InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously. The salvador (sav) 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 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). This biological overview will deal with the Pantalacci study, while subsequent sections will deal with the other studies.

The final size of each organ in an animal is determined by cell growth and by the balance between proliferation and cell death, which determines cell number. Each of these parameters is controlled and coordinated by developmental cues so that the body parts reach their characteristic sizes and shapes. It remains unclear how cells in a developing organ sense their limits and respond by modulating growth, division and apoptosis. The gene sav (also known as shar-pei) limits organ size in vivo. Initial loss-of function (LOF) analyses have shown that sav is required for restricting cell division rates, for timely exit from the cell cycle and for programmed cell death during Drosophila eye development. In the absence of sav, an increase in cell number generated through excessive proliferation and reduced apoptosis leads to an increase in organ size. Timely cell-cycle exit during Drosophila development is contingent on the downregulation of Cyclin E and cyclin-dependent kinase activity, which is achieved through transcriptional and posttranscriptional events. In sav mutant clones, increases in Cyclin E protein and messenger RNA persist after their disappearance from wild-type cells. Thus, the effect of sav on proliferation can, at least in part, be explained by its ability to repress cellular quantities of Cyclin E. Apoptosis in Drosophila is triggered by developmentally regulated expression of the pro-apoptotic proteins Head involution defective (Hid), Grim and Reaper. These act by sequestering DIAP1 through their inhibitor of apotosis protein (IAP)-binding motif, and also by repressing quantities of DIAP1 protein through various mechanisms. When inactivated, DIAP1 no longer represses upstream caspases such as Dronc, and cell death ensues. In sav mutant clones, Hid- or Reaper-induced cell death is impaired. In addition, DIAP1 protein is increased in sav clones. Thus, sav promotes apoptosis (at least in part) by repressing DIAP1. sav encodes a protein containing two WW domains and a coiled coil, suggesting that it carries out its functions by interacting with partner proteins (Pantalacci, 2003).

Mutations in the warts (wts; also known as Large Tumour Suppressor) Ser/Thr kinase were initially shown to cause tissue overgrowth. Further studies established that the wts mutations phenocopy the sav mutants, suggesting that these genes function in the same pathway. Indeed, Wts and Sav associate biochemically in vitro; however, the role of wts is unclear and provides little insight into sav function (Pantalacci, 2003).

To further understanding of the biochemical mechanisms underlying sav function, the yeast two-hybrid system to was used to search for Sav binding partners. One of the recovered clones encoded the carboxy-terminal 67 amino acids of the Drosophila ortholog of the mammalian Ste20-like kinases MST1 and MST2 (MST1/2); the Drosophila protein is referred to as Hippo. When expressed in Drosophila S2 tissue-culture cells, epitope-tagged Sav and Hpo co-imunoprecipitate, confirming that these two proteins associate in Drosophila cells. In addition, epitope-tagged Hpo co-precipitate with Wts, the other Sav partner kinase (Pantalacci, 2003).

RNA-mediated interference (RNAi) was used to analyse the hpo LOF phenotype. Transgenic animals were generated bearing a fragment of the hpo complementary DNA in the pSympUAS12 vector to drive the expression of a double-stranded RNA under the control of the Gal4 transcription factor. Large hpo RNAi clones marked with green fluorescent protein (GFP) were induced by using the flp-out GAL4 technique, and the consequences were examined of hpo LOF on tissue growth. hpo inactivation induces an increase in tissue and organ size, leading, for example, to oversized halteres, or to an overgrowth of imaginal discs, the epithelial structures that give rise to adult body parts. Thus, like sav, hpo restricts tissue and organ growth in vivo (Pantalacci, 2003).

The expression pattern of hpo mRNA was examined by in situ hybridization; hpo is expressed uniformly in larval eye imaginal discs. To test whether hpo is required for cellular differentiation, eye discs stained for the neuronal marker Elav were examined. Like sav mutations, hpo LOF does not impair neuronal differentiation. Cells in the eye imaginal disc normally proliferate actively during early larval development in the first mitotic wave, and then temporarily arrest in the morphogenetic furrow where differentiation begins. After the passage of the morphogenetic furrow, a subpopulation of cells undergoes one additional round of divisions (the second mitotic wave) before permanently arresting. BrdU incorporation and Cyclin E staining were used to monitor S phases in larval eye imaginal disc. In hpo RNAi clones, cells fail to arrest in the morphogenetic furrow and, after the second mitotic wave, undergo additional rounds of division, similar to sav mutant cells. As observed for sav mutants, hpo mutant cells fail to downregulate Cyclin E. Conversely, transgenic animals were generated to express hpo ectopically under the control of the GAL4 promoter. When overexpressed in clones, hpo prevents cells from entering the second mitotic wave (Pantalacci, 2003).

Apoptosis was examined in hpo RNAi clones. Overexpression of the proapoptotic protein Hid in the eye by using the GMR promoter potently induces apoptosis, which is blocked by the caspase inhibitor p35 or by sav mutations. GMR-hid-induced apoptosis, as assayed by TdT-mediated dUTp nick end labelling (TUNEL) staining, is also blocked in hpo RNAi clones. In addition, DIAP1 expression is increased in hpo RNAi clones in a wild-type background, as has been observed in sav clones. Conversely, ectopic expression of hpo potently induces apoptosis. Apoptosis triggered by hpo overexpression is not impaired in sav mutant clones, suggesting that hpo overexpression can bypass the absence of sav. Thus, hpo inactivation phenocopies sav LOF, suggesting that in vivo the two genes act in concert to control apoptosis and proliferation (Pantalacci, 2003).

MST1/2, like most Ste20 family members, are potent activators of the JNK pathway (Dan, 2001). The ability of Hpo to trigger Drosophila JNK (DJNK; Basket) activation was measured by using an enhancer trap inserted in the DJNK target gene puckered (puc). Overexpression of Hpo in the eye robustly activates the puc-lacZ reporter gene. The MAPK pathway has been reported to protect cells from apoptosis by downregulating hid function in the Drosophila eye. Using an antibody against phosphorylated MAPK, it was found that Hpo overexpression neither represses nor activates MAPK. Thus, like its mammalian counterparts, Hpo can activate JNK but not the MAPK pathway in vivo (Pantalacci, 2003).

Numerous studies have shown that the JNK pathway can participate in the apoptotic process in Drosophila and mammals. The possibility exists that Sav and Hpo might act through DJNK to promote apoptosis. Therefore, apoptosis markers were examined in clones mutant for basket (bsk), which encodes the only Drosophila JNK. Quantities of DIAP1 were found to be normal in bsk mutant cells. Accordingly, developmental apoptosis in the pupal retina, which is blocked in sav clones, is unimpaired in bsk mutant clones. Finally, apoptosis triggered by combined overexpression of Sav and its partner Wts in the eye is not affected by bsk mutations. These data suggest that, at least in the eye, sav and hpo induce apoptosis through a DJNK-independent pathway (Pantalacci, 2003).

