The neuronal transcription factor erect wing regulates specification and maintenance of Drosophila R8 photoreceptor subtypes

Signaling pathways are often re-used during development in surprisingly different ways. The Hippo tumor suppressor pathway is best understood for its role in the control of growth. The pathway is also used in a very different context, in the Drosophila eye for the robust specification of R8 photoreceptor neuron subtypes, which complete their terminal differentiation by expressing light-sensing Rhodopsin (Rh) proteins. A double negative feedback loop between the Warts kinase of the Hippo pathway and the PH-domain growth regulator Melted regulates the choice between 'pale' R8 (pR8) fate defined by Rh5 expression and 'yellow' R8 (yR8) fate characterized by Rh6 expression. This study shows that the gene encoding the homolog of human Nuclear respiratory factor 1, erect wing (ewg), is autonomously required to inhibit warts expression and to promote melted expression to specify pR8 subtype fate and induce Rh5. ewg mutants express Rh6 in most R8s due to ectopic warts expression. Further, ewg is continuously required to maintain repression of Rh6 in pR8s in aging flies. This study shows that Ewg is a critical factor for the stable down-regulation of Hippo pathway activity to determine neuronal subtype fates. Neural-enriched factors, such as Ewg, may generally contribute to the contextual re-use of signaling pathways in post-mitotic neurons (Hsiao, 2013).

The proper specification of photoreceptor subtypes including Rhodopsin expression is critical for proper color-detection and related behavior. This study found that the neural-specific transcription factor Ewg contributes to R8 subtype specification. In the absence of ewg, most pR8s are mis-specified as yR8s. This analysis shows that ewg acts autonomously in R8 to regulate the Warts-Melted feedback loop controlling subtype fate. Ewg appears to regulate pR8 fate by promoting melted expression and warts repression, suggesting that ewg is necessary to promote the complete pR8 fate rather than directly regulating Rh5 expression. Furthermore, epistasis experiments with merlin, warts and melted place ewg genetically upstream of the Warts-Melted feedback loop (Hsiao, 2013).

In addition to its role in R8 subtype establishment, ewg also functions in subtype maintenance in adult flies, as Rh6 is de-repressed in pR8 and is co-expressed with Rh5 in 4-week old ewg mutant flies. Expression of Rh5 still remains in old pR8s. ewg mutants also progressively lose melted expression and gain warts expression in R8s. The gradual disappearance of melted in old ewg mutant flies likely allows expression of warts and reactivation of Rh6. This represents another genetic program required to maintain gene expression in differentiated sensory neuron subtypes of adult animals. Previous studies have shown that the Hippo pathway is required both to specify and to maintain yR8 subtypes. Removing merlin after eclosion results in de-repression of Rh5 in all yR8s and co-expression with Rh6, a phenotype opposite to that of old ewg mutant flies. Furthermore, an active Rh6 protein is required to repress Rh5 to maintain its exclusive expression in yR8s, as loss of Rh6 results in the expansion of Rh5 to all R8s in old flies. These results are consistent with the model that establishment and maintenance programs are coupled by using the same genes, resulting in efficient long-term gene regulation (Hsiao, 2013).

How does the Ewg protein function in R8 subtype specification? Ewg has the same consensus DNA binding site as Nuclear respiratory factor 1. However, no motifs were found matching the Ewg consensus sequence in the regulatory regions of melted and warts. The diverse transcriptional targets of Ewg in various organisms also prevent a clear assignment of a conserved Ewg protein function. For example, Nuclear respiratory factor 1 acts as a transcriptional activator in the regulation of expression of cytochrome C and mitochondrial genes. However, the sea urchin Ewg homolog, P3A2, limits expression of the cytoskeletal cyIIIA actin gene. As human Nuclear respiratory factor 1 functions as an activator while sea urchin P3A2 negatively regulates cyIIIA, Ewg therefore appears to act either as an activator or as a repressor, consistent with the presence of a C-terminal activation domain and an N-terminal repression domain identified in Drosophila. Although Ewg functions upstream of the warts/melted loop, neither warts nor melted contain canonical Ewg binding motifs, suggesting that Ewg likely regulates these genes indirectly (Hsiao, 2013).

Several other genes are expressed in all photoreceptors and act as permissive factors to regulate specific Rhodopsins and photoreceptor subtypes. For example, Orthodenticle (Otd), the fly homolog of vertebrate Crx and Otx proteins, is a K50 homeoprotein expressed in all photoreceptors. Loss of otd results in the loss of Rh3 and Rh5 in p ommatidia and de-repression of Rh6 in outer photoreceptors. However, like Ewg, Otd is not sufficient to activate these genes when mis-expressed. Otd is therefore a permissive factor that likely acts with co-factors to specify their activating or repressive functions in particular photoreceptors. For Rh3, restricted Rh expression is achieved by repression by Dve, Senseless and Prospero. The same principle might apply for Ewg: since Ewg is expressed in all photoreceptors, it might recruit co-factors specific to pR8 to promote the expression of melted or to negatively regulate the Hippo pathway. Recently, ewg was shown to be required for the recruitment of the cell specific Armadillo-TCF adapter, Earthbound 1 (Ebd1), to specific chromatin sites to activate a subset of Wingless target genes. Ebd1 shares similar polytene chromatin binding sites with Ewg. It is possible that Ewg recruits a specific co-factor such as Ebd1 to function in pR8. However, no decrease was observed in Rh5 expression in ebd1 mutant retinas, suggesting that Ewg acts differently in the retina. Nevertheless, it is likely that another subtype specific co-factor functions with Ewg to specify pR8 fate (Hsiao, 2013).

In conclusion, ewg is autonomously required to specify the pR8 subtype and induce Rh5 expression. ewg appears to act upstream of the Hippo pathway, of melted, and the feedback loops to determine pR8 fate. Therefore, a neuronal specific transcription factor, Ewg, contributes to the regulation of the Hippo pathway either directly or indirectly through regulation of melted to specify the fate of R8 photoreceptors (Hsiao, 2013).

The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and maintenance

Precise patterning of dendritic fields is essential for neuronal circuit formation and function, but how neurons establish and maintain their dendritic fields during development is poorly understood. In Drosophila class IV dendritic arborization neurons, dendritic tiling, which allows for the complete but non-overlapping coverage of the dendritic fields, is established through a 'like-repels-like' behaviour of dendrites mediated by Tricornered (Trc), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila. The other NDR family kinase, the tumour suppressor Warts/Lats (Wts), regulates the maintenance of dendrites; in wts mutants, dendrites initially tile the body wall normally, but progressively lose branches at later larval stages, whereas the axon shows no obvious defects. Biochemical and genetic evidence is provided for the tumour suppressor kinase Hippo (Hpo) as an upstream regulator of Wts and Trc for dendrite maintenance and tiling, respectively, thereby revealing important functions of tumour suppressor genes of the Hpo signalling pathway in dendrite morphogenesis (Emoto, 2006).

Dendritic arborization patterns are critical to a neuron's ability to receive and process impinging signals. Whereas neurons normally maintain the gross morphology of their dendrites, cortical neurons of Down's syndrome patients gradually lose dendritic branches after initially forming normal dendritic fields. Thus, neurons appear to have separate mechanisms for establishment and maintenance of their dendritic fields (Emoto, 2006).

Dendritic tiling is an evolutionarily conserved mechanism for neurons of the same type to ensure complete but non-redundant coverage of dendritic fields. In the mammalian visual system, for instance, dendrites of each retinal ganglion cell type cover the entire retina with little overlap, like tiles on a floor. In Drosophila, the dendritic arborization sensory neurons can be divided into four classes (I-IV) based on their dendrite morphology, and the dendritic field of class IV dendritic arborization neurons is shaped, in part, through a like-repels-like tiling behaviour of dendrite terminals. The NDR family kinase Trc and its activator Furry (Fry) has been identified as essential regulators of dendritic tiling and branching of class IV dendritic arborization neurons. These proteins are evolutionarily conserved and probably serve similar functions in neurons of different organisms (Emoto, 2006).

In addition to Trc, Drosophila has one other NDR family kinase, Wts, which is a tumour suppressor protein that functions in the coordination of cell proliferation and cell death in flies. To uncover the cell-autonomous functions of Wts in neurons, MARCM (mosaic analysis with a repressive cell marker) was ised to generate mCD8-GFP-labelled wts clones in a heterozygous background. Wild-type class IV neurons elaborate highly branched dendrites that cover essentially the entire body wall. Compared to wild-type ddaC (dorsal dendrite arborization neuron C) neurons, wts clones showed a severe and highly penetrant simplification of dendritic trees, with significantly reduced number (wild type, 575.1; wts, 255.6) and length (wild type, 1,457.0; wts, 590.4) of dendritic branches, and hence a greatly reduced dendritic field (Emoto, 2006).

In contrast to the severe dendritic defects caused by loss of Wts function, wts mutant ddaC axons entered the ventral nerve cord at the appropriate position and showed arborization patterns very similar to wild-type controls, with their axons terminating on the innermost fascicle and sending ipsilateral branches anteriorly and posteriorly and sometimes also a collateral branch towards the midline. Thus, Wts seems to have a crucial role in dendrite-specific morphogenesis in post-mitotic neurons (Emoto, 2006).

In proliferating cells, Wts is part of a signalling complex for tumour suppression that includes the adaptor protein Salvador (Sav) and the serine/threonine kinase Hpo. sav mutant ddaC MARCM clones were examined and dendritic defects were observed similar to wts MARCM clones. In severely affected clones (3 of 15 clones), most of the high-order branches were missing, whereas moderately affected clones (12 of 15 clones) exhibited a partial loss of their fine branches and major branches (Emoto, 2006).

To confirm that Wts and Sav function in the same pathway, genetic interaction between wts and sav in regulating dendrite morphogenesis was tested. Whereas heterozygous wts or sav mutants had no obvious dendritic phenotype, trans-heterozygous combinations of wts and sav alleles resulted in simplified dendrites similar to moderately affected sav clones. Furthermore, sav wts double mutant clones showed a severe dendrite defect comparable to wts mutant clones. Thus, Wts and Sav most probably function together in class IV neurons to regulate dendrite morphogenesis (Emoto, 2006).

The dendritic phenotypes of wts mutants and sav mutants might result from defects in branch formation and/or elongation, or loss of normally formed dendrites. Therefore ddaC dendrites were examined at different time points of larval development using the pickpocket-EGFP reporter, which is specifically expressed in class IV dendritic arborization neurons. Wild-type ddaC neurons elaborated primary and secondary dendritic branches by 24-28 h after egg laying, but large regions of the body wall were not yet covered by dendrites. By 48-52 h after egg laying, the major branches reached the dorsal midline, and the open spaces between major branches were filled with fine branches, resulting in complete dendritic coverage of the body wall. This tiling of dendrites persisted throughout the rest of larval development. In wts and sav mutants, ddaC dendrites were indistinguishable from those of wild-type controls at 24-28 h after egg laying. By 48-52 h after egg laying, wts and sav dendrites tiled the body wall as in wild type. During the next 24 h, however, dendrites of wts and sav mutants no longer tiled the body wall. Therefore, wts and sav seem to be required for maintenance of the already established tiling of dendrites (Emoto, 2006).

The loss of dendrites was further documented in live mutant larvae imaged for 30 h starting in early second instar larvae (48-50 h after egg laying). In wild-type larvae, ddaC dendrites grew steadily; the number of terminal branches increased by 23.0 over this time period. By contrast, dendrites of wts and sav mutants gradually lost their fine branches (decrease of 27.5 and 31.5, respectively) as well as some of the major branches by 78-80 h after egg laying (Emoto, 2006).

Class-IV-neuron-specific expression of wts and sav largely rescued the dendritic phenotype of wts and sav mutants, respectively, confirming that Wts and Sav act cell autonomously in class IV neurons. Furthermore, no detectable defect in patterning of the epidermis (anti-Armadillo antibody) or muscle (Tropomyosin::GFP reporter) was observed in wts or sav mutant third instar larvae. Taken together, these results indicate that the Wts/Sav signalling pathway functions in class IV neurons to maintain dendritic arborizations (Emoto, 2006).

Wts kinase activity is regulated, at least in part, by the Ste20-like serine/threonine kinase Hpo. Indeed, ddaC clones mutant for hpo exhibited simplified dendritic trees in third instar larvae, similar to wts or sav mutant clones, but showed more extensive dendritic arborizations in earlier larval stages (second to early third instar), consistent with the involvement of Hpo in the maintenance of dendrites. Notably, in hpo mutant clones at earlier developmental stages, dendritic branches were often found to overlap. Both the dendritic tiling and maintenance phenotypes were rescued by hpo expression in MARCM clones, consistent with the cell-autonomous function of Hpo in class IV neurons. Because this tiling defect in hpo mutant clones was similar to the tiling defects of trc mutant clones, whether hpo could genetically interact with trc to regulate dendritic tiling was tested. Compared with wild-type controls, trans-heterozygous combinations of trc and hpo exhibited obvious iso-neuronal as well as hetero-neuronal tiling defects, whereas wts and hpo trans-heterozygotes displayed simplified dendrites similar to wts mutants. These dendritic defects were consistently observed in multiple allelic combinations between hpo and trc or wts. In contrast, trans-heterozygous combinations of trc and wts showed no significant dendritic phenotypes. Furthermore, overexpression of wild-type Trc, but not Wts, in hpo MARCM clones partially rescued the dendritic tiling defects in class IV neurons. Thus, Hpo acts through Trc and Wts to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).

Not only did Hpo interact genetically with Trc and Wts, its physical association with these NDR kinases could be detected in vivo. When Flag-tagged Trc was expressed using a nervous-system-specific Gal4 driver, anti-Flag antibodies immunoprecipitated Trc together with Hpo. Similarly, Myc-tagged Wts co-immunoprecipitated with Hpo expressed in embryonic nervous systems. Hpo co-immunoprecipitation appeared to be specific, because Misshapen, another Ste20-like kinase protein present in neurons, was not co-immunoprecipitated by anti-Flag or anti-Myc antibodies in similar experiments. These results suggest that Hpo associates with Trc and Wts in the Drosophila nervous system (Emoto, 2006).

To examine further the physical interaction between Trc and Hpo, analogous experiments were carried out in Drosophila S2 cells co-transfected with a haemagglutinin (HA)-tagged Trc construct and a Flag-tagged Hpo construct containing the full open reading frame, an amino-terminal fragment containing the kinase domain, or a carboxy-terminal fragment containing the regulatory domain. Full-length Hpo and the C-terminal portion of Hpo, but not the N-terminal fragment, were co-immunoprecipitated with Trc, suggesting that the C-terminal domain of Hpo is sufficient for Trc-Hpo complex formation (Emoto, 2006).

Hpo physically interacts with Wts and promotes Wts phosphorylation at multiple serine/threonine sites, including two sites, S920 and T1083 of Drosophila Wts, that appear to be necessary for Wts kinase activation. Indeed, Wts protein with mutations in the S920 and T1083 residues was unable to rescue the wts mutant dendritic phenotypes. Given that the corresponding phosphorylation sites in Trc are critical for Trc activation as well as control of dendritic tiling and branching, it was of interest to know whether Hpo may promote Trc phosphorylation at the critical serine and/or threonine residue. Wild-type Hpo, but not catalytically inactive Hpo or the Misshapen kinase, led to substantial incorporation of 32P-labelled phosphate into recombinant Trc or Trc with a mutation at the S292 site (S292A), but not the T449A mutant form of Trc. Analogous results were obtained with Wts. These results support a model in which Hpo associates with and phosphorylates Trc and Wts at a critical threonine residue to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).

Both genetic and biochemical evidence reveals that Hpo regulates complementary aspects of dendrite development through two distinct downstream signalling pathways: the Trc kinase pathway for tiling and the Wts kinase pathway for maintenance. These studies of class IV dendritic arborization neurons, together with the recent report that Wts signalling is required for cell fate specification of photoreceptor cells in Drosophila retina, demonstrate that the Wts signalling pathway is important for post-mitotic neurons. In proliferating cells, Wts phosphorylates Yorkie (Yki), a transcriptional co-activator, to regulate cell cycle and apoptosis in growing cells. However, Yki is dispensable for Hpo/Wts-mediated dendrite maintenance. Hpo probably functions as an upstream kinase for Trc, as well as Wts, in neurons by phosphorylating a functionally essential threonine, which may also be regulated by MST3, a Ste20-like kinase closely related to Hpo. Given the evolutionary conservation of known components in the Trc and Wts signalling pathways, it will be important to identify their relevant downstream targets and explore mechanisms that coordinate the establishment and maintenance of dendritic fields, and to determine the role of Trc and Wts signalling in the mammalian nervous system (Emoto, 2006).

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 (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 (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 (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 (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 (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 (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 (Chen, 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).

Drosophila CK2 promotes Wts to suppress Yki activity for growth control

Drosophila Hippo signaling regulates Wts activity to phosphorylate and inhibit Yki in order to control tissue growth. CK2 is widely expressed and involved in a variety of signaling pathways. This study reports that Drosophila CK2 promotes Wts activity to phosphorylate and inhibit Yki activity, which is independent of Hpo induced Wts promotion. In vivo, CK2 overexpression suppresses hpo mutant induced Ex upregulation and overgrowth phenotype while it cannot affect wts mutant. Consistent with this, knockdown of CK2 upregulates Hpo pathway target expression. It was also found that Drosophila CK2 is essential for tissue growth as a cell death inhibitor as knockdown of CK2 in developing discs induces severe growth defect as well as caspase3 (Drice) signal. Taken together, these results uncover a dual role of CK2: while its major role is promoting cell survive, it may potentially be a growth inhibitor as well (Hu, 2014).

The conserved Misshapen-Warts-Yorkie pathway acts in enteroblasts to regulate intestinal stem cells in Drosophila

Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. This study shows that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP (Li, 2014).

Previous studies have shown that endothelial cells (ECs) produce regulatory factors in response to infection and damage and function as part of the niche to regulate intestinal stem cell (ISC)-mediated regeneration. Meanwhile, recent reports show that enteroblasts (EBs) can also produce growth factors including EGF receptor ligands, Wingless and Upd3, although the pathways that regulate their production are not known. The current results demonstrate that differentiating EBs also function as an important part of the niche to regulate ISC division via the Msn pathway. EB-specific knockdown of msn leads to highly increased Upd3 expression and midgut proliferation. A previous report suggests that undifferentiated EBs if remain in contact with the mother ISC can inhibit proliferation. Although the hyperproliferating midguts after loss of Msn contain many EBs, these EBs do go into normal differentiation and express high level of Upd3, which may overcome any inhibitory effect of undifferentiated EBs on ISC proliferation (Li, 2014).

Msn is known to regulate a number of biological processes. During embryonic dorsal closure the MAP kinase pathway Slipper-Hemipterous-JNK is downstream of Msn, and Slipper is able to bind to Msn in vitro. In the adult midgut, JNK is a mediator of aging-related intestinal dysplasia and is a stress-activated kinase in ECs to positively regulate ISC division. While the current RNAi experiments show that JNK has a function in EBs to negatively regulate ISC proliferation, this phenotype is not dependent on Upd3 or Yki. No change of JNK phosphorylation was detected after loss of Msn. Mammalian MAP4K4 has also been shown to function independently of JNK in some biological contexts. Therefore, Msn and JNK probably have independent functions in the midgut (Li, 2014).

This study has instead uncovered an interaction of Msn with Wts and subsequently regulation of Yki. Hpo-Wts-Yki has been demonstrated to have a function in ECs for stress and damage-induced response. Gal4 driven experiments have many caveats including cell-type specificity, differences in promoter strengths, and knockdown efficiency in different cell types. Nonetheless, the results of many parallel experiments that this study conducted strongly suggest that Msn and Hpo independently regulate Wts-Yki in EBs and ECs, respectively. How the Msn and Hpo pathways in the two cell types are coordinately regulated to produce an appropriate amount of Upd3 to achieve desirable intestinal growth under different circumstances remains an important question to be answered (Li, 2014).

Previous experiments in developing discs suggest that Wts and Yki but not Hpo act downstream of cytoskeleton regulators. Similarly, the mammalian Hpo homologs MST1/2 appear not to be involved in LATS regulation after cytoskeletal perturbation in some cell types. In vivo assay in midgut suggests a function for Msn, Yki and Upd3 downstream of actin capping proteins in EBs. Similarly, the Latrunculin B effect on MEFs suggests that MAP4K4 is required for cytoskeleton-regulated LATS and YAP phosphorylation. The situation in mammalian cells may be more complicated because the Msn/MAP4K4 subfamily also includes two other closely related kinases TNIK and MINK1. Proper regulation of Wts by the cytoskeleton may require both positive and negative regulators, because recent work in flies identified the LIM-domain protein Jub as a negative regulator of Wts in response to cytoskeletal tension. It will be interesting in future studies to determine how positive and negative regulators of Wts act in a coordinated manner to regulate cell fate and proliferation in response to cytoskeletal tension (Li, 2014).

Protein Interactions

So far, relatively few mechanisms have been shown to be capable of regulating both cell proliferation and cell death in a coordinated manner. In a screen for Drosophila mutations that result in tissue overgrowth, salvador (sav), a gene that promotes both cell cycle exit and cell death was identified. Elevated Cyclin E and DIAP1 levels are found in mutant cells, resulting in delayed cell cycle exit and impaired apoptosis. Salvador contains two WW domains and binds to the Warts protein kinase. The human ortholog of salvador (hWW45) is mutated in several cancer cell lines. Thus, salvador restricts cell numbers in vivo by functioning as a dual regulator of cell proliferation and apoptosis (Tapon, 2002).

Clones of wts tissue generate outgrowths that resemble tumors. Nine alleles of wts were identified in the screen that identified wts, and the phenotype of sav3 is similar to that elicited by hypomorphic wts mutations. Null alleles of wts display a more severe phenotype. Like sav, wts clones in the pupal retina have additional interommatidial cells. Larval imaginal discs containing large wts clones are enlarged and convoluted. Larval eye discs that contain eyFLP-induced wts clones are composed mostly of mutant tissue with small regions of wild-type tissue. Many additional BrdU-incorporating nuclei are observed in mutant clones posterior to the SMW. As observed with sav, the stripe of cyclin E RNA expression is also broadened in these discs. Moreover, the normal cell death that occurs in the pupal retina is almost completely abolished in wts mutant clones. Thus, as for sav, wts mutations generate additional interommatidial cells resulting from both increased cell proliferation posterior to the SMW as well as reduced apoptosis in the pupal retina. In addition, Drice activation induced by GMR-hid is markedly diminished in wts clones (Tapon, 2002).

