hpo kinase activity. Furthermore, p53 expression alone, in the absence of IR, was sufficient to activate Hpo phosphorylation. Finally, it was determined whether p53-dependent Hpo activation could be observed in vivo by taking advantage of the fact that p53 is not required for viability. Dissected ovaries from p53 mutant and wild-type flies were treated with γ-rays and examin Hpo activity was examined by Western blotting. Interestingly, although γ-rays potently activated Hpo in wild-type flies, this response was abolished in p53 mutant animals. p53 expression in the ovaries was able to induce apoptosis, ovary degeneration, and total loss of fecundity. It is concluded that Hpo is activated as part of a p53-dependent DNA-damage response both in cultured cells and in vivo (Colombani, 2006).

MST1 and 2 are known to be activated by caspase 3 through proteolytic cleavage. Therefore, the possibility exists that the Hpo activation observed is merely a by-product of Rpr-dependent caspase activation. Several lines of evidence suggest that this is not the case. First, reaper overexpression in S2 cells did not increase Hpo activity. Second, depletion of DIAP1 from cultured cells, which potently induces caspase activation, fails to trigger detectable Hpo activation. Third, the phospho-Hpo signal detected corresponds to full-length Hpo rather than a caspase-cleaved fragment. In fact, the caspase cleavage site present in the MSTs is not thought to be conserved in Hpo, and no evidence was seen of Hpo cleavage upon apoptotic stimuli. Fourth, treatment of cultured cells with caspase inhibitors did not affect Hpo activation by IR. Thus, it is unlikely that Hpo is stimulated via p53-dependent caspase activation (Colombani, 2006).

The time course of Hpo activation by IR (2–3 hr for maximal activation) suggests that transcription may be required for this response. Indeed, treatment of cells with IR in the presence of the transcription inhibitor Actinomycin D (ActD) abolishes Hpo activation. Thus, Hpo activation in response to IR requires new gene transcription, which could be mediated, at least in part, by p53. Hpo activity is induced by p53 expression, but Hpo protein itself does not appear to be a target of p53 because Hpo levels are not detectably upregulated when p53 is expressed in the posterior portion of the eye imaginal disc or in Dmp53-expressing clones in the wing disc. Future studies will be aimed at determining the exact mechanism through which Dmp53 promotes Hpo activation (Colombani, 2006).

This study has demonstrate by genetic and biochemical approaches not only that the Hpo pathway is required for the full apoptotic response induced by γ-ray irradiation but also that DNA damage triggers Hpo kinase activity in a p53-dependent manner both in vivo and in vitro. The apoptosis induced by p53 overexpression is strongly affected in hpo, wts, and sav mutant clones and p53 does not modulate Hpo levels. This study constitutes the first description of an upstream activating signal of the Hpo complex in vivo and during organism development (Colombani, 2006).

It is noted that the blockage of p53-induced apoptosis is not complete in hpo clones; this incomplete blockage likely reflects the role of other pro-apoptotic proteins, such as Reaper, Hid, and Sickle, in this process. Thus, it is proposed that, after exposure to ionizing radiations, the ATM, Chk2, p53 signaling pathway is activated and induces apoptosis by targeting expression of pro-apoptotic effectors such as Reaper, as well as by activating the Hpo pathway. This cell-death response to irradiation requires the caspase DRONC and leads to upregulation of JNK activity in a p53-dependent manner. Because Hpo has been shown to induce JNK activation when overexpressed in vivo, it will be interesting to determine whether Hpo is necessary for IR-induced JNK activation (Colombani, 2006).

Several reports have suggested that the mammalian homologs of members of the Hpo pathway might behave as tumor suppressors in humans. In addition, mice lacking the Wts homolog mLats1 are more sensitive to tumor-inducing agents. The current data suggest that one effect of mutations in Hpo-pathway members may be to protect these cells from DNA-damage-induced apoptosis and thus promote tumor progression and the accumulation of additional mutations. Further work on the Hpo pathway should further understanding of the DNA-damage response and its role in the transformation process (Colombani, 2006).

Elucidation of a universal size-control mechanism in Drosophila and mammals

Coordination of cell proliferation and cell death is essential to attain proper organ size during development and for maintaining tissue homeostasis throughout postnatal life. In Drosophila, these two processes are orchestrated by the Hippo kinase cascade, a growth-suppressive pathway that ultimately antagonizes the transcriptional coactivator Yorkie (Yki). This study demonstrates that a single phosphorylation site in Yki mediates the growth-suppressive output of the Hippo pathway. Hippo-mediated phosphorylation inactivates Yki by excluding it from the nucleus, whereas loss of Hippo signaling leads to nuclear accumulation and therefore increased Yki activity. A mammalian Hippo signaling pathway has been delimited that culminates in the phosphorylation of YAP, the mammalian homolog of Yki. Using a conditional YAP transgenic mouse model, it has been demonstrated that the mammalian Hippo pathway is a potent regulator of organ size, and that its dysregulation leads to tumorigenesis. These results uncover a universal size-control mechanism in metazoan (Dong, 2007).

This study provides several lines of evidence demonstrating that Hippo signaling antagonizes Yki function by changing its subcellular localization. Hippo signaling promotes Yki cytoplasmic localization in cultured Drosophila cells, and accordingly, loss of Hippo signaling promotes nuclear accumulation of Yki in imaginal discs. This is further supported by the ability of phosphorylated Yki (but not unphosphorylated Yki) to bind to 14-3-3 proteins, which are known to promote the cytoplasmic shuttling of other transcription factors in a phosphorylation-dependent manner. Importantly, S168 was identified as a primary Hippo-responsive phosphorylation site on Yki both in vitro and in vivo: the S168A mutation not only abrogates Hippo-induced Yki phosphorylation and cytoplasmic shuttling in S2 cells but, more significantly, causes constitutive Yki activation in developing tissues. These results demonstrate that S168 mediates the growth-suppressive output of the Hippo signaling pathway (Dong, 2007).

Despite the presence of mammalian homologs for all the known components of the Drosophila Hippo pathway (Mst1/2 for Hpo, WW45 for Sav, Lats1/2 for Wts, and YAP for Yki), previous studies in mammals have failed to unite these proteins in a physiologically relevant signaling cascade. The conservation of of the S168 phosphorylation site in mammalian YAP provides the first opportunity to functionally link Mst1/2, WW45, and Lats1/2 in a single kinase cascade that culminates in YAP S127 phosphorylation. The mammalian Hippo signaling pathway antagonizes YAP function by promoting its cytoplasmic localization in a S127 phosphorylation-dependent manner. The identification of S168/S127 as Wts/Lats-mediated phosphorylation site in Yki/YAP is rather unexpected given that previous studies have implicated this residue as an Akt phosphorylation site. The observation that both YkiS168A and YAPS127A result in a loss-of-Wts rather than a loss-of-Akt phenotype in Drosophila strongly suggests that this site is regulated by the Hippo pathway rather than Akt under normal physiological conditions (Dong, 2007).

The identification of a single phosphorylation site as the functional output of the Hippo pathway, and the constitutive active Yki/YAP mutants described in this study, will greatly facilitate future investigation of this important size-control pathway in multiple species. For example, the constitutive active Yki/YAP mutants can be conveniently used to modulate the Hippo pathway in animal models and in genetic epistasis studies to characterize new components of the pathway; the phospho-Yki/YAP antibodies should provide a sensitive assay to link a specific protein to the Hippo pathway. These tools are especially important for the mammalian system, where a functional readout of the Hippo pathway has so far been unavailable. Indeed, this study placed hWW45 in the mammalian Hippo pathway using phospho-YAP as a convenient readout (Dong, 2007).

Despite the conservation of many Hippo pathway components between flies to mammals, previous studies have not revealed a direct role for this pathway in mammalian organ size control. Several recent studies have focused on their involvement in tumorigenesis. For example, YAP was recently shown to transform immortalized mammary epithelial cells in vitro and to accelerate tumorigenesis in conjunction with p53 loss and c-myc overexpression. While suggestive of an involvement of the Hippo pathway in mammalian tumorigenesis, these observations alone do not necessarily prove a direct requirement for the Hippo pathway in the control of organ size, since perturbations of many cellular processes in addition to growth control can contribute to tumorigenesis. It is worth noting that knockout mice have been generated for several components of the mammalian Hippo pathway. However, these mice are either viable, lacking any overt overgrowth characteristic of the respective Drosophila mutants (e.g., Lats1), or embryonic lethal, thus preventing a critical assessment of their involvement in organ size regulation (e.g., Lats2 and YAP (Dong, 2007).

The identification of YAP as the nuclear effector of the mammalian Hippo pathway provides a powerful tool to manipulate this pathway in mammals, in much the same way that Yki overexpression recapitulates loss of Hippo signaling in Drosophila. By manipulating YAP activity in a conditional and tissue-specific manner, this study demonstrates that modulating Hippo pathway activity is sufficient to cause a rapid and reversible change of organ size (up to 500%), therefore offering the first direct evidence implicating the Hippo pathway in mammalian organ size control. It is further demonstrated that, like its Drosophila counterpart, the mammalian Hippo pathway coordinately regulates both cell proliferation and apoptosis. The dual function of YAP in promoting cell proliferation and suppressing apoptosis distinguishes it from a conventional oncogene such as c-myc, whose mitogenic activity is coupled with a proapoptotic activity. It is suggested that this dual activity in promoting cell proliferation and suppressing apoptosis underlies the rapid and uniform expansion of liver mass in the ApoE/rtTA-YAP mice. The ability of YAP to induce organomegaly in postnatal mice is consistent with the notion that the Hippo pathway not only controls organ size during development as demonstrated in Drosophila but also regulates tissue homeostasis in postnatal life (Dong, 2007).

Initially isolated as a yes-associated protein, YAP has since been reported to bind to a large number of proteins in cultured mammalian cells, including EBP50, Smad7, ErbB4, p53BP-2, p73, and hnRNAP U, as well as Runt and TEAD transcription factors. However, it has been difficult to ascertain whether any of these binding partners mediate YAP function in vivo. The antiapoptotic activity observed in the transgenic mouse liver is clearly distinct from the reported ability of YAP to potentiate p73-mediated apoptosis in response to DNA damage in cultured mammalian cells. Given that p73-deficient mice are viable while YAP-deficient mice die at embryonic day 8.5, p73 is unlikely to be a critical partner for YAP in mouse development (Dong, 2007).

Studies from both insects and mammals support the existence of an intrinsic size checkpoint that monitors organ size at the tissue, rather than the cellular, level. For example, while constitutive activity of the myc oncogene drives the growth of individual Drosophila cells, it has little effect on the size of imaginal disc compartments. Therefore, increased cell growth or cell proliferation does not automatically lead to a corresponding increase in tissue size, unless the size checkpoint is simultaneously perturbed. It follows that such intrinsic size-control mechanism must be overridden to permit the sustained overgrowth of tumors. The finding that YAP overexpression leads to immediate organomegaly followed by tumor formation provides direct support for this hypothesis. The widespread upregulation of YAP in diverse tumor types further suggests that the Hippo pathway represents a common mutational target that allows cancer cells to evade the intrinsic size-control mechanisms that normally maintain tissue homeostasis in animals (Dong, 2007).

The observation of two distinct patterns of YAP distribution in tumor cells -- with or without nuclear accumulation -- implicates two possible mechanisms by which Hippo signaling may be dysregulated in cancer cells. Based on the mechanism of Yki/YAP inactivation by Hippo signaling as revealed by the current study, it is suggested that the former pattern could result from inactivation of tumor suppressors upstream of YAP, mutation of the S127 phosphorylation site, or perturbation of the nuclear-cytoplasmic shuttling machinery, whereas the latter pattern could be caused by YAP overabundance, either via gene amplification, increased transcription, or protein stabilization. It is further speculated that these mechanisms may also be employed in normal physiological contexts to regulate the activity of the Hippo pathway in flies and mammals. Thus, besides phosphorylation, mechanisms that regulate Yki/YAP transcription or stability are likely relevant to the modulation of Hippo signaling activity in vivo (Dong, 2007).

Mob as tumor suppressor is activated by Hippo kinase for growth inhibition in Drosophila

Tissue growth and organ size are determined by coordinated cell proliferation and apoptosis in development. Recent studies have demonstrated that Hippo (Hpo) signaling plays a crucial role in coordinating these processes by restricting cell proliferation and promoting apoptosis. Mob as tumor suppressor protein, Mats, functions as a key component of the Hpo signaling pathway. Mats associates with Hpo in a protein complex and is a target of the Hpo serine/threonine protein kinase. Mats phosphorylation by Hpo increases its affinity with Warts (Wts)/large tumor suppressor (Lats) serine/threonine protein kinase and ability to upregulate Wts catalytic activity to target downstream molecules such as Yorkie (Yki). Consistently, epistatic analysis suggests that mats acts downstream of hpo. Coexpression analysis indicated that Mats can indeed potentiate Hpo-mediated growth inhibition in vivo. These results support a model in which Mats is activated by Hpo through phosphorylation for growth inhibition, and this regulatory mechanism is conserved from flies to mammals (Wei, 2007).

Two protein kinases Hippo [Hpo and Warts (Wts)/large tumor suppressor (Lats)], and a scaffold protein Salvador (Sav)/Shar-pei, are key components of this pathway. Moreover, two FERM-domain proteins, Merlin (Mer) and Expanded (Ex), function upstream of Hpo, and Mob as tumor suppressor (Mats), associates with Wts to stimulate the catalytic activity of the Wts protein kinase. Recently, both putative receptor and ligand that function further upstream of, or in parallel with, Hpo signaling have been identified (Hariharan, 2006). A major signal output of this growth inhibitory pathway is to inactivate a transcription coactivator Yorkie (Yki) via phosphorylation by Wts kinase. In addition to Cyclin E and Drosophila inhibitor of apoptosis 1 (diap1), the bantam microRNA is also found to be a target of the Hpo pathway. Most components in this emerging signaling pathway are conserved from yeast to flies and humans, suggesting that this pathway plays a fundamental role in cellular regulation (Wei, 2007).