A common aspect of sav and hpo function seems to be the downregulation of DIAP1. This protein, like other IAP family members, is a RING-finger protein that directly binds to and inhibits upstream caspases, such as Dronc, and promotes their ubiquitination. DIAP1 quantities seem to be crucial in the apoptosis-triggering pathway and are therefore tightly regulated through distinct mechanisms controlled by the pro-apoptotic proteins Hid, Grim and Reaper, as well as by the caspases. (1) DIAP1 is highly unstable and is targeted for degradation by auto-ubiquitination as well as by at least two other ubiquitin ligases, Morgue and UbcD1. (2) Grim and Reaper repress the translation machinery, which primarily affects unstable proteins such as DIAP1. DIAP1 is also cleaved by the downstream caspases Drice and Dcp-1, which expose an N-terminal asparagine and make DIAP1 susceptible to degradation by the N-end rule pathway. It was therefore considered that the Hpo and Sav regulation of DIAP1 might be crucial to their pro-apoptotic function. Indeed, Hippo was shown to phosphorylate and decreases the stability of DIAP1 (Pantalacci, 2003).

Therefore, Hippo, a partner of the Sav, controls cell proliferation and apoptosis during Drosophila development. sav and hpo share similar LOF phenotypes, suggesting that they function in the same pathway. Downregulation of Hpo or Sav in vivo leads to an upregulation of DIAP1. Evidence in cultured cells is provided that Hpo-dependent phosphorylation of DIAP1 can occur, and that this phosphorylation is correlated with a reduction in DIAP1 stability. Further work will be required to determine whether DIAP1 phosphorylation is responsible for its destabilization; however, the results do not exclude additional modes of DIAP1 regulation by Hpo. Hpo interaction with Sav results in Sav phosphorylation and stabilization. However, because kinase-dead Hpo still elicits these effects, Hpo may not phosphorylate Sav directly. Thus, Hpo may recruit a kinase that phosphorylates Sav and stabilizes it. Alternatively, Hpo binding may prevent Sav degradation, leading to an increase in Sav levels that is independent of its phosphorylation. Because Sav may function in a complex with Hpo, this might constitute a positive feedback mechanism. Since Sav and Hpo have well-conserved mammalian orthologs, it will be interesting to investigate whether these orthologs trigger apoptosis by regulating IAPs. Sav, Hpo and Wts seem to form the basis of a previously unknown tissue-growth-suppressing pathway in flies, and the study of their mammalian orthologs will probably provide valuable insight into tumor formation (Pantalacci, 2003).

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

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

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

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

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

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

Rap1 negatively regulates the Hippo pathway to polarize directional protrusions in collective cell migration

In collective cell migration, directional protrusions orient cells in response to external cues, which requires coordinated polarity among the migrating cohort. However, the molecular mechanism has not been well defined. Drosophila border cells (BCs) migrate collectively and invade via the confined space between nurse cells, offering an in vivo model to examine how group polarity is organized. This study shows that the front/back polarity of BCs requires Rap1, hyperactivation of which disrupts cluster polarity and induces misoriented protrusions and loss of asymmetry in the actin network. Conversely, hypoactive Rap1 causes fewer protrusions and cluster spinning during migration. A forward genetic screen revealed that downregulation of the Hippo (Hpo) pathway core components hpo or mats enhances the Rap1^V12-induced migration defect and misdirected protrusions. Mechanistically, association of Rap1^V12 with the kinase domain of Hpo suppresses its activity, which releases Hpo signaling-mediated suppression of F-actin elongation, promoting cellular protrusions in collective cell migration (Chang, 2018).

How cells induce group polarity to form directional protrusions is a long-standing and intensively studied question in collective cell migration. In particular, which molecular mechanism induces individual cells to compete for the leading position or to communicate with other members of the migratory cluster has yet to be established. The finding that a Rap1 and Hpo complex polarizes actin protrusion provides a mechanism underlying group polarity in collective cell migration. First, the polarity of front end-enriched actin protrusions and inward contraction directed by non-muscle myosin II are continuously retained during migration. Rap1 hyperactivity disrupts the front/rear polarity of border cells (BCs), resulting in excessive misoriented protrusions. Furthermore, individual cells expressing Rap1^V12 gain an advantage in protrusion extension and take the lead in the migrating cluster, whereas Rap1^N17-expressing cells behave in the opposite way. As a consequence, BCs compete to move forward, making the cluster migrate in that direction. Thus, upon higher Rap1 activity, the 'winner' cell extends protrusions and moves to the front, whereas the others, having lower activity, are outcompeted and lag behind. Second, a forward genetic screen revealed that hypoactivity of Hpo signaling enhances the Rap1^V12-induced migration defect and protrusion numbers in either the backward or forward direction. Most intriguingly, pull-down assays illustrate a strong physical interaction between Rap1^V12 and Hpo protein, which significantly reduces Hpo activity. Suppression of Hpo signaling has long been demonstrated to cause abnormal F-actin polymerization, which, in turn, has been linked to enormous organ size in vertebrates and invertebrates. Manipulation of the actin cytoskeleton or stiffness of the extracellular matrix also affects nuclear localization of YAP (Yes-associated protein; i.e., the mammalian homolog of Yki), which supports the role of Hpo signaling in sensing the actin architecture or local environment. Therefore, the results link Rap1 to the Hpo pathway and unfold a mechanism through which group polarity and protrusions are organized by attenuating Hpo signaling activity at the leading edge of a migrating cluster (Chang, 2018).

In general, actin-based extension occurs at the front of motile cells and, subsequently, is followed by myosin II-mediated contraction, resulting in rhythmic cycles during locomotion. In external cue-directed cell movement, chemotaxis molecules bind to membrane receptors to activate Rho GTPase and its downstream effectors, which interact with the Arp2/3 complex to initiate actin polymerization. Previous studies in the social ameba Dictyostelium discoideum uncovered that, in response to chemoattractant stimulation, spatially activated Rap1 binds to RacGEF1 at the front of cells to activate Rac1-dependent actin polymerization. Accumulated evidence suggests that, during single cell migration, Rap1 is a vital part of the polarity system that accounts for the asymmetric actin cytoskeleton. However, in terms of group migration, whether Rap1 plays the same role had not been investigated until this study, in which the conserved function of Rap1 in polarizing individual cells to move during collective migration was demonstrated (Chang, 2018).