Overexpression of sav alone using the GMR promoter has no effect, and overexpression of wts generates subtle irregularities in ommatidial architecture. However, combined overexpression of sav and wts results in a smaller eye where the ommatidial pattern is highly irregular. This effect appears to reflect a synergistic increase in cell death in the eye discs of flies that express both transgenes as well as a minor effect on reducing cell proliferation assoiated with the SMW (Tapon, 2002).

Thus, Sav and Wts may function in the same pathway and may bind to each other. Indeed, the Sav protein has a Group I WW domain that is predicted to interact with the PPXY (PY) motif, five of which are found in the Wts protein. To test whether Drosophila Sav and Wts proteins could physically interact, a GST pull-down assay was employed. The region containing the two potential WW domains of Sav was fused to GST and incubated with cell lysates that expressed Myc-tagged Wts protein. Using this assay, Wts was found to interact specifically with the region of Sav that contained the WW domain. Furthermore, a 15 amino acid peptide, designed to mimic one of the PY motifs of Wts, was found to inhibit the interaction between the WW domain region of Sav and Wts. An identical peptide where the tyrosine residue that is required for interaction with type I WW domains had been replaced by an alanine did not prevent this interaction. Thus, at least under the conditions of this experiment, Sav and Wts interact in a WW domain- and PY motif-dependent fashion, suggesting that an analogous interaction could occur in vivo (Tapon, 2002).

Discs containing clones of the wts null allele, wtslatsX1, are much larger than discs containing sav3 clones. If all sav functions were wts dependent, the double mutant phenotype should not be more severe than the wts phenotype. When mutant clones were generated with eyFLP, average disc sizes were 39,669 pixels for sav3, wtslatsX1 double mutant discs and 31,360 pixels for wtslatsX1 discs. Thus, the double mutant discs were significantly larger than the wtslatsX1 discs. Thus, while sav and wts appear to function together in certain ways, they are also likely to have functions that are independent of each other (Tapon, 2002).

Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously. The salvador gene, which encodes a scaffold protein, restricts cell number by coordinating cell-cycle exit and apoptosis during Drosophila development. Hippo (Hpo), the Drosophila ortholog of the mammalian MST1 and MST2 serine/threonine kinases, is a partner of Sav. Hippo was described in five publications that appeared simutaneously: Pantalacci (2003) identified Hippo in a yeast two-hybrid screen in a search for Salvador interacting proteins, Udan (2003) identifed and positionally cloned hippo in a mutagenesis screen for genes that regulate tissue growth, and Harvey (2003), Jia (2003) and Wu (2003) identified hippo in screens for genes that restrict growth and cell number. Loss of hpo function leads to sav-like phenotypes, whereas gain of hpo function results in the opposite phenotype. Whereas Sav and Hpo normally restrict cellular quantities of the Drosophila inhibitor of apoptosis protein DIAP1 (Thread), overexpression of Hpo destabilizes DIAP1 in cell culture. DIAP1 is phosphorylated in a Hpo-dependent manner in S2 cells and that Hpo can phosphorylate DIAP1 in vitro. Thus, Hpo may promote apoptosis by reducing cellular amounts of DIAP1. In addition, Sav is an unstable protein that is stabilized by Hpo. It is proposed that Hpo and Sav function together to restrict tissue growth in vivo (Pantalacci, 2003; Harvey, 2003; Jia, 2003; Udan, 2003 and Wu, 2003).

The coordination between cell proliferation and cell death is essential to maintain homeostasis within multicellular organisms. The mechanisms underlying this regulation are yet to be completely understood. hippo has been identified as a gene that regulates both cell proliferation and cell death in Drosophila. hpo encodes a Ste-20 family protein kinase that binds to and phosphorylates the tumor suppressor protein Salvador, which is known to interact with the Warts protein kinase. Loss of hpo results in elevated transcription of the cell cycle regulator cyclin E and the cell-death inhibitor diap1, leading to increased proliferation and reduced apoptosis. Further, hpo, sav, and wts define a pathway that regulates diap1 at the transcriptional level. A human homolog of hpo completely rescues the overgrowth phenotype of Drosophila hpo mutants, suggesting that hpo might play a conserved role for growth control in mammals (Wu, 2003).

A yeast two-hybrid screen was carried out in the hope of identifying Hpo binding proteins. Approximately 1 million cDNA clones were screened using as bait the noncatalytic C-terminal portion of Hpo. Interestingly, 6 out of 12 positive clones isolated from the screen corresponded to Sav, representing 3 different classes of clones. These Hpo-interacting Sav clones define the C-terminal half of Sav (residues 362-607) as an Hpo binding region. This region contains predicted Sav WW and coiled-coil domains. Another yeast two-hybrid screen was carried out using the C-terminal half of Sav as the bait. In this screen, 5 out of 45 positive clones isolated from the screen corresponded to Hpo, representing 4 different classes of clone. These Sav-interacting Hpo clones define the C-terminal portion of Hpo (residues 474-669) as a Sav binding region. The identification of Hpo and Sav as interacting proteins in unbiased yeast two-hybrid screens provides strong evidence that these proteins interact with each other in vivo. Consistent with this hypothesis, Hpo and Sav associate with each other in vitro. GST fusion protein containing full-length Sav, but not a control GST fusion protein, is able to specifically pull-down endogenous Hpo protein from S2 cell extracts. Hpo and Sav also interact with each other in coimmunoprecipitation assays (Wu, 2003).

Next, whether Hpo can function as a Sav kinase was tested. For this purpose, a cotransfection assay was established in S2 cells. Coexpression of Hpo and Sav results in retarded mobility of Sav, leading to the formation of multiple slower migrating bands. Phosphatase treatment abrogates this shift, suggesting that the mobility shift is due to protein phosphorylation. In contrast, coexpression of Sav and Wts, also a Ser/Thr kinase, does not result in Sav mobility shift, nor does expression of Wts affect the phosphorylation of Sav by Hpo. In vitro, myc-tagged Hpo protein specifically phosphorylates a GST fusion protein containing the Hpo binding region of Sav. Thus, Hpo phosphorylates Sav. These results presented above suggest a model wherein the C-terminal domain of Hpo associates with Sav and presents Sav to the Hpo kinase. If so, a kinase-dead mutant of Hpo, or the C-terminal noncatalytic domain of Hpo expressed alone, should behave as dominant-negative forms, since these variants should associate nonproductively with endogenous Sav and interfere with signal propagation. Indeed this is the case (Wu, 2003).

Having established a functional link between Hpo and Sav and given the results from a genetic analyses implicating hpo, sav, and wts in a common pathway, whether Wts might be regulated by Hpo and/or Sav was tested. In S2 cells, expression of Hpo results in retarded mobility of Wts, while coexpression of Hpo and Sav results in a further mobility shift of Wts. For simplicity, this further shift of Wts upon coexpression of Hpo and Sav is referred to as 'supershift' to be distinguished from the mobility shift caused by expression of Hpo alone. Both shifts are largely abolished by phosphatase treatment, confirming that the shifts are due to phosphorylation. Taken together, these data suggest that Sav increases the ability of Hpo to phosphorylate Wts (Wu, 2003).

The mobility shift assay described the narrowing down of the domain of Wts that is the target of Hpo-mediated phosphorylation to a region at the N-terminal noncatalytic portion (residues 68-414) of the Wts protein. In vitro, a GST fusion protein containing this region of Wts is phosphorylated by Hpo. Consistent with Wts as a kinase substrate of Hpo, the mobility of endogenous Wts protein on SDS-PAGE is increased in Hpo mutant animals (Wu, 2003).

These results suggest a model wherein Hpo associates with and phosphorylates Sav and interactions between Hpo and Sav facilitate Wts phosphorylation by Hpo. This model is consistent with a direct physical interaction between Sav and Wts. Thus, Sav could be viewed as an adaptor protein that brings Hpo in proximity to Wts to facilitate Wts phosphorylation. Since the Sav WW domains have been implicated in Sav/Wts interaction, it is speculated that the coiled-coil domain of Sav, located C-terminal to the WW domains, might be involved in Sav/Hpo interaction. Interestingly, the shrp6 allele of sav causes a frameshift mutation that truncates just the coiled-coil domain but leaves the WW domains intact. To pinpoint the functional defect of the savshrp6 allele, a mutant Sav protein, Savshrp6, was engineered that lacks the C-terminal 79 residues as seen in savshrp6, and the ability of this mutant protein to associate with Hpo and to facilitate Wts phosphorylation by Hpo was examined. Unlike wild-type Sav, Savshrp6 can not associate with Hpo, suggesting that the coiled-coil domain of Sav is required for Hpo/Sav interaction. Importantly, coexpression of Savshrp6 and Hpo can no longer cause the supershift of Wts as seen when wild-type Sav and Hpo are coexpressed. Thus, Hpo/Sav interaction is required for Sav to facilitate the phosphorylation of Wts by Hpo (Wu, 2003).

The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP: Yorkie physically interacts with Warts

Coordination between cell proliferation and cell death is essential to maintain homeostasis in multicellular organisms. In Drosophila, these two processes are regulated by a pathway involving the Ste20-like kinase Hippo (Hpo) and the NDR family kinase Warts (Wts; also called Lats). Hpo phosphorylates and activates Wts, which in turn, through unknown mechanisms, negatively regulates the transcription of cell-cycle and cell-death regulators such as cycE and diap1. Yorkie (Yki), the Drosophila ortholog of the mammalian transcriptional coactivator yes-associated protein (YAP), has been identified as a missing link between Wts and transcriptional regulation. Yki is required for normal tissue growth and diap1 transcription and is phosphorylated and inactivated by Wts. Overexpression of yki phenocopies loss-of-function mutations of hpo or wts, including elevated transcription of cycE and diap1, increased proliferation, defective apoptosis, and tissue overgrowth. Thus, Yki is a critical target of the Wts/Lats protein kinase and a potential oncogene (Huang, 2005).

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).

Control of cell proliferation and apoptosis by Mob as tumor suppressor Mats: Mats associates with Wts to form a protein complex

As a unique group of the Mob superfamily, Mats orthologs exist in both plants and animals. Since Mats proteins are highly conserved, their function may be conserved across species. In support of this, human Mats1 was found to functionally substitute for mats in Drosophila. Importantly, loss-of-function mutations in Mats1 have been identified in a human skin cancer and a mouse breast tumor, suggesting that mammalian Mats genes may indeed act as tumor suppressors. Further molecular analysis of mammalian Mats genes from tumor tissues will be needed to test this hypothesis. On the basis of these data, it is speculated that all mats genes from animals and plants may negatively regulate cell number and tissue growth by restricting cell proliferation and promoting apoptosis (Lai, 2005).

Tumor suppressors normally act as inhibitors of cell proliferation or activators of apoptosis and use a variety of mechanisms in tissue growth suppression. This work provides evidence that mats functions to restrict cell proliferation and promote apoptosis in Drosophila. In this regard, functions of mats are similar to those of hpo, sav, and wts. Like hpo, sav, and wts, mats negatively regulates expression of CycE and DIAP1, two key regulators involved in cell cycle or apoptosis control. However, the overgrowth phenotypes of mats mutants appear to be stronger than those of hpo, sav, and wts and therefore cannot be explained simply by increased expression of Cyclin E and loss of apoptosis. It is suspected that mats might use other mechanisms to regulate cell number and organ size. For instance, Mats may negatively regulate cell cycle regulators such as Cdc25 protein phosphatase that are required for the G2-M transition. Since yeast Mob1 is able to form a complex with Mps1 (Mono polar spindle 1) kinase, Mats may also play a role in the spindle assembly checkpoint by acting together with Mps1. Mps1 has been previously shown to be involved in the spindle assembly checkpoint in yeast, and Mps1 is also implicated in this process in vertebrate cells. Involvement of Mats in the spindle assembly checkpoint would help explain the dramatic overgrowth phenotypes of mats mutants. Clearly, further investigations are needed to test these hypotheses (Lai, 2005 and references therein).

Consistent with a model that Mats functions as a critical component of the Hpo-Sav-Wts pathway, the data show that Mats associates with Wts to form a protein complex. Supporting this, crystal structure analysis of human Mats1/Mob1A reveals that several evolutionarily conserved acidic residues are exposed on the surface to provide a strong electrostatic potential for mediating protein-protein interactions (Stavridi, 2003). Based on this finding, Mats binding regions are expected to be basic and indeed such regions do exist in Wts family proteins. It remains to be addressed as to how exactly Mats interacts with Wts and whether the Mats-Wts complex can be associated with Hpo and Sav. Excitingly, it was found that Mats functions as an activating subunit to stimulate Wts kinase activity. In this way, Wts activation can be effectively controlled by the availability of Mats protein through differential distribution of Mats in different tissues, cells, or subcellular locations. With Mats acting as an activator of Wts kinase, the relationship between Mats and Wts mimics that of Cyclin and Cyclin-dependent kinases, which are essential for cell cycle control (Lai, 2005).

How does Mats association lead to Wts activation? In a model, association with Mats may allow Wts to undergo an allosteric conformational change critical for Wts activation or to simply relieve an autoinhibition of Wts. Interestingly, the N-terminal region of Wts was shown to be able to associate with its C-terminal kinase domain through intramolecular binding, and this interaction may be inhibitory for the Wts kinase activity. Thus, association with Mats may activate Wts by disrupting this intramolecular binding within Wts. In the case of human Ndr kinase, an autoinhibitory sequence has been identified and binding of the hMats1/hMob1A protein induces a release of this autoinhibition (Bichsel, 2004). In another model, Mats association may allow the Mats-Wts complex to recruit additional coactivators or to prevent coinhibitors from being recruited in order for Wts to be activated. Clearly, any model of Wts activation would have to consider the effect of Wts phosphorylation. (1)Wts has been shown to be phosphorylated in a cell cycle-dependent manner. Because Wts kinase activity can be increased through treatment of phosphatase inhibitors, phosphorylation appears to be critical for Wts kinase activity. (2) The Drosophila homolog of C-terminal Src kinase (dCsk) gene has been shown to genetically interact with wts to inhibit cell proliferation, and dCsk phosphorylates Wts in vitro. (3) Human Wts2 is a phosphorylation target of Aurora-A kinase, and this phosphorylation plays a role in regulating centrosomal localization of hWts2. (4) Hpo can directly target Wts for phosphorylation, and this event is facilitated by Sav. At present, it is unclear how Mats may affect Wts phosphorylation by Hpo or how Mats-Wts complex may be regulated by Hpo through phosphorylation. In yeast, Mob1 is essential for the phosphorylation of Dbf2 kinase by an upstream kinase Cdc15. Further studies on Wts phosphorylation are expected to provide a better understanding of how Wts is regulated (Lai, 2005).

While functions of most Mob superfamily proteins are still poorly understood, this work on Mats supports that a common feature of Mats proteins is to function as coactivators of protein kinases such as Wts. Identification and functional studies of Mats have revealed a mechanism for the control of Wts tumor suppressor activity. Because Mats-mediated growth inhibition and tumor suppression appear to be evolutionarily conserved, it extends the understanding of tissue growth and cell number control during development and tumorigenesis and raises the possibility that Mats-dependent growth inhibition may have important implications for the understanding and treatment of human cancers (Lai, 2005).

Individual Mob family proteins also interact with Tricornered (Trc), the Drosophila Ndr (Nuclear Dbf2-related) serine/threonine protein kinase that is required for the normal morphogenesis of a variety of polarized outgrowths including epidermal hairs, bristles, arista laterals, and dendrites. In yeast the Trc homolog Cbk1 needs to bind Mob2 to activate the RAM pathway. Genetic and biochemical data is provided that Drosophila Trc interacts with and is activated by Drosophila Dmob proteins, specifically Mats and Dmob2 (FlyBase terms the gene Dmob2 Mob1). Evidence is provided that Drosophila Mob proteins also interact with the related Warts/Lats kinase, which functions as a tumor suppressor in flies and mammals. In trc mutants the overall pattern of denticles is partly disorganized and many denticles are split. Split denticles are infrequent in wild-type larvae. The denticle pattern of mats mutant larvae is also disorganized and contains many split denticles. Interestingly, the overgrowth tumor phenotype that results from mutations in Dmob1 (mats) is only seen in genetic mosaics and not when the entire animal is mutant. Unlike in yeast, in Drosophila individual Mob proteins interact with multiple kinases and individual NDR family kinases interact with multiple Mob proteins; in particular, Mats interacts physically and genetically with trc and mats phenotype resembles that of trc. Notably, trc:mats double mutant larvae do not have a more severe phenotype than the single mutants. This lack of additivity argues that trc and mats function in a common pathway during denticle development. These observations also suggest that mats functions with both Trc and Wts (He, 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).

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).

Regulation of Hippo signaling by EGFR-MAPK signaling through Ajuba family proteins

EGFR and Hippo signaling pathways both control growth and, when dysregulated, contribute to tumorigenesis. This study found that EGFR activates the Hippo pathway transcription factor Yorkie and demonstrates that Yorkie is required for the influence of EGFR on cell proliferation in Drosophila. EGFR regulates Yorkie through the influence of its Ras-MAPK branch on the Ajuba LIM protein Jub. Jub is epistatic to EGFR and Ras for Yorkie regulation, Jub is subject to MAPK-dependent phosphorylation, and EGFR-Ras-MAPK signaling enhances Jub binding to the Yorkie kinase Warts and the adaptor protein Salvador. An EGFR-Hippo pathway link is conserved in mammals, as activation of EGFR or RAS activates the Yorkie homolog YAP, and EGFR-RAS-MAPK signaling promotes phosphorylation of the Ajuba family protein WTIP and also enhances WTIP binding to the Warts and Salvador homologs LATS and WW45. These observations implicate the Hippo pathway in EGFR-mediated tumorigenesis and identify a molecular link between these pathways (Reddy, 2013).

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).

Kibra is a regulator of the Salvador/Warts/Hippo signaling network

The Salvador (Sav)/Warts (Wts)/Hippo (Hpo) (SWH) network controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core of the pathway consists of a MST and LATS family kinase cascade that ultimately phosphorylates and inactivates the YAP/Yorkie (Yki) transcription coactivator. The FERM domain proteins Merlin (Mer) and Expanded (Ex) represent one mode of upstream regulation controlling pathway activity. This study identified Kibra as a member of the SWH network. Kibra, which colocalizes and associates with Mer and Ex, also promotes the Mer/Ex association. Furthermore, the Kibra/Mer association is conserved in human cells. Finally, Kibra complexes with Wts and kibra depletion in tissue culture cells induces a marked reduction in Yki phosphorylation without affecting the Yki/Wts interaction. It is suggested that Kibra is part of an apical scaffold that promotes SWH pathway activity (Genevet, 2010).

An in vivo screen was performed in the fly wing in order to identify genes implicated in growth control. Transgenic flies bearing RNA interference (RNAi) constructs generated by the Vienna Drosophila RNAi Centre (VDRC) were crossed to the hedgehog-GAL4 (hh-GAL4) driver, leading to target gene silencing in the posterior compartment of the wing. A collection was screened of 12,000 lines targeting genes conserved between Drosophila and mammals. Expressing an RNAi line directed against kibra induced overgrowth of the posterior wing compartment compared to control flies. This phenotype was also observed upon wts depletion. Driving the same kibra RNAi line in the eye also led to increased organ size, similarly to a wts RNAi line. Adult eye sections revealed that kibra knockdown retinas present an excess of interommatidial cells (IOCs). The IOCs, the last population of cells to differentiate in the eye primordium, give rise to the secondary and tertiary pigment cells that optically isolate the ommatidia in the compound eye from each other. Extra IOCs are produced during normal development but are then eliminated by apoptosis at the pupal stage to give rise to the adult lattice. The presence of extra IOCs is a hallmark of SWH network loss of function, which reduces retinal apoptosis, as seen in wts RNAi adult eye sections. Thus, depletion of kibra elicits a similar phenotype to SWH network mutants, suggesting a potential role for Kibra in Hpo signaling (Genevet, 2010).

To study kibra loss of function, the kibraΔ32 allele loss of function allele was generated by imprecise excision of the EP747 transposon. This deletion allele, which removes the translation initiation site, is homozygous lethal and may be a null allele for kibra. kibraΔ32 FLP/FRT mutant clones in 40 hr after-puparium-formation (APF) retinas present extra IOCs, similarly to what was observed in adult eyes with kibra knockdown. Duplication of bristles or missing bristles can also be observed. Apoptotic indexes were determined during the retinal apoptosis wave (28 hr APF) in pupal retinas containing kibra mutant clones stained with an anti-active Caspase-3 antibody. kibra mutant tissue presents a reduced apoptotic index compared with wild-type (WT) areas in the same retinas. Thus, extra IOCs persist in kibra mutant clones as a result of decreased developmental apoptosis (Genevet, 2010).

The proliferation rate of kibra mutant cells was assessed in imaginal discs, the larval precursors to the adult appendages. By using the FLP/FRT system under the control of the heat-shock promoter, kibra mutant cells and their WT sister clones were generated through single recombination events from heterozygous mother cells. After several rounds of divisions, the sizes of mutant clones (no GFP) and WT twin spots (two copies of GFP) were compared, allowing estimation of the relative proliferation rates of mutant versus WT cells. The total kibra clone area is 1.57-fold larger than the control twin spot area, compared to a ratio of 0.98 when both clones and twin spots are WT, indicating that kibraΔ32 mutant cells grow 1.6 times faster than WT cells (Genevet, 2010).

In addition to cell cycle rates, the timing of cell cycle exit can readily be measured in the eye disc, where cell divisions follow a spatially determined pattern. During the third larval instar, the morphogenetic furrow, a wave of differentiation, sweeps the eye disc from posterior to anterior. Anterior to the furrow cells still proliferate asynchronously, while in the furrow cells synchronize in G1. Immediately posterior to the furrow, cells enter a final round of synchronous S phases, the second mitotic wave (SMW). Posterior to the SMW, most cells permanently exit the cell cycle. Thus, in WT discs, no S phases can be observed posterior to the SMW. As expected, hpo mutant cells fail to exit from the cell cycle in a timely manner and present ectopic EdU-positive staining posterior to the SMW. kibra mutant cells exhibit a less pronounced but similar phenotype. Thus, kibra mutant tissues have a proliferative advantage and an apoptosis defect, consistent with an involvement in the SWH network. The overgrowth defect appears more subtle than that of core pathway members such as wts and is more akin to upstream regulators (e.g., ex and mer) (Genevet, 2010).