The function of Mob proteins has been better studied in yeast, Drosophila and mammalian cells, which revealed a conserved property of Mob proteins as a binding partner as well as a coactivator of protein kinases of the Ndr (nuclear Dbf2-related) family (Hergovich, 2006b). As stated above, Drosophila Mats/dMob1 is required for mediating Hpo signaling by regulating Wts kinase activity in growth inhibition and tumor suppression. All four Drosophila mob genes dMob1-4 genetically interact with trc (tricornered) (He, 2005a), the fly Ndr homolog important for maintaining integrity of epidermal outgrowths and regulating dentritic tiling and branching (Emoto, 2004; He, 2005b). In the budding yeast Saccharomyces cerevisiae, Mob1 binds to and activates Dbf2/Dbf20 protein kinases for controlling mitotic exit and cytokinesis (Komarnitsky, 1998; Lee, 2001; Mah, 2001). Similarly, Mob1 is required for the activation of Sid2, an Ndr family kinase in the fission yeast Schizosaccharomyces pombe essential for cytokinesis (Hou, 2000; Hou, 2004). In human, hLats1 preferentially interacts with hMob1/hMats, but not hMob2 protein, and appeared to be required for promoting mitotic exit (Bothos, 2005), as well as cytokinesis (Yang, 2004). Importantly, the function of Mob proteins has been highly conserved in evolution. For instance, the human Mob1A/Mats1 protein has been shown to act as a kinase activator and can rescue the lethality and tumor phenotypes ofDrosophila mats mutants (Lai, 2005; Wei, 2007 and references therein).

Structural analysis of a human Mob1 protein, Mob1A/Mats1, revealed several important features of Mob family proteins (Stavridi, 2003). One is that several highly conserved residues are responsible for generating an atypical Cys2-His2 zinc-binding site, which is predicted to contribute to the stability of the Mob protein. Another striking feature is that there is a flat surface rich in acidic residues on one side of the protein. This property provides the structural basis for a Mob protein to interact with its partner, such as Ndr family kinases through electrostatic forces. Indeed, a 65-amino-acid region rich in basic residues exists in the N-terminal side of the kinase domain of Ndr family kinases, and alterations in the basic residues can prevent the kinases from binding to Mob proteins (Bichsel, 2004; Bothos, 2005; Hergovich, 2006b). Finally, hMob1A adopts a globular structure involving residues throughout the polypeptide. Mob proteins are small and usually do not carry any other structural motifs other than the Mob domain (Wei, 2007).

Although previous studies suggest that Ndr family kinases can be activated by upstream regulators such as Cdc15, Hpo and Mst kinases via phosphorylation in yeast, flies or human cells, very little is known about how Mob is regulated. Studies carried out in yeast and mammalian cells suggested that Mob proteins may be regulated through phosphorylation. For instance, yeast Mob1 was shown to be essential for the phosphorylation of Dbf2 by an upstream protein kinase Cdc15 and Mob1 itself was also phosphorylated by Cdc15 (Mah, 2001). However, the functional significance of this modification has not been elucidated. Work on human Mob1A/Mats1 also suggested that phosphorylation might provide a mechanism for regulating hMob1A activity (Bichsel, 2004). This study has tested a hypothesis that Mats is directly activated by Hpo kinase to regulate Wts kinase activity for growth inhibition and tumor suppression. Using the Drosophila system, it was found that Mats can be complexed with Hpo and is a target of the Hpo protein kinase. Similarly, human Mats1 is also a target protein of mammalian Mst kinases. Mats phosphorylation by Hpo increases its affinity with Wts protein kinase and ability to increase Wts activity to target Yki. Moreover, epistatic analysis suggested that mats acts downstream of hpo. Genetic analysis indicated that Mats functions together with Hpo for mediating growth inhibition of developing organs. Therefore, the Mob as tumor suppressor protein, Mats, functions as a critical component of the Hpo signaling pathway. The results support a model in which Mats is activated by Hpo through phosphorylation for growth inhibition, and this regulatory mechanism is conserved from flies to mammals (Wei, 2007).

Recent studies have defined an emerging growth inhibitory pathway mediated by Fat, Mer/Ex, Hpo/Sav and Wts/Mats proteins in tissue growth and organ size control in Drosophila. Previous work has shown that Mats functions as a coactivator of the Wts protein kinase (Lai, 2005). This study has focused on addressing how Mats is activated to regulate Wts kinase activity. Fenetic analysis suggests that Mats acts downstream of Hpo and is a critical component of the Hpo signaling pathway. Moreover, evidence is provided that Hpo-mediated phosphorylation increases Mats's activity as a coactivator of the Wts protein kinase, and this regulatory mechanism is conserved from flies to humans. Therefore, Hpo-mediated phosphorylation of Mats significantly contributes to Wts activation. In a simple model, Hpo needs to directly phosphorylate Wts as well as Mats in order for Wts kinase to be fully activated. Although both Wts and Mats are activated by Hpo-mediated phosphorylation, further investigations are needed to address how Hpo phosphorylation and Mats binding are coordinated for Wts activation (Wei, 2007).

This report provides evidence that Mats is a target of Hpo/Mst protein kinases and Hpo/Mst-mediated phosphorylation positively regulates Mats protein's coactivator activity for Wts protein kinase. Importantly, it was found that Mats exists as a phosphoprotein in living cells, indicating that Mats phosphorylation occurs under physiological conditions. In addition to Hpo/Mst, Wts kinase has also been shown to target Mats for phosphorylation (Lai, 2005), although the physiological effect of this modification has not been elucidated. In S. cerevisiae, the founding member of the Mob superfamily Mob1 was found to be a phosphoprotein and a substrate for the Mps1 kinase. Mob1 is also phosphorylated by an upstream regulator Cdc15 kinase (Mah, 2001). However, the role of Cdc15 in Mob1 phosphorylation has not been revealed even though Mob1 is known to be required for Cdc15-mediated activation of its binding partner Dbf2 kinase. In mammalian cells, protein phosphatase 2A inhibition by OA treatment caused phosphorylation of a Mob family protein (Moreno, 2001). Moreover, OA-induced modification on hMob1 was shown to be critical for its binding to its partner Ndr kinase (Bichsel, 2004). Thus, phosphorylation appears to be a common mechanism for Mob regulation (Wei, 2007).

Consistent with the finding that Mats is activated by Hpo via phosphorylation for upregulating Wts kinase activity, epistatic analysis suggests that Mats is acting downstream of Hpo. This is the first case that Ste20 family protein kinase-mediated phosphorylation of Mob is critical for regulating the catalytic activity of Ndr family protein kinase such as Wts. At this point, it is not clear how Mob proteins function to activate Ndr family kinases. Based on the results from recent studies of human Mob1 and Ndr family kinases, a potential mechanism is that Ndr family kinase is rapidly recruited by hMob1 to the plasma membrane for activation (Hergovich, 2005; Hergovich, 2006a). It is speculate dthat Hpo phosphorylation might facilitate Mats to associate to the membrane through an unknown mechanism, which in turn recruits Wts to the membrane as evidenced by the observation that Hpo phosphorylated Mats has an increased affinity to Wts. Subsequently, Wts is activated by phosphorylations mediated by protein kinases such as Hpo. Mats as a target of Hpo kinase, is able to associate with Hpo in a protein complex. Since Hpo/Mst1 kinase was not present in the Mats/Wts protein complex (Lai, 2005), it appears that Mats simultaneously cannot associate with Hpo and Wts in the same protein complex (Wei, 2007).

In addition to the membrane recruitment model, the data also support an active and more direct role of Mats in upregulating Wts kinase. From in vitro kinase assays, it was found that Hpo-mediated phosphorylation increases the affinity between Mats and Wts, as well as the ability of Mats to activate Wts kinase activity in the absence of any membrane structures. The results support a model in which Mats binding likely causes a conformational change of Wts for Wts activation. In the case of human Ndr kinase, an autoinhibitory effect of hNdr can be released by hMob1 binding (Bichsel, 2004), which presumably induces a conformational change in hNdr for its activation. Finally, it was found that Mats increases the steady level of Wts protein, which contributes to the increase in Wts activity. Further investigation is needed to understand how Mats is able to stabilize and/or increase the production of Wts protein (Wei, 2007).

Previous work has shown that Mats negatively regulates tissue growth by binding to another tumor suppressor Wts and subsequently activating the catalytic activity of Wts kinase (Lai, 2005). Since loss of mats function leads to tissue overgrowth and tumor development, it suggests that Wts alone is not sufficient to inhibit tissue growth in the absence of Mats. Therefore, Mats is an indispensable component of the Hpo pathway, and Wts activation is dependent not only on Hpo-mediated phosphorylation, but also on Mats binding. Further studies are needed to understand how exactly Wts activation is coordinated by Hpo phosphorylation and Mats binding. This work has provide evidence that Mats activation can be mediated by Hpo phosphorylation (Wei, 2007).

The Hpo signaling pathway plays an important role in growth inhibition and tumor suppression in Drosophila, and this pathway appears to be also critical for tissue growth control and tumor suppression in mammals. For instance, mammalian NF2 tumor suppressor is a homolog of Drosophila Mer and Ex proteins, which are upstream regulators of the Hpo signaling pathway. Moreover, loss of Lats1 function in mouse causes soft tissue sarcomas and ovarian tumors. Recently, it was found that hMats1 can functionally replace fly Mats to suppress tumor development, and Mats1 is mutated in mammalian tumors (Lai, 2005). Thus, mechanisms for the control of Hpo signaling might be commonly used across species, and understanding such mechanisms should provide insights into tumor development in mammals. As shown in this report, one mechanism by which Hpo functions to control tissue growth is to target Mats for phosphorylation, and, consequently, Mats is activated to upregulate Wts kinase. Because mammalian Hpo orthologs, Mst kinases, regulates hMats1 in a similar manner, this mechanism is likely used in mammalian cells as well. Therefore, by understanding how Hpo/Mst kinases regulate Mats and Wts/Lats in normal as well as tumor cells, valuable insights will be gained into tissue growth inhibition and tumor suppression (Wei, 2007).

Scalloped interacts with Yorkie, the nuclear effector of the hippo tumor-suppressor pathway in Drosophila

In Drosophila, Scalloped (Sd) belongs to a family of evolutionarily conserved proteins characterized by the presence of a TEA/ATTS DNA-binding domain. Sd physically interacts with the product of the vestigial (vg) gene, where the dimer functions as a master gene controlling wing formation. The Vg-SD dimer activates the transcription of several specific wing genes, including sd and vg themselves. The dimer drives cell-cycle progression by inducing expression of the dE2F1 transcription factor, which regulates genes involved in DNA replication and cell-cycle progression. Yorkie (Yki) is a transcriptional coactivator that is the downstream effector of the Hippo signaling pathway, which controls cell proliferation and apoptosis in Drosophila. This study identified Sd as a partner for Yki. Interaction between Yki and Sd increases Sd transcriptional activity both ex vivo in Drosophila S2 cells and in vivo in Drosophila wing discs and promotes Yki nuclear localization. Yki overexpression induces vg and dE2F1 expression, and proliferation induced by Yki or by a dominant-negative form of Fat in wing disc is significantly reduced in a sd hypomorphic mutant context. Contrary to Yki, Sd is not required in all imaginal tissues. This indicates that Yki-Sd interaction acts in a tissue-specific fashion and that other Yki partners must exist (Goulev, 2008).

Yorkie (Yki) encodes a transcriptional coactivator that is the downstream effector of the Salvador-Warts-Hippo signaling pathway. This pathway consists of the two serine-threonine kinases, Hippo and Warts (Wts, also known as Large Tumor Suppressor), the adaptator molecules Salvador (Sav) and Mob as Tumor Suppressor (MATS), the FERM domain proteins Expanded (Ex) and Merlin (Mer), and the protocadherin Fat (Ft). In this pathway, the Hpo kinase phosphorylates Wts, which in turn phosphorylates and inactivates Yki by excluding it from the nucleus. Mer and Es colocalize at the cell cortex and act upstream of Hpo to regulate the activity of Yki. Fat acts upstream of the Hippo signaling cascade by recruiting Ex to the apical plasma membrane and modulating the abundance of Wts. Inactivation of ft, hpo, wts, and sav, inactivation of both ex and mer, and overexpression of yki all lead to increased proliferation and reduced apoptosis, This pathway acts through Yki to regulate the expression of cyclin E (cycE) and the Drosophila inhibitor of apoptosis 1 (diap-1), which are involved in cell-cycle progression and cell-death inhibition, respectively, and the microRNA bantam, which can promote growth and inhibit apoptosis. The Yki peptide shares significant homology with the mammalian Yes Associated Protein 65 (YAP) (31% identity with human YAP). A particular region near the amino terminus of Yki (amino acids 57-114) displays the highest homology with YAP (56.2% identity). This region in YAP corresponds to a domain that binds all mammalian transcription-enhancer factors (TEFs), which are the homologs of the Drosophila Sd protein (Goulev, 2008).

Conservation between mammalian YAP and Yki of the YAP domain that interacts with TEF suggests that Yki might interact with Sd. To investigate this possibility, glutathione S-transferase (GST)-pull-down experiments were conducted. 35S-labeled Yki specifically binds to GST-Sd beads (Goulev, 2008).

These results are in good accordance with a large two-hybrid screen in which Sd was identified as a binding partner for Yki. To refine the Sd domain that binds Yki and the region of Yki involved in the interaction with Sd, the binding of deleted Sd or Yki peptides was examined. Results that were obtained indicate a requirement of the N terminus of Yki and the C terminus domain of Sd. These results implicate Yki as an auxiliary protein that specifically interacts with Sd and demonstrate that this ability to interact is conserved between TEA factors (TEF-1 and Sd) and YAP factors (YAP and Yki) (Goulev, 2008).

To determine whether Sd, which possesses a putative nuclear localization signal (NLS) within the TEA domain, can modify Yki localization, transfection experiments were performed with plasmids expressing Sd fused to FLAG sequence and Yki fused to hemagglutinin (HA) sequence. In Drosophila S2 cells, Yki is mainly cytoplasmic. S2 cells were transfected with Yki and Sd and it was observed that Yki is exclusively nuclear. The nuclear translocation of Yki when Sd is expressed argues in favor of the idea that Sd and Yki interact ex vivo. Co-transfection experiments indicate that Yki interacts in a cellular context with Sd to stimulate the transcriptional activity conferred by Sd and additional experiments indicate that Vg and Yki do not compete for binding to Sd (Goulev, 2008).

It has been shown that the transcriptional activity of Yki is suppressed by coexpression of upstream components of the Hippo pathway. The transcriptional activity of Sd-Vg complex was analyzed when Hpo was overexpressed. Coexpression of GAL4db-Sd and Vg results in a powerful induction of luciferase activity. This induction was suppressed when Hpo was overexpressed. Although it cannot be excluded that Hpo used a mechanism other than Yki inhibition, the most likely expanation for these results is that the Hippo pathway, and thus Yki function, is required for Sd-Vg activity (Goulev, 2008).

It has been proposed that, in the Sd-Vg complex, Vg provides the activating component, and that the binding of Vg to Sd switches the target selectivity of Sd. If it is assumed that the Sd-Vg-Yki complex is able to form into a cell, one attractive explanation for the role of interaction between Yki and Sd is that Yki provides the main activating component and Vg undergoes a conformational change in Sd ensuring accurate DNA target selection (Goulev, 2008).