Hpo signaling was first identified as being responsible for cell growth in Drosophila, but it has since been shown to be conserved in humans and to be pivotal in various fundamental aspects of biology, including embryonic development, tissue homeostasis, and disease. Intensive genetic screens and biochemical studies have explored the complexity of the Hpo network, but the mechanism of Hpo regulation remains less understood. The Hpo pathway can be initiated by phosphorylation of the activation loop of Hpo/MST, which is accomplished by transphosphorylation by Tao-1/TAO kinase or autophosphorylation via dimerization of the SARAH domains. Active Hpo/MST2 subsequently autophosphorylates its linker domain to create docking sites for Mats/Mob1, whose interaction with Hpo/MST2 enables it to relay the kinase cascade to Warts/large tumor suppressor kinases 1/2 (LATS1/2). The current results suggest a role for Rap1 as a Hpo signaling suppressor, impairing Hpo activation by binding to the Hpo kinase domain. It would be of great interest to investigate whether a similar mechanism operates in other systems, such as cancer metastasis, and which signals/stimuli activate Rap1 in such contexts (Chang, 2018).

In Drosophila, Hpo is activated by multiple upstream scaffold proteins such as Kib, Mer, and Ex, but the screen did not reveal any of them to have genetic interaction with Rap1 in BCs. This study also examined whether Rap1^V12 affects Yki transcriptional activity using a well-characterized reporter (ex-lacZ) but no alteration in BCs was observed even in combination with one hpo mutant allele. In fact, neither wild-type nor constitutively active Yki alone delayed migration. In light of this work and other independent research showing that depletion of yki does not affect BC migration, it is concluded that Rap1 functions in the Hpo pathway independent of yki to induce biased protrusions and facilitate BC migration. Interestingly, although yki has no role in normal BC migration, a strong genetic interaction was observed between Rap1^V12 and wild-type or hyperactive yki, which only arose in double gain-of-function scenarios. This finding may represent a potential mechanism by which Yki/YAP participates in Rap1-mediated migration in an oncogenic context (Chang, 2018).

Over the past decade, several upstream regulators of Hpo signaling have been identified, including adhesion junction proteins, actin-binding proteins, molecules determining apical/basal/planar cell polarity, and proteins involved in cell matrix attachment. Most of these regulators are not required to regulate the core kinase activity of Hpo through Kib, Mer, or Ex. For example, loss of capping protein (which restricts actin polymerization) leads to a reduction of Hpo signaling activity and tissue outgrowth. F-actin-destabilizing treatment in NIH 3T3 cells enhances Mst1 activity, which is sufficient to stabilize p21, a key cell cycle regulator. This evidence relating to actin cytoskeleton integrity and cell morphology has led to Hpo signaling being proposed as a mechanosensor for monitoring the local environment. However, it remains unclear whether Hpo core kinase components are directly regulated by actin architecture or whether additional adaptor proteins, such as actin-binding proteins or membrane-associated proteins, are required to transduce mechanical cues. Rap1 has been implicated in E-cadherin-dependent morphogenesis and integrin-mediated cell attachment in several tissues. The results might imply that the stress generated from BC invasion through nurse cells can be transduced to interact with Rap1 through the membrane-tethering cadherin complex, the integrin/focal adhesion complex, or even a membrane-associated protein, suppressing Hpo activity and promoting protrusion (Chang, 2018).

Rap1 has been reported to interact with the mammalian Hpo, Mst1, through RapL; i.e., the downstream effector of Rap1 that regulates T lymphocyte polarization and migration. Both these latter processes require the kinase activity of Mst1, activation of which relies on recruitment of this protein to the leading edge by Rap1 and RapL. Combined with the evidence presented in this study, the association of Rap1 with Hpo appears to be an evolutionarily conserved mechanism for advancing cellular motion, but the effect of Rap1 on Hpo/MST1 varies in different contexts. This study reveals a role for Rap1; rather than acting as a positive regulator of Hpo, it inhibits Hpo suppression of the actin polymerization involved in cell cluster migration (Chang, 2018).

In summary, this study provides cellular and molecular insights into how Rap1 regulates directional protrusions to achieve collective cell movement. In BCs, the intermolecular autophosphorylation essential for Hpo activation is hampered by its association with activated Rap1, which suppresses Hpo signaling activity and, thereby, promotes cellular protrusions (Chang, 2018).

STRIPAK-PP2A regulates Hippo-Yorkie signaling to suppress retinal fate in the Drosophila eye disc peripodial epithelium

The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. This study identified Protein Phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice.These factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hpo kinase, and consequently its substrate Wts, to release the transcriptional co-activator Yki into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate (Neal, 2020).

Broad expression of the Pax6-type retinal determination factors Eyeless and Twin-of-eyeless initially prime the disc to give rise to the eye primordium. This event is closely linked to the acquisition of distinct morphological characteristics between the facing portions of the disc epithelium. Cells on one side of the epithelium adopt a columnar morphology and begin to express the retinal determinant Eyes absent (Eya), thus initiating eye formation; cells on the apposed side adopt the squamous and cuboidal morphology typical of the peripodial epithelium (PE). This binary choice between PE and retina developmental potential is a first step in a complex regional specification program that progresses in larvae over a 4-day period while the disc epithelium grows from fewer than 100 cells to more than 50,000 cells (Neal, 2020).

While the development of the eye, from primordium establishment to differentiation of the various retinal cell types, has been extensively studied, much less is known about PE formation. During metamorphosis, the PE is necessary for the morphogenetic events that transform a pair of apparently simple epithelia into the complex fly head. However, there is evidence that PE is more broadly required for the development of eye-antennal disc-derived organs, in particular the retina. In fact, the PE is essential for cellular growth in the eye primordium and for retinal neurogenesis; for example, PE ablation during photoreceptor neuron formation results in an arrest of retinogenesis. Notwithstanding these important functions of the PE, very little is known about the genes and molecular mechanisms that specify PE fate. This study has focused on the initial step, when retina potential is fostered within a portion of the eye disc epithelium but suppressed elsewhere, in favor of PE formation (Neal, 2020).

Heterotrimeric protein phosphatase 2A (PP2A) holoenzymes are an abundant group of cellular phosphatases that are highly conserved across Metazoa. They consist of A, B and C subunits; the catalytic (C) subunit and adaptor subunit (A) are each encoded by unique genes in Drosophila (mts and PP2A-29B, respectively). In contrast, the interchangeable substrate-specifying and activity-gating B subunits are divided into four families (B, B', B'' and B''') and are encoded by numerous genes in flies and vertebrates. Cka encodes the sole atypical B''' subunit that is orthologous to the mammalian striatins. PP2A holoenzymes containing Cka are found in the striatin-interacting phosphatase and kinase (STRIPAK) complex, which also includes other scaffolding and substrate-associated factors such as Strip. The contribution of this major cellular phosphatase to developmental fate decisions is yet to be determined (Neal, 2020).

This study demonstrates that STRIPAK-PP2A is essential for PE cell fate specification in the Drosophila eye disc. Loss of function of either Cka or Strip results in the emergence of an ectopic retinal field within the presumptive PE. STRIPAK-PP2A acts by negatively regulating the activity of the Hippo kinase (Hpo) and its effector Warts (Wts), thereby releasing the PE-promoting activity of the transcriptional co-activator Yorkie (Yki). Thus, this study elucidates the genetic pathway through which STRIPAK-PP2A promotes PE fate and provide a first example of a STRIPAK-PP2A-controlled cell fate decision in metazoan development (Neal, 2020).