Several transcriptional targets of the SWH network have been identified, such as the Drosophila Inhibitor of Apoptosis 1 (DIAP1) gene, the cell cycle regulator cycE, the miRNA bantam, as well as ex. In kibra mutant wing or eye discs, no strong change in DIAP1, CycE, or ex-lacZ reporter levels could be detected. Since overgrowth of kibra mutant cells in the wing is subtle compared to wts mutants, it is possible that Kibra plays a relatively minor role in SWH signaling in the wing. Accordingly, using an anti-Kibra antibody, it was noted that Kibra staining in the wing disc is weak and consists of a punctate apical staining which can clearly be observed when kibra is overexpressed in a stripe of cells. Thus, the extent to which Kibra is required may vary in different tissues (Genevet, 2010).

Ovarian posterior follicle cells (PFCs) are particularly sensitive to SWH loss of function, leading to a study the kibraΔ32 phenotype in the ovary. First, it was noted that Kibra protein levels are higher in follicle cells than in the wing discs. Kibra staining is mainly apical and is severely reduced in kibraΔ32 clones. Similarly to hpo or wts loss of function, kibra loss of function in the PFCs induces an upregulation of the ex-lacZ reporter. hpo or wts mutant PFCs also show a misregulation of the Notch (N) pathway and ectopic cell divisions. The N target Hindsight (Hnt) is normally repressed in all follicle cells up to stage 6 and switched on from stage 7 to stage 10B. Cut, which is repressed by Hnt, presents an opposite pattern of expression. In kibra mutant PFCs from stage 7-10B egg chambers, Hnt expression is lost, while Cut is ectopically expressed. This indicates that N signaling is downregulated in kibra mutant PFCs. Loss of kibra also leads to perturbation of epithelial integrity, as mutant PFCs show an accumulation of the apical polarity protein aPKC and the N receptor as well as multilayering of the follicular epithelium. Ectopic mitotic divisions are also observed in PFCs clones after stage 6, as detected by phospho-histone H3 (PH3) staining. Together, these phenotypes are identical to those observed in hpo or wts loss of function, suggesting that Kibra is indeed a member of the SWH network (Genevet, 2010).

To further explore the role of Kibra in the SWH network, genetic interaction and epistasis experiments were performed. Overexpressing kibra in the eye under the GMR (Glass Multimer Reporter) promoter elicits the formation of a small rough eye with frequent ommatidial fusions. This phenotype can be partially rescued by removing one copy of the hpo gene. In contrast, overexpressing kibra could not rescue the hpo-like overgrowth phenotype induced by yki overexpression, suggesting that Kibra may be an upstream regulator of the pathway (Genevet, 2010).

To conduct epistasis experiments between kibra and yki, the MARCM system was used to generate clones of mutant cells while simultaneously overexpressing or depleting other pathway components. MARCM clones expressing yki RNAi generated with eyFLP lead to the formation of a normal eye, because yki-depleted cells are eliminated by apoptosis and replaced by WT cells. As expected, eyFLP kibra MARCM clones cause eye overgrowth. This overgrowth is rescued by yki depletion in the mutant cells, indicating that the kibra overgrowth phenotype is yki dependent. Furthermore, overexpressing kibra in the eye under the GMR promoter induces apoptosis in third instar eye discs, which is suppressed by loss of hpo. Together, these epistasis experiments are consistent with Kibra being a member of the SWH network acting upstream of Yki and Hpo (Genevet, 2010).

Genetic interactions between kibra, mer, and ex, upstream members of the SWH network, were then investigated. Expressing a kibra, an ex, or a mer RNAi line in the eye under the GMR promoter induces eye overgrowth. Combined depletion of either Ex/Kibra or Mer/Kibra shows stronger phenotypes than individual depletion of these proteins. The MARCM technique was used to evaluate epistatic relationships between those three genes. hsFLP MARCM clones of various genotypes were generated and scored according to the severity of the wing overgrowth phenotypes, with type 0 representing normal wings and type 4 the strongest overgrowth. Overexpressing ex or mer in kibra mutant clones significantly rescues the overgrowth of kibra mutant clones. Reciprocally, kibra overexpression was also able to suppress the ex overgrowth phenotype. Thus, a strict epistatic relationship between kibra, ex, and mer could not be determined, consistent with a model whereby kibra, ex, and mer cooperate to control SWH pathway activity (Genevet, 2010).

As well as being an upstream regulator of the SWH network, ex is also one of its transcriptional targets, as are other upstream regulators (e.g., mer, four-jointed, dachsous). Since epistasis experiments place Kibra at the level of Mer and Ex, it was of interest to test whether this is also the case for kibra. Kibra levels were highly upregulated in mer;ex or hpo clones, showing an apical localization. The same is true in hpo clones in follicle cells. Similarly, hpo-depleted cultured Drosophila S2R+ cells have increased Kibra levels. To determine whether kibra is a transcriptional SWH network target, quantitative RT-PCR experiments were performed on yki-overexpressing and control wing imaginal discs. As expected, ex mRNA levels were increased in yki-expressing discs compared to control discs. Interestingly, kibra mRNA levels were also upregulated in yki-expressing discs, confirming that kibra is a Yki transcriptional target and suggesting the existence of a possible negative feedback loop regulating Kibra expression (Genevet, 2010).

Because hpo clones present increased levels of Kibra as well as Mer and Ex, these constitute a good system to evaluate the colocalization of those proteins. Indeed, Kibra colocalizes with Mer in the wing disc. As expected, Mer and Ex also colocalize. Thus, Kibra, Mer, and Ex colocalize apically in imaginal disc cells, but are dispensable for each other's apical sorting, because Kibra is still apical in mer;ex clones and Mer/Ex are normally localized in kibra clones (Genevet, 2010).

Because Kibra colocalizes with Mer/Ex, a possible association between those proteins was examined by conducting coimmunoprecipitation (co-IP) assays in S2R+ cells. Kibra was found to co-IP with Ex and Mer, but not with Hpo or with the negative regulator of Hpo, dRASSF. Kibra possesses two WW domains, which are predicted to mediate protein-protein interactions by binding to PPXY motifs. Furthermore, the first WW domain of human KIBRA was shown to recognize the consensus motif RXPPXY in vitro. In flies, Mer does not contain any PPXY sites, while Ex has two PPXY sites (P786PPY and P1203PPY) and an RXPPXY site (R842DPPPY). The association between Kibra and Ex was investigated by mutating amino acids that are known to be required for WW domains and PPXY sites to interact. A Kibra protein mutant for its first WW domain (P85A) could no longer co-IP WT Ex. Reciprocally, WT Kibra could not co-IP an Ex protein deficient for its RXPPXY site (P845A). Thus, Kibra associates with Ex through its first WW domain and the Ex RXPPXY motif (Genevet, 2010).

Because Kibra complexes with Ex and a Yki/Ex interaction has recently been described, attempts were made to determine whether Kibra can affect Yki activity. S2R+ cells were treated with RNAi against several SWH pathway components, and Yki phophorylation on Ser168 was monitored by western blotting. The phosphorylation of Yki by Wts at Ser168 leads to Yki inactivation and sequestration in the cytoplasm, where it has been reported to bind Ex, Wts, Hpo, and 14.3.3. lacZ RNAi-treated cells show a high basal level of phospho-Yki (P-Yki). As expected, Yki phosphorylation is abolished when Wts is depleted, and mildly reduced when the Wts cofactor Mats is depleted. In wts treated RNAi cells, a Yki downward shift can also be observed using a pan-Yki antibody. ex RNAi treatment has only a mild effect on P-Yki levels. Interestingly, kibra depletion leads to a marked reduction in P-Yki. When depleted in conjunction with ex, the P-Yki signal becomes even further reduced (Genevet, 2010).

This suggests that Kibra and Ex are required for Wts activity on Yki, which prompted an investigation of whether Kibra could associate with Wts. Co-IP assays reveal that Kibra interacts with Wts. Wts does not seem to compete with Ex for Kibra association, because it could still complex with a form of Kibra mutant for its first WW domain. Because Kibra associates with Wts and Ex interacts with Yki, whether Wts requires Kibra/Ex to bind Yki was investigated. Endogenous IPs between Yki and Wts were performed in S2 cells treated with various dsRNAs. In these conditions, the effect of kibra and ex depletion on Yki phosphorylation can also be observed. In control cells, Wts binding to Yki is detected after immunoprecipitating Yki. This endogenous interaction is unaffected by the individual or combined depletion of ex and kibra. These results suggest that Ex and Kibra are required to activate the SWH pathway by nucleating an active Hpo/Wts kinase cassette, rather than promoting the Wts/Yki interaction (Genevet, 2010).

These data identify Kibra as a regulator of the SWH network that associates with Ex and Mer, with which it is colocalized apically and transcriptionally coregulated. Given that the apical surface of epithelial cells is instrumental in both cell-cell signaling and tissue morphogenesis, it is speculated that Kibra may cooperate with Ex and Mer to transduce an extracellular signal, or relay information about epithelial architecture, via the SWH network, to control tissue growth and morphogenesis (Genevet, 2010).

Recent data have suggested that an apical scaffold machinery containing Hpo, Wts, and Ex recruits Yki to the apical membrane, facilitating its inhibitory phosphorylation by Wts. Since Kibra associates with Ex and is also apically localized, it is hypothesized that Kibra is also part of this scaffold and participates in nucleating an active Hpo/Wts complex and recruiting Yki for inactivation. This view is supported by the finding that Kibra complexes with Wts and that combined depletion of Kibra and Ex leads to a strong decrease in Yki phosphorylation, but does not disrupt the Wts/Yki interaction. The data also suggest that the importance of Kibra may be tissue-specific since robust phenotypes were observed in ovaries and hemocyte-derived S2R+ cells, but weaker effects in imaginal discs. Thus, considering the relative levels of expression of Ex, Mer, and Kibra may be important in determining pathway activation. Finally, since mammalian KIBRA complexes with the NF2/MER tumor suppressor, these findings raise the possibility that human KIBRA may contribute to tumor suppression in human neurofibromas and potentially other tumors (Genevet, 2010).

Riquiqui and Minibrain are regulators of the Hippo pathway downstream of Dachsous

The atypical cadherins Fat (Ft) and Dachsous (Ds) control tissue growth through the Salvador-Warts-Hippo (SWH) pathway, and also regulate planar cell polarity and morphogenesis. Ft and Ds engage in reciprocal signalling as both proteins can serve as receptor and ligand for each other. The intracellular domains (ICDs) of Ft and Ds regulate the activity of the key SWH pathway transcriptional co-activator protein Yorkie (Yki). Signalling from the FtICD is well characterized and controls tissue growth by regulating the abundance of the Yki-repressive kinase Warts (Wts). This study identified two regulators of the Drosophila melanogaster SWH pathway that function downstream of the DsICD: the WD40 repeat protein Riquiqui (Riq) and the DYRK-family kinase Minibrain (Mnb). Ds physically interacts with Riq, which binds to both Mnb and Wts. Riq and Mnb promote Yki-dependent tissue growth by stimulating phosphorylation-dependent inhibition of Wts. Thus, this study describes a previously unknown branch of the SWH pathway that controls tissue growth downstream of Ds (Degoutin, 2013).

The related cadherins Ft and Ds control tissue growth by regulating SWH pathway activity, and also control PCP and morphogenesis. Intriguingly, Ft and Ds regulate SWH pathway activity by engaging in reciprocal signalling as a ligand-receptor pair. Signalling downstream of Ft is reasonably well defined, but signalling downstream of Ds in growth control has remained uncharacterized until the discovery of a membrane-to-nucleus signalling pathway controlling SWH pathway activity downstream of the DsICD, described in this study. Genetic and biochemical data imply that the WD40 repeat protein Riq complexes with the DsICD and can bind to the Mnb and Wts kinases. Riq promotes Mnb-dependent phosphorylation and inhibition of Wts, and thereby promotes Yki-dependent tissue growth. Further investigation is required to define biologically relevant Wts residues that are phosphorylated by Mnb. This study shows that Mnb phosphorylates Wts on several residues in its amino-terminal third, although it is formally possible that other regions of Wts are also phosphorylated by Mnb (Degoutin, 2013).

Therefore, Ds-Ft ligation induces two seemingly opposing growth-regulatory events: Ds activates Ft, which represses Yki by modulating Dachs whereas Ft signals through Ds, Riq and Mnb to activate Yki. At first glance it seems counter-intuitive that Ft-Ds binding would both promote, and repress Yki-dependent tissue growth but raises several interesting possibilities. One option is that the timing of signalling from both the DsICD and the FtICD is different and varies throughout the cell cycle. For example, DsICD might deliver a pulse of Yki activity to induce transcriptional events associated with tissue growth. Subsequently, to ensure that Yki activity does not perdure and cause tissue overgrowth, it could be repressed by signalling from FtICD. Alternatively, DsICD or FtICD signalling might predominate over the other in different regions of imaginal discs or at different stages of development, to regulate Yki. Such regulation could occur through several mechanisms; 1) it could possibly stem from polarized activity of Ft and Ds that occurs in cells of growing imaginal discs in response to graded expression of Ds and Fj, 2) it could occur if the influence of signalling downstream of FtICD or DsICD on Wts activity was quantitatively different, 3) it could result from non-uniform activity of additional proteins that mediate Ft and Ds signalling. Alternatively, repression of Yki by the FtICD, and activation by the DsICD, could quantitatively oppose each other and serve to set a fine threshold of Yki activity that is highly sensitive to regulation by other branches of the SWH pathway such as the Kibra-Ex-Merlin complex, the Hpo activating kinase Tao-1 or apicobasal polarity proteins. In future studies it will be important to define the spatiotemporal activity profile of FtICD and DsICD signalling and the relative influence of the Ds and Ft branches of the SWH pathway on tissue growth (Degoutin, 2013).

Given that Ft and Ds also engage in bi-directional signalling to control PCP and morphogenesis, it will be important to determine whether Riq and Mnb control these processes downstream of the DsICD. In addition, it will be important to investigate whether the signalling events described in this study are conserved in mammals. Interestingly, a reverse regulatory event to that described in this study, between the human orthologues of Wts (LATS2) and Mnb (DYRK1A), has been reported. LATS2 was shown to phosphorylate DYRK1A and promote senescence of cultured cells, raising the possibility that Wts/LATS1/2 and Mnb/DYRK1A/1B kinases engage in mutual regulatory relationships (Degoutin, 2013).

Finally, given the emergence of the SWH pathway as an important regulator of different human tumours, the present study raises the possibility that in a pathological setting the human orthologues of Riq (DCAF7) and Mnb (DYRK1A and DYRK1B) could function as oncogenes. Cell culture studies have provided conflicting reports on whether DYRK1A and DYRK1B act as oncogenes or tumour suppressor genes However, in vivo studies in both flies and mice, and genetic studies in humans, have described only positive roles for Mnb/DYRK1A/DYRK1B in tissue growth: dyrk1a heterozygous mice exhibit growth retardation and impaired brain development, DYRK1A mutations cause microcephaly and growth retardation in humans, whereas Mnb promotes D. melanogaster tissue growth ). These in vivo studies support the possibility that DYRK1A, DYRK1B and DCAF7 could be oncogenic in human cancers (Degoutin, 2013).

Spatial Organization of Hippo Signaling at the Plasma Membrane Mediated by the Tumor Suppressor Merlin/NF2

Although Merlin/NF2 was discovered two decades ago as a tumor suppressor underlying Neurofibromatosis type II, its precise molecular mechanism remains poorly understood. Recent studies in Drosophila revealed a potential link between Merlin and the Hippo pathway by placing Merlin genetically upstream of the kinase Hpo/Mst. In contrast to the commonly depicted linear model of Merlin functioning through Hpo/Mst, this study shows that in both Drosophila and mammals, Merlin promotes downstream Hippo signaling without activating the intrinsic kinase activity of Hpo/Mst. Instead, Merlin directly binds and recruits the effector kinase Wts/Lats to the plasma membrane. Membrane recruitment, in turn, promotes Wts phosphorylation by the Hpo-Sav kinase complex. This study further shows that disruption of the actin cytoskeleton promotes Merlin-Wts interactions, which implicates Merlin in actin-mediated regulation of Hippo signaling. These findings elucidate an important molecular function of Merlin and highlight the plasma membrane as a critical subcellular compartment for Hippo signal transduction (Yin, 2013).

Since its initial discovery as a human disease gene underlying NF2, the tumor suppressor Merlin has been the subject of intense investigation. Besides the Hippo pathway, Merlin has been linked to a variety of mechanisms such as transmembrane receptor endocytosis/localization (EGFR and CD44) and signaling by Ras, Rac/PAK, and PI3K pathways. Paradoxically, as a membrane-associated tumor suppressor, Merlin was also reported to suppress tumorigenesis in mammalian cells by translocating to the nucleus to inhibit a specific E3 ubiquitin ligase. Among these proposed targets, the linkage between Merlin and Hippo signaling has attracted much attention given the similarity of the respective mutant phenotypes in Drosophila and the dosage-sensitive genetic suppression of Merlin mutant phenotypes by heterozygosity of the Hippo effector YAP in multiple mouse tissues (Yin, 2013).

Despite the genetic evidence implicating Merlin in Hippo signaling, the molecular basis of this functional link was unknown. The current study addresses this outstanding issue in two important ways. First, molecular evidence is provided showing that Merlin promotes downstream Hippo signaling without activating the intrinsic kinase activity of Hpo/Mst. These studies therefore disprove the prevailing assumption that Merlin functions biochemically upstream of Hpo activation. Along this line, it is noted that current models of Hippo signaling are actually a composite of true molecular relationships (such as Hpo acting upstream of Wts or Wts acting upstream Yki) and genetic epistasis relationships (such as Mer acting upstream of Hpo). In light of the current study, it is cautioned that biochemical and epistasis relationships should be clearly distinguished in signaling diagrams because mixing and matching them can be misleading. Second, direct physical interactions between Merlin and Wts/Lats were elucidated, and it was shown that such interactions promote Hippo signaling by recruiting Wts/Lats to the plasma membrane. The discovery of physical interactions between Merlin and a key component of the Hippo pathway therefore provides molecular support for a Merlin-Hippo connection that has so far been based largely on genetics and indirect evidence. Interestingly, interactions between Merlin and Wts are regulated by the actin cytoskeleton, underscoring Merlin as a potential mediator of actin-regulated Hippo signaling (Yin, 2013).

Besides identifying a conserved molecular function for Merlin, these studies also revealed quantitative differences between Drosophila and mammalian Merlin. WT Mer normally does not associate with Wts in Drosophila S2R+ cells, yet WT NF2 suffices to bind Lats1/2 in human cells. Such differences correlate with an intrinsically more open conformation of NF2 compared to Mer. These findings agree with previous reports that the intramolecular interaction in NF2 is relatively weak and dynamic. It is noted that the intrinsically more active/open state of NF2 is consistent with the role of S518 phosphorylation in antagonizing NF2 activity and the absence of this negative regulatory site in Drosophila Mer. Obviously, such negative regulation would be of more functional relevance in the context of an intrinsically more active Merlin protein as in mammals (Yin, 2013).

The plasma membrane is the entry point of diverse environmental stimuli and is intimately involved in spatial organization of signaling proteins. Although many reported upstream regulators of the Hippo pathway in Drosophila are transmembrane proteins (e.g., Fat and Crumbs) or are localized in apical membrane domains (e.g., Mer, Ex, and Kibra), how these membrane-associated inputs spatially organize the Hippo kinase cassette was poorly understood. This question is further complicated by the possible evolutionary divergence of upstream inputs into the pathway between Drosophila and mammals. Notably, among these upstream inputs, Merlin is the only protein whose contribution to Hippo signaling has been genetically validated in both flies and mammals (Yin, 2013).

This study demonstrates that an important and evolutionarily conserved molecular function of Merlin is to promote the membrane association of Wts/Lats. Sav is also implicated as a membrane-associated scaffold that promotes the membrane association of Hpo/Mst, the upstream kinase of Wts/Lats. Thus, two predominantly membrane-associated proteins, Merlin and Sav, are involved in targeting the two essential kinases of the Hippo kinase cassette to the plasma membrane. It is tempting to speculate that at least some of the other upstream regulators of Hippo signaling may function in a similar manner by promoting the membrane association of the Hippo kinase cassette. It is noted that a functional role for Sav in membrane association of Hpo does not preclude the other previously described roles for the Sav scaffold in Hippo signaling, such as tethering Hpo and Wts. It is possible that Sav potentiates Hippo signaling both by tethering multiple signaling components and by localizing signaling activity to specific subcellular compartments, as shown in other well-studied scaffold signaling proteins such as Ste5 and KSR. Nevertheless, this study has uncovered a role for Sav in spatial organization of the Hippo pathway (Yin, 2013).

Wts/Lats is known to be subjected to two modes of regulation, including phosphorylation and protein stability. This study extends previous studies by showing that the membrane association of Wts represents an additional mode of regulation. In addition, this study suggests that Wts may be activated by alternate upstream kinase(s) besides Hpo. Identifying the kinases that mediate Hpo-independent activation and understanding the regulation of such kinases should greatly expand knowledge about the physiological regulation of Hippo signaling. With its activity subjected to multiple modes of regulation, it is becoming increasingly clear that Wts/Lats represents as a critical node in the Hippo signaling network. These different modes of regulation are not exclusive of each other and are indeed functionally intertwined, as membrane association of Wts/Lats also enhances its phosphorylation. Understanding how the multiple regulatory inputs into Wts/Lats are coordinated will shed light on the physiological regulation of Hippo signaling in normal development and offer new strategies for therapeutic intervention in pathological conditions such as NF2 (Yin, 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).

Localization of Hippo signalling complexes and Warts activation in vivo

Hippo signalling controls organ growth and cell fate by regulating the activity of the kinase Warts. Multiple Hippo pathway components localize to apical junctions in epithelial cells, but the spatial and functional relationships among components have not been clarified, nor is it known where Warts activation occurs. This study reports that Hippo pathway components in Drosophila wing imaginal discs are organized into distinct junctional complexes, including separate distributions for Salvador, Expanded, Warts and Hippo. These complexes are reorganized on Hippo pathway activation, when Warts shifts from associating with its inhibitor Ajuba LIM protein (Jub) to its activator Expanded, and Hippo concentrates at Salvador sites. This study identify mechanisms promoting Warts relocalization, and using a phospho-specific antisera and genetic manipulations, where Warts activation occurs was identified: at apical junctions where Expanded, Salvador, Hippo and Warts overlap. These observations define spatial relationships among Hippo signalling components and establish the functional importance of their localization to Warts activation (Sun, 2015).