To explore the possibility that Yki function is evolutionarily conserved, the effects of Yki on TEF transcriptional activity was tested in a HeLa human cell line. Yki, similarly to YAP, stimulate TEF1 transcriptional activity. These results reveal an evolutionarily conserved regulatory mechanism. Interestingly, it has recently been shown that YAP is the effector of the mammalian Hippo pathway. It is amplified in mouse tumor models and has several oncogenic properties. Furthermore, YAP can functionally substitute for Yki in Drosophila. The relevance of the YAP-TEF interaction in this pathway, however, remains to be determined (Goulev, 2008).

To determine whether Yki-Sd interaction regulates Sd transcriptional activity in vivo in Drosophila, a sensor transgene, 'hsp70-GAL4db-sd', expressing a modified Sd protein was used, in which the TEA DNA-binding domain has been replaced by that of the yeast GAL4 DNA-binding domain, under the control of the hsp70 promoter. GAL4::Sd activity, monitored by the UAS-lacZ-responsive reporter transgene, is restricted to the wing pouch, where Sd dimerizes with Vg. A significant reduction in lacZ activity was observed in wing discs heterozygous for the amorphic ykiB5 allele compared to control wing discs. The fact that Sd activity is sensitive to yki dosage in wing imaginal discs supports the idea that Yki is required for Sd transcriptional activity in vivo (Goulev, 2008).

To better understand the functional relationship between Yki and Sd during wing development, genetic interactions between these genes was assessed. The results indicate that reduction of both vg and yki dosage enhances the sd phenotype and support the hypothesis that Yki works together with Sd-Vg during wing development (Goulev, 2008).

It has been shown that binding sites for Sd and Sd-Vg are necessary for regulation of vg expression and thus may regulate by a feedback mechanism vg expression. Results obtained in S2 cells indicate that Yki increases Sd transcriptional activity. This prompted an investigation of whether yki overexpression in imaginal discs enhances Vg-Sd activity, thus regulating vg expression and vgQE activity. yki overexpression was drived by using a UAS-GAL4 system or by flip-out (FLP-FRT) recombination in clones. In both cases, overexpression of yki was shown to induce vg and vgQE expression in a cell-autonomous manner in wing discs and in haltere discs (Goulev, 2008).

Several sets of data argue strongly in favor of an involvement of Vg-Sd in cell proliferation and cell-cycle progression in wing imaginal discs. vgnull cell clones do not proliferate in the wing pouch, whereas they can be recovered in the notum region, where vg is not expressed, indicating that vg is required for the proliferation of wing blade cells or for their survival. In contrast, vg ectopic expression induces wing outgrowths and dE2F1 expression. dE2F1 is the Drosophila homolog of the E2F transcription-factor family that plays a pivotal role during cell-cycle progression in controlling expression of different genes involved in G1-S transition (including cycE, a target gene of the Hippo pathway) and DNA replication. It was found that Yki overexpression with the ptc-GAL4 driver clearly upregulates expression of the dE2F1-LacZ reporter strain in a cell-autonomous manner. This indicates that Yki and Vg-Sd both activate dE2F1 expression and might work together in this process. It can be hypothesized either that Sd-Vg is required for dE2F1 induction by Yki or that Yki induces dE2F1 independently from Vg-Sd and reinforces dE2F1 expression through Sd-Vg induction (Goulev, 2008).

To determine more precisely the relationship between Sd and Yki in cell proliferation, the effect of Yki overexpression was tested in a mutant context for sd. Strong hypomorph alleles of sd are characterized by an absence of wing pouch cells. In contrast, sdETX4 corresponds to a weak allele of sd in which a large number of wing pouch cells are still present. This allele was used to exclude the possibility that the observed effect resulted from an absence of cell proliferation in the wing pouch because of a lack of sd expression. Wing discs overexpressing yki with the en-GAL4 driver exhibit clear increases in size of the posterior region, resulting in massive overgrowth. In contrast, the size of the posterior compartment was almost normal in wing discs from flies hemizygous for sdETX4 and overexpressing yki with the en-GAL4 driver. This result shows that Yki needs Sd to fully induce cell proliferation. Next, it was asked whether Sd-Yki interaction is acting downstream of the Hippo pathway. To test this, use was made of a dominant-negative form of Ft that lacks the intracellular domain (FTΔICD) . Indeed, Ft is an upstream component of the Hippo pathway, and overexpression of FTΔICD induces overgrowth and the expression of Hippo target genes. A significant reduction was observed of induced overgrowth in sdETX4 wing discs. This result strongly suggests that Sd-Yki interaction acts downstream of the Hippo pathway in wing discs (Goulev, 2008).

This paper has addressed the role of Sd-Yki interaction in the wing disc. The role of Sd in other imaginal tissues is poorly understood. Strong alleles of sd are associated with lethality in early larval stage. In the eye disc, clones homozygous for a strong allele of sd die or poorly survived, whereas they are associated with truncated legs when generated in the leg disc, suggesting that Sd should play a role in cell survival in these tissues. In the wing disc, sd clones die in the wing pouch but can be easily recovered in regions that will give rise to the notum, whereas Yki is required in the entire wing disc. This implies that other partners must interact with Yki to promote tissue growth in other structures. However, the induction of vg that was observed in cells overexpressing yki and the results showing that proliferation induced by Yki in the wing disc is significantly reduced in sd mutant context suggest that Sd-Vg is required for Yki function in the wing disc (Goulev, 2008).

The TEAD/TEF family protein Scalloped mediates transcriptional output of the Hippo growth-regulatory pathway

The Hippo (Hpo) kinase cascade restricts tissue growth by inactivating the transcriptional coactivator Yorkie (Yki), which regulates the expression of target genes such as the cell death inhibitor diap1 by unknown mechanisms. The TEAD/TEF family protein Scalloped (Sd) is a DNA-binding transcription factor that partners with Yki to mediate the transcriptional output of the Hpo growth-regulatory pathway. The diap1 (th) locus harbors a minimal Sd-binding Hpo Responsive Element (HRE) that mediates transcriptional regulation by the Hpo pathway. Sd binds directly to Yki, and a Yki missense mutation that abrogates Sd-Yki binding also inactivates Yki function in vivo. sd is required for yki-induced tissue overgrowth and target gene expression, and that sd activity is conserved in its mammalian homolog. These results uncover a heretofore missing link in the Hpo signaling pathway and provide a glimpse of the molecular events on a Hpo-responsive enhancer element (Wu, 2008).

The Hpo signaling pathway has emerged as a central and highly conserved mechanism that regulates organ size in animals. At the core of this pathway is a kinase cascade that impinges on the transcriptional coactivator Yki to regulate the transcription of target genes involved in cell growth, proliferation, and survival. Given Yki's pivotal position in the Hpo pathway, understanding the mechanisms by which Yki regulates target gene expression should provide important mechanistic insights that can facilitate therapeutic manipulation of this crucial size-control pathway (Wu, 2008).

A major gap in understanding of the Hpo signaling pathway concerns how Yki regulates target gene transcription. This study shows that Sd represents a crucial missing link between Yki and the regulatory DNA of Hpo pathway target genes. First, an unbiased dissection of diap1 regulatory region revealed a minimal Sd-binding enhancer element (HRE) that confers Hpo-responsive regulation. The HRE not only responded to Yki activity in vivo, but also conferred Sd-dependent and Yki-dependent transcriptional activity in cultured Drosophila cells. In a parallel line of experiments, Sd was identified in an unbiased screen for proteins that bind to a critical N-terminal Yki domain defined by a missense allele, ykiP88L. The fact that this missense mutation disrupted binding of Yki to Sd supports the physiological relevance of a Sd-Yki transcription complex in vivo. The identification of Sd as a cognate Yki partner using two unbiased approaches, combined with the genetic interactions between sd and yki, provide strong evidence that Sd is a critical DNA-binding factor that mediates the transcriptional output of the Hpo signaling pathway. It is worth noting that the requirement for sd in yki-driven overgrowth is highly specific, since the same sd mutation had no effect on overgrowth driven by the activated Ras oncogene. The observation that Yki, but not Sd, can be overexpressed or mutated to elicit tissue overgrowth further suggests that Sd is normally present in excess, and the activity of the Sd-Yki complex is regulated through modulation of Yki activity effected by the Hpo kinase cascade (Wu, 2008).

The founding member of the TEAD family transcription factors, TEAD-1/TEF-1, was initially identified based on its binding to the GTIIC motif of the simian virus 40 (SV40) enhancer. The TEAD family transcription factors have been mostly studied in the context of muscle-specific gene transcription, and their roles in cell proliferation and cell survival are poorly understood. The observation that TEAD-2 and YAP have similar activity to Sd and Yki, respectively, suggests that the growth-regulatory activity of the Sd-Yki complex is likely conserved in the mammalian Hpo pathway. It is also worth noting that besides growth regulation, the Hpo pathway has also been implicated in controlling other biological processes such as rhodopsin gene expression in mature photoreceptors and dendrite morphogenesis in postmitotic neurons. It remains to be determined whether Yki partners with Sd or other (unknown) DNA-binding factors in such nongrowth contexts (Wu, 2008).

Despite their elevated transcription upon inactivation of Hpo pathway tumor suppressors or activation of the Yki oncoprotein, it was previously unknown whether Yki regulates the known Hpo pathway target genes directly or indirectly through intermediary transcriptional regulators. This study has taken an unbiased approach to this question by isolating a HRE for diap1. This DNA element provided several important insights into how Hpo signaling activity is converted into transcriptional output. First, the Sd protein directly binds to the HRE and activates an HRE-luciferase reporter in cell culture in conjunction with Yki, supporting the notion that Yki directly regulates the transcription of the diap1 gene. Second, the minimal diap1 HRE contains non-Sd-binding sequence that is indispensable for HRE activity, suggesting that the HRE likely binds to additional transcription factors besides Sd in vivo. This latter characteristic is not unique to the HRE, but is a general feature that has been observed for many signaling pathways in Drosophila. For example, Notch-regulated enhancers contain not only binding sites for the signal-regulated transcription factor Suppressor of Hairless, but also binding sites for additional cofactors whose activity is Notch independent. It will be important to identify the factors that bind to the non-Sd sequence in the diap1 HRE, and to investigate whether such factors play a general role in mediating the Hpo responsiveness of other target genes (Wu, 2008).

The identification of a minimal HRE makes possible several new avenues of investigation to better understand the Hpo signaling pathway. The minimal HRE revealed in this study should facilitate a comprehensive cataloguing of Hpo pathway target genes, many of which remain to be identified. It also provides a useful tool for constructing reporters that can be used to monitor the specific activity of the Hpo pathway in vivo. Furthermore, this work will facilitate cell-based RNAi screens for components or modulators of the Hpo pathway, as illustrated by the successful use of pathway-specific luciferase reporters for interrogating other signaling pathway (Wu, 2008).

An interesting and somewhat unexpected finding from this study concerns the differential requirement of yki for the basal expression of diap1 and expanded (ex), with the former being yki dependent and the latter being yki independent, respectively. Thus, different Hpo target genes, and by inference different enhancer elements or their combinations, can respond to different threshold levels of Yki activity. It is suggest the basal expression of ex is mediated by non-HRE sequence in the ex locus and is therefore independent of yki. Excessive yki activity, either directly via an HRE in the ex locus or indirectly by turning on another factor, promotes ex transcription above the basal level. In contrast, Yki (through Sd, non-Sd DNA-binding factors, or both), regulates the basal level transcription of diap1. It is noted that the basal transcription of diap1 is not necessarily regulated through the HRE, which was identified by virtue of reporter expression under hyperactive Yki activities. Indeed, it was found that although the HRE is responsive to yki overexpression, it is largely unresponsive to loss of yki. Thus, the diap1 HRE revealed in this study is uniquely sensitive to unrestrained Yki activity (Wu, 2008).

The exquisite sensitivity of yki-induced overgrowth to sd dosage suggests that Sd/TEAD could be specifically targeted to ablate certain unwanted tissue growth, such as that caused by aberrant Hpo signaling, with minimal effect on normal growth. Thus, Sd/TEAD belongs to a growing list of genes that cause 'non-oncogene addition' -- genes that cannot be mutated or overexpressed to an extent that directly promotes tumorigenesis, but are still rate limiting to their specific signaling pathways. The requirement of such non-oncogenes in tumor cells makes them excellent targets for the development of new cancer therapeutics (Wu, 2008).

The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control

The Hippo (Hpo) signaling pathway governs cell growth, proliferation, and apoptosis by controlling key regulatory genes that execute these processes; however, the transcription factor of the pathway has remained elusive. This study provides evidence that the TEAD/TEF family transcription factor Scalloped (Sd) acts together with the coactivator Yorkie (Yki) to regulate Hpo pathway-responsive genes. Sd and Yki form a transcriptional complex whose activity is inhibited by Hpo signaling. Sd overexpression enhances, whereas its inactivation suppresses, tissue overgrowth caused by Yki overexpression or tumor suppressor mutations in the Hpo pathway. Inactivation of Sd diminishes Hpo target gene expression and reduces organ size, whereas a constitutively active Sd promotes tissue overgrowth. Sd promotes Yki nuclear localization, whereas Hpo signaling retains Yki in the cytoplasm by phosphorylating Yki at S168. Finally, Sd recruits Yki to the enhancer of the pathway-responsive gene diap1, suggesting that diap1 is a direct transcriptional target of the Hpo pathway (Zhang, 2008).

The Hpo pathway has emerged as a conserved signaling pathway that plays a critical role in controlling tissue growth and organ size. Despite the growing recognition of the importance of this pathway in development and cancer, the transcription factor that links the cytoplasmic components to the nuclear events has remained elusive and thus represents a major gap in the pathway. This study demonstrates Sd is the missing transcription factor of the Hpo pathway based on several lines of genetic and biochemical evidence. (1) Sd and Yki form a transcriptional complex to activate a reporter gene in S2 cells and this transcriptional activity is inhibited by Hpo signaling. Furthermore, Sd and Yki synergize in vivo to promote Hpo target gene expression and tissue overgrowth. (2) More importantly, loss of Sd function suppresses tissue overgrowth induced by Yki overexpression or loss-of-function mutations in hpo, sav, and wts. In addition, Sd inactivation either by RNAi or a genetic mutation blocks the ectopic expression of Hpo responsive genes induced by excessive Yki activity. (3) RNAi knockdown of Sd phenocopies knockdown of Yki, which is manifested by reduced organ size and diminished expression of Hpo pathway-responsive genes. (4) A constitutively active form of Sd activates multiple Hpo pathway-responsive genes and promotes tissue overgrowth. (5) Sd promotes Yki nuclear translocation and recruited Yki to the diap1 enhancer (Zhang, 2008).