The results demonstrate a clear role for STRIPAK-PP2A in regulating the adoption of distinct cell fates in the eye disc. PE was absent when Cka function was reduced, and instead the development of a mirror-image ectopic retina was detected within the presumptive PE cell layer. Similar phenotypes were observed when the loss of function of the STRIPAK-PP2A subunits Strip and SLMAP were induced. Furthermore, a dominant enhancement was observed of the Cka RNAi phenotype by two different loss-of-function alleles of the PP2A catalytic subunit, Mts. In all tested cases, including temperature modulation and the co-expression of pro-proliferation and anti-apoptotic factors, changes in disc size did not correlate with the PE-to-retina transformation and development of the ectopic retinal field proceeded in an apparently normal pattern (Neal, 2020).

Interestingly, PE fate was restored by the removal of one wild-type copy of hpo in Cka- or SLMAP-deficient discs, and by hpo RNAi in Strip-deficient discs, strongly suggesting that the latter factors act genetically upstream of Hpo. Given the direct roles of Strip and SLMAP in the recruitment of Hpo as a PP2A substrate in cell culture, the findings strongly point to the Hpo kinase as the target of negative regulation by STRIPAK-PP2A in the control of PE cell fate (Neal, 2020).

Furthermore, the loss-of-Cka phenotype was also suppressed by genetic reduction of Wts and by overexpression of Yki. As the Wts kinase is a direct intermediate between Hpo and Yki, this evidence links STRIPAK-PP2A to the eye disc PE fate-promoting factor Yki. Therefore, it is postulated that during the induction of PE fate, high levels of Yki activity are maintained in the presumptive PE via the negative regulation of the Hpo-Wts kinase axis by a STRIPAK-PP2A complex containing Cka, Strip and SLMAP. Notably, the ubiquitous role of the Hpo-Yki signaling axis in regulating tissue size appears to be in parallel to, and genetically separable from, its role in determining PE fate. Hence, STRIPAK-PP2A represents one effector of the developmental regulation of Yki activity in fate specification (Neal, 2020).

PE fate specification was first characterized in the wing imaginal disc, where the factor Bowl is a driver of PE fate. In addition, ectopic activation of Wg signaling or expression of Lines within the presumptive PE domain suppress PE fate by repressing the accumulation of Bowl (Neal, 2020).

Previous work demonstrated that the transcription co-activator Yki, with its DNA-binding partners Sd and Hth, appeared to be necessary for the establishment of PE in the eye disc, based on phenotypes induced by RNAi-mediated gene silencing (Zhang, 2011). This was confirmed by showing that ykiB5/ykiB5 mutant eye discs generated by MARGE are nearly identical to yki-RNAi discs, and also demonstrate that Yki's role in the PE-retina cell fate choice is independent of its nearly universal role in proliferation and cell survival (Neal, 2020).

Interestingly, these mechanisms of PE versus non-PE fate specification are not conserved between the eye and wing discs. Whereas a similar role for Wg has been proposed for the leg and haltere PE, based on the induction of columnar cell morphology by ectopic Wg in the PE, the same effect is not observed in the eye antennal disc. Lines overexpression within the eye disc PE also has no effect on cell fate, whereas in the retinal progenitor region of the disc, Lines repression of Bowl initiates precocious eye morphogenesis. Thus, Wg, Lines and Bowl do not appear to be required for PE specification in the eye disc. Conversely, extensive analyses of Yki and Sd function in the wing imaginal disc have not revealed roles for Yki in the PE versus wing-proper fate choice, consistent with the specific role for Yki in suppressing ectopic retina formation that is documented in this study (Neal, 2020).

Thus, the acquisition of PE fate results from different genetic determinants in the eye and wing discs, and is mechanistically distinct (Neal, 2020).

Given the plethora of cellular kinases that regulate myriad biological processes, it has been appreciated that cellular phosphatases might play equally important and diverse biological roles. However, there are far fewer cellular phosphatases, and each is known to target a broad range of cellular substrates. PP2A is no exception to this; it is essential for cell proliferation and viability. Hence, it is difficult to investigate potential roles of this phosphatase in more specific biological processes, such as cell fate specification. This paper reports the involvement of STRIPAK-PP2A in regulating Hippo-Yki signaling in the PE fate choice within the developing eye disc. This role of STRIPAK-PP2A was revealed by targeted silencing of complex-specific PP2A co-factors (Cka-B'', Strip, SLMAP), and the link to PP2A was confirmed by genetic interaction testing using alleles of the vital catalytic subunit (mts) (Neal, 2020).

Few studies in metazoans have identified contexts in which PP2A complexes may play a role in fate specification during development. Screens in Drosophila and Caenorhabditis elegans have identified mutant alleles of fly mts (PP2A-C) and worm sur-6 (a PP2A-B-proper subunit) as modifiers of the MAPK pathway in the R7 photoreceptor and vulval fate choices, respectively. In the fly, mts mutant alleles dominantly enhanced the effects of activated Ras signaling and dominantly suppressed the effects of activated Raf signaling, whereas in the worm, sur-6 mutant alleles dominantly suppressed the effects of activated Ras signaling. Yet, homozygous loss-of-function conditions for these PP2A components could not be shown to affect either fate in an otherwise wild-type background (Neal, 2020).

Two additional phenotypes suggestive of potential changes in cell fate have also been linked to loss-of-function alleles of tws, which encodes the sole Drosophila PP2A-B-proper subunit. First, loss of tws led to the overgrowth and duplication of the pouch in the developing wing imaginal disc, and second, hypomorphic alleles of tws were shown to yield flies with atypical sensory bristles that display supernumerary shaft and socket cells with coincident loss of their single neuronal and sheath cells. However, neither study substantiated a true switch in cell fate vis-à-vis aberrant proliferation or reorganization of patterning cues, nor did they establish a connection between the PP2A catalytic subunit and the putative fate-switch phenotypes. The molecular substrates of PP2A-Tws also remain to be determined in these contexts. Nonetheless, these data hint at the possibility that specific PP2A holoenzymes, STRIPAK-PP2A and PP2A-Tws/Sur-6, may direct different cell fate choices and do so via the modulation of distinct signaling pathways (Neal, 2020).

Whereas there is a lack of evidence in the literature for PP2A- and/or STRIPAK-regulated fate decisions, Hippo (MST1/2) signaling and Yki (YAP/TAZ) have been implicated in fate decisions in different developmental contexts. Arguably the best studied example also involves the Drosophila eye, where Hpo-Yki-Sd signaling has been shown to instruct the choice between Rh5- versus Rh6-expressing R8 photoreceptor subtypes (Neal, 2020).