Wts is a key control point within the Hippo pathway, where multiple upstream regulatory processes converge. A fundamental gap in understanding of Hippo signal transduction has been the cellular location of Wts activation. This study established that Wts activation in wing disc epithelial cells occurs at sub-apical junctions where Hpo, Sav, Ex and Wts overlap. Co-recruitment of Hpo and Wts kinases to a common scaffold is implicated as a central feature of Hippo pathway activation, and this helps to explain why genes required for apical junctions and apical-basal polarity promote Hippo signalling and can act as tumour suppressors (Sun, 2015).

These studies indicate that a key step in Wts activation in disc epithelia is its relocalization from Jub to Ex. No special mechanism is needed to transport Wts from Jub to Ex, as Wts localization could simply be governed by equilibrium binding with a limited cytoplasmic pool. That is, if Wts normally binds relatively strongly to Jub, and relatively weakly to Ex, it could, depending on its concentration, accumulate at Jub sites but not at Ex sites. Expression of activated Yki induced a robust relocalization of Wts from Jub to Ex, and these studies identify three factors that contribute to the visible accumulation of Wts at Ex sites under these conditions. First, Yki activation appears to increase Hpo activity. It was also found that hpo RNAi suppresses the relocalization of Wts from Jub to Ex, and that increased Hpo activity promotes Ex-Wts binding, as assayed by co-immunoprecipitation experiments. These observations are consistent with the hypothesis that Wts shifts from Jub sites towards Ex sites due to an increased Ex-Wts binding affinity induced by Hpo. Second, Yki activation increases levels of Ex, which under equilibrium binding would also increase the recruitment of Wts to Ex sites. The relocalization of Wts back to adherens junctions in the absence of Ex indicates that the shift in Wts localization is Ex dependent, and implies that Jub and Ex can compete for association with Wts. A third factor that contributes to detection of Wts-Ex co-localization is the increase in Wts protein levels induced by activated Yki, which could lead to Wts concentrations high enough to bind even lower-affinity Ex sites, and indeed it was observed that simply overexpressing Wts was sufficient to induce Wts-Ex overlap, without removing Wts from adherens junctions where it co-localizes with Jub. It is suggested that an additional consequence of increased Wts levels that enables detection of Wts and pWts overlapping Ex could be a saturation of pWts removal. While at present this remains speculative, all signal transduction pathways require mechanisms to turn off after they have been activated, so there should exist mechanisms that either degrade or dephosphorylate pWts. Relatively low levels of pWts due to rapid turnover could also help explain why pWts was undetectable in wild-type wing discs (Sun, 2015).

The discovery of Ex-Wts binding, together with earlier studies that identified Ex-Hpo binding, implicate Ex as a scaffold that could promote Wts activation by co-localizing it with Hpo, and thus define a role for Ex distinct from previous suggestions that it functions as an activator of Hpo. Similarly, recent studies in cultured cell models showed that activated forms of Mer could bind Wts, and suggested a model in which Mer promotes Wts activation by recruiting it to membranes where it could be activated by Hpo. This suggests that in tissues where Mer, rather than Ex, plays key roles in Wts activation, such as glia, Mer, which can also associate with Hpo, through Sav, could play an analogous role in assembling a Wts activation complex. It is thus noteworthy that the best characterized upstream branches of Hippo signalling characterized in Drosophila (Fat, Ex and Mer) can all now be said to act principally at the levels of Wts regulation rather than Hpo regulation. Moreover, it is noted that Kibra, which has been suggested to act at a similar point in the Hippo pathway as Mer and Ex, has also been reported to be able to physically interact with both Hpo and Wts, and thus might also act principally as a scaffold that links them together rather than as a promoter of Hpo activation (Sun, 2015).

Indeed, external signals that impinge directly on Hpo activity have not yet been identified. The current discovery that Hpo localization to Sav is greatly increased by Yki activation reveals that regulators of Hpo localization exist, and implies that they are subject to negative feedback regulation downstream of Yki. As Hpo kinase activity can be promoted by Hpo dimerization, it is proposed that the increased recruitment of Hpo to Sav could elevate Hpo activity by increasing its local concentration, and thereby its dimerization. Relocalization of Hpo might also affect its interactions with kinases that can modulate Hpo activity. Recruitment of Hpo to Sav also concentrates Hpo near Ex, where it would more efficiently phosphorylate Ex-bound Wts. However, since most junctional Wts in disc epithelia is normally complexed with Jub rather than Ex, a mechanism-based solely on Hpo recruitment to apical junctions would not be expected to induce robust Wts activation. Importantly, then, these studies revealed that Hpo can increase Ex-Wts binding, possibly by phosphorylating Ex. Increased Ex-Wts binding would help recruit Wts to Ex, where it could then be phosphorylated by Hpo. Thus, it is now possible to suggest a sequential model for Hippo pathway activation in which Hpo is first recruited to membranes and activated, activated Hpo then phosphorylates Ex to recruit Wts and finally Hpo phosphorylates and activates Wts complexed with Ex. While further studies will be required to validate this model, it provides a framework that could guide future investigations, and these current studies clearly emphasize the importance of determining the in vivo localization of endogenous pathway components (Sun, 2015).



Northern analysis shows there are more maternally deposited 4.7 kb transcripts than 5.7 kb transcripts in young embryos. The 5.7 kb transcript, known to be zygotically expressed at the embryonic stage (4-6 hours), becomes the dominant message (Xu, 1995).

Warts and Yorkie mediate intestinal regeneration by influencing stem cell proliferation

Homeostasis in the Drosophila midgut is maintained by stem cells. The intestinal epithelium contains two types of differentiated cells that are lost and replenished: enteroendocrine (EE) cells and enterocytes (ECs). Intestinal stem cells (ISCs) are the only cells in the adult midgut that proliferate, and ISC divisions give rise to an ISC and an enteroblast (EB), which differentiates into an EC or an EE cell. If the midgut epithelium is damaged, then ISC proliferation increases. Damaged ECs express secreted ligands (Unpaired proteins) that activate Jak-Stat signaling in ISCs and EBs to promote their proliferation and differentiation]. This study shows that the Hippo pathway components Warts and Yorkie mediate a transition from low- to high-level ISC proliferation to facilitate regeneration. The Hippo pathway regulates growth in diverse organisms and has been linked to cancer. Yorkie is activated in ECs in response to tissue damage or activation of the damage-sensing Jnk pathway. Activation of Yorkie promotes expression of unpaired genes and triggers a nonautonomous increase in ISC proliferation. These observations uncover a role for Hippo pathway components in regulating stem cell proliferation and intestinal regeneration (Staley, 2010).

Hippo signaling can have both autonomous and nonautonomous effects on growth, and this study reports that in the adult Drosophila midgut, Yki has profound nonautonomous effects on growth via the Jak-Stat pathway. Jak-Stat signaling is important for proliferation control and stem cell biology, not only in the Drosophila intestine, but also in other tissues, both in Drosophila and in vertebrates. Members of the interleukin (IL) family of cytokines are homologous to Upd ligands, and a microarray study in cultured mammalian cells found that the Yki homolog Yap could regulate IL cytokines, which raises the possibility that a regulatory connection between Hippo signaling and Jak-Stat signaling might be conserved. Increased levels and nuclear localization of Yap have been reported in colon cancer patient samples, and ubiquitous Yap1 overexpression causes overproliferation of progenitor cells in the murine intestine. These observations suggest that future considerations of the potential contributions of Hippo signaling to colon cancer should include evaluations both of its possible regulation by Jnk signaling and of possible nonautonomous effects mediated by cytokines (Staley, 2010).

The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary

The Hippo pathway is a key signaling cascade in controlling organ size. The core components of this pathway are two kinases, Hippo (Hpo) and Warts (Wts), and a transcriptional coactivator Yorkie (Yki). YAP (a Yki homolog in mammals) promotes epithelial-mesenchymal transition and cell migration in vitro. This study used border cells in the Drosophila ovary as a model to study Hippo pathway functions in cell migration in vivo. During oogenesis, polar cells secrete Unpaired (Upd), which activates JAK/STAT signaling of neighboring cells and specifies them into outer border cells. The outer border cells form a cluster with polar cells and undergo migration. This study found that hpo and wts are required for migration of the border cell cluster. In outer border cells, over-expression of hpo disrupts polarization of the actin cytoskeleton and attenuates migration. In polar cells, knockdown of hpo, wts, or over-expression of yki impairs border cell induction and disrupts migration. These manipulations in polar cells reduce JAK/STAT activity in outer border cells. Expression of upd-lacZ is increased and decreased in yki and hpo mutant polar cells, respectively. Furthermore, forced-expression of upd in polar cells rescues defects of border cell induction and migration caused by wts knockdown. These results suggest that Yki negatively regulates border cell induction by inhibiting JAK/STAT signaling. Together, these data elucidate two distinct mechanisms of the Hippo pathway in controlling border cell migration: 1) in outer border cells, it regulates polarized distribution of the actin cytoskeleton; 2) in polar cells, it regulates upd expression to control border cell induction and migration (Lin, 2014).

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).

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).

Effects of Mutation or Deletion

Homozygous loss of the warts (wts) gene of Drosophila, caused by mitotic recombination in somatic cells, leads to the formation of cell clones that are fragmented, rounded, and greatly overgrown compared with normal controls. Therefore, the gene is required for the control of the amount and direction of cell proliferation as well as for normal morphogenesis. The absence of wts function also results in apical hypertrophy of imaginal disc epithelial cells. Secretion of cuticle over and between the domed apical surfaces of these cells leads to a honeycomb-like structure and gives the superficial wart-like phenotype of mitotic clones on the adult. One wts allele allows survival of homozygotes to the late larval stage, and these larvae show extensive imaginal disc overgrowth. Because of the excess growth and abnormalities of differentiation that follow homozygous loss, wts is considered to be a tumor suppressor gene (Justice, 1995).

Disrupting mechanisms that control cell proliferation, cell size and apoptosis can cause changes in animal and tissue size and contribute to diseases such as cancer. The LATS family of serine/threonine kinases control tissue size by regulating cell proliferation and function as tumor suppressor genes in both Drosophila and mammals. In order to understand the role of lats in size regulation, a genetic modifier screen was performed in Drosophila to identify components of the lats signaling pathway. Mutations in the Drosophila homolog of C-terminal Src kinase (dcsk) were identified as dominant modifiers of both lats gain-of-function and loss-of-function phenotypes. Homozygous dcsk mutants have enlarged tissue phenotypes similar to lats and FACS. An immunohistochemistry analysis of these tissues revealed that dcsk also regulates cell proliferation during development. Animals having mutations in both dcsk and lats display cell overproliferation phenotypes more severe than either mutant alone, demonstrating these genes function together in vivo to regulate cell numbers. Furthermore, homozygous dcsk phenotypes can be partially suppressed by overexpression of lats, indicating that lats is a downstream mediator of dcsk function in vivo. It was shown that dCSK phosphorylates LATS in vitro at a conserved C-terminal tyrosine residue, which is critical for normal LATS function in vivo. Taken together, these results demonstrate a role for dCSK in regulating cell numbers during development by inhibiting cell proliferation and suggest that lats is one of the mediators of the dcsk phenotype (Stewart, 2003).

The growth regulators warts/lats and melted interact in a bistable loop to specify opposite fates in Drosophila R8 photoreceptors

Color vision in Drosophila relies on the comparison between two color-sensitive photoreceptors, R7 and R8. Two types of ommatidia in which R7 and R8 contain different rhodopsins are distributed stochastically in the retina and appear to discriminate short (p-subset) or long wavelengths (y-subset). The choice between p and y fates is made in R7, which then instructs R8 to follow the corresponding fate, thus leading to a tight coupling between rhodopsins expressed in R7 and R8. warts, encoding large tumor suppressor (Lats) and melted, encoding a PH-domain protein, play opposite roles in defining the yR8 or pR8 fates. By interacting antagonistically at the transcriptional level, they form a bistable loop that insures a robust commitment of R8 to a single fate, without allowing ambiguity. This represents an unexpected postmitotic role for genes controlling cell proliferation (warts and its partner hippo and salvador) and cell growth (melted) (Mikeladze-Dvali, 2005b).

The fly eye provides a powerful system to study cell-fate decisions: it develops from a flat epithelium into a complex three-dimensional structure of multiple cell types in less than a week. The adult eye allows the fly to perform various visual tasks, ranging from motion detection and the discrimination of colors to measuring the orientation of polarized light for navigation (Mikeladze-Dvali, 2005b).

In the fly compound eye, each of the 800 ommatidia is a single optical unit that contains 8 photoreceptor cells (PRs). The 8 PRs form widely expanded membrane structures, the rhabdomeres, which contain the photosensitive Rhodopsins (Rh). The rhabdomeres of the six outer PRs (R1-R6) form a trapezoid. R1-R6 all express the broad spectrum rhodopsin1 (rh1 or ninaE) and are morphologically and functionally invariant in all ~800 ommatidia (Mikeladze-Dvali, 2005b).

The center of the trapezoid is occupied by the two inner PRs, R7 and R8. The rhabdomeres of R7 are positioned on top of R8, so that they share the same optic path. Inner PRs are involved in color vision and can be viewed as equivalent to vertebrate cones. Each R7 and R8 expresses only one of the four rhodopsins, rh3, rh4, rh5, or rh6 in a highly regulated manner, defining three different subtypes of ommatidia: 'yellow' (y), 'pale' (p) (for their appearance under UV illumination), and the 'dorsal rim area' (DRA). Ommatidia in the DRA express rh3 in both R7 and R8 and are specified in a very restricted region by the gene homothorax. They are believed to function as polarized light detectors (Mikeladze-Dvali, 2005b).

In contrast, color vision depends on the y and p ommatidial subtypes that are randomly distributed through the main part of the retina, with a bias of y (~70%) over p subtype (~30%). In the p subtype, R7 expresses the UV-sensitive Rh3 and R8 the blue-sensitive Rh5. In the y subtype, R7 expresses a distinct UV-sensitive Rh4 while R8 expresses the green-sensitive Rh6. As in many other sensory systems, expression of a given Rhodopsin excludes all others to prevent sensory overlap. While the p subtype is better suited to discriminate among shorter wavelengths, the y subtype should discriminate amongst longer wavelengths (Mikeladze-Dvali, 2005b).

The choice between the p and y fate is first made in R7: once an R7 commits to the p fate and expresses rh3, it sends an instructive signal to the underlying R8, which then also commits to the p fate and expresses rh5. In the absence of the R7 signal (i.e., when R7 expresses rh4 or in a sevenless mutant), R8 commits to the y fate and expresses rh6. The stochastic choice appears to be made by each R7 independently of its neighbors, resulting in the biased random distribution of p and y ommatidia throughout the main part of the retina (for review see Mikeladze-Dvali, 2005a).

Four genes required in R8 cells for ensuring the correct choice of y versus p cell fate have been identified. The warts (wts) gene, which encodes the Drosophila large tumor suppressor (also known as lats) and melted (melt) play a critical role in the specification of p and y R8 cells, without affecting the R7 choice. wts encodes a Ser/Thr kinase, while melt encodes a Pleckstrin Homology (PH) domain protein. wts is necessary and sufficient for R8 to adopt the y fate, while melt plays the opposite role and specifically induces the p fate in R8. wts and melt are expressed in a complementary manner in the yR8 and pR8 subsets, respectively. Evidence is presented that the two genes repress each other's transcription to form a bistable loop. melt seems to respond to the R7 signal, while wts appears to regulate the output of the loop. The tumor-suppressor genes hippo (hpo) and salvador (sav), which encode the two molecular partners of Wts/Lats, have phenotypes identical to wts. Interestingly, melt has been reported to regulate growth and fat metabolism in Drosophila. Thus, genes known to regulate both cell growth (melt) and proliferation (wts, sav, hpo) interact antagonistically during retinal patterning (Mikeladze-Dvali, 2005b).

To identify genes involved in the differentiation of p or y PR subsets, a Gal4 (pGawB) enhancer trap screen was performed in adult flies using GFP expression as a reporter. One insertion produced a strong GFP signal in inner PRs. Staining of sectioned adult eyes for the UAS-lacZ reporter gene revealed Gal4 expression in a large subset of R8 cells. Additional expression was found in DRA R7 and R8, as well as in outer PRs in the ventral half of the eye. Occasionally, weak expression was also found in some R7 cells, but not in any PR subset-specific pattern. Staining of the same enhancer trap (driving UAS-lacZnuc expression) with antibodies against β-Gal, Rh6 (α-Rh6), and Rh5 (α-Rh5) in whole-mounted retinas revealed that the reporter was specific to Rh6-positive R8 and was excluded from the Rh5-positive R8, indicating that the targeted gene is expressed in the yR8 subtype (Mikeladze-Dvali, 2005b).

The genomic DNA flanking the pGawB transposon, which is inserted upstream of the third exon of the gene warts (wts), was identified. An existing wts nuclear lacZ enhancer trap line P[lacZ,w+] was stained. lacZ expression in this line (wtsZn) was also specific to the y subset of R8 cells as well as the DRA and some ventral outer PRs, confirming the restricted expression pattern of wts (Mikeladze-Dvali, 2005b).

wts-Gal4 appears to be activated by a late eye-specific enhancer of wts, which first directs expression long after R8 has exited the cell cycle. wts therefore appears to play two distinct roles: a ubiquitous role in proliferating cells and a more restricted role in terminally differentiated PR (Mikeladze-Dvali, 2005b).

Flies with wts-Gal4 insertion were homozygous viable and did not exhibit any visible growth phenotype. However, it was noticed that heterozygous wts-Gal4 flies always exhibited a strong rh phenotype when present in combination with one specific UAS-lacZ reporter construct (P w[+mC] = UAS-lacZ.B Bg4-2-4b, FlyBase #1777). The y/p R8 ratio was dramatically affected: most R8 expressed rh5, while rh6 expression was almost completely lost, with wts-Gal4 expression reduced to the remaining rh6 expressing R8. However, specification of R7 and of outer PRs was unaffected. This phenotype was only observed with this specific UAS-lacZ transgene, and not with UAS-GFP or other UAS-lacZ transgenes. When homozygous (in the absence of wts-Gal4), this UAS-lacZ line manifested an even more severe R8 opsin phenotype: about 90% of R8 expressed rh5 at the expense of rh6. This suggested that this particular insertion disrupted a gene affecting the p/y choice in R8 (Mikeladze-Dvali, 2005b).

This UAS-lacZ P element was found to be inserted 21 bp upstream of the transcriptional start site of the gene melted (melt). The Melt protein has a C-terminal PH domain and is conserved from C. elegans to humans. Insertions in melt were initially identified in a screen for genes affecting peripheral nervous system development. Thus the role of melt in R8 subtype specification and its interaction with wts was examined (Mikeladze-Dvali, 2005b).

Since R7 and R8 in a given ommatidium share the same optic path, their fates must be tightly regulated. The decision of a given ommatidium to become y or p is initially made by R7. Once R7 has chosen its fate, it imposes it onto the underlying R8. To coordinate opsin expression between R7 and R8, R8 has to respond to the R7 signal with high fidelity (Mikeladze-Dvali, 2005b).

This study shows that wts and melt act in R8 to prevent an ambiguous response to the instructive R7 signal. wts and melt play opposite roles in the specification of R8 subtypes. In the absence of wts, the yR8 subtype is completely misspecified into pR8. By contrast, in melt mutants, the pR8 subtype is lost with expansion of yR8. Overexpression of wts or melt leads to the transformation of all R8 into the y or p fate, respectively. The complementary expression patterns of the two genes in y or p R8 subtypes are set up in response to the pR7 signal. Therefore, wts and melt appear to interpret the signal from R7, and mutations in wts and melt render R8 insensitive to this signal without influencing R7 or outer PR (Mikeladze-Dvali, 2005b).

The decision to express wts or melt in R8 is determined by R7, but the two genes repress each other's transcription. Thus, wts and melt act in a loop of negative crossregulation. However, if R7 imposes its fate upon R8, what then is the role of this crossregulation? It is suggested that the bistable loop allows only an unambiguous readout while R7 provides an asymmetric bias of this choice (Mikeladze-Dvali, 2005b).

In a negative bistable crossregulatory loop, the input signal biasing cell-fate choice might act at any level. Similarly, any member of the loop can serve as the output. For instance, wts could positively regulate rh6 expression (yR8 fate), while melt could activate rh5 (pR8 fate). Double misexpression and double loss-of-function experiments suggest that wts is the output regulator of the loop. When both wts and melt are ectopically expressed, all R8 acquire the y fate, i.e., the fate imposed by wts. In melt, wts double mutants, all R8 acquire the p fate. These phenotypes resemble the single gain- or loss-of-function phenotypes of wts, which appears to be necessary and sufficient for rh6 expression. In contrast, while melt is sufficient to induce rh5 in yR8, rh5 remains expressed in the absence of melt in the double mutant. This argues that melt is not necessary for the pR8 fate (rh5). In melt, wts double-mutant eyes, rh5 does not depend on instruction from pR7, which confirms that rh5 expression is a consequence of the absence of wts (a derepression rather than activation by the pR7 signal) (Mikeladze-Dvali, 2005b).

The following model is proposed: in the absence of an instructive pR7 signal, i.e., in y ommatidia, the loop is biased in favor of wts expression, which represses melt. In p ommatidia, the R7 signal either induces melt expression in R8 or represses expression of wts in R8. In either case, the balance of the loop is shifted, leading to upregulation of melt and complete suppression of wts expression. This system is able to amplify a weak or transient signal to ensure that the cell-fate decision is made unambiguously (Mikeladze-Dvali, 2005b).