Several sd null alleles were generated to further explore the consequence of loss of Sd. sd null clones located in the wing pouch region were found to exhibit growth deficit such that early-induced clones (48-72 hrs AEL) were eliminated by the end of late third instar. However, late-induced clones (72-96 hrs AEL) survived and exhibited diminished expression of diap1. In contrast, early-induced clones were recovered in the notal region of wing discs and in eye discs without showing discernible change in Diap1 levels. However, a previous study showed that yki mutant clones exhibited reduced diap1 expression in eye discs. It is possible that low levels of residual Sd activity persist in sd mutant clones, which are sufficient to support the basal expression of the Hpo target genes. Alternatively, Yki may act through another transcription factor to maintain the basal expression of Hpo target genes. Nevertheless, sd null mutation suppresses the overgrowth phenotype and ectopic cycE expression induced by excessive Yki activity, suggesting the residual Sd in sd mutant clones is insufficient to support the elevated Yki activity (Zhang, 2008).

The identification of Hpo pathway transcription factor provided an opportunity to assess direct transcriptional targets of the pathway. To this end, the diap1 enhancer was characterized, and a 1.8 kb enhancer element critical for diap1 expression was identified. This region contains a total of seventeen predicted Sd binding sites. Using the ChIP assay, it was demonstrated that both Sd and Yki physically interact with the 1.8 kb diap1 enhancer and the association of Yki with the diap1 enhancer is mediated by Sd. These results suggest that Sd recruits Yki to the diap1 enhancer to activate its transcription (Zhang, 2008).

It has been shown that Sd acts in conjunction with Vg to promote wing development by directly regulating the expression of wing patterning genes. This study has demonstrated that Sd acts in conjunction with Yki to control organ size by regulating the expression of genes involved in cell proliferation, cell growth, and apoptosis. These observations raise an important question of how Yki-Sd and Vg-Sd transcriptional complexes specifically select their targets. One possibility is that Vg-Sd and Yki-Sd prefer to interact with distinct Sd binding sites. Indeed, a previous study showed that binding of Vg to Sd modulates the DNA binding selectivity of Sd. Another possibility is that target selectivity could be influenced by cofactors that bind in the vicinity of Sd binding sites. In support of this notion, previous studies have shown that wing specific enhancers contain both Sd binding sites and binding sites for transcription factors that mediate specific signaling pathways. It is also possible that Vg-Sd and Yki-Sd may share common targets. For example, diap1 could be activated by Vg-Sd in the wing pouch, which might explain why sd mutant clones in this region exhibits diminished diap1 expression (Zhang, 2008).

In principle, the Hpo pathway could regulate the activity of Yki-Sd transcriptional complex at several levels. For example, Hpo signaling could regulate the formation Yki-Sd complex or the recruitment of other factor(s) to the Yki-Sd transcriptional complex. Alternatively, Hpo signaling could regulate the nuclear-cytoplasmic transport of Yki. In support of the latter possibility, Yki exhibits elevated nuclear localization in wts or hpo mutant clones. In addition, coexpression of Hpo with Yki depletes nuclear Yki in S2 cells, suggesting that Hpo signaling impedes nuclear localization of Yki and thereby limits the amount of active Yki-Sd transcriptional complex (Zhang, 2008).

Mutating Yki S168 to Ala increases nuclear localization and growth promoting activity of Yki. In addition, it has been demonstrated that phosphorylation of Yki S168 was stimulated by Hpo. Phosphorylation of Yki by Hpo signaling increases their association with 14-3-3, which is abolished by mutating Yki S168 to Ala. Since 14-3-3 often regulates nuclear-cytoplasmic shuttling of its interacting proteins, these observations suggest that Hpo signaling inhibits Yki at least in part by phosphorylating Yki S168, which promotes 14-3-3 binding and cytoplasmic sequestration of Yki (Zhang, 2008).

The Hpo pathway appears to restrict cell growth and control organ size in mammals. The finding that Sd is critical for Yki-induced tissue growth has raised the interesting possibility that the effect of YAP in promoting tissue growth may rely on the TEAD/TEF family of transcription factors. Corroborating this hypothesis, TEAD-2/TEF-4 protein purified from mouse cells was associated predominantly with YAP (Vassilev, 2001). Furthermore, YAP can bind to and stimulate the trans-activating activity of all four TEAD/TEF family members (Vassilev, 2001). The TEAD/TEF family members exhibit overlapping but distinct spatiotemporal expression patterns and thus may have redundant but unique roles during development . It will be important to determine which TEAD/TEF family members are involved in the mammalian Hpo pathway and whether YAP employs distinct sets of TEAD/TEF transcription factors in different tissues. Since abnormal activation of YAP is associated with multiple types of cancer, disrupting YAP-TEAD/TEF interaction may provide a new strategy for cancer therapeutics (Zhang, 2008).

TEAD mediates YAP-dependent gene induction and growth control

The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. This study demonstrates that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial-mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). This study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP (Zhao, 2008).

To investigate the function of TEAD in YAP-induced growth control, transgenic flies were generated that express human YAP-S127A (an active form) or YAP-S94A/S127A in developing eyes. YAP-S127A overexpression significantly increased eye size and the number of interommatidial cells. Mutation of S94A dramatically decreased the activity of YAP-S127A in promoting tissue growth. Scalloped (Sd) is the only TEAD homolog in Drosophila. Yki was found to directly interacted with Sd in an in vitro binding assay. Furthermore, Yki S97A mutation (equivalent to YAP-S94A) diminished its interaction with Sd. Moreover, this Sd-binding-defective Yki-S97A mutant was less potent in stimulating growth in vivo compared with wild-type Yki. The functional defect of the TEAD-binding-deficient YAP/Yki was further confirmed by generating overexpression flip-out clones in the Drosophila larval wing discs as labeled by positive GFP expression. Both YAP-S127A and Yki are potent in stimulating tissue growth as individual clones, and the whole discs were generally larger than wild-type clones or discs. However, neither YAP-S94A/S127A nor Yki-S97A showed a similar level of growth-promoting effect. These data indicate that TEAD/Sd binding is important for the physiological function of YAP/Yki (Zhao, 2008).

The genetic interaction between Yki and Sd was tested. A strong loss-of-function allele of sd dominantly suppressed the enlarged and rough eye phenotypes caused by Yki overexpression. Thus, the level of Sd is critical for Yki to promote tissue growth. Overexpression of Sd caused small eyes, presumably due to a dominant-negative effect, but it did not result in lethality. This phenotype was strongly enhanced by reduction of yki levels, such that all of these flies died at the late pupal stage and had no eyes. Furthermore, coexpression of Yki with Sd suppressed the reduced eye phenotype caused by Sd overexpression. In fact, the eyes of animals overexpressing both Yki and Sd were enlarged more than those of animals that only expressed Yki. Therefore, Sd overexpression enhanced the Yki overexpression phenotypes. Together, these results indicate that Sd is a critical functional partner of Yki, a conclusion consistent with TEAD as a critical downstream target transcription factor of YAP (Zhao, 2008).

The FERM-domain protein Expanded regulates Hippo pathway activity via direct interactions with the transcriptional activator Yorkie

The Hippo kinase pathway plays a central role in growth regulation and tumor suppression from flies to man. The Hippo/Mst kinase phosphorylates and activates the NDR family kinase Warts/Lats, which phosphorylates and inhibits the transcriptional activator Yorkie/YAP. Current models place the FERM-domain protein Expanded upstream of Hippo kinase in growth control. To understand how Expanded regulates Hippo pathway activity, affinity chromatography and mass spectrometry were used to identify Expanded-binding proteins. Surprisingly it was found that Yorkie is the major Expanded-binding protein in Drosophila S2 cells. Expanded binds Yorkie at endogenous levels via WW-domain-PPxY interactions, independently of Yorkie phosphorylation at S168, which is critical for 14-3-3 binding. Expanded relocalizes Yorkie from the nucleus, abrogating its nuclear activity, and it can regulate growth downstream of warts in vivo. These data lead to a new model whereby Expanded functions downstream of Warts, in concert with 14-3-3 proteins to sequester Yorkie in the cytoplasm, inhibiting growth activity of the Hippo pathway (Badouel, 2009).

Current models propose that ex and mer function together to restrict tissue growth upstream of hpo. Mer and Ex colocalize with cortical actin in the apical region of the cell. Both genetic and physical interactions have been observed between Mer and Ex: loss of one copy of mer dominantly enhanced wing overgrowth in ex mutants. In addition, fragments of Mer and Ex protein can interact physically with each other in far-western experiments or when overexpressed in cultured cells. Clones doubly mutant for mer and ex have more dramatic overgrowth than either single mutant, and mer,ex double mutants phenocopy hpo mutants (Badouel, 2009).

However, despite the widespread acceptance that ex and mer function upstream of Hpo, some data are difficult to reconcile with ex acting strictly upstream of hpo in activation of the pathway. Genetic analysis indicates that ex is downstream of dachs, which has been shown to be directly upstream of wts in growth control. Biochemical analysis of the effects of overexpressing Mer and Ex also suggested that ex may not act simply upstream of hpo. For example, overexpression of Mer in S2 cells leads to a shift in Wts mobility, whereas overexpression of Ex does not alter Wts mobility. In vivo analysis has also suggested that mer and ex may have different roles in controlling growth and apoptosis (Badouel, 2009).

Using biochemical purification and mass-spectrometic analysis, Yki as a major Ex-binding protein in Drosophila S2 cells was identified. Binding of Yki to Ex is direct and is mediated by a WW domain-PPxY interaction. This interaction is independent of Wts-dependent phosphorylation at S168, a site previously shown to be essential for strong interactions of Yki with 14-3-3 proteins. Consistent with the biochemical analysis, it was shown that ex can act downstream of wts in the regulation of growth in eye imaginal discs and can repress the pupal lethality caused by excessive growth of wts clones. In addition, it was found that loss of hpo does not alter the ability of ex to regulate yki activity, as indicated by transcriptional assays in S2 cells, and that expression of Ex is sufficient to relocalize Yki to the cytoplasm. These data lead to a model in which Ex functions to repress Yki activity at least in part by keeping Yki out of the nucleus. Intriguingly, the Yki homolog, YAP, was first identified as a protein that binds Src family kinases at the cell membrane. Subsequent studies have focused on the role of Yki in the nucleus. Interestingly, immunohistochemical analysis reveals that a portion of Yki colocalizes with Ex at the cell membrane in Drosophila imaginal discs (Badouel, 2009).

Once Yki is phosphorylated by Wts, it can bind 14-3-3 proteins and can be transported out of the nucleus. However, since 14-3-3-bound Yki can also shuttle back into the nucleus, Ex binding Yki provides an anchor that can effectively dampen Yki activity. 14-3-3 shuttling activity results in an equilibrium of distribution of Yki between the nucleus and the cytoplasm. This equilibrium is biased in favor of Yki in the cytoplasm in the presence of Ex acting as an anchor. The presence of a tether of Yki in the cytoplasm was already suggested based on the distribution of YkiS168A between the cytoplasm and the nucleus, instead of predominantly in the nucleus (Badouel, 2009).

The strong nuclear localization of Yki is seen in Drosophila tissues only in cases of pathological stimulation of growth, such as in wts loss-of-function clones, which lead to massive overgrowth. The lack of detectable Yki nuclear localization during normal growth regulation suggests that Yki is an exceedingly potent growth regulator, and points to why there are many layers of regulation of Yki localization. The need for Ex to dampen Yki signaling in the nucleus is reflected by the increase of Cyclin E and Diap1 transcription in ex mutant clones (Badouel, 2009).

It is speculated that the regulation of the Hpo pathway by combined loss of Ex and Mer is so potent because one acts as the brake and the other controls the accelerator. Ex restricts Yki to the cytoplasm, thus blocking activity downstream, whereas Mer activates Hpo activity, thereby restricting Yki via inhibitory phosphorylation. Thus, loss of Ex on its own does not have a dramatic effect on cell proliferation and apoptosis, since the activity of the kinase cascade is regulated via Mer. Conversely, as long as Ex is present, excessive pathway activity induced by loss of Mer can be effectively modulated by the dampening activity of Ex (Badouel, 2009).

The data strongly suggest that Ex regulates Yki activity downstream of wts, by directly binding Yki and inhibiting Yki nuclear localization and transcriptional activity. The possibility cannot, however, be excluded that Ex also has additional upstream roles in regulating Hpo activity. Interestingly, overexpressed Ex does not induce apoptosis in a wts mutant background, although it can block growth, suggesting that ex is upstream of wts in apoptosis control, yet downstream of wts in growth control. Genetic dissection of this pathway is complicated by the well-documented feedback loops in the Hpo pathway: for example, Yki regulates the expression of both mer and ex. In addition, genetic evidence suggests that ex and mer function together to regulate endocytosis and growth factor signaling. Further biochemical dissection of Hpo pathway activity will be required to fully elucidate the diverse ways in which growth and apoptosis are controlled in response to various developmental and environmental signals (Badouel, 2009).

The binding of Ex to Yki is likely to be 14-3-3 independent, as mutation of S168, the predominant Wts phosphorylation site, impairs 14-3-3 binding, yet does not affect the ability of Ex to bind Yki. Thus, Ex binding to Yki could provide a pool of Yki that is nonphosphorylated and poised for release by upstream growth regulators. The binding of 14-3-3 to Yki can protect Yki from dephosphorylation. This provides a problem for the cell, since 14-3-3 must dissociate from Yki to allow it to become dephosphorylated, thus releasing a potent activator of proliferation and inhibitor of cell death, allowing it to re-enter the nucleus. The ability of Ex to bind both phosphophorylated and dephosphorylated Yki provides a mechanism by which to anchor dephosphorylated Yki in the cytoplasm (Badouel, 2009).

An appealing model is that the binding of Ex to Yki may be modulated, once in the cytoplasm, as an additional control point for the Hpo pathway. FERM-domain proteins frequently form inhibitory intramolecular associations, blocking the activity of the protein until the repression is relieved. Modifications of the Ex FERM domain or linker region could thus alter the ability of Ex to bind Yki in vivo. Ex localization at apical junctions is at least partially dependent upon the atypical cadherin Fat, which can regulate Hpo pathway activity. The recruitment of Ex complexes (directly or indirectly) to Fat may modify the ability of Ex to interact with Yki (Badouel, 2009).

The in vivo analysis indicates that the N-terminal FERM domain of Ex contains apical localization elements, whereas the C-terminal region contains junctional localization elements. Thus, each of these localization elements might be regulated independently and might impact on the ability of Ex to sequester Yki. Identification of which protein(s) Ex binds at junctions may illuminate the mechanisms by which Ex responds to external inputs to regulate Yki activity (Badouel, 2009).