Perhaps more intriguing is that, during the development of the vertebrate eye, a fate paradigm strikingly similar to the PE/retina fate choice has been described to involve the Yki orthologs YAP and TAZ. In vertebrates, the primordia of the retina and the retinal pigment epithelium (RPE) are specified at the optic vesicle stage, when the vesicle epithelium is partitioned into two fields, one that generates neural retina and one that develops into non-neural RPE in the optic cup. Miesfeld and colleagues demonstrated that YAP and its paralog TAZ play important roles in promoting RPE fate in zebrafish, as loss of these factors leads to transdifferentiation of RPE into retina-like cells (Miesfeld, 2015). A similar role for YAP in RPE fate specification was subsequently demonstrated in mice (Kim, 2016), where loss of YAP produces optic cups with mirror-image retinae, consisting of a pseudostratified epithelium that expresses markers of retinal progenitor cells in the presumptive RPE (Neal, 2020).

The contribution of STRIPAK-PP2A to cell fate decisions in these models has not been investigated. However, given the similar fate-determining roles for Yki and YAP/TAZ, particularly in the latter example, it will be of great interest to determine whether STRIPAK-PP2A-mediated regulation of YAP and/or TAZ activity through the MST2 (Hpo)-LATS1/2 (Wts) kinase cascade is equally important in the patterning of the vertebrate optic vesicle, and possibly in other Yki/YAP/TAZ-linked cell fate decisions (Neal, 2020).

REGULATION

The Hippo pathway as a target of the Drosophila DRE/DREF transcriptional regulatory pathway

The DRE/DREF transcriptional regulatory system has been demonstrated to activate a wide variety of genes with various functions. In Drosophila, the Hippo pathway is known to suppress cell proliferation by inducing apoptosis and cell cycle arrest through inactivation of Yorkie, a transcription co-activator. This study found that half dose reduction of the hippo (hpo) gene induces ectopic DNA synthesis in eye discs that is suppressed by overexpression of DREF. Half reduction of the hpo gene dose reduced apoptosis in DREF-overexpressing flies. Consistent with these observations, overexpression of DREF increased the levels of hpo and phosphorylated Yorkie in eye discs. Interestingly, the diap1-lacZ reporter was seen to be significantly decreased by overexpression of DREF. Luciferase reporter assays in cultured S2 cells revealed that one of two DREs identified in the hpo gene promoter region was responsible for promoter activity in S2 cells. Furthermore, endogenous hpo mRNA was reduced in DREF knockdown S2 cells, and chromatin immnunoprecipitation assays with anti-DREF antibodies proved that DREF binds specifically to the hpo gene promoter region containing DREs in vivo. Together, these results indicate that the DRE/DREF pathway is required for transcriptional activation of the hpo gene to positively control Hippo pathways (Vo, 2014: PubMed).

Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis

Merlin, the protein product of the Neurofibromatosis type-2 gene, acts as a tumour suppressor in mice and humans. Merlin is an adaptor protein with a FERM domain and it is thought to transduce a growth-regulatory signal. However, the pathway through which Merlin acts as a tumour suppressor is poorly understood. Merlin, and its function as a negative regulator of growth, is conserved in Drosophila, where it functions with Expanded, a related FERM domain protein. Drosophila Merlin and Expanded are shown to be components of the Hippo signalling pathway, an emerging tumour-suppressor pathway. Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs. Genetic and biochemical data place Merlin and Expanded upstream of Hippo and identify a pathway through which they act as tumour-suppressor genes (Hamaratoglu, 2006).

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).

bantam is a target of the hippo tumor-suppressor pathway

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

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

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

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

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

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

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

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

The 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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Drosophila PI4KIIIalpha is required in follicle cells for oocyte polarization and Hippo signaling

In a genetic screen mutations were isolated in CG10260, which encodes a phosphatidylinositol 4-kinase (PI4KIIIalpha). PI4KIIIalpha was found to be is required for Hippo signaling in Drosophila ovarian follicle cells. PI4KIIIalpha mutations in the posterior follicle cells lead to oocyte polarization defects similar to those caused by mutations in the Hippo signaling pathway. PI4KIIIalpha mutations also cause misexpression of well-established Hippo signaling targets. The Merlin-Expanded-Kibra complex is required at the apical membrane for Hippo activity. In PI4KIIIalpha mutant follicle cells, Merlin fails to localize to the apical domain. This analysis of PI4KIIIalpha mutants provides a new link in Hippo signal transduction from the cell membrane to its core kinase cascade (Yan, 2011).

DV asymmetry of the Drosophila oocyte is established during mid-oogenesis through a repolarization process initiated in the posterior follicle cells PFCs. In response to an unknown signal from the PFCs the oocyte nucleus migrates from the posterior end to the dorsal-anterior corner of the oocyte. As a consequence, the Gurken (Grk) protein no longer accumulates at the posterior cortex of the oocyte, but is now found in the dorsal-anterior membrane overlying the oocyte nucleus where it activates EGFR to initiate DV patterning. In a genetic screen directed at FC components affecting this repolarization process, a complementation group with six lethal mutant alleles was isolated, and initially named after a representative allele, GS27. When the PFCs were mutant for the GS27 gene product, the oocyte nucleus frequently remained at the posterior end of the oocyte. This phenotype was confirmed by the abnormal posterior localization of Grk in late egg chambers (Yan, 2011).

The lethality of the GS27 complementation group was mapped through duplication and deficiency mapping to the X-chromosomal region 3A4-3A8, which contains 16 genes. Sequencing of candidate genes showed that four alleles of the GS27 complementation group contained mutations that lead to premature stop codons in the coding region of CG10260, a predicted phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinases (PI4Ks) catalyze the generation of PIP₄. Phosphoinositides, including PIP₄, are important phospholipids in the cell membrane that participate in numerous signaling events. Four classes of PI4Ks have been identified in mammalian cells that localize to different cellular compartments and are likely to perform non-redundant functions. Three PI4K genes have been annotated in the fly genome: four wheel drive (fwd; PI4KIIIbeta), CG2929 (PI4KIIalpha) and CG10260 (PI4KIIIalpha) (Yan, 2011).

To investigate the oocyte polarization defects caused by PI4KIIIalpha mutations, the localization of well-established oocyte polarity markers was examined. The microtubule cytoskeleton is polarized in the oocyte. The microtubule plus-end marker Kinesin (Kin, or Khc) fused to β-gal (Kin-β-gal), which normally forms a crescent at the posterior of the oocyte after stage 8, was examined. When the PFCs were mutant for PI4KIIIalpha, Kin-β-gal either localized to the center of the oocyte or was diffuse in the oocyte. Staufen localizes to the posterior pole of wild-type oocytes after stage 8 and is required for the localization of maternal RNAs. In PFC clones mutant for PI4KIIIalpha, Staufen also frequently mislocalized to the center of the oocyte or became dispersed in the oocyte. Therefore, in combination with the mislocalization of the oocyte nucleus, these results demonstrate that PI4KIIIalpha is required in the PFCs for all aspects of the establishment of correct oocyte polarity (Yan, 2011).