There are clearly a number of examples of bistable loop that often reinforce stochastic decisions or transient differentiation stimuli. Bistable systems require positive feedback loops as proposed for the BMP signaling during dorso-ventral patterning in Drosophila or double-negative feedback loops as in the case of the wts-melt loop. The left-right choice by chemosensory ASE neurons in C. elegans is a similar example where a negative bistable loop is involved in making an unambiguous cell-fate decision. This loop includes two transcription factors and two microRNAs. In the left ASE, this loop is strongly biased toward Na+-sensitive fate and in the right ASE, toward Cl sensitivity (Johnston, 2005). This strong bias is likely imposed by a factor outside of the loop. In R8 cells, the wts-melt loop is inherently biased toward y fate. The signal from R7 in p ommatidia biases the choice toward the pR8 fate. The transcription loop described in this study is clearly incomplete since neither Wts nor Melt is a transcription factor. A mutation, daltonien (don), has been identified which genetically interacts with melt, activates the expression of melt (unpublished data cited in Mikeladze-Dvali, 2005b), and appears to encode a component of this loop. Another potential member of the loop is the newly identified transcriptional coactivator Yorkie (Yki), a direct target of the Wts kinase (Mikeladze-Dvali, 2005b).

The bistable loop is specific to those R8 that are involved in color vision: in DRA ommatidia, melt misexpression does not lead to wts downregulation. This is not surprising since R7 and R8 in DRA are specified independently by positional information and do not appear to communicate (Mikeladze-Dvali, 2005b).

The transcriptional regulation of wts and melt expression is surprising, since kinases and PH domain proteins are usually regulated by changes in their activity or subcellular localization. For instance, Wts/Lats kinase activity is regulated through phosphorylation by Hpo in the presence of Sav. However, the nature of the signal that triggers activation of the Wts/Hpo/Sav proliferation control pathway has remained elusive. Thus, identification of the signal from pR7 to R8 could provide important insights into the mechanism by which this tumor-suppressor complex is regulated to control proliferation and cell death (Mikeladze-Dvali, 2005b).

The ability of wts to indirectly regulate transcription of other genes (here melt) is less surprising. wts, sav, and hpo have been reported to negatively regulate the transcription of Cyclin E and DIAP1, leading to a decrease in cell cycle progression and to an increase in cell death. The same (unknown) transcription factor required downstream of wts could therefore also play a role in repressing melt and rh5, and possibly in activating rh6 (Mikeladze-Dvali, 2005b).

Cbk1, the Lats/Wts homolog in S. cerevisiae has been shown to regulate a broad range of daughter specific genes during budding. The asymmetric gene expression between mother and daughter cells is due to Cbk1-dependent activation and nuclear localization of the transcription factor Ace2 in daughter cells. Cbk1 kinase activity requires another gene, Mob2. Recently, a member of the Mob family in Drosophila, Mats, has been shown to bind and synergistically interact with Wts/Lats to control proliferation and apoptosis. Although Melt is not known to regulate the transcription of other target genes, it can affect subcellular localization of FOXO and the TSC1/TSC2 complex to regulate fat metabolism. However, the members of the TOR or InR do not seem to play a role in the specification of R8 subtypes (Mikeladze-Dvali, 2005b).

Wts, together with the Ser/Thr kinase Hpo and the adaptor protein Sav, acts as a potent tumor suppressor. All three genes play a critical role for the establishment of the R8 subtypes. The function described in this study for hpo/sav/wts represents an unexpected new role unrelated to their tumor-suppression function: R8 PRs have exited the cell cycle for at least 4 days when they choose to express a particular rhodopsin, and these cells are not prone to die (PRs are particularly difficult to kill through induction of the cell death pathway). Furthermore, there is no detectable difference in cell size or shape between y and p R8, which specifically express or exclude wts or melt expression. However, it is interesting to note that p and y inner photoreceptors are morphologically distinguishable in Calliphora blowflies. Perhaps Wts and Melt represent an evolutionary remnant of a system in large flies where subtypes required different morphologies. Therefore, specification of the correct R8 fate utilizes two signaling cassettes used for different purposes earlier in development, after these cassettes are no longer in use in these highly differentiated PR cells (Mikeladze-Dvali, 2005b).

Lats1, the human ortholog of Wts, is able to rescue the lethality of wts in flies. Canine Lats1 splice variant is specifically expressed in the retina. Moreover, a gene responsible for an autosomal dominant cone dystrophy (involving impaired color vision, sensitivity to light, and gradual loss of visual activity) has been mapped close to the Lats1 locus. Thus, it might be expected that the hpo/sav/wts pathway functions in the human retina as well. Although, melt knockout mice are viable and fertile, it will be interesting to test whether they are defective in cone differentiation or vision (Mikeladze-Dvali, 2005b).

Polycomb genes interact with the tumor suppressor genes hippo and warts in the maintenance of Drosophila sensory neuron dendrites

Dendritic fields are important determinants of neuronal function. However, how neurons establish and then maintain their dendritic fields is not well understood. Polycomb group (PcG) genes are required for maintenance of complete and nonoverlapping dendritic coverage of the larval body wall by Drosophila class IV dendrite arborization (da) neurons. In esc, Su(z)12, or Pc mutants, dendritic fields are established normally, but class IV neurons display a gradual loss of dendritic coverage, while axons remain normal in appearance, demonstrating that PcG genes are specifically required for dendrite maintenance. Both multiprotein Polycomb repressor complexes (PRCs) involved in transcriptional silencing are implicated in regulation of dendrite arborization in class IV da neurons, likely through regulation of homeobox (Hox) transcription factors. Genetic interactions and association between PcG proteins and the tumor suppressor kinase Warts (Wts) is demonstrated, providing evidence for their cooperation in multiple developmental processes including dendrite maintenance (Parrish, 2007).

Dendrite arborization patterns are a hallmark of neuronal type; yet how dendritic arbors are maintained after they initially cover their receptive field is an important question that has received relatively little attention. The Drosophila PNS contains different classes of sensory neurons, each of which has a characteristic dendrite arborization pattern, providing a system for analysis of signals required to achieve specific dendrite arborization patterns. Class IV neurons are notable among sensory neurons because they are the only neurons whose dendrites provide a complete, nonredundant coverage of the body wall. This study found tha the function of Polycomb group genes is required specifically in class IV da neurons to regulate dendrite development. In the absence of PcG gene function, class IV dendrites initially cover the proper receptive field but subsequently fail to maintain their coverage of the field. Time-lapse analysis of dendrite development in esc or Pc mutants suggests that a combination of reduced terminal dendrite growth and increased dendrite retraction likely accounts for the gradual loss of dendritic coverage in these mutants. Maintenance of axonal terminals in class IV da neurons is apparently unaffected by loss of PcG gene function, suggesting that PcG genes function as part of a program that specifically regulates dendrite stability (Parrish, 2007).

Establishment of dendritic territories in class IV neurons is regulated by homotypic repulsion, and this process proceeds normally in the absence of PcG function. In PcG mutants, class IV neurons tile the body wall by 48 h AEL, similar to wild-type controls. However, beginning at 48 h AEL, likely as a result of reduced dendritic growth and increased terminal dendrite retraction, class IV neurons of PcG mutants gradually lose their dendritic coverage. In contrast, the axon projections and terminal axonal arbors of PcG mutants show no obvious defects. Although an early role for PcG genes in regulating axon development cannot be ruled out, MARCM studies showed that PcG genes are required for the maintenance of dendrites but not axons in late larval development. Thus, different genetic programs appear to be responsible for the establishment and maintenance of dendritic fields, and for the maintenance of axons and dendrites (Parrish, 2007).

It is well established that PcG genes participate in regulating several important developmental processes including expression of Hox genes for the specification of segmental identity. In comparison, much less is known about the function of PcG genes in neuronal development. Studies of the expression patterns of PcG genes and the consequences of overexpression of PcG genes suggest that PcG genes may affect the patterning of the vertebrate CNS along the anterior-posterior (AP) axis, analogous to their functions in specifying the body plan. A recent study demonstrates that the PcG gene Polyhomeotic regulates aspects of neuronal diversity in the Drosophila CNS. The current study now links the function of PcG genes to maintenance of dendritic coverage of class IV sensory neurons. Thus it will be interesting to determine whether PcG genes play a conserved role in the regulation of dendrite maintenance (Parrish, 2007).

Since Hox genes function in late aspects of neuronal specification and axon morphogenesis, it seems possible that regulation of Hox genes by PcG genes may be important for aspects of post-mitotic neuronal morphogenesis, including dendrite development. The PcG genes esc and E(z) are required for proper down-regulation of BX-C Hox gene expression in class IV neurons. The timing of this change in BX-C expression corresponds to the time frame during which PcG genes are required for dendritic maintenance. Furthermore, post-mitotic overexpression of BX-C genes in class IV da neurons, but not other classes of da neurons, is sufficient to cause defects in dendrite arborization, thus phenocopying the mutant effects of PcG genes. Finally, it was found that Hox genes are required cell-autonomously for dendrite development in class IV neurons, and loss of Hox gene function causes defects in terminal dendrite dynamics that are opposite to the defects caused by loss of PcG genes. Therefore, it seems likely that PcG genes regulate dendrite maintenance in part by temporally regulating BX-C Hox gene expression (Parrish, 2007).

Several recent studies have focused on the identification of direct targets of PcG-mediated silencing, demonstrating that PcG genes regulate expression of distinct classes of genes in different cellular contexts. During Drosophila development, PRC proteins likely associate with >100 distinct loci, and the chromosome-associate profile of PRC proteins appears dynamic. Therefore, identifying the targets of PcG-mediated silencing in a given developmental process has proven difficult. Thus far, alleles of >20 predicted targets of PcG-mediated silencing have been analyzed for roles in establishment or maintenance of dendritic tiling and a potential role has been found for only Hox genes. Future studies will be required to identify additional targets of PcG-mediated silencing in regulation of dendrite maintenance (Parrish, 2007).

PcG genes are broadly expressed, so it seems likely that interactions with other factors or post-translational mechanisms may be responsible for the cell type-specific activity of PcG genes. Indeed, PcG genes genetically interact with components of the Wts signaling pathway to regulate dendrite development specifically in class IV neurons. Based on the observations that wts mutants also show derepression of Ubx in class IV neurons and that Wts can physically associate with PcG components, it seems likely that Wts may directly or indirectly influence the activity of PcG components. In proliferating cells, Wts phosphorylates the transcriptional coactivator Yorkie to regulate cell cycle progression and apoptosis, demonstrating that Wts can directly influence the activity of transcription factors. In support of a possible role for Wts directly modulating PcG function, several recent reports have documented roles for phosphorylation in regulating PcG function both in Drosophila and in vertebrates. Thus, it is possible that some of the components involved in PcG-mediated silencing are regulated by Wts phosphorylation. Alternatively, association of Wts with PcG proteins may facilitate Wts-mediated phosphorylation of chromatin substrates (Parrish, 2007).

The tumor suppressor kinase Hpo regulates both establishment and maintenance of dendritic tiling in class IV neurons through its interactions with Trc and Wts, respectively, but how Hpo coordinately regulates these downstream signaling pathways is currently unknown. Similar to mutations in wts, mutations in PcG genes interact with mutations in hpo to regulate dendrite maintenance but show no obvious interaction with trc, consistent with the observation that PcG gene function is dispensable for establishment of dendritic tiling. Although it is possible that different upstream signals control Hpo-mediated regulation of establishment and maintenance of dendritic tiling, the nature of such signals remain to be determined. Another possibility is that the activity of the Wts/PcG pathway could be antagonized by additional unknown factors that promote establishment of dendritic tiling (Parrish, 2007).

In addition to their interaction in regulating dendrite maintenance, PcG genes and wts interact to regulate expression of the Hox gene Scr during leg development. This finding suggests that the Hpo/Wts pathway may play a general role in contributing to PcG-mediated regulation of Hox gene expression. The presence of ectopic sex combs provides a very simple and sensitive readout of wts/PcG gene interactions and should form the basis for conducting large-scale genetic screens to identify other genes that interact with wts or PcG genes and participate in this genetic pathway (Parrish, 2007).

Delineation of a Fat tumor suppressor pathway

Recent studies in Drosophila of the protocadherins Dachsous and Fat suggest that they act as ligand and receptor, respectively, for an intercellular signaling pathway that influences tissue polarity, growth and gene expression, but the basis for signaling downstream of Fat has remained unclear. This study characterizes functional relationships among Drosophila tumor suppressors and identifies the kinases Discs overgrown and Warts as components of a Fat signaling pathway. fat, discs overgrown and warts regulate a common set of downstream genes in multiple tissues. Genetic experiments position the action of discs overgrown (dco) upstream of the Fat pathway component dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs and discs overgrown in vivo, consistent with its placement as a downstream component of the pathway. The tumor suppressors Merlin, expanded (ex), hippo, salvador (sav) and mob as tumor suppressor (mats) also share multiple Fat pathway phenotypes but regulate Warts activity independently. These results functionally link what had been four disparate groups of Drosophila tumor suppressors, establish a basic framework for Fat signaling from receptor to transcription factor and implicate Warts as an integrator of multiple growth control signals (Cho, 2006).

Since Dachs is required for loss of Wts protein in fat mutants, and Dachs encodes a large Myosin protein, a model was considered in which Dachs acts as a scaffold to link Wts to proteins that promote Wts proteolysis, analogous to the roles of Costal2 in Hedgehog signaling, or APC in Wnt signaling. This model predicts that Dachs should be able to bind to Wts. To evaluate this possibility, tagged forms of Dachs and Wts were coexpressed in cultured cells and assayed for coimmunoprecipitation. These experiments identified a specific and reproducible interaction between Dachs and Wts (Cho, 2006).

Recent studies have identified the transcriptional coactivator Yorkie (Yki) as a downstream component of the Hippo pathway and a substrate of Wts kinase activity. Phosphorylation of Yki by Wts inactivates Yki, and overexpression of Yki phenocopies wts mutation. The determination that the Fat tumor suppressor pathway acts through modulation of Wts thus predicts that Yki should also be involved in Fat signaling. When the influence of Yki overexpression was examined on Fat target genes, expression of Wg in the proximal wing, Ser in the proximal leg and fj in the wing and eye were each upregulated by Yki overexpression, consistent with the inference that Fat tumor suppressor pathway signaling acts through Yki (Cho, 2006).

In order to identify additional components of the Fat tumor suppressor pathway, advantage was taken of the observation that loss of fat in clones of cells is associated with an induction of Wingless (Wg) expression in cells just proximal to the normal ring of Wg expression in the proximal wing, reflective of its role in distal-to-proximal wing signaling. It was reasoned that this influence on Wg expression could be used to screen other Drosophila tumor suppressors for their potential to contribute to Fat signaling. Analysis of mutant clones in the proximal wing identified dco, ex, mats, sav, hpo and wts as candidate components of the Fat tumor suppressor pathway. As for fat, mutation of each of these genes is associated with induction of Wg expression specifically in the proximal wing, whereas Wg expression is not affected in more distal or more proximal wing cells. Although Wg expression often seems slightly elevated within its normal domain, the effect of these mutations is most obvious in the broadening of the Wg expression ring. The induction of Wg expression does not seem to be a nonspecific consequence of the altered growth or cell affinity associated with these mutations, since Wg expression is unaffected by expression of the growth-promoting microRNA gene bantam or by expression of genes that alter cell affinity in the proximal wing (Cho, 2006).

dco encodes D. melanogaster casein kinase I delta/epsilon. The overgrowth phenotype that gave the gene its name is observed in allelic combinations that include a hypomorphic allele, dco3, and it is this allele that is associated with induction of Wg. Null mutations of dco actually result in an 'opposite' phenotype: discs fail to grow, and clones of cells mutant for null alleles fail to proliferate. This is likely to reflect requirements for dco in multiple, distinct processes, as casein kinase I proteins phosphorylate many different substrates, and dco has been implicated in circadian rhythms, Wnt signaling and Hedgehog signaling (Cho, 2006).

Mer and ex encode two structurally related FERM domain-containing proteins. ex was first identified as a Drosophila tumor suppressor, whereas Drosophila Mer was first identified based on its structural similarity to human Merlin. Mutation of Mer alone causes only mild effects on imaginal disc growth, but Mer and ex are partially redundant, and double mutants show more severe overgrowth phenotypes than either single mutant. Consistent with this, elevation of Wg expression was observed in ex mutant clones (7/10 proximal wing clones induced Wg) and not in Mer mutant clones (0/8 clones), whereas Mer ex double mutant clones showed even more severe effects on Wg than ex single mutant clones. Because of the partial redundancy between Mer and ex, when possible, focus was placed for subsequent analysis on Mer ex double mutant clones (Cho, 2006).

Wts, Mats, Sav and Hpo interact biochemically, show similar overgrowth phenotypes and regulate common target genes. Mats, Sav and Hpo are all thought to act by regulating the phosphorylation state and thereby the activity of Wts. Mutation of any one of these genes is associated with upregulation of Wg in the proximal wing. The effects of sav (47/84 clones in the proximal wing induced Wg) and hpo (23/31 clones) were weaker than those of mats (19/19 clones) and wts (92/97 clones), but this might result from differences in perdurance or allele strength. Because sav, hpo and mats all act through Wts, focus for most of the subsequent analysis was placed on wts (Cho, 2006).

The observation that mutation of dco, Mer, ex, mats, sav, hpo or wts all share the distinctive upregulation of Wg expression in the proximal wing observed in fat mutants suggests that the functions of these genes are closely linked. To further investigate this, the effects of these tumor suppressors were characterized on other transcriptional targets of Fat signaling. Expression of the Notch ligand Ser is upregulated unevenly within fat mutant cells in the proximal region of the leg disc. A very similar upregulation occurred in dco3, Mer ex, and wts mutant clones. fj is a target of Fat signaling in both wing and eye imaginal discs, and fj expression was also upregulated in dco3, Mer ex, or wts mutant clones. The observation that these genes share multiple transcriptional targets in different Drosophila tissues implies that they act together in a common process (Cho, 2006).

The hypothesis that Fat pathway genes and Hippo pathway genes are linked predicts that not only should Fat target genes be regulated by Hippo pathway genes, but Hippo pathway target genes should also be regulated by Fat pathway genes. The cell cycle regulator CycE and the inhibitor of apoptosis Diap1 (encoded by thread) have been widely used as diagnostic downstream targets to assign genes to the Hippo pathway. Notably, then, clones of cells mutant for fat showed upregulation of both Diap1 and CycE protein expression. Genes whose expression is upregulated within fat mutant cells (such as wg, Ser and fj) have been shown previously to be induced along the borders of cells expressing either fj or dachsous (ds), and Diap1 is also upregulated around the borders of ds- or fj-expressing clones. That thread is affected by fat at a transcriptional level was confirmed by examining a thread-lacZ enhancer trap line. The regulation of Diap1 by the Hippo pathway is thought to be responsible for a characteristic eye phenotype in which an excess of interommatidial cells results from their failure to undergo apoptosis; an increase was also observed in interommatidial cells in fat mutant clones. Upregulation of both Diap1 and CycE is also observed in Mer ex double mutant clones. In dco3 mutant clones, consistent upregulation was detected only for Diap1, and CycE was upregulated only weakly and inconsistently. dco3 also has weaker effects on Wg and fj expression; the weaker effects of dco3 could result from its hypomorphic nature. ex has recently been characterized as another Hippo pathway target, and an ex-lacZ enhancer trap that is upregulated in wts or Mer ex mutant clones is also upregulated in fat or dco3 mutant clones. Analysis of ex transcription by in situ hybridization also indicated that ex is regulated by fat. Altogether, this analysis of Hippo pathway targets further supports the conclusion that the functions of the Fat pathway, the Hippo pathway and the tumor suppressors Mer, ex and dco are linked (Cho, 2006).

Genetic epistasis experiments provide a critical framework for evaluating the functional relationships among genes that act in a common pathway. The relationships was evaluated between each of the tumor suppressors linked to the Fat pathway and dachs, using both wing disc growth and proximal Wg expression as phenotypic assays. dachs is the only previously identified downstream component of the Fat tumor suppressor pathway. It acts oppositely to fat and is epistatic to fat in terms of both growth and gene expression phenotypes (Cho, 2006).

dachs is also epistatic to dco3 for overall wing disc growth and for proximal Wg expression. The epistasis of dachs to dco3 implies that the overgrowth phenotype of dco3 is specifically related to its influence on Fat signaling, as opposed to participation of dco in other pathways. By contrast to the epistasis of dachs to dco3, both wts and ex are epistatic to dachs for disc overgrowth phenotypes, and wts and Mer ex are epistatic to dachs in their influence on proximal Wg expression. Together, these epistasis experiments suggest that dco acts upstream of dachs, whereas Mer ex and wts act downstream of dachs (Cho, 2006).

Because wts and Mer ex have similar phenotypes, their epistatic relationship cannot be determined using loss-of-function alleles. However, overexpression of ex inhibits growth and promotes apoptosis, which suggests that ex overexpression affects ex gain-of-function. Clones of cells overexpressing ex are normally composed of only a few cells, and over time most are lost, but coexpression with the baculovirus apoptosis inhibitor p35 enabled recovery of ex-expressing clones. These ex- and p35-expressing clones were associated with repression of proximal Wg expression during early- to mid-third instar, as has been described for dachs2, consistent with ex overexpression acting as a gain-of-function allele in terms of its influence on Fat signaling. In epistasis experiments using overexpressed ex and mutation of wts, wts was epistatic; Wg was induced in the proximal wing. Additionally, when wts is mutant, coexpression with p35 was no longer needed to ensure the viability and growth of ex-expressing clones, indicating that wts is also epistatic to ex for growth and survival. Consistent with this conclusion, others have recently described phenotypic similarities between Mer ex and hpo pathway mutants and have reported that hpo is epistatic to Mer ex (Cho, 2006).

When Fat was overexpressed, a slight reduction was detected in Wg expression during early- to mid-third instar, suggesting that overexpression can result in a weak gain-of-function phenotype. Clones of cells overexpressing Fat but mutant for dco3 still showed reduced Wg levels, whereas clones of cells overexpressing Fat but mutant for warts showed increased Wg levels. Although experiments in which the epistatic mutation is not a null allele cannot be regarded as definitive, these results are consistent with the conclusion that wts acts downstream of fat and suggest that dco might act upstream of fat (Cho, 2006).