All of the components of the Hpo pathway are well conserved in mammals and have been shown to have conserved functions in regulating growth. Loss of Hpo and Wts orthologs and overexpression of the Yki ortholog, YAP, have been implicated in a variety of human cancers. FERM6, the human ortholog of Ex, also regulates Hpo pathway activity in mammals. Future studies will determine if FERM6 directly binds YAP, and if disrupting YAP-FERM6 interactions is a tumor-promoting event (Badouel, 2009).

A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCFβ-TRCP

The Yes-associated protein (YAP) transcription coactivator (a homolog of Drosophila Yorkie) is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14-3-3 binding and cytoplasmic retention. This study reports that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1delta/epsilon (Drosophila homolog: Discs overgrown) in a phosphodegron. The phosphorylated phosphodegron then recruits the SCFβ-TRCP E3 ubiquitin ligase (see Drosophila Slmb), which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. This study identified CK1delta/epsilon as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation) (Zhao, 2010).

Accumulating evidence supports the role of YAP as a key controller of organ size and as a human oncogene. Elucidating the mechanisms regulating YAP activity will have implications in the normal physiology of organ size regulation and pathogenesis of human cancer. The Hippo pathway is the only inhibitor of YAP known to date. It has been shown to play a key role in limiting organ size in Drosophila, and deregulation of several components of this pathway, such as NF2 mutation, has been implicated in human cancer. It has been shown that the Hippo pathway inhibits YAP by S127 phosphorylation-mediated 14-3-3 binding and cytoplasmic retention, therefore providing a mechanism of spatial separation of YAP from its nuclear target transcription factors, such as TEAD (Zhao, 2010).

YAP has been shown to be ubiquitinated, although the mechanism was unknown. The data presented in this study elucidated another layer of YAP regulation. By phosphorylation on S381, the Hippo pathway primes YAP for phosphorylation by CK1delta/epsilon, and subsequent ubiquitination and degradation. This provides a mechanism of temporal regulation of YAP protein levels upon activation of the Hippo pathway. Under physiological conditions like high cell density, the S381 phosphorylation-mediated degradation might be the major cause for YAP degradation. Relative S381 phosphorylation dropped dramatically when cell density increased, although relative S127 phosphorylation of YAP was increased, indicating that the S381-phosphorylated YAP could not be accumulated, possibly due to degradation. However, it is possible that there exists a S127 phosphorylation-dependent fail-safe mechanism for YAP destabilization when S381-mediated degradation is not working properly. Such a mechanism may explain why both S127 and S381 mutations are required for YAP stabilization. This study reveals that inhibition of YAP by the Hippo pathway is more complex than expected, with both spatial and temporal mechanisms. It is speculated that the spatial regulation could provide a reversible short-term inhibition of YAP, while the temporal regulation through YAP degradation may provide an irreversible long-term inhibition. Dysregulation of both mechanisms could lead to oncogenic transformation (Zhao, 2010).

It is worth noting that the S381-initiated degradation of YAP is not conserved in Drosophila Yki, because this phosphorylation site and the phosphodegron are not present in Yki, although they are conserved through vertebrates. However, this does not exclude the possibility that Yki protein stability is controlled by the Hippo pathway through other mechanisms. The phosphodegron is conserved in TAZ, a YAP paralog, and also modulates TAZ stability in a similar manner (Zhao, 2010).

Are there additional mechanisms of YAP regulation by the Hippo pathway? The possibility exists. The current studies confirmed three other Lats phosphorylation sites in YAP, but their functions are unknown. Although these sites do not seem to play an obvious role in controlling the oncogenic activity of YAP, as indicated by NIH-3T3 cell transformation assay, they may function in other contexts (Zhao, 2010).

The similarity between YAP and β-catenin is quite interesting. β-catenin is also a transcription coactivator implicated in malignant transformation. Without Wnt signaling, β-catenin is constantly degraded through SCFβ-TRCP-mediated ubiquitination. Similar to YAP, β-catenin binding with SCFβ-TRCP depends strictly on multistep phosphorylation of the phosphodegron involving CK1α and GSK-3. Perturbation of this process leads to β-catenin accumulation in colorectal cancer, HCCs, and malignant melanomas. There are similarities between YAP and β-catenin in many aspects, including their function as transcription coactivators with growth-promoting activity and as latent oncogenes. They are both subject to multistep phosphorylation and phosphodegron-dependent ubiquitination by SCFβ-TRCP, and deregulation of the degradation leads to oncogenic transformation (Zhao, 2010).

Extensive studies have been done to analyze mutations leading to β-catenin stabilization, which should shed light on future studies of YAP. In the case of β-catenin, its stabilization in cancer is frequently due to failure to recruit GSK3 as a result of inactivating mutations of adenomatous polyposis coli (APC) or axin. In some cases, stabilization of β-catenin also results from mutation in the phosphodegron and its priming phosphorylation sites. Interestingly, elevated YAP protein levels have been observed in some cancers. It will be interesting to survey possible YAP mutations in cancer samples and identify proteins regulating YAP phosphodegron phosphorylation. It will also be important to examine deregulation of YAP protein levels as a result of Hippo pathway component mutations in cancer (Zhao, 2010).

CK1 is a family of multifunctional kinases with unique substrate specificity as pS/T-X1-2-S/T. Phosphorylation by CK1 requires preceding phosphorylation of residue at the -2 or -3 position of the target residue. This requirement of a priming phosphorylation by another kinase provides a possible mechanism of signal integration in complex biological processes. For example, in the case of YAP destabilization, the requirement of CK1delta/epsilon phosphorylation following Lats phosphorylation may integrate other signals besides the Hippo pathway to regulate YAP. CK1 is often referred to as constitutively active kinase. However, it has also been reported that CK1 is regulated by subcellular localization and inhibitory autophosphorylation by stimuli such as γ irradiation and Wnt signaling. At high cell density, a clear drop of relative YAP-S381 phosphorylation and an increase of relative YAP-S127 phosphorylation are observed. The fact that both sites are phosphorylated by Lats kinase suggests that phosphorylation of S384 might induce YAP degradation. It will be interesting to investigate if cell density increases CK1 activity (Zhao, 2010).

In Drosophila, the CK1delta/epsilon homolog discs overgrown (dco) has been positioned in the Hippo pathway upstream of dachs by its regulation of the Hippo pathway downstream target genes and by genetic epistasis experiments. Recently, dco has further been shown to phosphorylate Fat, although it has not been determined if this phosphorylation directly affects Fat function and the Hippo pathway activity. However, the function of CK1delta/epsilon in regulating YAP-β-TRCP interaction is not due to inhibition of the Hippo pathway, as both YAP-4SA/S381 and YAP-S381D mutants are still inhibited by IC261. Conversely, the mechanism of CK1delta/epsilon in regulating YAP stability is unlikely to be conserved in dco, as the phosphodegron is not conserved in Yki. Nevertheless, the function of dco/CK1delta/epsilon in inhibiting Yki/YAP is conserved between Drosophila and mammals, although different mechanisms may be employed (Zhao, 2010).

YAP contains a phosphodegron, DSGXS, that is highly similar to but does not exactly match the canonical DSGXXS phosphodegron. However, the requirement of the second serine residue for β-TRCP binding is less stringent compared with the first one. In the reported phosphodegron variants, some of them require the second serine to be further away from the DSG, and, in certain cases like CDC25A, the second S is not even required. In the case of YAP, the second serine (S387) is not absolutely required, but contributes to YAP interaction with β-TRCP and YAP ubiquitination. This was shown by the residual binding between β-TRCP and the phosphorylation-deficient S387A, and the largely normal binding between β-TRCP and the phosphomimetic S387D (Zhao, 2010).

The exact YAP sequence S(-3)TDS(0)G, where S(-3) (S381) serves as a priming phosphorylation site for S(0) (S384), is conserved in some other β-TRCP substrates like CDC25A, which contains S(-6)XXS(-3)TDS(0)G. In this case, the -6 position serine phosphorylation by Chk1 is shown to be required for CDC25A binding with β-TRCP and subsequent degradation in vivo. However, in an in vitro binding assay, a peptide with phosphorylation on the S(0) but not S(-3) showed a strong binding to β-TRCP, which was not further enhanced by phosphorylation on S(-3). This in vitro binding assay using peptides sharing similar phosphodegron structure with YAP helps to exclude the function of YAP-S381 as an integral part of the phosphodegron directly involved in β-TRCP binding, but rather supports S381 as a priming phosphorylation site for S384 phosphorylation by CK1delta/epsilon. Compared with YAP, it is speculated that the main function of the S(-3) in the CDC25A phosphodegron might be a phosphorylation-relaying residue passing the signal from the -6 position to the 0 position instead of being directly involved in β-TRCP binding. Phosphodegron with a phosphorylated -3 position serine also exists in other known SCFβ-TRCP substrates, such as RE-1 silencing transcription factor (REST). Together with YAP, they may represent a class of SCFβ-TRCP substrates containing a SXDSG phosphodegron, in which the first serine serves as a priming phosphorylation site. In the case of CDC25A and REST, the kinase responsible for phosphorylating the second serine residue is unknown. The CK1 family kinases are attractive candidates for this function because of their pS/T-X1-2-S/T target consensus. It is speculated that there may be a broader role for the CK1 family in SCFβ-TRCP-mediated protein ubiquitination and degradation (Zhao, 2010).

In close proximity with the YAP phosphodegron, there is a tyrosine residue (Y391) reported to be phosphorylated by c-Abl in response to DNA damage, which results in YAP stabilization. Future studies are needed to test if the Y391 phosphorylation modulates SCFβ-TRCP-mediated YAP ubiquitination and degradation (Zhao, 2010).

Cooperation between dE2F1 and Yki/Sd defines a distinct transcriptional program necessary to bypass cell cycle exit

The Hippo signaling pathway regulates organ size homeostasis, while its inactivation leads to severe hyperplasia in flies and mammals. The transcriptional coactivator Yorkie (Yki) mediates transcriptional output of the Hippo signaling. Yki lacks a DNA-binding domain and is recruited to its target promoters as a complex with DNA-binding proteins such as Scalloped (Sd). In spite of recent progress, an open question in the field is the mechanism through which the Yki/Sd transcriptional signature is defined. This study reports that Yki/Sd synergizes with and requires the transcription factor dE2F1 to induce a specific transcriptional program necessary to bypass the cell cycle exit. Yki/Sd and dE2F1 bind directly to the promoters of the Yki/Sd-dE2F1 shared target genes and activate their expression in a strong cooperative manner. Consistently, RBF, a negative regulator of dE2F1, negates this synergy and limits the overall level of expression of the Yki/Sd-dE2F1 target genes. Significantly, dE2F1 is needed for Yki/Sd-dependent full activation of these target genes, and a e2f1 mutation strongly blocks yki-induced proliferation in vivo. Thus, the Yki transcriptional program is determined through functional interactions with other transcription factors directly at target promoters. It is suggested that such functional interactions would influence Yki activity and help diversify the transcriptional output of the Hippo pathway (Nicolay, 2011).

While recent work has provided insight into how the regulation of Yki occurs via the location within the cell through protein-protein interactions, less is known about how Yki-mediated transcription is regulated. The results presented in this study suggest that Yki may rely on a combinatorial network of transcription factors to modulate transcriptional output in response to Hippo pathway signaling. One such transcription factor is dE2F1, which is required for the full activation of specific target genes by Yki/Sd (Nicolay, 2011).

These studies were prompted by the strong enhancement of the wts mutant phenotype by an rbf mutation. Both the pRB and Hippo pathways are negative regulators of cell proliferation. In flies, RBF functions to limit the activity of the transcriptional activator dE2F1, while the Wts kinase inhibits the transcriptional coactivator Yki. Therefore, one possibility is that, in rbf wts double mutants, dE2F1 and Yki are left unchecked to independently induce genes that promote cell proliferation. However, the data do not support such a trivial explanation. Microarray profiling followed by gene ontology analysis demonstrated that the rbf wts double mutant gene expression signature was distinct from that of either rbf or wts single mutants. Importantly, the rbf wts double mutant signature contained a significant number of up-regulated genes involved in cell cycle progression and cell proliferation that were not present in the rbf or wts single mutant signatures. Thus, an alternative explanation, one that is favored, is that, in rbf wts double mutants, hyperactivated dE2F1 and Yki synergistically up-regulate a novel set of genes and establish the distinct gene expression signature needed to overcome terminal cell cycle exit upon differentiation. Importantly, the synergy results from a direct binding and cooperation between the two factors on the target promoters, since both can be detected by ChIP on dE2F1-Yki/Sd coregulated genes. Consistently, inhibition of dE2F1 by RBF, which is also present on the same set of promoters, is sufficient to limit this synergistic activation by dE2F1 and Yki/Sd (Nicolay, 2011).

Previous studies demonstrated that, in the absence of de2f1, Yki fails to drive inappropriate proliferation, indicating that Yki alone is not sufficient to induce the transcriptional program to prevent cell cycle exit. Importantly, Yki is still active and capable of inducing other Yki-dependent target genes, such as dIAP1. Thus, it appears that the interplay between Yki/Sd and dE2F1 is highly specific to the activation of a distinct set of target genes and is not simply a reflection of a Yki transcription program gone awry. It is suggested that Yki requires an assist from dE2F1 to up-regulate some, if not all, of the dE2F1-Yki/Sd target genes. This assist is critical, since, in the absence of dE2F1, Yki is unable to fully activate these genes to a level sufficient to bypass the cell cycle exit and undergo inappropriate proliferation. Such an interpretation is supported by the transcriptional reporter assays demonstrating that the activation potential of Yki/Sd is reduced in dE2F1-depleted cells. It is noteed that the dE2F1-Yki/Sd target genes are regulated primarily through activation. It remains unclear why RBF/dE2F2 complexes are bound at promoters that are regulated by dE2F1, yet these genes remain insensitive to RBF/dE2F2-mediated repression. Interestingly, two of the dE2F1-Yki/Sd target genes, dDP and cdc2c, were isolated in a genome-wide RNAi screen for factors that are required for Yki to activate a synthetic reporter (Ribeiro, 2010). Given that de2f1 is a transcriptional target of Yki activity as well, it is tempting to speculate that a positively reinforcing signaling loop occurs between Yki/Sd and dE2F1 (Nicolay, 2011).