Oocyte polarization relies on the integrity of four signaling pathways in the PFCs: Notch, JAK/STAT, EGFR and Hippo. To examine whether the polarization defect observed in PI4KIIIalpha mutants was caused by disruption of one of these signaling pathways, well-established downstream targets of each pathway were examined in PI4KIIIalpha mutants (Yan, 2011).

The EGFR signaling reporter kekkon-lacZ (kek-lacZ) is highly expressed in the PFCs at stage 7 and 8 as a result of EFGR activation by Grk. In PFCs mutant for PI4KIIIalpha, the kek-lacZ expression level was comparable to that of wild-type PFCs, indicating that EFGR signaling was unaffected. The JAK/STAT signaling reporter 10×STAT92E-GFP is normally turned on in the PFCs during stage 7 and 8 in response to JAK/STAT activation. Apparently normal levels of GFP were detected in the nuclei of PI4KIIIalpha mutant PFCs, suggesting that JAK/STAT signaling was also intact (Yan, 2011).

Notch signaling is required for FCs to exit the mitotic cell cycle at stage 6 and switch to an endocycle. PI4KIIIalpha mutant PFCs maintained a mitotic cell cycle after stage 6, as indicated by the sustained staining of the mitotic marker phosphorylated Histone H3 (PH3), which is only seen up to stage 6 in wild-type FCs. Consistent with a failure to exit the mitotic cycle, the PI4KIIIalpha mutant PFCs often lost their monolayered epithelial structure and had smaller nuclei than neighboring cells. The expression of two Notch signaling targets, Cut and Hindsight (Hnt; Pebbled) was examined. In wild-type FCs, Cut expression is downregulated whereas Hnt expression is upregulated upon Notch activation at stage 6. PI4KIIIalpha mutant PFCs frequently failed to downregulate Cut and upregulate Hnt expression. Interestingly, PI4KIIIalpha mutant cells on the lateral side of the egg chambers showed no defect in Notch signaling. These results suggest that PI4KIIIalpha mutations compromise Notch signaling in the PFCs only (Yan, 2011).

The phenotypes described above are similar to those caused by mutations in Hippo pathway components. In particular, the observation that only PFCs appear affected is characteristic of mutations in the Hippo pathway, which are reported to affect Notch signaling only in this group of FCs. When the expression of a Hippo pathway target, ex, was checked using the enhancer trap line ex-lacZ, a much higher level of β-galin PI4KIIIalpha mutant FCs was detected than in wild-type cells. This upregulation was observed in all FCs, regardless of their position. Another Hippo pathway target, Diap1, monitored with the enhancer trap line diap1-lacZ, was mildly upregulated in the PI4KIIIalpha mutant FCs. These results indicate that the polarization defect in the PI4KIIIalpha mutants is likely to be caused by defective Hippo signaling (Yan, 2011).

Multiple lines of evidence suggest that the apical localization of the Expanded-Merlin-Kibra complex is crucial for Hippo signaling activity as it is proposed to function as a platform to bring the core Hippo components into close proximity and facilitate the phosphorylation reactions. In addition, it has been reported that Expanded directly interacts with Yki and functions to sequester Yki in the cytoplasm (Yan, 2011).

To investigate how mutations in PI4KIIIalpha lead to defective Hippo signaling, the apical localization of the Merlin-Expanded-Kibra complex was exmined. The complex is confined to the apical domain in wild-type FCs. In the PI4KIIIalpha mutant cells, a loss of apical Merlin staining was observed, whereas Expanded and Kibra were upregulated at the apical membrane. In addition to being Hippo pathway regulators, Expanded and Kibra are also targets of the Hippo signaling pathway. Mutations in Hippo pathway components lead to upregulation of Expanded and Kibra. In addition, it has been reported that the apical sorting of Merlin, Expanded and Kibra occur independently of each other. Therefore, the absence of Merlin from the apical membrane in PI4KIIIalpha mutant cells is the likely cause of the signaling defect, and the upregulation of Expanded and Kibra would be an expected secondary consequence of the disrupted Hippo signaling (Yan, 2011).

When PI4KIIIalpha mutant clones were examined in the imaginal eye discs of early second instar larvae, an absence of Merlin from the apical and junctional region was observed. However, no overgrowth phenotype typical of Hippo pathway mutations was observed. In fact, adults with mutant eye clones had smaller eyes than wild-type adults. Eye discs from late L2 larvae exhibited pyknotic nuclei staining in PI4KIIIalpha mutant clones, indicating death of the mutant cells (data not shown) (Yan, 2011).

Multiple classes of PI4Ks exist in eukaryotic cells that participate in producing various phosphoinositide species in distinct cellular compartments. Three PI4K genes have been annotated in the fly genome. When the intracellular distribution and level of PIP₂ was examined using a Ubi-PH-PLCδ-GFP reporter, a complete absence of PIP₂ from PI4KIIIalpha mutant FCs was observed in rare cases. In most cases, the PIP₂ reporter was specifically lost from the apical plasma membrane in the mutant cells. The yeast homolog of PI4KIIIalpha, Stt4p, localizes to patches on the plasma membrane where it is required for normal actin cytoskeleton organization. When the actin cytoskeleton of PI4KIIIalpha mutant FCs was examined by phalloidin staining, they exhibited abnormal actin-enriched spike structures on their apical domain that were positively marked by the microvillus marker Cad99C, suggesting that the spikes were malformed microvilli. As mutations in the Hippo pathway have been reported to lead to apical domain expansion, one possibility is that the malformed microvilli are caused by defective Hippo signaling. However, the morphology of the actin-enriched spikes in PI4KIIIalpha mutant cells is distinct from that caused by mutations in the Hippo pathway, suggesting that the loss of PI4KIIIalpha might also have a Hippo-independent effect on apical membrane structure (Yan, 2011).

How could PI4KIIIalpha mutations cause Merlin mislocalization? Expanded and Merlin are ERM (Ezrin, Radixin and Moesin)-related proteins, which are key linkers of the plasma membrane and cytoskeleton. Classical ERM proteins bind to PIP₂ in the membrane to switch from a closed to an open conformation for their activation. Significantly, in PI4KIIIalpha mutant cells, phosphorylated ERM proteins were absent from the apical microvilli region as indicated by a phospho-ERM-specific antibody. The malformed microvillus structure might therefore indicate a general failure of ERM protein activation in the PI4KIIIalpha mutant cells. For Merlin, the closed conformation is the active form, opposite to other ERM proteins. Nevertheless, Merlin undergoes a similar conformational switch to the other ERM proteins and contains an ERM PIP₂-binding site. Given these observations, it is possible that PIP₂ binding activates and/or stabilizes Merlin in the apical membrane, and a depletion of this lipid species due to the absence of PI4KIIIalpha might directly lead to the loss of Merlin (Yan, 2011).