The epistasis results described above suggest an order of action for Fat tumor suppressor pathway genes in which dco acts upstream of fat, fat acts upstream of dachs, dachs acts upstream of Mer and ex, and Mer and ex act upstream of wts. However, the determination that one gene is epistatic to another does not prove that the epistatic gene is biochemically downstream, as it is also possible that they act in parallel but converge upon a common target. Thus, to better define the functional and hierarchical relationships among these genes, experiments were initiated to investigate the possibility that genetically upstream components influence the phosphorylation, stability or localization of genetically downstream (that is, epistatic) components. Focus in this study was placed on the most downstream of these components, Wts. As available antibodies did not specifically recognize Wts in imaginal discs, advantage was taken of the existence of functional, Myc-tagged Wts-expressing transgenes (Myc:Wts) to investigate potential influences of upstream Fat pathway genes on Wts protein. In wing imaginal discs, Myc:Wts staining outlines cells, suggesting that it is preferentially localized near the plasma membrane, and it was confirmed that expression of Myc:Wts under tub-Gal4 control can rescue wts mutation. Notably, mutation of fat results in a reduction of Myc:Wts staining. As Myc:Wts is expressed under the control of a heterologous promoter in these experiments, this must reflect a post-transcriptional influence on Wts protein. fat does not exert a general influence on the levels of Hippo pathway components; fat mutant clones had no detectable influence on the expression of hemagglutinin epitope-tagged Sav (HA:Sav) (Cho, 2006).

The decrease in Wts protein associated with mutation of fat contrasts with studies of the regulation of Wts activity by the Hippo pathway, which have identified changes in Wts activity due to changes in its phosphorylation state. To directly compare regulation of Wts by Fat with regulation of Wts by other upstream genes, Myc:Wts staining was examined in ex, sav and mats mutant clones. In each of these experiments, the levels and localization of Myc:Wts in mutant cells was indistinguishable from that in neighboring wild-type cells (Cho, 2006).

Since Myc:Wts appears preferentially localized near the plasma membrane, it was conceivable that the apparent decrease in staining reflected delocalization of Wts, rather than destabilization. To investigate this possibility, Wts levels were examined by protein blotting. Antisera against endogenous Wts recognized a band of the expected mobility in lysates of wing imaginal discs or cultured cells, and this band was enhanced when Wts was overexpressed. The intensity of this band was reproducibly diminished in fat or dco3 homozygous mutant animals but was not diminished in fat or dco3 heterozygotes or in ex mutants. Conversely, levels of Hpo, Sav, Mer or Mats were not noticeably affected by fat mutation (Cho, 2006).

The determination that Wts is affected by Fat, together with the genetic studies described above, place Wts within the Fat signaling pathway, as opposed to a parallel pathway that converges on common transcriptional targets. Indeed, given that even hypomorphic alleles of wts result in disc overgrowth, the evident reduction in Wts levels might suffice to explain the overgrowth of fat mutants. As a further test of this possibility, Wts levels were examined in fat dachs double mutants. As the influence of Fat on gene expression and growth is absolutely dependent upon Dachs, if Fat influences growth through modulation of Wts, its influence on Wts levels should be reversed by mutation of dachs. Examination of Myc:Wts staining in fat dachs clones and of Wts protein levels in fat dachs mutant discs confirmed this prediction (Cho, 2006).

Prior observations, including the influences of fat and ds on gene expression, and the ability of the Fat intracellular domain to rescue fat phenotypes, suggested that Fat functions as a signal-transducing receptor. By identifying kinases that act both upstream (Dco) and downstream (Wts) of the Fat effector Dachs and by linking Fat to the transcriptional coactivator Yki, these results have provided additional support for the conclusion that Fat functions as a component of a signaling pathway and have delineated core elements of this pathway from receptor to transcription factor. Fat activity is regulated, in ways yet to be defined, by Ds and Fj. The influences of Fat on gene expression, growth, and cell affinity, as well as on Wts stability, are completely dependent on Dachs, indicating that Dachs is a critical effector of Fat signaling. Since Dachs can associate with Wts or a Wts-containing complex, it is suggested that Dachs might act as a scaffold to assemble a Wts degradation complex. The observations that Fat, Ds and Fj modulate the subcellular localization of Dachs, that Wts is preferentially localized near the membrane and that Dachs accumulates at the membrane in the absence of Fat, suggest a simple model whereby Fat signaling regulates Wts stability by modulating the accumulation of Dachs at the membrane and thereby its access to Wts. The working model is that dco3 is defective in the phosphorylation of a substrate in the Fat pathway, but the recessive nature of dco3, the genetic epistasis experiments, and biochemical experiments argue that this substrate is not Wts, and further work is required to define the biochemical role of Dco in Fat signaling (Cho, 2006).

In addition to identifying core components of the Fat pathway, the results establish close functional links between the Fat pathway, the Hippo pathway and the FERM-domain tumor suppressors Mer and Ex. The common phenotypes observed among these tumor suppressors can be explained by their common ability to influence Wts. However, they seem to do this in distinct ways, acting in parallel pathways that converge on Wts rather than a single signal transduction pathway. The Fat pathway modulates levels of Wts, apparently by influencing Wts stability. By contrast, the Hippo pathway seems to regulate the activity of Wts by modulating its phosphorylation state. Thus, Wts seems to act as an integrator of distinct growth signals, which can be transmitted by both the Fat pathway and the Hippo pathway. It has been suggested that Mer and Ex also act through the Hippo pathway, although present experiments cannot exclude the possibility that Mer and Ex act in parallel to Hpo. Moreover, it should be noted that Mats might regulate Wts independently of Hpo and Sav and hence function within a distinct, parallel pathway. Although it is simplest to think of parallel pathways, there is also evidence for cross-talk. fj and ex are both components and targets of these pathways. Thus, they can be regarded as feedback targets within their respective pathways, but their regulation also constitutes a point of cross-talk between pathways. Another possible point of cross-talk is suggested by the observation that levels of Fat are elevated within Mer ex mutant clones. Although the potential for cross-talk complicates assessments of the relationships between tumor suppressors, the observations that fat, dco3 and dachs affect Warts protein levels in vivo, whereas ex, hippo, sav and mats do not, argues that there are at least two distinct pathways that converge on Warts. This conclusion is also consistent with the observations that ex, hippo, sav and mats can influence Wts phosphorylation in cultured cell assays, but Fat, Dachs and Dco do not (Cho, 2006).

Although the Fat and Hippo pathways converge on Wts, Hippo pathway mutants seem more severe. Thus, hpo, wts or mats mutant clones show a distinctive disorganization and outgrowth of epithelial tissues that is not observed in fat mutant clones, and they show a greater increase in interommatidial cells. This difference presumably accounts for the previous failure to recognize the tight functional link between Fat and Hippo signaling, and it can be explained by the finding that Wts levels are reduced but not completely absent in fat mutant cells. Thus, fat would be expected to resemble a hypomorphic allele of wts rather than a null allele, and consistent with this, a hypomorphic allele, wtsP2, results in strong overgrowth phenotypes. The effects of Yki overexpression on growth and target gene expression can be even stronger than those of fat or wts mutations, which suggests that Yki levels become limiting when upstream tumor suppressors are mutant (Cho, 2006).

fat encodes a protocadherin, which in the past has led to speculation that its influences on growth and cell affinity might result from Fat acting as a cell adhesion molecule. However, all of the effects of fat on growth and affinity require dachs, which is also required for the effects of fat on transcription. Additionally, targets of Fat signaling include genes that can influence growth and affinity; recent studies identified an influence of fat on E-cadherin expression, and as describe in this study, Fat influences CycE and Diap1 expression. Thus, one can account for the influence of fat on growth and affinity by its ability to regulate gene expression. fat interacts genetically with other signaling pathways, including EGFR and Wnt, and in some cells Fat signaling also influences the expression of ligands (such as Wg and Ser) for other signaling pathways. Regulation of these ligands contributes to fat overgrowth phenotypes, but since clonal analysis indicates that fat is autonomously required for growth control in most imaginal cells, the principal mechanism by which fat influences growth presumably involves the regulation of general targets (Cho, 2006).

Normal tissue growth and patterning depend on a relatively small number of highly conserved intercellular signaling pathways. The Fat pathway is essential for the normal regulation of growth and PCP in most or all of the external tissues of the fly and also participates in local cell fate decisions. In this regard, its importance to fly development can be considered comparable to that of other major signaling pathways. Although the biological roles and even the existence of a Fat pathway in mammals remain to be demonstrated, there is clear evidence that the mammalian Warts homologs Lats1 and Lats2 act as tumor suppressors and that a mammalian Yorkie homolog, YAP, can act as an oncogene. Moreover, other genes in the Drosophila Fat pathway have apparent structural homologs in mammals. Thus, it is likely that mammals also have a Fat tumor suppressor pathway that functions in growth control (Cho, 2006).

The salvador-warts-hippo pathway is required for epithelial proliferation and axis specification in Drosophila

In Drosophila, the body axes are specified during oogenesis through interactions between the germline and the overlying somatic follicle cells. A Gurken/TGF-alpha signal from the oocyte to the adjacent follicle cells assigns them a posterior identity. These posterior cells then signal back to the oocyte, thereby inducing the repolarization of the microtubule cytoskeleton, the migration of the oocyte nucleus, and the localization of the axis specifying mRNAs. However, little is known about the signaling pathways within or from the follicle cells responsible for these patterning events. It study shows that the Salvador Warts Hippo (SWH) tumor-suppressor pathway is required in the follicle cells in order to induce their Gurken- and Notch-dependent differentiation and to limit their proliferation. The SWH pathway is also required in the follicle cells to induce axis specification in the oocyte, by inducing the migration of the oocyte nucleus, the reorganization of the cytoskeleton, and the localization of the mRNAs that specify the anterior-posterior and dorsal-ventral axes of the embryo. This work highlights a novel connection between cell proliferation, cell growth, and axis specification in egg chambers (Meignin, 2007).

Multicellular organisms develop through an orchestrated temporal and spatial pattern of cell behavior, which is controlled by cell-to-cell signaling. In Drosophila melanogaster, the establishment of the embryonic axes occurs in the oocyte and depends on a sequence of signals between the germline and the somatic cells. First, Gurken (Grk) signals from the oocyte to the adjacent follicle cells (FCs), in which Torpedo (Top, EGFR) is activated, and this signal instructs them to adopt a posterior identity. The posterior FCs (PFCs) then send an unidentified signal back to the oocyte, leading to the movement of the nucleus from the posterior to the dorsoanterior (DA) corner and the repolarization of the microtubule (MT) cytoskeleton, with the minus ends at the anterior and lateral cortex and the plus ends at the posterior. This repolarization results in the localization of the mRNAs that encode key patterning factors. grk mRNA is next to the nucleus at the DA corner of the oocyte. At this corner, Grk instructs the overlying FCs to adopt dorsal fates. In contrast, oskar (osk) and bicoid (bcd) mRNAs are localized at the posterior and anterior pole, respectively, thus defining the anterior posterior (AP) embryonic axis and the germ cells. Although several genes are required in the FCs to control these events, little is known about the signaling pathways within and from the FCs (Meignin, 2007).

One of the genes required for axis formation during oogenesis is the tumor suppressor merlin (mer). However, it is not known whether Mer influences axis specification directly or what signaling pathways lie downstream of Mer. In other tissues, Mer is known to activate the Salvador Warts Hippo (SWH) pathway, which is a tumor-suppressor pathway. Inhibition of the SWH pathway leads to a characteristic overgrowth phenotype in adult organs because of an overproliferation of cells, increased cell growth, and defects in apoptosis. To test whether the SWH pathway is required in the function of Mer in axis formation, the localization of grk, bcd, and osk mRNA was examined in egg chambers with warts (wts) and hippo (hpo) mutant FCs. wts and hpo encode two serine/threonine kinases that are core components of this pathway. In both cases, grk mRNA is mislocalized at the posterior, osk mRNA is mislocalized at the center, and bcd mRNA is mislocalized at the posterior and anterior poles. The mislocalization of these mRNAs could be due to failure of the MTs to repolarize, as has been previously shown in grk/EGFR and mer mutants. In wild-type oocytes, the MTs are organized in an AP gradient. In contrast, in egg chambers with hpo mutant FCs, the MTs are distributed diffusely all over the oocyte cytoplasm. Considering these results, together with previous characterizations of similar phenotypes, it is concluded that the oocyte cytoskeleton in mutant egg chambers for the SWH pathway is disorganized with the MT plus ends at the center and the minus ends at the anterior and posterior poles. These defects resemble those described in oocytes lacking the Grk signal. In wts mutants, however, Grk protein is detected at the posterior pole, where grk mRNA is mislocalized. This demonstrates that the axis-specification defects in wts mutant egg chambers are not a consequence of the absence of Grk protein (Meignin, 2007).

It was shown that mer is required in the FCs for the repolarizing signal back to the germline and consequently for the migration of the oocyte nucleus from the posterior to the DA corner. Similarly, when mutant FC clones were generated for wts, hpo, and expanded (ex), an activator of the SWH pathway, the oocyte nucleus fails to migrate to the anterior. Another protein that is upstream of the SWH pathway is the giant atypical cadherin fat (ft). However, egg chambers with ft mutant FCs show no defects in oocyte polarity, and both the nucleus and Staufen (Stau) [a marker for osk mRNA] are always properly localized. In other epithelia, hpo and wts are required to repress the activity of Yorkie (Yki) and overexpression of yki phenocopies loss-of-function mutations of hpo and wts. Similarly, it was found that overexpression of yki in the FCs also causes the mislocalization of Stau and the oocyte nucleus. These results indicate that the SWH pathway, with the exception of Ft, might be required for the repolarizing signal back from the FCs to the oocyte (Meignin, 2007).

Because this signal is sent by the PFCs, whether the SWH pathway is required only in these cells was analyzed. In egg chambers with wild-type PFCs within an otherwise hpo or wts mutant epithelium, as well as in hpo, wts, and ex germline clones, the oocyte polarity is unaffected. However, in egg chambers with hpo mutant PFCs in an otherwise wild-type epithelium, the oocyte nucleus is mislocalized. It was also observed that when only a few cells at the posterior are mutant, Stau localizes in the region of the oocyte that faces the posterior wild-type cells. The SWH pathway is not required in the polar cells for axis determination because egg chambers with hpo or wts mutant PFCs and wild-type polar cells show oocyte polarity defects. It is concluded that the SWH pathway is required only in the PFCs to induce axis specification in the oocyte (Meignin, 2007).

In contrast to the monolayered wild-type epithelium, anterior and posterior, but not lateral, hpo and wts mutant cells form a bilayered, and occasionally a multilayered, epithelium. Given that the SWH pathway is required to control proliferation in epithelia of imaginal discs, whether the bilayered epithelium is a result of overproliferation was analyzed. At stage 6 of oogenesis, wild-type FCs undergo a Notch-dependent switch from a mitotic cell cycle to an endocycle. For this reason, phosphohistone 3 (PH3), a marker for mitotic cells, is detected only until that stage and never later. In contrast, hpo mutant anterior and posterior FCs are often positive for PH3 at stage 7-10B, indicating that these cells are still dividing. Similar results are obtained in yki overexpressing FCs. Taken together, these findings show that the SWH pathway is required for the control of proliferation at the anterior and posterior FCs (Meignin, 2007).

The formation of a multilayered epithelium was also observed in stage 3-5 mutant FCs, although the number of dividing cells is similar to that of the wild-type. It has been recently shown that the aberrant orientation of the mitotic spindle in the FCs results in the formation of a multilayered epithelium. Therefore the orientation of the mitotic spindle was analyzed in wild-type and hpo mutant cells. It was observed that, contrary to wild-type cells, the mitotic spindle in mutant FCs is often at an angle or perpendicular to the membrane. This aberrant orientation disrupts the remaining daughter cells within the same plane, thereby resulting in a bilayered epithelium (Meignin, 2007).

Often, tumor suppressors are important for the polarity of the epithelia. To determine whether this is the case for the SWH pathway, the atypical (novel) Protein Kinase C (nPKC), an apical marker, and Disc large (Dlg), a lateral marker, were examined in the FCs. In wild-type cells, as well as in hpo mutant FCs that maintain a monolayer epithelium, nPKC and Dlg localize at the apical and lateral membrane, respectively. However, when the mutant epithelium forms several layers of cells, nPKC and Dlg are often mislocalized, with a reduction of the nPKC staining and an expansion of the Dlg-positive membrane. Nevertheless, a certain degree of the polarity in these cells is maintained because nPKC is always apical in the cells that are in contact with the oocyte (Meignin, 2007).

Because SWH pathway mutant cells do not exit mitosis and keep dividing, it is possible that their differentiation is impaired. To address this question, the expression of Fasciclin III (FasIII) and eyes absent (eya) were analyzed in wild-type and wts and hpo mutant FCs. FasIII and Eya are downregulated in a Notch-dependent manner in the main-body FCs after stage 6 of oogenesis. However, the levels of FasIII in hpo mutant PFCs and Eya in wts mutant PFCs remain high after stage 6. To further assess the effect of the SWH pathway on the Notch-dependent maturation of the FCs, the expression of Hindsight (Hnt), a transcription factor that is upregulated by Notch signaling in all FCs was examined.. In hpo posterior FC clones, this Hnt upregulation is blocked. Contrary to notch clones, however, hpo lateral and anterior clones do not show defects in FasIII, Eya, or Hnt expression. Furthermore, border, centripetal, and stretched cells that are mutant for hpo migrate normally. Considering all these results together, it is concluded that the SWH pathway is essential for the PFCs to fully differentiate (Meignin, 2007).

The findings described above, together with the proliferation defects in hpo and wts mutant cells, suggest that the SWH pathway is required for Notch signaling. To test whether this is the case, the expression of universal Notch transcriptional reporters was analyzed in wild-type and hpo mutant FCs. In wild-type egg chambers, the Notch reporter E(spl)mß7-lacZ is expressed in all FCs upon Notch activation at stage 6 of oogenesis. In contrast, it was found that in hpo mutant cells, the levels of E(spl)mß7-lacZ are weakly reduced in 53% of the clones and normally expressed in the rest. It has been shown that in wing imaginal discs, mer and ex are required to control Notch localization in the cell and consequently its activity. Similarly, the subcellular distribution of Notch is affected in hpo mutant FCs. Contrary to the wild-type, in which Notch accumulates in the apical membrane, Notch expands to other membranes and is often detected in clusters in hpo clones. The results point out that hpo is essential in the PFCs for the Notch-dependent expression of several differentiation markers, such as FasIII, Eya, and Hnt, and for Notch subcellular localization. These observations and the weak defects on the Notch reporters support a function of the SWH pathway in modulating Notch signaling (Meignin, 2007).

Because the SWH pathway is required for the polarization of the oocyte, as well as for the differentiation of the PFCs, whether the mutant cells are competent to respond to Grk and indeed adopt a posterior fate was analyzed. Dystroglycan (DG) is expressed in all FCs at early stages of oogenesis, but upon Grk signaling, DG forms an AP gradient with lower levels at the PFCs. The fact that this Grk-dependent gradient of DG is also observed in the hpo mutant epithelia suggests that the mutant cells are responsive to the Grk signaling. Similarly, when hpo clones affect only a portion of the PFCs, the posterior fate marker pointed is expressed as in the wild-type in 40% of the cases. However, in 60% of the egg chambers with partial hpo posterior clones, and in all cases when all the PFCs are mutant, the expression of pointed is abolished. These results illustrate that hpo is required to fully process the Grk/EGFR signal in the PFCs. Conversely, in grk mutant egg chambers, the Hpo-dependent expression of Hnt is not affected, suggesting that the EGFR pathway is not required for the activation of the SWH pathway in the PFCs (Meignin, 2007).

Considering all these results together, it is concluded that the SWH pathway is involved in the Notch- and Gurken-dependent maturation of the PFCs. Whether the SWH pathway modulates this maturation directly or indirectly, for example by affecting membrane properties, needs to be further investigated (Meignin, 2007).

To study whether the oocyte polarity defects in egg chambers with FCs mutants for the SWH pathway are a consequence of the FCs proliferation and differentiation defects, egg chambers with ex and ft mutant PFCs were analyzed. Egg chambers with ft PFCs occasionally form a bilayer, although they never have defects in oocyte polarity, suggesting that the morphological disruption of the epithelia in itself does not block the repolarizing signal. Egg chambers with ex PFCs show weak defects in the epithelium, with a bilayer rarely formed and restricted to only a few mutant cells, but Stau is never properly localized. However, Hnt is not properly expressed in stage 7 ex mutant FCs, suggesting that the mislocalization of Stau is a consequence of the ex mutant cells being undifferentiated at the stage when the repolarizing signal is sent to the oocyte. These results suggest that the defects in oocyte polarity are probably due to a lack of proper differentiation of FCs in SWH mutant egg chambers (Meignin, 2007).

This study has analyzed the requirement of the SWH pathway during oogenesis. Several of the components of this pathway, but not ft, are required in the PFCs to induce the axis specification in the germline. The defects in oocyte polarity, however, are probably due to a lack of proper differentiation of the PFCs in SWH mutant egg chambers. In addition, the pathway is required in the terminal cells to control their proliferation. It has already been shown that terminal follicle cells are different from lateral follicle cells. The distinct spatial requirement of the SWH pathway for differentiation and proliferation is another feature that distinguishes the terminal from the lateral FCs, and the posterior from the anterior FCs. These results point out that this dual function of the SWH pathway might be achieved by modulation of the Notch and EGFR signals. In conclusion, the SWH pathway lies at the intersection of two signaling pathways and is permissive for the signal that is sent from the follicle cells to repolarize the oocyte.

Salvador-warts-hippo signaling promotes Drosophila posterior follicle cell maturation downstream of notch

The Salvador Warts Hippo (SWH) network limits tissue size in Drosophila and vertebrates. Decreased SWH pathway activity gives rise to excess proliferation and reduced apoptosis. The core of the SWH network is composed of two serine/threonine kinases Hippo (Hpo) and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). Two band 4.1 related proteins, Merlin (Mer) and Expanded (Ex), have been proposed to act upstream of Hpo, which in turn activates Wts. Wts phosphorylates and inhibits Yki, repressing the expression of Yki target genes. Recently, several planar cell polarity (PCP) genes have been implicated in the SWH network in growth control. This study shows that, during oogenesis, the core components of the SWH network are required in posterior follicle cells (PFCs) competent to receive the Gurken (Grk)/TGFβ signal emitted by the oocyte to control body axis formation. These results suggest that the SWH network controls the expression of Hindsight, the downstream effector of Notch, required for follicle cell mitotic cycle-endocycle switch. The PCP members of the SWH network are not involved in this process, indicating that signaling upstream of Hpo varies according to developmental context (Polesello, 2007).