Yki is a potent oncogene and can elicit a dramatic effect on cell proliferation and apoptosis. Therefore Yki is tightly regulated at multiple levels, including its transcriptional activity, nuclear localization, and degradation. Additionally, it appears that Yki target gene specificity is determined by the transcription factors that interact with Yki and tether it to DNA. For example, Yki partners with Sd and Hth transcription factors. Notably, Hth/Yki transcriptional complexes appear to be important for promoting cell proliferation and survival within the anterior compartment of the eye disc, while in the posterior of the eye disc, Yki switches to partner with Sd to regulate a different set of target genes. The ability of Yki to partner with different DNA-binding proteins in different contexts is thought to provide a basis for altering the transcriptional output of the Hippo pathway. The current results exemplify how, under oncogenic conditions, another transcription factor, such as dE2F1, helps to set up a specific Yki/Sd gene expression signature that is needed to overcome the cell cycle exit. Thus, one conclusion drawn from these results is that the Yki transcriptional program is determined not only by DNA binding proteins that recruit Yki to its target genes, but additionally through interactions with other transcription factors directly at specific target genes. Such functional interactions would influence Yki activity and essentially help to further shape the transcriptional output of the Hippo pathway (Nicolay, 2011).

Another implication of the results is that not only does dE2F1 help to engage a Yki/Sd transcriptional program, but, conversely, a hyperactive Yki/Sd complex contributes to the deregulation of E2F transcription in rbf wts double mutant cells. Given that E2F-dependent transcription is often deregulated in tumor cells, this is an important point. Thus, depending on the identity of other cooperating mutations in pRB-deficient tumor cells, E2F can potentially synergize with a distinct repertoire of transcription factors to engage in transcriptional programs unique to tumor cells of different origins (Nicolay, 2011).

Although initially Yki-induced ectopic proliferation was characterized by an up-regulation in the expression of cyclin E, cyclin A, and cyclin B in flies, this mechanism does not appear to be conserved. In mammals, the up-regulation of cyclin D1 by YAP (the Yki mammalian homolog) is thought to be more critical in promoting inappropriate cell divisions. Thus, it is possible that, in mammals, YAP relies on a different network of transcription factors to promote cell cycle progression than Yki does in flies. Indeed, although YAP has been shown to partner with the Sd homologs TEAD1-4 in mammals, it is also known to interact with other transcription partners (SMAD1 and p73) under specific contexts. Thus, it appears that, similar to Yki, YAP may rely on a distinct repertoire of transcription factors to relay the response to various cellular stimuli (Nicolay, 2011).

Intriguingly, it has been demonstrated that the pRB and Hippo pathways are functionally integrated in human cells. However, the precise mechanism of interaction has seemingly evolved, as it has been shown that inactivation of the Wts homolog LATS2 interferes with the formation of the p130/DREAM repressor complex at E2F target promoters. The inability to repress E2F targets in the absence of LATS2 prevents pRB-induced senescence in human cells . In contrast, the Drosophila dREAM complex appears to be functional in wts mutants (data not shown), and instead the cross-talk between the two pathways occurs at the level of cooperation between Yki and dE2F1. Nonetheless, although the mechanistic paths taken may have diverged between flies and humans, the end point is the same: limit E2F transcriptional activity to prevent inappropriate proliferation (Nicolay, 2011).

To date, the most well-defined oncogenic role for YAP, in the context of Hippo pathway signaling, is in the formation of hepatocellular carcinoma (HCC). However, YAP is also capable of transforming immortalized human mammary epithelial cells, which appears to be through an interaction with the EGFR signaling pathway. In the future, it will be interesting to determine how many other signaling networks oncogenic YAP activity is dependent on, and with what degree these interactions are tissue- or cell type-specific. Finally, these findings support a conserved function of the pRB and Hippo pathways and suggest that a complex coordination of gene expression by these two pathways may underlie a key mechanism during oncogenic proliferation (Nicolay, 2011).

Homeodomain-interacting protein kinase regulates Yorkie activity to promote tissue growth

The Hippo (Hpo) tumor suppressor pathway regulates tissue size by inhibiting cell proliferation and promoting apoptosis. The core components of the pathway, Hpo, Salvador, Warts (Wts), and Mats, form a kinase cascade to inhibit the activity of Yorkie (Yki), the transcriptional effector of the pathway. Homeodomain-interacting protein kinases (Hipks) are a family of conserved serine/threonine kinases that function as regulators of various transcription factors to regulate developmental processes including proliferation, differentiation, and apoptosis. Hipk can induce tissue overgrowth in Drosophila. This study demonstrates that Hipk is required to promote Yki activity. Hipk affects neither Yki stability nor its subcellular localization. Moreover, hipk knockdown suppresses the overgrowth and target gene expression caused by hyperactive Yki. Hipk phosphorylates Yki and in vivo analyses show that Hipk's regulation of Yki is kinase-dependent. This is the first kinase identified to positively regulate Yki (J. Chen, 2012; see graphical abstract).

These findings indicate that Hipk promotes Yki transcriptional activity to regulate tissue growth during Drosophila development. Because Hipk functions in multiple signaling pathways, the possibility cannot be excluded that Hipk regulates Hpo signaling and Yki via multiple pathways; however, it is thought that this study has ruled out a regulation through known targets. Furthermore, it was shown that Hipk phosphorylates Yki, implying a direct regulatory role of Hipk on Yki. Although Hipk promotes Yki activity by phosphorylation, the functional significance of this modification has yet to be further investigated. These analyses have applied the same genetic criteria and obtained similar results as were used to demonstrate that Sd was an essential factor for Yki-mediated growth. Given that Hipk exerts its effects on nuclear Yki and is enriched in nuclear speckles , it may play an important role to facilitate the interactions between Yki and its transcriptional cofactors. Further studies should reveal whether this growth regulation by Hipk family members is evolutionarily conserved. Trapasso (2009) demonstrate that hipk2 mutant mice have a reduced body size and hipk2-/- mouse embryo fibroblasts show reduced proliferation rates, suggesting that Hipks may also promote Yap activity to regulate tissue growth in vertebrates. Using mammalian cell culture, it has been observed that overexpressing WT Hipk2, but not kinase-dead Hipk2, is able to induce elevated Yap level. This finding suggests that vertebrate Hipks may regulate Hpo signaling through Yap stability and possibly also by regulating Yap nuclear activity (J. Chen, 2012).

Homeodomain-interacting protein kinase regulates Hippo pathway-dependent tissue growth

The Salvador-Warts-Hippo (SWH) pathway is an evolutionarily conserved regulator of tissue growth that is deregulated in human cancer. Upstream SWH pathway components convey signals from neighboring cells via a core kinase cassette to the transcription coactivator Yorkie (Yki). Yki controls tissue growth by modulating activity of transcription factors including Scalloped (Sd). To date, five SWH pathway kinases have been identified, but large-scale phosphoproteome studies suggest that unidentified SWH pathway kinases exist. To identify such kinases, this study performed an RNA interference screen and isolated homeodomain-interacting protein kinase (Hipk). Unlike previously identified SWH pathway kinases, Hipk is unique in its ability to promote, rather than repress, Yki activity and does so in parallel to the Yki-repressive kinase, Warts (Wts). Hipk is required for basal Yki activity and is likely to regulate Yki function by promoting its accumulation in the nucleus. Like many SWH pathway proteins, Hipk's function is evolutionarily conserved as its closest human homolog, HIPK2, promotes activity of the Yki ortholog YAP in a kinase-dependent fashion. Further, HIPK2 promotes YAP abundance, suggesting that the mechanism by which HIPK2 regulates YAP has diverged in mammals (Poon, 2012).

Drosophila activated Cdc42 kinase has an anti-apoptotic function

Activated Cdc42 kinases (Acks) are evolutionarily conserved non-receptor tyrosine kinases. Activating somatic mutations and increased ACK1 protein levels have been found in many types of human cancers and correlate with a poor prognosis. ACK1 is activated by epidermal growth factor (EGF) receptor signaling and functions to regulate EGF receptor turnover. ACK1 has additionally been found to propagate downstream signals through the phosphorylation of cancer relevant substrates. Using Drosophila as a model organism, it has been determined that Drosophila Ack possesses potent anti-apoptotic activity that is dependent on Ack kinase activity and is further activated by EGF receptor/Ras signaling. Ack anti-apoptotic signaling does not function through enhancement of EGF stimulated MAP kinase signaling, suggesting that it must function through phosphorylation of some unknown effector. Several putative Drosophila Ack interacting proteins were isolated, many being orthologs of previously identified human ACK1 interacting proteins. Two of these interacting proteins, Drk and Yorkie, were found to influence Ack signaling. Drk is the Drosophila homolog of GRB2, which is required to couple ACK1 binding to receptor tyrosine kinases. Drk knockdown blocks Ack survival activity, suggesting that Ack localization is important for its pro-survival activity. Yorkie is a transcriptional co-activator that is downstream of the Salvador-Hippo-Warts pathway and promotes transcription of proliferative and anti-apoptotic genes. yorkie and Ack were found to synergistically interact to produce tissue overgrowth, and yorkie loss of function interferes with Ack anti-apoptotic signaling. These results demonstrate how increased Ack signaling could contribute to cancer when coupled to proliferative signals (Schoenherr, 2012).

The founding member of tha ACK family is human ACK1, which was identified as a protein that binds to CDC42 in its active GTP bound form. Since this discovery Ack homologs have been found in chordates, arthropods and nematodes. Ack family members can be divided into three structural categories based on the presence or absence of four conserved domain motifs. All Ack family members contain an amino-terminal tyrosine kinase domain that is flanked by a sterile alpha motif (SAM) and a Src homology 3 (SH3) domain. The carboxy-terminal half of these kinases contains short proline rich sequences, but lacks any identifiable domains, with the exception of two tandemly repeated ubiquitin-associated (UBA) domains located at the extreme carboxy-terminus. ACK1 UBA domains have been shown to interact with both mono and poly-ubiquitinated proteins and are thought to play a role in ACK1 protein turnover. The Caenorhabditis elegans Ack homolog, Ark-1, contains no UBA domains, placing it in a class by itself. The other two Ack structural classes can be distinguished by the presence or absence of a Cdc42/Rac interactive binding (CRIB) domain. Human ACK1 and Drosophila PR2 are representative members of the CRIB domain containing structural class, while human TNK1 and Drosophila Ack are members of the structural class lacking a conserved CRIB domain. Variants containing a CRIB domain bind GTP liganded CDC42, but this interaction does not appear to directly influence Ack activity in vitro (Schoenherr, 2012).

Human ACK1 is the most well characterized member of the Ack family. Early studies uncovered a role for ACK1 in the promotion of internalization and down-regulation of activated epidermal growth factor (EGF) receptor. ACK1 tyrosine phosphorylation is enhanced and ACK1 is co-localized with EGF receptor after EGF stimulation. Knockdown of ACK1 reduces the rate of EGF receptor degradation following EGF stimulation. While on the surface these data suggest that ACK1 merely serves as a negative regulator of growth factor signaling, ACK1 activation may additionally propagate downstream signaling. Recent studies support this latter alternative by uncovering a role for ACK1 as a positive transducer of cell surface receptor signaling that promotes growth and survival by ACK1 mediated phosphorylation and activation of downstream components, including AKT (Mahajan, 2010) and the androgen receptor (Schoenherr, 2012 and references therein).

A pro-survival role for Ack function is consistent with reported links between activation of Ack family members and cancer genesis and metastasis. Several somatic missense mutations have been identified in ACK1 from cancer tissue samples that increase ACK1 autophosphorylation and promote cellular proliferation and migration. Amplification of the ACK1 gene in tumors correlates with a poor prognosis, and ACK1 overexpression in cancer cell lines increases invasiveness in a mouse metastasis model, while knockdown of ACK1 reduces the migration of human breast cancer cells (Schoenherr, 2012).

Activated ACK1 has been detected in advanced human prostate cancers where it has been shown to phosphorylate three cancer relevant substrates in prostate cancer cell lines: WWOX, AKT, and androgen receptor. WWOX spans the FRA16D chromosomal fragile site that is frequently disrupted in human cancers. While the molecular function of WWOX is not known, it has been shown that the growth of tumor cells lacking WWOX is strongly inhibited by restoring WWOX expression. ACK1 phosphorylation of WWOX leads to the polyubiquitination and degradation of WWOX, which correlates with a tumorigenic role. AKT is a serine/threonine kinase whose activity promotes cell survival and proliferation, while deregulation of the AKT signaling pathway is commonly associated with cancer. ACK1 activation results in tyrosine phosphorylation and apparent activation of AKT in a PI3K independent mechanism. Finally, the activity of the androgen receptor is required for growth of prostate cells. In advanced stages of prostate cancer, these cells lose their dependence on androgens for activation of this receptor to become androgen independent prostate cancer. ACK1 has been found to phosphorylate the androgen receptor, promote androgen independent growth of prostate cells, and activate transcription of androgen inducible genes in the absence of androgen (Schoenherr, 2012).

Less is known about the function of Ack family members lacking CRIB domains, and published studies on TNK1 describe conflicting functions. TNK1 overexpression in cell culture lines inhibits cell growth in a kinase dependent manner. Mutant mice having deletions in the kinase domain of TNK1 develop spontaneous tumors at a high frequency, which is thought to originate from hyperactivation of Ras signaling and suggests that TNK1 functions as a tumor suppressor. In contrast to this function, TNK1 was identified as a potentially oncogenic tyrosine kinase in a mutagenesis screen and activated TNK1 was found in Hodgkin's lymphoma. It is possible that these conflicting findings reflect tissue specific responses or complex dosage sensitivity to TNK1 loss and gain of function (Schoenherr, 2012 and references therein).

In order to better understand the physiological role of Ack family members and determine how Ack might contribute to cancer, genetic and biochemical experiments were conducted in the model organism Drosophila melanogaster. These studies focus on Drosophila Ack, which has a domain structure resembling human TNK1, but shares significantly higher sequence identity with ACK1 in all conserved domains including the kinase domain activation loop. It was found that Drosophila Ack possesses potent anti-apoptotic properties that function downstream of EGF receptor signaling through an unknown mechanism. This activity is dependent on Ack kinase function and can be further stimulated by increased Ras signaling. A protein interaction study was conducted, and it was found that many of the same proteins that associate with human ACK1 also bind to fly Ack. The influence of these proteins on Ack anti-apoptotic activity was tested, and it was determined that the adapter protein Drk (GRB2) is required for this activity, while the transcriptional co-activator protein Yki (YAP) functions synergistically with Ack to promote cell survival and massive tissue overgrowth. These findings support both anti-apoptotic and proliferative roles for Ack family members, which may contribute to cancer genesis and progression (Schoenherr, 2012).

Drosophila has two Ack family members: Ack and PR2. Ack possesses anti-apoptotic properties, while PR2 either does not possess anti-apoptotic properties or requires activators not present in the assay system. While Ack may appear to be more closely related to vertebrate TNK1 because both proteins lack a CRIB domain, Ack is most similar to vertebrate ACK1 based on sequence identity of all shared protein domains. Additionally it was found that many of the proteins that interact with ACK1 also interact with fly Ack. Therefore, the conclusions drawn in this study will likely be applicable to vertebrate ACK1 function (Schoenherr, 2012).