In summary, this study has shown that PI4KIIIalpha is required in the FCs for Merlin localization and Hippo signaling. PI4KIIIalpha mutations in the PFCs lead to a Notch signaling defect and the subsequent failure of oocyte repolarization, which are precisely the phenotypes reported for Hippo mutations in the FCs. This effect is likely to be caused by a change in lipid composition in the membrane. How the abnormal actin structures are generated in the mutant cells, and whether they have a direct role in Merlin localization, remain to be investigated (Yan, 2011).

Protein Interactions

Hippo/dMST physically interacts with Sav and Wts

The dMST mutant phenotypes closely resemble those caused by sav or wts mutations. It has been shown that Sav and Wts physically and genetically interact, suggesting that they may act in common pathways. To determine if dMST could act in the same pathways, coimmunoprecipitation assays with Sav and Wts were performed. S2 cells were transfected with DNA constructs expressing HA-tagged Sav and Flag-tagged full-length dMST (dMSTf), its N-terminal fragment containing the kinase domain (dMSTn), or its C-terminal fragment containing the regulatory domains (dMSTc). Both dMSTf and dMSTc, but not dMSTn, coimmunoprecipitate with Sav, suggesting that dMST binds Sav through its C-terminal regulatory region. The dimerization domain at the C terminus of dMST appears to be essential for interaction as deletion of this domain from dMSTc abolishes its ability to bind Sav (Jia, 2003).

To define the domain in Sav that binds dMST, a series of truncated forms of Sav were generated. Both C-terminal fragments, SavC1 and SavC2, bind dMST. In contrast, all the C-terminally truncated fragments, including SavDeltaC1, SavDeltaC2, and SavDeltaC3, fail to bind dMST, suggesting that dMST binds the C-terminal region of Sav and the coiled-coil domain of Sav is essential (Jia, 2003).

dMST also binds Wts. S2 cells were transfected with DNA constructs expressing Myc-tagged Wts and Flag-tagged dMSTf, dMSTn, or dMSTc. Wts binds dMSTf and dMSTn, but not dMSTc, suggesting that Wts interacts with the N-terminal region of dMST. The interaction between Wts and dMST is not affected by Sav, since coexpression of Sav does not increase the amount of dMSTf coimmunoprecipitated with Wts. However, it remains possible that Sav might regulate dMST/Wts interaction in vivo at physiological concentration. Taken together, these results suggest that dMST, Wts, and Sav form a complex in which Wts and Sav bind the kinase and regulatory domains of dMST, respectively (Jia, 2003).

Sav quantities increase markedly in the presence of Hpo. Furthermore, Sav mobility on acrylamide gels shifts toward higher molecular weights. Sav immunoprecipitated from lysates containing Hpo was treated with phosphatases and it was found that this band shift disappeared, confirming that Sav becomes phosphorylated in the presence of Hpo. Sav is also stabilized by treatment with the proteasome inhibitor LLnL, suggesting that Sav is normally targeted for degradation by the proteasome. Unexpectedly, kinase-dead Hpo also induces stabilization and a mobility shift of Sav, whereas Hpo lacking the Sav-binding domain has little effect (Pantalacci, 2003).

Hippo phosphorylates and decreases the stability of DIAP1

Because Hpo is a kinase, the possibility that it might phosphorylate DIAP1 was investigated. Epitope-tagged Hpo was immunoprecipitatedfrom S2 cell lysates, and the ability of these immunoprecipitates to phosphorylate bacterially expressed DIAP1 was tested. Whereas Hpo induces DIAP1 phosphorylation in this assay, a kinase-dead (K71R) mutant of Hpo (HpoKD) does not. This activity is unimpaired by removal of the Sav-binding domain (amino acids 602-669) of Hpo (HpoC). DIAP1 phosphorylation by Hpo might be direct because bacterially expressed Hpo is also able to phosphorylate DIAP1 tagged with glutathione S-transferase (GST) in vitro. In addition, Hpo can autophosphorylate, as has been reported for its mammalian ortholog (Dan, 2001). Constructs expressing epitope-tagged Hpo and DIAP1 were transfected into S2 cells, and the cells were subjected to metabolic labelling. When normalized for the reduction in DIAP1 expression on cotransfection of Hpo, DIAP1 labelling increased 2.5-fold in the presence of Hpo. Thus, Hpo-dependent phosphorylation of DIAP1 can occur in live cells (Pantalacci, 2003).

The effect of Hpo on DIAP1 quantities was examined. Constructs expressing epitope-tagged DIAP1 plus Hpo and/or Sav, as well as a LacZ control plasmid, were transfected into S2 cells. On co-transfection of DIAP1 and Hpo, quantities of DIAP1 were reduced, and this effect was increased to more than twofold in the presence of Sav. By contrast, Sav alone or plus HpoC had only a minor effect on DIAP1, whereas Wts and HpoKD had no effect. To characterize further the effect of Hpo and Sav on DIAP1, S2 cells transfected with constructs expressing DIAP1 plus/minus Hpo and Sav were treated with the translation inhibitor cycloheximide (CHX). This experiment was done in the presence of caspase inhibitors to block caspase-induced DIAP1 degradation. The efficiency of caspase blockage was verified by Western blot analysis of DIAP1. Under these conditions, overexpressed DIAP1 is stable (95% remaining after 6 h). In the presence of Hpo and Sav, however, DIAP1 is less abundant and considerably destabilized (35% remaining after 6 h). Thus, Hpo decreases the stability of DIAP1 in S2 cells (Pantalacci, 2003).

Two independent clones of hpo were isolated in a yeast two-hybrid screen by using Sav as bait. Both clones contained the autoregulatory and dimerization domains. To characterize this interaction further, 35S-labelled Hpo and Sav proteins were produced by coupled in vitro transcription and translation, and they were assayed for complex formation by co-immunoprecipitation. Full-length Sav co-purifies Hpo, and systematic deletion analyses show that the dimerization domain of Hpo and the conserved Sav-specific domain (SD) of Sav are both necessary and sufficient for this interaction. The dimerization domain of Hpo is also necessary and sufficient for dimerization of Hpo itself, similar to its vertebrate homolog MST1. However, Sav and Hpo do not form tetrameric complexes in this assay. The interaction between Hpo and Sav is probably direct because it is also observed in yeast, which lacks this signalling pathway. All four hpo alleles produce C-terminally truncated proteins in which the region that interacts with Sav is deleted, indicating that the interaction between Hpo and Sav is important in vivo. The previously identified sav^shrp allele specifically deletes the SD domain and causes phenotypes that are as strong as those of sav null alleles, further supporting this conclusion. The other conserved domains of Sav, the two WW domains, bind to Wts. Thus, Sav may act as a scaffold to assemble signalling complexes comprising Hpo and Wts kinases (Udan, 2003).