Body axis formation is a critical stage of development in most multicellular organisms. In Drosophila melanogaster, the anteroposterior (AP) body axis is determined by the polarization of the developing oocyte. The egg chamber is composed of 16 germ cells (15 nurse cells plus the oocyte) and the follicular epithelium. Specification of the AP axis requires active transport of several mRNAs along the microtubule network, thereby resulting in asymmetric mRNA and protein localization inside the oocyte. For example, bicoid (bcd) and oskar (osk) mRNAs localize to and control the formation of the anterior and posterior poles, respectively. This process is initiated through bidirectional signaling between the oocyte and the adjacent follicle cells. In midoogenesis egg chambers, grk mRNA is localized between the oocyte nucleus and the plasma membrane at the presumptive posterior pole and targets the Grk signal to the posterior follicle cells (PFCs) only. Grk is believed to be the ligand for the Torpedo/DER (EGFR) signaling pathway, which controls PFC identity. Once they are specified, the PFCs send an unknown signal back to the oocyte; this signal is required to establish oocyte posterior polarity (Polesello, 2007).

Mer, which has recently been proposed to be part of the SWH network in tissue-size control, has been suggested to play a role in signal back. Therefore whether other members of this network could play a role in body axis formation was addressed. It was tested whether hpo, like mer, is required in PFCs to control oocyte polarity by generating FLP/FRT mitotic clones of mutant cells in the egg chamber was tested with either a kinase-dead (hpoJM1) or a truncating (hpoBF33) allele of hpo. These two alleles behave similarly in all subsequent experiments (Polesello, 2007).

In wild-type egg chambers, the RNA-binding proteins Staufen (Stau) and Osk are localized in a crescent at the posterior pole of the oocyte. When the PFCs were mutant for hpo (visualized by the lack of GFP), both Osk and Stau are mislocalized. If all PFCs were mutant, both Stau and Osk were found in the middle of the oocyte or were absent in some cases for Osk. When hpo clones affected only a portion of the PFCs, Stau was mislocalized almost exclusively in the mutant part, showing the importance of the crosstalk between PFCs and the oocyte (Polesello, 2007).

In hpo germline clones, Stau localization is unaffected if the PFCs are wild-type, suggesting that Hpo is not required for secretion of the Grk signal by the oocyte. Similarly, hpo activity in polar cells is not sufficient to rescue hpo PFC phenotypes because chambers with mutant PFCs and wild-type (GFP-positive) polar cells show disrupted Stau localization. Together, these data suggest that hpo is required in the PFCs to control oocyte polarity (Polesello, 2007).

By using Stau localization as a readout, it was found that like mer and hpo, ex, sav, mats, wts, and yki are playing a role in PFCs to control oocyte polarity, suggesting that 'canonical' Hpo signaling is responsible for the observed phenotype. In contrast, fat (ft) and discs overgrown (dco) are not required in PFCs to control oocyte polarity. This suggests that the core components of the SWH network but not the SWH-associated PCP genes are required for anteroposterior axis formation (Polesello, 2007).

The microtubule cytoskeleton plays an active role in the correct localization of posterior determinants such as Osk mRNA and Stau. Therefore, whether the microtubules are normally organized when the PFCs were mutant for hpo was tested. The oocyte nucleus is initially positioned at the posterior pole (up to stage 6) and migrates to an anterodorsal localization in a microtubule-dependent manner after the signal back from the PFCs (stages 7-14). The oocyte nucleus fails to migrate to an anterodorsal position in 50% of egg chambers with PFC hpo clones. The expression of a tubulin-GFP fusion protein was drived in the germline to visualize the microtubule network. In control oocytes, tubulin-GFP forms a regular network of filaments with a stronger accumulation at the anterior pole corresponding to the nucleation site. Egg chambers with hpo mutant PFCs present ectopic Tubulin-GFP accumulation at the posterior pole of the oocyte. Apart from this defect, the general aspect of the microtubule network is normal in egg chambers with hpo PFC clones, even when the oocyte nucleus has failed to migrate to the anterior end. Finally, microtubule polarity was examined by using both Nod-βGalactosidase (Nod-βGal, minus end marker-anterior) and Kinesin-βGalactosidase (Kin-βGal, plus end marker-posterior) fusion proteins. When the PFCs were mutants for hpo, Nod-βGal was present at both poles or only at the posterior of the oocyte when the nucleus failed to migrate. When all PFCs were hpo mutant, Kin-βGal localization was in a diffuse cloud in the middle of the ooplasm. As for Stau, only half of the Kin-βGal was normally localized when only part of the PFCs were hpo mutant. Together these data support the idea that core components of the SWH pathway are required in the PFCs to build oocyte polarity, controlling microtubule-network orientation (Polesello, 2007).

Because the SWH network is known to control cell number, a phosphorylated Histone 3 (PH3) antibody was used to follow cell division in the follicle cells. During egg-chamber development, follicle cells undergo normal mitotic divisions up to stage 6, thereby giving rise to ~650 follicle cells surrounding the germ cells. Follicle cells then switch from mitotic cycles to three rounds of endoreplication cycles (endocycles) during stages 7-10A. Thus, follicle cells normally stop proliferating after stage 6, as assayed by the absence of PH3-positive cells. hpo PFC clones still contained PH3-positive cells until stage 10B. This excess proliferation observed in hpo mutant cells gives rise to both a reduction of the size of follicle cell nuclei (reduced endocycling) and formation of double layers of cells at the posterior of the egg chamber. Formation of extra layers in the follicular epithelium has been reported to result from misorientation of the mitotic spindle. Normally, the mitotic spindle is parallel to the surface of the germline cells but appears randomly oriented in hpo mutant PFCs because both parallel and perpendicularly oriented spindles were observed. This defect in the mitotic-spindle orientation is probably responsible for the double-layer formation. The proliferation defect specifically affects PFCs because reduced nuclei, ectopic PH3 foci or double layers were not obvious elsewhere. Finally, it was found that loss of the core components of the SWH network, but not of ex for which the proliferation defect is weaker, produced a double cell layer (Polesello, 2007).

In imaginal discs, loss of SWH pathway genes leads to increased expression of Yki target genes. Whether this is also the case in PFCs was tested. As expected, disruption of SWH activity in PFCs gave rise to an increase in ex expression, although no changes were detected in DIAP1 or cycE expression. ex upregulation was restricted to the PFCs in both wts mutant cells and yki gain-of-function experiments. These results suggest that core components of the SWH network specifically control proliferation of a particular subset of follicle cells required for body axis establishment (Polesello, 2007).

Because hpo mutant PFCs were still dividing after stage 6, whether hpo loss of function could affect PFC polarity was assessed. Armadillo (Arm) and Discs large (Dlg) normally label the adherens junctions and the lateral region of the cell, respectively. In hpo mutant PFCs, these were found all around the cells. In addition, the level of Arm, atypical Protein Kinase C (aPKC), and phosphorylated Moesin (P-Moe) were increased. Nevertheless, some aspects of the polarity in these cells were preserved because aPKC was still localized in the apical domain facing the oocyte (Polesello, 2007).

Grk signals via the EGF receptor Torpedo (Top) and activates the Ras signaling pathway, specifying the PFC identity. The PFC fate can be followed by the expression of the Ras target pointed (pnt-LacZ). In the absence of hpo, pnt-LacZ expression was disrupted in most but not all PFC clones. Nevertheless, hpo mutant PFCs were still able to activate the Jak/STAT pathway in response to a signal emerging from the polar cells, (monitored with a STAT reporter suggesting that the polarity defect observed in hpo mutant PFCs does not affect their ability to receive secreted signals in general. wts mutant PFCs were negative for the dpp-LacZ reporter, a specific marker of the anterior follicle cell fate (stretch and centripetal cells), suggesting that when the SWH pathway is compromised, the PFCs are not merely transformed into anterior cells. In addition, it was found that hpo mutant PFCs present characteristics of immature cells such as maintenance of Fasciclin III (FASIII) and eyes absent (eya) expression. Normally, the level of these two genes is downregulated when the follicle cells switch from mitotic cycles to endocycles. It is noted that, when hpo mutant PFCs were FASIII positive, they did not express pnt-LacZ and vice versa. In addition, it was found that pnt-LacZ-positive hpo mutant PFCs have normal Stau localization. This suggests that the primary defect in hpo mutant cells is the failure to mature. In the rare cases where hpo mutant PFCs mature properly, they are competent to transduce the Grk signal, and oocyte polarity is normal (Polesello, 2007).

Notch (N) is required in the follicle cells for the mitotic-endocycle switch that occurs at stage 6 and for controlling follicle cell identity. N mutant follicle cells, like hpo mutant PFCs, keep proliferating because they are stuck in an immature state and continue to express undifferentiated markers such as FASIII. Recently, members of the SWH network were reported to modulate N activity by affecting its subcellular localization. N protein, which localizes to the apical part of the follicle cells, is downregulated at midoogenesis. This downregulation is delayed in wts and hpo mutant PFCs, possibly causing a defect in N signaling. Hindsight (Hnt), a target of N, which starts to be expressed in all follicle cells at stage 7 after N activation, was examined. Expression of Hnt in hpo mutant PFCs is compromised. In addition, it was found that the expression of Cut, which is normally inhibited by Hnt at stage 7, was maintained in hpo and wts clones up to stage 10. Finally, whether the modulation of N activity by the SWH network was direct was tested by looking at the expression of direct N reporters. no obvious reduction of the m7-LacZ reporter was found in hpo PFC clones. However, because of the perdurance of the β-galactosidase protein, this type of reporter is more suitable to follow increases rather than decreases in signaling. It therefore cannot be entirely rule out that the SWH network might directly affect Notch activity. Nevertheless, together these data show that inactivation of the SWH network compromises the regulation of downstream targets of Notch such as Hnt and Cut. As is the case for FASIII, misregulation of these genes is restricted to the PFCs in a SWH mutant background (Polesello, 2007).

Because of this spatial restriction of SWH activity to PFCs, whether the SWH network could be part of the Torpedo/Ras pathway acting downstream of the Grk signal was tested. ras, wts double loss-of-function clones were examined. ras, wts clones present characteristics of both ras and wts single-mutant clones, namely upregulation of Dystroglycan (DG), as observed in ras clones, and maintenance of FASIII protein, as observed in wts clones. In addition, grk mutant egg chambers present only DG upregulation but no FASIII modification and no substantial change in ex expression. It is therefore concludes that the SWH network and EGFR/Ras signaling are likely to act in parallel to control respectively PFC maturation and identity and that Grk is not the ligand that controls the SWH network activation (Polesello, 2007).

A last concern was to test whether the SWH network is involved in the PFC signal back that controls oocyte polarity. To tackle this point, attempts were made to uncouple the possible signal back to the oocyte from the PFC maturation phenotypes. ex loss of function, which affects Stau localization but presents a very reduced proliferation rate and double-layer formation compared to other SWH members, was examined. Unfortunately, ex loss of function still affected Arm, FASIII, and Cut protein levels in the PFCs, in particular at midoogenesis, when both the N and Grk signals act. Therefore mer, cut double mutants were generated. In theory, this should force the cells to differentiate (lack of cut) and still affect SWH activity (lack of mer). As expected, whereas mer loss of function alone elicited both Cut upregulation and Stau mislocalization, mer/cut PFC clones were able to induce normal oocyte polarity, manifested by correct Stau localization. It is concluded that the activity of the SWH network is required to control PFC maturation, but this pathway is probably not involved in the signal-back process (Polesello, 2007).

In conclusion, this study has shown that the core components of the SWH network are required specifically to allow the maturation of the PFCs receiving the Grk signal, thus controlling AP body axis formation. The PFC defect is due to a lack of Hnt expression in response to Notch signaling. Because the function of the SWH network is restricted to the PFCs, one interesting speculation is that it is an added layer of Notch regulation specific to PFCs, which, given their crucial role in initiating body axis formation, need robust control of signaling. Placing this regulatory element in complement and in parallel to the signal that initiates PFC specification (Grk) would ensure, in cooperation with the Unpaired signal (Jak/STAT pathway) from the polar cells, a tight and robust boundary between the PFCs and the rest of the follicle cells (Polesello, 2007).

Finally the results make a clear distinction between the core components of the SWH network (hpo, sav, wts, mats, and yki) and mer, ex on one hand and the PCP genes (ft and dco) on the other. It is speculated that the core components are used in a variety of contexts during development, whereas the PCP genes are restricted to organ-size specification (Polesello, 2007).

Basolateral junctions utilize warts signaling to control epithelial-mesenchymal transition and proliferation crucial for migration and invasion of Drosophila ovarian epithelial cells

Fasciclin2 (Fas2) and Discs large (Dlg) localize to the basolateral junction (BLJ) of Drosophila follicle epithelial cells and inhibit their proliferation and invasion. To identify a BLJ signaling pathway a genome-wide screen was performed for mutants that enhance dlg tumorigenesis. Two genes were identified that encode known BLJ scaffolding proteins, lethal giant larvae (lgl) and scribble (scrib), and several not previously associated with BLJ function, including warts (wts) and roughened eye (roe/rotund), which encode a serine-threonine kinase and a transcription factor, respectively. Like scrib, wts and roe also enhance Fas2 and lgl tumorigenesis. Further, scrib, wts, and roe block border cell migration, and cause noninvasive tumors that resemble dlg partial loss of function, suggesting that the BLJ utilizes Wts signaling to repress EMT and proliferation, but not motility. Apicolateral junction proteins Fat (Ft), Expanded (Ex), and Merlin (Mer) either are not involved in these processes, or have highly spatio-temporally restricted roles, diminishing their significance as upstream inputs to Wts in follicle cells. This is further indicated in that Wts targets, CyclinE and DIAP1, are elevated in Fas2, dlg, lgl, wts, and roe cells, but not Fat, ex, or mer cells. Thus, the BLJ appears to regulate epithelial polarity and dynamics not only as a localized scaffold, but also by communicating signals to the nucleus. Wts may be regulated by distinct junction inputs depending on developmental context (Zhao, 2008).

The purpose of this work was to gain greater insight into how the BLJ suppresses epithelial tumorigenesis and invasion by identifying and understanding the function of new genes important for BLJ function. To do so, a genomewide screen was completed for enhancers of dlg, which encodes a scaffolding protein that is a crucial organizer of the BLJ and is a potent repressor of follicle epithelial cell tumorigenesis and invasion. Deficiencies that cumulatively span ∼80% of the autosomes, or 64% of the Drosophila genome were systematically screened. A relatively small number of enhancers, ∼1 per 1000 genes screened, were detected indicating that the screen selected for loci specifically required for dlg function. Thus, the novel dlg enhancer genes that were identified, wts, roe, ebi, as well as at least two genes yet to be identified, are likely to be key collaborators with dlg in suppressing epithelial invasion. The specificity of the interactions between dlg and these enhancers is further indicated in that more than one allele of each gene showed an interaction, in several dlg backgrounds, and the strengths of enhancement were similar to deficiencies defining each locus. wts, roe, and ebi also enhanced Fas2 and lgl, indicating that they are not just important for dlg function, but for the function of the BLJ as a whole. In addition, overexpression of all enhancers except ebi suppressed dlg and Fas2 tumorigenesis, further confirming that the identified genes function in a BLJ network (Zhao, 2008).

BLJ pathway components in the nucleus and their putative relationship to Notch: ebi encodes an F-box protein with WD repeats that promotes protein degradation of specific targets. The failure of ebi overexpression to suppress Fas2 or dlg, and the relatively mild ebi phenotypes (midoogenesis small-nucleus and epithelial-organization defects, but no defects in germinal vesicle localization), suggest that ebi may function in only one of the three branches of BLJ signaling or in a parallel pathway to the BLJ. In the eye, ebi is important for promoting differentiation and inhibiting proliferation, which appear to be separable functions. Thus ebi could enhance Fas2 and dlg tumorigenesis by functioning within the proliferation-repressing branch of the BLJ, or the importance of ebi for differentiation suggests that it could function in the EMT branch of the BLJ or both. In contrast, ebi promotes protein degradation in response to Notch (N) and Drosophila EGF receptor (EgfR) signals, suggesting that it may act in a parallel pathway. Both Ebi and its mammalian homolog, TBL1, function in a corepressor complex through association with nuclear hormone transcriptional corepressor SMRTER/SMRT (Zhao, 2008).

Interestingly, although most N appears to be localized on the apical surface of follicle cells, some N is also localized in BLJs. Thus, it is possible that N localized to the BLJ may signal directly to Ebi. Consistent with this possibility, it was found that all of the genes in the BLJ network share some midoogenesis defects with N, including the small nucleus phenotype, epithelial stratification defects, and mislocalization of the germinal vesicle. The epithelial defects are also reminiscent of N-pathway mutants brainiac and egghead, which are required in the germ line for regulating N that is localized on the apical surface of the follicle cells abutting the germ line. Thus one possibility is that N signaling activity is regulated by its localization to apical vs. basolateral junctions in response to several signaling pathways acting during midoogenesis (Zhao, 2008).

The other modest dlg enhancer that was identified, roe, encodes a Krüppel-family zinc-finger protein that appears to be a transcription factor. Roe is also implicated in Notch signaling and thus may function with Ebi in N-dependent processes as proposed above. However, in contrast to ebi, roe loss caused follicle cell tumors, suggesting that roe may function more directly in a BLJ pathway than ebi. Consistent with a direct role for Roe in BLJ signaling, it was found that roe overexpression suppressed Fas2 and dlg tumorigenesis. Further, as for Fas2, dlg, and wts, roe represses CycE and DIAP1 expression (Zhao, 2008).

Warts was of special interested because of the many similarities observed in the quality and strength of wts and scrib phenotypes, suggesting that they are components in a BLJ signaling pathway, rather than a parallel pathway that cross talks with BLJ signaling. wts encodes a serine/threonine kinase that is an ortholog of human tumor suppressors Lats1 and Lats2, both of which have been linked to highly aggressive breast cancers. The prevailing model for Wts signaling in Drosophila is based on signaling in eye and wing tissue. Wts appears to relay signals from apicolateral junction proteins Ft, Ex, and Mer in wing and eye tissues. However, the results from almost every assay, including early tumor formation, border cell migration, BrdU, PH3, CycE, and DIAP1 expression, indicated little functional overlap between Ft, ex, mer, or mer; ex and wts, thus diminishing the importance of apicolateral Ft-Ex-Mer for Wts activation in follicle cells. The exceptions were that during midoogenesis, Mer is required for border cell migration and Ex is required for the endocycle switch, while both are required for maintenance of epithelial integrity and positioning of the germinal vesicle. However, the involvement of Ex and Mer in these processes are fundamentally distinct from how they act in Wts-dependent processes in other tissues. (1) Ft is not involved; (2) no indication was observed of Ex-Mer synergism; (3) ex, mer, and mer; ex phenotypes are relatively mild when compared to wts. It is concluded that the model for Wts activation in which apicolateral junction proteins Ft, Ex, and Mer play the predominant role cannot be universally applicable in all cell types. Rather, the relative importance of Ex and Mer for Wts regulation appears to depend on developmental context (Zhao, 2008).

Consistent with this proposal, strong functional interdependence and phenotypic similarities were found between Fas2, dlg, lgl, scrib, and wts, thus indicating that the BLJ, not the apicolateral junction, plays the predominant role in Wts regulation during oogenesis. Although genetic evidence alone cannot completely rule out that Wts may act in a parallel pathway to the BLJ and impinge on a set of downstream targets that overlap with those targeted by the BLJ, the following observations favor a model in which the BLJ is more directly involved in Wts regulation (it is noted that these are not mutually exclusive alternatives): (1) over 50 tumor suppressor genes have been identified in Drosophila, but lgl, scrib, and wts were the only strong dlg enhancers identified in this genomewide screen; (2) wts showed strong genetic interactions with Fas2, dlg, and lgl, similar to or stronger than scrib, which encodes a known BLJ protein; (3) wts has early tumor phenotypes similar to dlg partial loss of function and to scrib; (4) wts has the same border cell migration phenotype as scrib; (5) wts has similar small nucleus, epithelial stratification, and germinal vesicle defects as Fas2, dlg, lgl, and scrib; (6) like lgl and scrib, wts overexpression suppressed Fas2 and dlg tumorigenesis; (7) Fas2, dlg, and wts have similar proliferation defects, and (8) Fas2, dlg, and wts similarly repress CycE and DIAP1 expression, which is especially crucial, because CycE and DIAP1 are downstream targets of Wts signaling, and ex and mer had no impact on their expression, contrary to results in other tissues. Thus, the data strongly indicate that the BLJ signals through Wts, and may impinge on Roe in the nucleus, thus suggesting the first BLJ signaling pathway in animal cells. This implies that the BLJ not only acts as a localized scaffold, but also signals to the nucleus to control gene expression, both of which cooperate to regulate epithelial polarity and dynamics (Zhao, 2008).

How can these results in follicle cells, which suggest that Wts acts predominantly downstream of the BLJ, be reconciled with findings in eye tissue, which indicate that Wts acts downstream of the apicolateral junction? Interestingly, the genetic data in the eye suggest that Ft, Ex, and Mer cannot account for all of the signals that activate Wts, because wts overgrowth and tissue disorganization phenotypes are more severe than ft or mer; ex. On the basis of these findings in follicle cells, it is possible that Wts activation in the eye requires additional input from the BLJ. This possibility may have been overlooked thus far because dlg does not appear to have an overgrowth phenotype in the eye. dlg may be essential for additional functions in the eye that are epistatic to its tumor suppressor function, thus preventing loss of cells from the epithelium that could mask an overgrowth phenotype. Consistent with this, when activated Rasv12 is combined with dlg loss, dramatic tumors develop that are larger and more invasive than those produced by Rasv12 alone (Zhao, 2008).

In contrast, Dlg may have a diminished role in Wts signaling in the eye, much as the evidence indicates a diminished role for Ex and Mer in Wts signaling in the ovary. According to this model, Wts receives predominant input from distinct lateral junctions depending on tissue context. One distinction is that ovarian follicle cells are derived from a mesodermal lineage, while the eye and wing tissues are from ectodermal lineages. Further, many genes that disrupt apical-basal polarity and epithelial morphology have only subtle phenotypes in the eye by comparison to the ovary or embryo. Finally, the follicular epithelium requires input from junctions on all three follicle cell surfaces, lateral, apical, and basal, whereas most epithelia require only two, lateral and apical or basal. Thus, ovarian and imaginal tissues are likely to organize signaling pathways acting downstream of epithelial junctions in similar, yet fundamentally different ways to meet the unique organizational requirements of their cell-tissue morphologies. Some or all of these differences may contribute to the suggested specificity observed in Wts signaling downstream of BLJs in follicle cells. In general, these findings raise the possibility for future investigation that depending on the cell-tissue morphologies of a given organ, one lateral junction may play a predominant organizational role, and Wts signaling may act as a universal signaling adapter for mediating contact inhibition from that junction (Zhao, 2008).