Ack function can suppress programmed cell death induced by Hid or Rpr expression in the developing eye and wing discs. Overexpression studies reveal that Ack kinase activity is required for suppression of apoptosis induced by hid but is unnecessary for suppression of rpr-induced apoptosis. It was further shown that Ack loss of function enhances cell death induced by expression of both of these genes, and it was determined that Ack is critically required for the survival of rpr expressing eye tissue. The molecular mechanisms underlying these differential requirements are not known. hid and rpr are known to function in a multimeric protein complex on the mitochondria outer membrane to promote apoptosis. Both hid and rpr are able to stimulate apoptosis by competing with initiator and effector caspases for DIAP binding, but rpr additionally induces DIAP auto-ubiquitination leading to DIAP degradation. While Ack kinase activity may be important for aspects of hid complex regulation, it is tempting to speculate that the UBA domains of Ack may play a critical role in the modulation of DIAP or Rpr ubiquitination and stability (Schoenherr, 2012).

EGF receptor signaling has been shown to activate ACK1 in vertebrates, and EGF signaling was found to enhance the anti-apoptotic function of Ack in Drosophila. ACK1 negatively regulates EGF receptor signaling by stimulating endocytosis of activated receptor complexes. The evidence supports the idea that Drosophila Ack, in conjunction with SH3PX1, functions in a similar manner, which will be described elsewhere. In Drosophila, EGF signaling is anti-apoptotic through the activation of MAPK, which phosphorylates and inactivates Hid. If Ack affected apoptosis exclusively through attenuation of EGF signaling, then it would be expected that Ack loss of function would be anti-apoptotic while gain of function would be pro-apoptotic, which is opposite to what was observed. By using the hidAla5 mutant, it was demonstratde that Ack does not modulate programmed cell death through activation of MAPK (Schoenherr, 2012).

The anti-apoptotic function of Ack is surprisingly robust compared to other proteins that were have tested. The studies show that activity of the kinase domain contributes to Ack anti-apoptotic function. Based on this, it is concluded that Ack propagates anti-apoptotic signals by phosphorylating downstream targets. Several ACK1 substrates have been identified that are attractive candidates for the regulation of programmed cell death: the putative tumor suppressor WWOX, the apoptosis inhibiting protein kinase AKT and the caspase-cleaved ubiquitin E3 ligase NEDD4. Akt1 loss and gain of function alleles and Nedd4 RNAi fail to significantly modify hid induced small eye phenotypes. Wwox RNAi is able to suppress hid induced apoptosis but not nearly as robustly as Ack expression. Since ACK1 mediated phosphorylation of WWOX leads to WWOX destruction, it would be predicted that Wwox RNAi would phenocopy Ack expression in the assay system, but it is unable to reproduce the magnitude of Ack anti-apoptotic function. This does not rule out Wwox as an anti-apoptotic substrate target of Ack, but it demonstrates that Wwox is not the only cell death relevant substrate of Ack. It is worth noting that hid and rpr act fairly late within the programmed cell death pathway, being just a step upstream of initiator caspase activation. Therefore, Ack must target substrates that have activities influencing hid, rpr or events at the level of caspase activation (Schoenherr, 2012).

Several Ack physically interacting proteins were identified using a tandem affinity purification strategy. Many of these have vertebrate homologs that have been previously determined to interact with ACK1. Drk and Yki have the most pronounced effect on Ack's anti-apoptotic properties, and their contribution to Ack signaling was further characterized. Drk is the fly ortholog of vertebrate GRB2, which has previously been described as an ACK1 and TNK1 interacting protein. In the case of TNK1, GRB2 is tyrosine phosphorylated by TNK1, which disrupts the ability of GRB2/SOS complexes to activate Ras. This does not appear to be the case for Drosophila Ack, because even though Drk forms a complex with tyrosine phosphorylated Ack, no evidence of tyrosine phosphorylation on Drk was found. Rather, the data support that Drk association with Ack is required for Ack anti-apoptotic properties. It is proposed that Drk SH3 domains likely interact with PXXP motifs in the C-terminal half of Ack similar to the interaction described in vertebrates. This interaction could then lead to the recruitment of Ack into protein complexes required for Ack activation (Schoenherr, 2012).

Yki is a transcriptional co-activator that regulates expression of genes with proliferative and anti-apoptotic functions. The vertebrate homolog of Yki is Yes Associated Protein (YAP), which has not previously been identified as an ACK1 or TNK1 interacting protein. Yki and YAP studies have focused primarily on the pathways that regulate their function as transcription factors. Given the role of Yki and YAP in transcriptional control of proliferative and anti-apoptotic genes, it would seem likely that Ack activity leads to enhancement of Yki function. However, this does not appear to be the case because Ack overexpression does not lead to increased Yki nuclear localization or increased expression of yki target genes. Rather, the data indicate that Yki directly interacts with Ack in the cytoplasm and functions to regulate Ack activity. In support of this, it was found that Ack colocalizes with Yki, and yki dosage reduction suppresses Ack anti-apoptotic function. Yki contains two WW domains, which may interact with conserved PPXY motifs that are present in the region flanked by the SH3 and UBA domains of Ack family members. In vertebrates, these PPXY motifs have been shown to interact with WWOX, which also contains two WW domains. Further studies are required to define how Yki and Ack interact (Schoenherr, 2012).

Yki expression in the fly eye produces an overgrowth phenotype that is indicative of its role in regulating proliferation. Ack overexpression produces a slightly larger eye due to inhibition of apoptotic events that occur during normal eye development. Simultaneous expression of yki and Ack results in a synergistic effect that produces enormous eyes. These results reveal that in addition to anti-apoptotic function, Ack can also enhance proliferation. This illustrates how increased Ack signaling could contribute to cancer when coupled to proliferative signals. Indeed, the results are consistent with recent reports of ACK1 activating somatic mutations and gene amplification being associated with human cancers. At present the key anti-apoptotic substrates of Ack and their mechanisms of action remain to be determined. With their discovery will come a better understanding of Ack signaling and potentially new targets for cancer interventions (Schoenherr, 2012).

Genome-wide association of Yorkie with chromatin and chromatin-remodeling complexes

The Hippo pathway regulates growth through the transcriptional coactivator Yorkie, but how Yorkie promotes transcription remains poorly understood. This was addressed by characterizing Yorkie's association with chromatin and by identifying nuclear partners that effect transcriptional activation. Coimmunoprecipitation and mass spectrometry identify GAGA factor (GAF), the Brahma complex, and the Mediator complex as Yorkie-associated nuclear protein complexes. All three are required for Yorkie's transcriptional activation of downstream genes, and GAF and the Brahma complex subunit Moira interact directly with Yorkie. Genome-wide chromatin-binding experiments identify thousands of Yorkie sites, most of which are associated with elevated transcription, based on genome-wide analysis of messenger RNA and histone H3K4Me3 modification. Chromatin binding also supports extensive functional overlap between Yorkie and GAF. These studies suggest a widespread role for Yorkie as a regulator of transcription and identify recruitment of the chromatin-modifying GAF protein and BRM complex as a molecular mechanism for transcriptional activation by Yorkie (Oh, 2013).

The Hippo signaling pathway interactome

The Hippo pathway controls metazoan organ growth by regulating cell proliferation and apoptosis. Many components have been identified, but knowledge of the composition and structure of this pathway is still incomplete. Using existing pathway components as baits, mass spectrometry was used to a high-confidence Drosophila Hippo protein-protein interaction network (Hippo-PPIN) consisting of 153 proteins and 204 interactions. Depletion of 67% of the proteins by RNA interference regulated the transcriptional coactivator Yorkie (Yki) either positively or negatively. A new member of the alpha-arrestin family, CG4674/Leash, was selected for further characterization. It was shown to promote degradation of Yki through the lysosomal pathway. Given the importance of the Hippo pathway in tumor development, the Hippo-PPIN will contribute to understanding of this network in both normal growth and cancer (Kwon, 2013).

The Hippo-PPIN contains many potential new components of Hippo signaling. To initiate their characterization, focus was placed on an interactor of Yki, CG4674, which belongs to the arrestin domain containing (Arrdc) protein family. CG4674 is of particular interest as its mammalian orthologs, including Arrdc3, are implicated in tumor suppression by regulating cell proliferation and survival (Kwon, 2013).

Co-IP further established the association between CG4674 and Yki. Depletion of CG4674 by RNAi increased Yki-reporter activity, and overexpression had the opposite, suggesting that CG4674 restrains Yki activity (thus leading to CG4674 being termed leash). Although Yki was found in the cytoplasm, nucleus and in cytoplasmic vesicles, Leash predominantly localized to cytoplasmic vesicles. However, when Yki and Leash were expressed together, strong colocalization was observed. Importantly, overexpression and RNAi of leash decreased and increased vesicular localization of Yki, respectively. Further, the late endosome marker, Rab9, colocalized with a subset of Yki-positive vesicles, and the lysosomal maker, Lamp1, encircled some Yki-positive punctae. Overexpression of wts significantly increased the abundance of Yki-containing vesicles, suggesting that Hippo signaling regulates the vesicular localization of Yki. Arrdc proteins have conserved arrestin domains at their N-termini that interact with their cargos. Expression of Leash N-terminal arrestin domains alone was sufficient to regulate Yki activity by forming a complex, indicating that Yki is a Leash cargo (Kwon, 2013).

Additional Leash interactors were identified by AP/M; Leash strongly binds to Nedd4 and a few other HECT ubiquitin ligases. Moreover, three ubiquitinated lysines on Leash were identified that were important for complex formation with Yki. To address if human Arrdcs could down-regulate Yki, 5 human Leash orthologs were tested. Expression of Arrdc1 or Arrdc3 reduced Yki-reporter activity and Yki protein abundance. Since Nedd4 induces degradation of its substrate through the endosomal-lysosomal pathway, the effect of Bafilomycin, an inhibitor of lysosome acidification, was tested. Bafilomycin treatment increased Yki activity and reversed the effect of overexpression of full-length Leash or Arrestin domains, indicating that Leash decreases Yki abundance through the lysosomal degradation pathway (Kwon, 2013).

Yki activity is a critical determinant of growth, and Hippo signaling restricts growth by suppressing Yki. Consistent with the role of Leash in inhibiting Yki, overexpression of leash or arrdc3 reduced wing size significantly. Further, depletion of leash in wing discs affected neither wing size nor Yki protein abundance significantly, suggesting functional redundancy between family members or additional regulatory mechanisms. In the midgut, under normal homeostasis, Yki is inhibited. However, when an active form of yki (yki3S/A) is expressed or an upstream regulator is inactivated, midgut stem cells overproliferate. Depletion of leash enhanced proliferation induced by hippo knockdown without affecting proliferation under normal homeostasis. Furthermore, overexpression of leash or arrdc3 suppressed yki3S/A-induced proliferation. Altogether, these results indicate that leash is a negative regulator of Yki (Kwon, 2013).

In summary, this study has generated a PPIN for the Hippo pathway. Analysis of the interactions using functional RNAi screen and COMPLEAT revealed a snapshot of the overall organization of the Hippo signaling network. Further, Leash, a novel component of the Hippo pathway was characterized, and shown to down-regulate Yki through lysosomal degradation. Altogether, the Hippo-PPIN provides a resource for further in depth characterization of new and existing components of the Hippo pathway (Kwon, 2013).

Ubiquitin E3 ligase dSmurf is essential for Wts protein turnover and Hippo signaling>

The Hippo pathway has been implicated in controlling organ size and tumorigenesis and the underlying molecular mechanisms have attracted intensive attentions. This work identified dSmurf as a new regulator of Wts, a core component of the Hippo pathway, in Drosophila. The data revealed that Wts and dSmurf colocalize to cytoplasm and physically form an immunoprecipitated complex in S2 cells. Sufficient knock-down of dSmurf increases the protein abundance of Wts and thus increases phosphorylation level at S168 of Yki, the key downstream target of Wts in the Hippo pathway. Genetic epistasis assays showed that halving dosage of dSmurf dominantly enhances the phenotype caused by overexpression of Wts and restrains Yki activity in Drosophila eyes. This works defines a novel role of dSmurf in animal development through modulating Wts turnover and thereby Hippo signal transduction, implying that targeting dSmurf may be a promising therapeutic strategy to manipulate the Hippo pathway in pathological conditions (Cao, 2014).

Cabut/dTIEG associates with the transcription factor Yorkie for growth control

The Drosophila transcription factor Cabut/dTIEG (Cbt) is a growth regulator, whose expression is modulated by different stimuli. This study determined Cbt association with chromatin and identified Yorkie (Yki), the transcriptional co-activator of the Hippo (Hpo) pathway as its partner. Cbt and yki co-localize on common gene promoters, and the expression of target genes varies according to changes in Cbt levels. Down-regulation of Cbt suppresses the overgrowth phenotypes caused by mutations in expanded (ex) and yki over-expression, whereas its up-regulation promotes cell proliferation. These results imply that Cbt is a novel partner of yki that is required as a transcriptional co-activator in growth control (Ruiz-Romero, 2015).

Gene expression is regulated through the integrated action of, among others, many cis-regulatory elements, including core promoters and enhancers located at greater distances from transcription start sites (TSS). The combinatorial binding of transcription factors (TF) to these elements can lead to diverse types of transcriptional output, and an understanding of this mechanism is crucial, for example, in the context of development. In fact, the final size and shape of an organism require a complex genetic network of signaling molecules, the final outcome of which must be finely regulated in space and time to ensure a proper response (Ruiz-Romero, 2015).

The transcription factor Cabut/dTIEG (Cbt) is the fly ortholog of TGF-β-inducible early genes 1 and 2 (TIEG1 and TIEG2) in mammals, which belong to the evolutionary conserved TIEG family. TIEGs are zinc finger proteins of the Krüppel-like factor (KLF) family that can function as either activators or repressors depending on the cellular context, the promoter to which they bind or the interacting partners. TIEG proteins participate in a wide variety of cellular processes, from development to cancer, and regulate genes that control proliferation, apoptosis, regeneration or differentiation (Ruiz-Romero, 2015).

Drosophila cbt was identified and characterized from an overexpression screen of EP lines conducted to determine genes involved in establishing epithelial planar cell polarity. This TF is ubiquitously expressed in the wing disk, and its expression increases in response to metabolic, hormonal and stress signals. Cbt levels rise upon inhibition of TOR signaling, and it is among the most highly Mlx-regulated genes. Among its functions, it is known that Cbt is required during dorsal closure downstream of JNK signaling, that it is a modulator of different signaling pathways involved in wing patterning and proliferation and that it promotes ectopic cell cycling when overexpressed. Moreover, Cbt is a crucial downstream mediator gene of the JNK signaling required during wing disk regeneration. In spite of this, little is known about its downstream target genes or its precise mechanism of action. This study reports a novel function for Cbt as a partner of yki (Yorkie). yki is the key effector of growth control and the downstream element of the highly conserved Hpo (Hippo) signaling pathway. The Hpo pathway limits organ size by phosphorylating and inhibiting Yki, a key regulator of proliferation and apoptosis. yki can also act as an oncogene, since it has potent growth-promoting activity. The results show a role for Cbt as a transcriptional activator with the capacity to modulate yki growth response (Ruiz-Romero, 2015).