An especially interesting aspect of Mer and Ex function that was uncovered in follicle cells is that it appears to be restricted to predominantly postmitotic, differentiated cells, in contrast to the role of Mer and Ex in other tissues. Further, given the absence of an involvement of Ft and lack of Mer-Ex synergism it is concluded that if Mer and Ex would be involved in Wts activation in follicle cells, they would have to function via a fundamentally distinct mechanism than in other tissues. It is proposed that during early oogenesis, the BLJ alone may provide the predominant input to Wts. Then, during midoogenesis, Ex and Mer may become involved in novel interactions with Dlg or other components of the BLJ to activate Wts in spatiotemporally distinct populations of differentiating cells to help achieve their unique developmental functions (Zhao, 2008).

How do wts, scrib, and roe promote motility? It is proposed that Scrib, Wts, and Roe are all crucially involved in EMT. In EMT, cells (1) loose apical-basal polarity and become mesenchymal-like, and (2) adopt a polarity conducive to movement. scrib, wts, and roe cells clearly lose epithelial polarity and become mesenchymal-like as indicated by their rounded morphology and lateralized phenotype. However, scrib, wts, and roe tumors do not invade, and scrib, wts, and roe border cells do not move, suggesting that the second aspect of EMT, adoption of a polarity conducive to movement, is defective. Consistent with this, mammalian Scrib is required for migration and epithelial wound healing of cultured human breast epithelial cells, and is also required in vivo for wound healing in mice. Human Scrib directs migration by organizing several polarities crucial for migration, including the orientation of the microtubule and Golgi networks and the localization of Cdc42 and Rac1 to the cell's leading edge. Thus Scrib has a conserved function in directed cell migration by organizing a polarity conducive to movement. In mammalian PC12 cells Scrib is in complex with Rac1. Fly Rac1 is essential for border cell migration and invasion of Fas2 and dlg tumors, suggesting that an essential role of Scrib in Rac1 function may be of crucial importance for movement. The apparent conserved role of BLJ proteins in organizing EMT, and both promoting and repressing movement, reemphasizes the suggestion that BLJ proteins do more than merely maintain apical-basal polarity, but rather repress a cellular transformation from epithelial polarity to a mesenchymal, lateralized signature conducive to movement (Zhao, 2008).

How is the function of scrib, wts, and roe in promoting border cell movement consistent with the requirement of Fas2, dlg, and lgl in repressing border cell movement? Further, how do scrib and wts act as enhancers of dlg tumor invasion even though scrib and wts tumors are noninvasive? For border cell movement, Fas2 and dlg mutations not only accelerate movement, but also delay border cell delamination. The delay in border cell delamination suggests that the BLJ normally promotes motility, but this promoting function can be bypassed when the repression of motility branch of the BLJ pathway is simultaneously lost. Cumulative data indicate that scrib, wts, and roe act predominantly within the EMT and proliferation branches of the BLJ pathway, and not the repression of motility branch. It is suggested that without simultaneous loss of the repression of motility branch of the BLJ pathway, scrib and wts border cells cannot bypass the essential requirement for the second step of EMT, thus border cell motility is blocked (Zhao, 2008).

This interpretation is also consistent with the seemingly paradoxical function of scrib and wts as enhancers of dlg tumor invasion, even though Scrib and Wts promote rather than repress border cell movement. The noninvasive scrib and wts tumor phenotypes indicate that they are crucial for repressing the first step of EMT, loss of epithelial polarity and adoption of a lateralized, mesenchymal-like phenotype. It has been suggested that scrib and wts enhance dlg invasive tumorigenesis by increasing the rate at which dlg mutant follicle cells undergo EMT and further facilitate invasion by depressing proliferation control and increasing the number of follicle cells available for movement. Thus, even though scrib and wts are required to promote movement, it is suggested that in dlg; scrib/+ or dlg; wts/+ tumors this requirement can be bypassed because the branch of the BLJ pathway that represses motility is simultaneously disrupted (Zhao, 2008).

The noninvasive tumor phenotypes of scrib and wts are very similar to the phenotypes of dlg mutants that specifically disrupt Dlg SH3 and GuK domains. Thus Scrib and Wts may act specifically downstream of the Dlg SH3 and GuK domains. Consistent with this, Scrib appears to associate with the Dlg GuK domain in neuronal synapses via the linker protein GuK-holder. Further, whereas Fas2, dlg, and lgl cause faster border cell migration, border cell migration is very similar to wild type in the dlg SH3/GuK-specific mutants, suggesting that Dlg SH3/GuK predominantly represses the first step of EMT and proliferation but not motility. On the basis of this specificity, it is suggest that one reason that lgl may be a stronger dlg enhancer than scrib and wts is that lgl represses motility in addition to EMT and proliferation. For example, the de novo tumor formation observed when one copy of lgl, scrib, or wts is removed in dlghf/dlgsw ovaries suggests that a threshold level of BLJ activity essential for maintenance of polarity has been lost. However, the lgl interaction may be much stronger than scrib and wts because lgl additionally represses motility (Zhao, 2008).

Increased expression of CycE and DIAP1, known Wts targets, was observed in Fas2, dlg, lgl, scrib, wts, and roe cells. Thus the importance of CycE for proliferation control, and DIAP1 for control of EMT and motility, suggests that part of the mechanism by which Fas2-Dlg represses tumorigenesis is through activating Wts signaling. DIAP1 is in a complex with Rac1 and Profilin and enables border cell motility apparently by promoting actin turnover. Further, in the embryo, DIAP1 loss leads to Dlg cleavage and cellular rounding and dispersal. Too much DIAP1 also appears to be deleterious to movement, because targeted overexpression of DIAP1 specifically in border cells slows their migration (data not shown). Thus maintaining the proper balance of DIAP1 is critical for directed movement, and it may be part of the mechanism by which Scrib and Wts influence border cell movement, suggesting that interaction with Dlg and Rac1 may be another level at which Scrib regulates EMT and movement, consistent with the possibility that it functions downstream of Scrib and Wts in follicle cells to repress both EMT and proliferation (Zhao, 2008).

In contrast to the strong enhancement of dlg by scrib, Fas2 was only weakly enhanced by scrib. Given the complexity of coordinating EMT, proliferation, and motility within an epithelial field, perhaps the simplest model is that multiple Dlg complexes reside within the BLJ, each with a distinct set of ligands that control one or more morphogenetic activities (Zhao, 2008).

Another interesting difference in the enhancement of dlg and Fas2 by lgl, scrib, wts, and roe was that they all enhanced both dlg tumorigenesis and invasion, but only enhanced Fas2 tumorigenesis, without invasion. An important difference between these experiments may be that in Fas2null follicle cells, Dlg is missing Fas2 as a ligand, whereas in dlghf/dlgsw, dlghf/dlgip20, and dlghf/dlglv55 follicle cells, Fas2 is localized at sites of contact between follicle cells in both the native epithelium and in streams of invading cells, suggesting that Fas2 continues to act as a Dlg ligand in these cells. This is probably an important difference because Fas2-Dlg binding is expected to control the conformation of Dlg. Dlg conformations in turn may specify Dlg intra- and intermolecular interactions that determine the relative balance of EMT, proliferation, and invasion factors that associate with the BLJ scaffold. For example, in neuronal cells intramolecular interactions between Dlg SH3 and GuK domains regulate the strength of intermolecular binding of GuK-holder, which binds Scrib. The SH3-GuK intramolecular interaction is further modulated by intramolecular interactions with PDZ3, which are regulated by intermolecular interactions with neurolignin, a transmembrane ligand for PDZ3 (Zhao, 2008).

On the basis of this molecular model, it is proposed that in the absence of Fas2, Dlg has a distinct conformation that tilts the balance toward EMT and proliferation over invasion, when Lgl, Scrib, Wts, or Roe are reduced. This study has shown that lgl, scrib, wts, and roe are expected to act predominantly downstream of Dlg SH3 and GuK domains to repress EMT and proliferation. Thus, removal of one copy of lgl, scrib, wts, or roe in Fas2 cells may tip the ratio of factors controlling EMT, motility, and proliferation toward derepression of EMT and proliferation, masking the Fas2 requirement for invasion. One possibility is that lgl, scrib, wts, or roe are especially important for expression of a protein in the apicolateral junction, such as Par-3/Bazooka, which is essential for dlg invasion. Consistent with this, Ex upregulation is seen in both dlg and wts clones. Further, lgl enhancement at the lglts permissive temperature showed essentially the opposite trend from Fas2. Rather than enhance tumorigenesis over invasion, removal of one copy of Fas2, dlg, scrib, wts, or roe in lgl egg chambers favored invasion. Thus, it is suggested that tumor invasiveness associated with particular combinations of mutated BLJ proteins may be masked or unmasked on the basis of the balance of activities that are disrupted, rather than disruption of particular activities per se (Zhao, 2008).

In summary, this study has identified the first signaling pathway that acts downstream of the BLJ that specifically controls EMT and proliferation, and important clues have been gained as to how this signaling may be organized. Like the Drosophila follicular epithelium, the human ovarian surface epithelium, which is thought to be the site of origin of most ovarian cancers, is derived from a mesodermal lineage. The data suggest that the BLJ plays an especially crucial role in the follicle cells compared to ectodermal lineages in repressing epithelial invasion and that the follicular epithelium appears to organize signaling from epithelial junctions in distinct ways compared to other epithelia. Given the conservation in the lineage of the fly and human epithelia, and the sensitivity of this screen for detecting molecules important for invasive carcinogenesis, it is proposed that the fly egg chamber may serve as a prototype for identifying early molecular events that are crucial for invasion of human ovarian cancer and possibly other malignancies that remain undetected before they start to invade (Zhao, 2008).

Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila

Cell growth arrest and autophagy are required for autophagic cell death in Drosophila. Maintenance of growth by expression of either activated Ras, Dp110, or Akt is sufficient to inhibit autophagy and cell death in Drosophila salivary glands, but the mechanism that controls growth arrest is unknown. Although the Warts (Wts) tumor suppressor is a critical regulator of tissue growth in animals, it is not clear how this signaling pathway controls cell growth. This study shows that genes in the Wts pathway are required for salivary gland degradation and that wts mutants have defects in cell growth arrest, caspase activity, and autophagy. Expression of Atg1, a regulator of autophagy, in salivary glands is sufficient to rescue wts mutant salivary gland destruction. Surprisingly, expression of Yorkie (Yki) and Scalloped (Sd) in salivary glands fails to phenocopy wts mutants. By contrast, misexpression of the Yki target bantam is able to inhibit salivary gland cell death, even though mutations in bantam fail to suppress the wts mutant salivary gland-persistence phenotype. Significantly, wts mutant salivary glands possess altered phosphoinositide signaling, and decreased function of the class I PI3K-pathway genes chico and TOR suppressed wts defects in cell death. Although it has been shown that salivary gland degradation requires genes in the Wts pathway, this study provides the first evidence that Wts influences autophagy. These data indicate that the Wts-pathway components Yki, Sd, and bantam fail to function in salivary glands and that Wts regulates salivary gland cell death in a PI3K-dependent manner (Dutta, 2008).

Wts was identified as a protein that is expressed during autophagic cell death of Drosophila larval salivary glands with a high-throughput proteomics approach. This was surprising, given that wts RNA was not detected with DNA microarrays. Therefore, this study investigated whether Wts is present in salivary glands, and it was determined to be constitutively expressed at stages before and after the rise in ecdysone that triggers autophagic cell death. Animals that are homozygous for the hypomorphic wtsP2 allele, which is caused by a P element insertion, are defective in salivary gland cell death (Martin, 2007). Significantly two forms of Hpo are expressed during stages preceding salivary gland cell death, suggesting that phosphorylated Hpo is present in these cells and that this signaling pathway is activated (Dutta, 2008).

These studies indicate that Wts and other core components of this tumor-suppressor pathway are required for autophagic cell death of Drosophila salivary glands. wts is required for cell growth arrest and for proper regulation of caspases and autophagy, which contribute to the destruction of salivary glands. Although it is well known that cell division, cell growth, and cell death are important regulators of tissue and tumor size, it has been unclear whether a mechanistic relationship exists between cell growth and control of cell death (Dutta, 2008).

It is possible that wts and associated downstream growth-regulatory mechanisms could suppress cell death in other animals and cell types. Autophagic cell-death morphology has been reported in diverse taxa, but little is known about the mechanisms that control this form of cell death, and this lack of understanding is probably related to the limited investigation of physiologically relevant models of this process (Dutta, 2008).

This study used steroid-activated autophagic cell death of salivary glands as a system to study the relationship between cell growth and cell death. It is logical that cell growth influences cell death in salivary glands, given that autophagy is known to be regulated by class I PI3K signaling, which contributes to the death of these cells (Berry, 2007). It is unclear whether growth arrest is a determinant of autophagic cell death in other cell types and animals, and this question is important to resolve because of the importance of growth and autophagy in multiple disorders, including cancer. wts mutant salivary gland cells fail to arrest growth at the onset of puparium formation, and this suppresses the induction of autophagy. The inhibitor of apoptosis DIAP1 influences salivary gland cell death and is one of the best-characterized target genes of the Wts signaling pathway, but DIAP1 levels are not altered in wts mutant salivary glands. Significantly, the data provide the first evidence that Wts regulates autophagy and support previous studies indicating that caspases and autophagy function in an additive manner during autophagic cell death. Given the importance of both the Wts pathway and autophagy in human health, it is critical to determine whether this relationship exists in other cells (Dutta, 2008).

Cell growth and division are often considered to be synonymous, even though they are controlled by independent mechanisms. The Wts signaling pathway must influence cell growth, but most studies have emphasized the influence of this pathway on cell division and death. bantam is the only previously studied gene that is regulated by the Wts pathway and that is known to regulate cell growth. However, the mechanism of bantam action remains obscure. The current studies suggest the possibility that Wts may regulate growth via different mechanisms and that the nature of this regulation may depend on cell context. It is premature to conclude that bantam regulates a completely novel cell growth program, but the fact that misexpression of bantam stimulates cell growth in the absence of changes in a phosphoinositide marker and that chico and TOR fail to suppress the bantam-induced salivary gland-persistence phenotype minimally suggests that this microRNA regulates genes downstream of TOR. Significant progress has been made in the identification of microRNA targets, and future studies should resolve the mechanism underlying bantam regulation of cell growth (Dutta, 2008).

Recent studies of Wts signaling in Drosophila have identified a linear pathway that terminates with Yki and Sd regulation of effector genes that influence cell growth, cell division, and cell death. These studies indicate that the Wts pathway may not always regulate downstream effector genes via Yki and Sd, given that Yki expression was not able to phenocopy the wts mutant salivary gland destruction and expression of Sd induced premature degradation of salivary glands. Although bantam expression is sufficient to induce growth and inhibit cell death in salivary glands, bantam function is not required for the wts mutant phenotype. wts mutant salivary glands possess altered markers of PI3K signaling, and their defect in cell death is suppressed by chico and TOR. Combined, these results indicate that Wts regulates cell growth and cell death via a PI3K-dependent, and Yki- and Sd-independent, mechanism. Future studies will determine whether Wts regulates cell growth in a PI3K-dependent manner in other cells and animals (Dutta, 2008).

Differential requirement of Salvador-Warts-Hippo pathway members for organ size control in Drosophila melanogaster

The Salvador-Warts-Hippo (SWH) pathway contains multiple growth-inhibitory proteins that control organ size during development by limiting activity of the Yorkie oncoprotein. Increasing evidence indicates that these growth inhibitors act in a complex network upstream of Yorkie. This complexity is emphasised by the distinct phenotypes of tissue lacking different SWH pathway genes. For example, eye tissue lacking the core SWH pathway components salvador, warts or hippo is highly overgrown and resistant to developmental apoptosis, whereas tissue lacking fat or expanded is not. This study explores the relative contribution of SWH pathway proteins to organ size control by determining their temporal activity profile throughout Drosophila eye development. Eye tissue lacking fat, expanded or discs overgrown displays elevated Yorkie activity during the larval growth phase of development, but not in the pupal eye when apoptosis ensues. Fat and Expanded do possess Yorkie-repressive activity in the pupal eye, but loss of fat or expanded at this stage of development can be compensated for by Merlin. Fat appears to repress Yorkie independently of Dachs in the pupal eye, which would contrast with the mode of action of Fat during larval development. Fat is more likely to restrict Yorkie activity in the pupal eye together with Expanded, given that pupal eye tissue lacking both these genes resembles that of tissue lacking either gene. This study highlights the complexity employed by different SWH pathway proteins to control organ size at different stages of development (Milton, 2010).

The SWH pathway controls Drosophila eye size by limiting growth during the larval stage of development and by restricting proliferation and promoting apoptosis during pupal development. Eyes lacking core SWH pathway components (e.g. sav, wts or hpo) are significantly larger than eyes lacking the non-core components ft, ex, dco or Mer. Owing to this disparity, it has been hypothesized that ft and ex only partially affect SWH pathway activity, whereas sav, wts and hpo have stronger effects, or, alternatively, that non-core components affect pathway activity in a temporally restricted fashion. Analysis of tissue recessive for ft, ex or dco3 revealed that Yki activity was elevated during larval eye development when tissues are actively growing and proliferating, but not during pupal development when apoptosis ensues, supporting the idea that Ft, Ex and Dco influence SWH pathway activity in a temporally restricted fashion. However, when tissue lacking both Mer and ft, or Mer and ex, was analysed, Yki activity was found to be elevated during both larval and pupal development, similar to the Yki activity profile observed in tissue lacking core SWH pathway proteins. This is consistent with previous reports showing that Mer acts in parallel to both Ft and Ex, and that these proteins can compensate for each other to control SWH pathway activity. Therefore, Ft and Ex do contribute to SWH pathway regulation in the pupal eye to ensure appropriate exit from the cell cycle and developmental apoptosis, but these functions can be executed by Mer in their absence, suggesting a degree of plasticity in the regulation of Yki activity by non-core SWH pathway proteins. The ability of Mer to compensate for Ft or Ex cannot simply be explained by compensatory increases in Mer protein in pupal eye tissues lacking ft or ex, since Mer expression levels were found to be unaltered in these tissues (Milton, 2010).

Previous analyses of tissue lacking both ft and ex showed that these proteins function, at least in part, in parallel to control growth of larval imaginal discs. The current analysis of ft,ex double-mutant tissue suggests that these proteins are likely to function together to control Yki activity in the pupal eye. Yki activity was not elevated in tissue lacking ft, ex or both genes, showing that these genes cannot compensate for each other in the pupal eye. This is consistent with the notion that Ft influences the activity of downstream SWH pathway proteins by multiple mechanisms, an idea that is supported by THE analysis of the requirement of the atypical myosin, Dachs, for Ft signalling in the pupal eye. During larval imaginal disc development, Ft can influence Yki activity by repressing Dachs activity, which in turn can repress the core SWH pathway protein Wts. Analysis of pupal eye tissue that lacks both Mer and ft, or Mer, ft and dachs, showed that Yki activity was elevated in each scenario. This shows that in the pupal eye, the ability of Ft to compensate for Mer is not reliant on Dachs, and implies that Ft can employ different modes of signal transduction throughout eye development. However, because Ft and Mer can compensate for each other it is not possible to formally conclude that normal signal transduction by Ft in the pupal eye occurs independently of Dachs (Milton, 2010).

Expression of Ex is tightly controlled in response to alterations in SWH pathway activity at both the transcriptional and post-transcriptional levels. Interestingly, it was also found that Ex expression is controlled in a temporal fashion throughout eye development; Ex is expressed at relatively high levels in the larval eye, but at very low levels in the pupal eye. Despite the fact that Ex expression is very low in the pupal eye, it clearly retains function at this stage of development because it can compensate for loss of Mer to restrict Yki activity. The dynamic expression profile of Ex suggests that factors that influence its expression play an important role in defining overall eye size in Drosophila. At present, only two transcriptional regulatory proteins have been shown to influence the expression of ex: Yki and Sd. There are conflicting reports on whether Yki and Sd control basal expression of ex in larval imaginal discs. It is clear, however, that Yki and Sd collaborate to drive ex expression when the activity of the SWH pathway is suppressed, presumably as part of a negative-feedback loop. Despite the fact that basal ex expression is low in the pupal eye, the ex promoter is still responsive to Yki, as Ex expression is substantially elevated in pupal eye clones lacking hpo or Mer and ex. Future investigation of the ex promoter will help to clarify understanding of the complex fashion by which expression of the ex gene is controlled, and should aid understanding of eye size specification in Drosophila (Milton, 2010).

This study emphasises the complexity of the means by which the activity of core SWH pathway proteins is regulated by non-core proteins such as Ft, Ex, Mer and Dco. The signalling mechanisms employed by non-core proteins appear to differ at discrete stages of development in order to achieve appropriate organ size during the larval growth period of eye development, and to subsequently sculpt the eye by regulating apoptosis during pupal development (Milton, 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).

Alcohol interacts with genetic alteration of the hippo tumor suppressor pathway to modulate tissue growth in Drosophila

Alcohol-mediated cancers represent more than 3.5% of cancer-related deaths, yet how alcohol promotes cancer is a major open question. Using Drosophila, this study identified novel interactions between dietary ethanol and loss of tumor suppressor components of the Hippo Pathway. The Hippo Pathway suppresses tumors in flies and mammals by inactivating transcriptional co-activator Yorkie, and the spectrum of cancers associated with impaired Hippo signaling overlaps strikingly with those associated with alcohol. Therefore, these findings may implicate loss of Hippo Pathway tumor suppression in alcohol-mediated cancers. Ethanol enhanced overgrowth from loss of the expanded, hippo, or warts tumor suppressors but, surprisingly, not from over-expressing the yorkie oncogene. It is proposed that in parallel to Yorkie-dependent overgrowth, impairing Hippo signaling in the presence of alcohol may promote overgrowth via additional alcohol-relevant targets. Interactions between alcohol and Hippo Pathway over-activation were also identified. It is proposed that exceeding certain thresholds of alcohol exposure activates Hippo signaling to maintain proper growth control and prevent alcohol-mediated mis-patterning and tissue overgrowth (Ilanges, 2013)


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warts: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 December 2015

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