To characterize Cbt target genes, chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) were performed from third instar larval wing imaginal disks. Analysis of Cbt-bound regions in the entire genome revealed that approximately 70% of its peaks were located in proximal promoters or introns, consistent with its role as a transcriptional regulator. Thus, 2,060 putative target genes were identified in the wing disk. Gene Ontology (GO) analysis indicated that this subset of genes was enriched in transcriptional activity, cell migration, mitotic cell cycle and signaling pathways known to play a role in imaginal disk development. As expected, among Cbt targets previously described genes were found such as salm (spalt major) or cbt itself, but also several unidentified target genes such as wg (wingless) or vg (vestigial) (Ruiz-Romero, 2015).

Cbt association around the TSS may be an indication of its function as a primary regulatory element, but does not provide any information about its role as an activator or a repressor. To elucidate this question, published data on chromatin modifications as well as recently obtained RNA-Seq data from the wing disk were examined and Cbt targets were ranked according to their expression level. Although at different levels, target genes are mostly expressed in the wing disk. This positive correlation with gene expression was also detected in the extensive overlap between Cbt occupancy and trimethylated histone 3 lysine 4 (H3K4me3). In contrast, only 13% of Cbt target genes correlated with the repressive chromatin mark H3K27me3. Although 200 Cbt targets seemed to present both modifications, these may be coupled to the differential expression pattern of several genes in the wing disk (Ruiz-Romero, 2015).

To clarify whether Cbt binds to active or inactive genes, Cbt occupancy was examined of genes known to be differentially expressed in a subpopulation of cells within the wing disk tissue. The gene nub (nubbin) is expressed in the wing primordium. GFP expression in the wing pouch was examined using a nub-GAL4 driver and ChIP assays followed by quantitative PCR (qPCR) were performed in sorted cells. In the vicinity of the TSS of genes expressed in the wing pouch, such as rn (rotund) and nub, Cbt was only found in GFP-positive cells. Cbt was also present in the promoter of cycA (cyclin A), both in GFP-positive and GFP-negative cells, in accordance with its expression throughout the entire wing disk (wing pouch and notum). These observations indicate that Cbt might act as a positive activator of transcription in this tissue. To further confirm this, the expression of selected targets was examined after cbt overexpression. Induction of cbt in the dorsal domain of the wing using an ap-GAL4 (apterous) driver led to a clear increase in the expression levels of target genes, as detected by qPCR. cbt was also ectopically expressed in the ptc (patched) domain of the wing disk using the ptc-GAL4 driver, and the pattern of Wg (normally restricted to cells adjacent to the D/V boundary in the wing blade and to two rings in the hinge region) and Vg (expressed throughout the wing blade) was examined by immunostaining. After cbt induction, spread staining of Wg was observed in the boundary and ring regions. Likewise, analysis of Vg revealed increased protein levels in the region where cbt was upregulated. No ectopic expression of Wg or Vg was detected in regions far from where they are normally expressed, suggesting that cbt alone is not sufficient to ectopically activate transcription of these genes but can modulate or cooperate with factors that promote their basal expression. Taken together, these results suggest that Cbt functions as a transcriptional activator in the wing disk. Nevertheless, its contribution to repression in some contexts or through binding to different partners cannot be disregarded, as previous experiments have demonstrated the relevance of the Sin3A interaction domain for Cbt's repressive role (Ruiz-Romero, 2015).

TIEG factors contain three conserved C-terminal zinc finger motifs that seem to bind to GC-rich sequences in vertebrates. To characterize the set of motifs enriched within Cbt binding sites, different pattern discovery methods were used. Among others, GC sequences and the Sp1 motif, were detected as expected for a TIEG family member, but in addition, one of the most enriched motifs comprised GAGA-binding sequences. No enrichment of the proposed consensus TIEG motif 5'GGTGTG3' was found, which suggests that Cbt binds to degenerated or alternative motifs or may function through its interaction with other TFs. A recent study identified a novel Mad-like motif in promoters of Cbt-regulated genes. However, this new motif does not coincide with previously reported Cbt binding data (Ruiz-Romero, 2015).

Many studies have emphasized the complexity of yki and its mammalian homologs YAP and TAZ regulation, including multiple combinations with associate proteins in distinct target genes. Besides DNA-binding partners such Sd (Scalloped) and Hth (Homothorax) in Drosophila, yki can cooperate with other factors directly on target promoters, such as the cell cycle-related gene dE2F1. Remarkably, a recent report shows that Cbt and dE2F1 regulate an overlapping set of cell cycle genes. In the Dpp pathway, Mad (Mothers against decapentaplegic) and yki interact to form a transcription complex to activate their common targets. This association is conserved through evolution, as YAP and TAZ interact with Smad proteins to potentiate transcriptional activity. Recent studies have also identified Mask (Multiple ankyrin repeats single KH domain) as a novel cofactor for Yki/YAP, required to induce target gene expression. The results highlight the role of Cbt as a new yki partner involved in the activation of some yki target gene expression. This function of Cbt may occur in part through association with GAF as well as chromatin remodeler complexes (Ruiz-Romero, 2015)

Since overexpression of cbt results in an increase in proliferation as well as wing size, it was hypothesized that Cbt's role in size control could be mediated through its association with Yki. To address this question, cbt levels were depleted, and the effect on the growth of ex mutant clones and in clones overexpressing yki in wing and eye-antenna imaginal disks was examined. The yki target gene ex acts as an upstream positive modulator of the Hpo pathway, and in accordance with its role as a tumor suppressor, its loss-of-function mutation results in large clones. Expression of cbt RNAi in this mutant background caused a clear reduction in the clone size. In the same direction, the overgrowth known to occur by overexpression of a yki-activated form is prevented in a mutant cbt background as well as expressing cbt RNAi. Moreover, impaired growth caused by yki depletion could not be rescued increasing cbt levels and overexpression of yki and cbt triggered massive growth in imaginal tissues. Finally, depletion of cbt in adult organs (wings and eyes) also reduced Yki-mediated overgrowth, indicating a general function for Cbt in the regulation of Hippo pathway-mediated tissue growth (Ruiz-Romero, 2015).

In addition to its role during development, it has been shown that Cbt expression is highly regulated by stress and metabolic conditions. Cbt has also been identified as a JNK-inducible gene during dorsal closure, and this study has shown that JNK and tissue damage trigger cbt transient overexpression to promote wing disk regeneration, indicating that its levels must be finely controlled during regenerative growth. Moreover, cbt heterozygous mutant disks fail to proliferate and do not regenerate, and it is known that during regeneration, the JNK pathway triggers yki translocation to the nucleus to promote the proliferative response. Altogether, these data support a model for Cbt acting as a modulator of yki activity in the transcriptional regulatory mechanisms that control tissue growth (Ruiz-Romero, 2015).

Mask loss-of-function rescues mitochondrial impairment and muscle degeneration of Drosophila pink1 and parkin mutants

PTEN-induced kinase 1 (Pink1) and ubiquitin E3 ligase Parkin function in a linear pathway to maintain healthy mitochondria via regulating mitochondrial clearance and trafficking. Mutations in the two enzymes cause the familial form of Parkinson's disease (PD) in humans, as well as accumulation of defective mitochondria and cellular degeneration in flies. This study shows that loss of function of a scaffolding protein Mask, also known as ANKHD1 (Ankyrin repeats and KH domain containing protein 1) in humans, rescues the behavioral, anatomical and cellular defects caused by pink1 or parkin mutations in a cell-autonomous manner. Moreover, similar rescue can also be achieved if Mask knock-down is induced in parkin adult flies when the mitochondrial dystrophy is already manifested. It was found that Mask genetically interacts with Parkin to modulate mitochondrial morphology and negatively regulates the recruitment of Parkin to mitochondria. Also, loss of Mask activity promotes co-localization of the autophagosome marker with mitochondria in developing larval muscle, and that an intact autophagy pathway is required for the rescue of parkin mutant defects by mask loss of function. Together, these data strongly suggest that Mask/ANKHD1 activity can be inhibited in a tissue- and timely-controlled fashion to restore mitochondrial integrity under PD-linked pathological conditions (Zhu, 2015).

Recent studies suggest that PINK1 activates Parkin E3 ubiquitin ligase activity by phosphorylating both Parkin and ubiquitin, and that PINK1 recruits Parkin to the damaged mitochondrial membrane, where Parkin ubiquitinates a pool of outer mitochondrial membrane proteins and promotes mitophagy. These data suggest that mitochondrial dysfunction observed in PD may be the result of compromised mitochondrial quality control mechanisms. Therefore, understanding the pathways of mitochondrial quality control holds the key to unravelling the pathogenesis of PD and other disorders associated with mitochondrial dysfunction (Zhu, 2015).

Flies carrying pink1 or parkin mutations show severe mitochondrial morphological and functional defects in multiple tissues as well as age-dependent  dopaminergic (DA) dysfunction, making it a great genetic model to study mechanisms of mitochondrial homeostasis. Using this model system, previous studies in Drosophila have identified a number of pathways that can be manipulated to rescue the parkin and/or pink1 mutant phenotype. First, increasing mitochondrial fission or decreasing fusion rescues the phenotypes of muscle degeneration and mitochondrial abnormalities in pink1 or parkin mutants. However, manipulation of mitochondrial dynamics causes the opposite effect on loss of parkin or pink1 function in mammalian cells, indicating that Pink1 and Parkin may regulate mitochondrial dynamics in a context-dependent manner. Second, promoting mitochondrial electron transport chain CI activity by overexpressing a yeast NADH dehydrogenase, the CI subunit NDUFA10, the GDNF receptor Ret, Sicily, dNK or Trap1 rescue pink1 mutant mitochondrial defects without affecting parkin mutant phenotypes, suggesting a distinct role of Pink1 in regulating CI activity in addition to its role in Parkin-mediated mitophagy (Zhu, 2015).

This study shows that a highly conserved scaffolding protein Mask, whose normal function is to regulate mitochondrial morphology and selectively inhibit mitophagy, can be targeted in a tissue- and temporal-specific manner to suppress both pink1 and parkin mutant defects in Drosophila. It also shows that such a rescue requires the presence of a functional autophagy pathway. Although tissue- and temporal-specific knock-down of Mask was performed with mainly one mask RNAi line, the mask loss-of-function analysis with mask genetic mutants and another independent RNAi line support the same notion that Mask dynamically regulates mitochondrial morphology. Together, these data suggest that enhancing mitochondrial quality control may serve as a common approach to mitigate mitochondrial dysfunction caused by PD-linked genetic mutations. Consistent with this notion, recent studies show that inhibition of deubiquitinases USP30 and USP15 enhances mitochondrial clearance and quality control, and rescues mitochondrial impairment caused by pink1 or parkin mutations (Zhu, 2015).

It was found that loss of mask function enhances the formation of autophagosome surrounding mitochondria. However, the increase of mCherry-ATG8 did not result in significant increase of free mCherry, suggesting the flux of autophagic degradation is not affected. Further studies are required to elucidate the molecular details by which Mask regulates mitochondrial morphology and function. Recent studies on the connection between Mask and the Hippo pathway demonstrates that Mask physically interacts with the Hippo effector Yorkie, and functions as an essential cofactor of Yorkie in promoting downstream target-gene expression. Interestingly, the Yorkie pathway was also shown to regulate mitochondrial structure and function during fly development. Together, these findings bring up an intriguing possibility that Mask and Yorkie together regulate mitochondrial size during development and disease. It was also shown that reducing Mask activity at the relatively progressed stage of parkin-dependent muscle degeneration mitigates the mitochondrial defects and impairs muscle function, indicating that the human Mask homolog ANKHD1 may serve as a potential therapeutic target for treating PD caused by pink1/parkin mutations (Zhu, 2015).

The Spectrin cytoskeleton regulates the Hippo signalling pathway

The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. This study shows that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta-heavy dimers) produced tissue overgrowth and mis-regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM-domain protein Expanded (Ex). Apical beta-heavy Spectrin bound to Ex and co-localised with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb were dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) were essential. Finally, the Spectrin cytoskeleton was required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. These findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors (Fletcher, 2015).

The ecdysone receptor coactivator Taiman links Yorkie to transcriptional control of germline stem cell factors in somatic tissue

The Hippo pathway is a conserved signaling cascade that modulates tissue growth. Although its core elements are well defined, factors modulating Hippo transcriptional outputs remain elusive. This study shows that components of the steroid-responsive ecdysone (Ec) pathway modulate Hippo transcriptional effects in imaginal disc cells. The Edysone receptor coactivator Taiman (Tai) interacts with the Hippo transcriptional coactivator Yorkie (Yki) and promotes expression of canonical Yki-responsive genes. Tai enhances Yki-driven growth, while Tai loss, or a form of Tai unable to bind Yki, suppresses Yki-driven tissue growth. This growth suppression is not correlated with impaired induction of canonical Hippo-responsive genes but with suppression of a distinct pro-growth program of Yki-induced/Tai-dependent genes, including the germline stem cell factors nanos and piwi. These data reveal Hippo/Ec pathway crosstalk in the form a Yki-Tai complex that collaboratively induces germline genes as part of a transcriptional program that is normally repressed in developing somatic epithelia (Zhang, 2015).

The Hippo pathway promotes cell survival in response to chemical stress

Cellular stress defense mechanisms have evolved to maintain homeostasis in response to a broad variety of environmental challenges. To identify novel players acting in stress response pathways, a cell culture RNA interference (RNAi) screen was conducted using caffeine as a xenobiotic stress-inducing agent, as this compound is a well-established inducer of detoxification response pathways. Specifically, how caffeine affects cell survival was evaluated when Drosophila kinases and phosphatases were depleted via RNAi. Using this approach, ten kinases and 4 phosphatases that are essential for cell survival were identified. Remarkably, the screen yielded an enrichment of Hippo pathway components, indicating that this pathway regulates cellular stress responses. Indeed, it was shown that the Hippo pathway acts as a potent repressor of stress-induced cell death. Further, it was demonstrate that Hippo activation is necessary to inhibit a pro-apoptotic program triggered by the interaction of the transcriptional co-activator Yki with the transcription factor p53 in response to a range of stress stimuli. These in vitro and in vivo loss-of-function data therefore implicate Hippo signaling in the transduction of cellular survival signals in response to chemical stress (Di Cara, 2015).

yorkie: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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