The similarity of the wts, sav, and hpo mutant phenotypes suggests that the three genes function in the same pathway and that all three proteins may exist in the same protein complex. The Sav protein has domains capable of interacting with other proteins, including a WW domain and a C-terminal portion that is predicted to form a coiled-coil. Sav may therefore function as a scaffold in a multiprotein complex. The WW domains of Sav have been shown to bind to the PPXY motifs in the N-terminal portion of Wts. Whether Hpo can also bind to Sav was tested. Sav was expressed in E. coli as a fusion with maltose binding protein (MBP). After stringent washing, MBP-tagged Sav (MBP-Sav) coupled to amylose resin showed significant binding to in vitro translated Hpo but MBP coupled to amylose resin did not. Hpo also bound to in vitro translated Sav expressed as a Myc-tagged fusion (MTSav). Parallel binding experiments with proteins corresponding to the truncated versions of Hpo generated by the hpo^MGH1, hpo^MGH2, and hpo^MGH3 alleles showed that all three mutant proteins had markedly reduced in vitro binding to both MT-Sav and MBP-Sav. These experiments indicate that the C-terminal portion of Hpo is necessary for binding to Sav. Since the protein encoded by the hpo^MGH3 allele lacks only the C-terminal 49 amino acids, it is likely that Sav interacts with the conserved C-terminal domain of Hpo. Alternatively, conformational changes induced by the deletion of the C-terminal portion may preclude interaction with Sav (Harvey, 2003).

To determine if Sav and Hpo interact directly or via an accessory protein found in reticulocyte lysates, MBP-Sav coupled to amylose resin was incubated with either partially purified bacterial His-tagged Hpo or a control bacterial lysate. Immunoblotting with an anti-His antibody showed that Hpo interacted with MBP-Sav but not to MBP alone, suggesting that Hpo and Sav can interact with each other directly (Harvey, 2003).

To define the portion of Sav that is capable of interaction with Hpo, different domains of the Sav protein were expressed as fusions with MBP. These fusion proteins were tested for their ability to bind in vitro translated Hpo. Binding of the portion of Sav N-terminal to the WW domain (Sav-N) was comparable to that of full-length Sav (Sav), however no binding was detected with the fusion proteins containing the WW domains of Sav (Sav-WW) or the C-terminal coiled-coil domain (Sav-CC). Thus, Hpo appears to bind predominantly to the N-terminal portion of Sav. In further experiments using in vitro translated MTSav domains, some binding of full-length Hpo to the coiled-coil region of Sav was observed. This might imply a second site of interaction between the two proteins or be the result of the 'stickiness' of the coiled-coil domain (Harvey, 2003).

To examine whether Hpo and Wts bind competitively to Sav or at distinct sites, increasing amounts of in vitro translated Wts were added to the binding reaction. Would this addition of Wts reduce the binding of Hpo to Sav? Even the addition of four times the amount of Wts as Hpo had no effect on the binding of Hpo to Sav. This is consistent with the notion that Hpo and Wts bind to different portions of Sav, i.e., the N-terminal portion and the WW domains respectively and is consistent with the possibility that the three proteins can form a ternary complex (Harvey, 2003).

The interaction of hpo with sav and wts was examined in vivo. Combined overexpression of sav and wts in the eye generates a small rough eye phenotype, which is mostly due to increased cell death. When hpo clones were generated in eyes overexpressing both sav and wts, eye size was significantly restored. This suggests that hpo is required for the ability of sav and wts to induce cell death. If, as the binding data suggest, Hpo functions in a complex with Sav and Wts, then Hpo function appears necessary for the proapoptotic activity of the complex (Harvey, 2003).

At a stage of development (38 hr APF) when excess interommatidial cells are eliminated by apoptosis, most of the cell death in discs containing hpo clones was restricted to wild-type portions of the disc. The DIAP1 protein, an antagonist of caspases, is elevated in sav clones. hpo clones posterior to the MF also have elevated DIAP1 levels. Regulation of DIAP1 by hpo is likely to occur largely at the posttranscriptional level since DIAP1 RNA expression, as assessed by either in situ hybridization or by RT-PCR, was not obviously elevated in eyes composed almost entirely of hpo mutant tissue. DIAP1 RNA expression was also examined in S2 cells treated with RNAi designed to reduce expression of Hpo. While DIAP1 protein levels were increased 1.5-fold in cells treated with Hpo RNAi, no obvious difference in DIAP1 RNA expression was seen. DIAP1 levels are also elevated in wts cells. Therefore the defect in apoptosis in sav, wts, and hpo tissue is likely due to the result of elevated levels of DIAP1 in mutant clones that renders caspases inactive in these cells (Harvey, 2003).

Proteins such as Hid, Rpr, and Grim are thought to downregulate DIAP1 levels by stimulating its autoubiquitination or by repressing general protein translation, which has the greatest effect on short-lived proteins such as DIAP1. To investigate whether hpo can modify the function of such proteins, hpo clones were generated in flies overexpressing the grim gene under the control of the GMR promoter. When overexpressed in the Drosophila eye, grim induces extensive cell death as visualized by TUNEL, which results in a small, rough eye. When hpo clones were generated in eyes overexpressing Grim, eye size was significantly restored and Grim-induced cell death was greatly reduced in hpo mutant clones. sav and wts clones are relatively resistant to cell death induced by Rpr or Hid. The increased basal level of DIAP1 found in sav, wts, or hpo clones may make it more difficult for proteins such as Rpr, Hid, or Grim to reduce DIAP1 levels sufficiently to activate caspases in these cells (Harvey, 2003).

Since DIAP1 protein levels are elevated in hpo clones and in S2 cells treated with hpo RNAi, the possibility that hpo might regulate DIAP1 stability was tested. When hpo is overexpressed in Drosophila S2 cells, endogenous DIAP1 protein is consistently reduced to approximately 60%-70% of normal levels, when normalized to loading controls. Since Wts and Hpo are both predicted to have kinase activity, it is possible that a complex consisting of Sav, Hpo, and Wts regulates the phosphorylation state of DIAP1 and hence regulates its turnover. Indeed, both Sav-associated and Hpo-associated complexes are capable of phosphorylating DIAP1 in vitro, and DIAP1 is destabilized in the presence of Hpo, presumably as a result of Hpo-dependent phosphorylation of DIAP1 (Harvey, 2003).

Hippo binds to and phosphorylates the tumor suppressor protein Salvador: Salvador in turn increases the ability of Hpo to phosphorylate Wts

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 sav^shrp6 allele, a mutant Sav protein, Sav^shrp6, was engineered that lacks the C-terminal 79 residues as seen in sav^shrp6, and the ability of this mutant protein to associate with Hpo and to facilitate Wts phosphorylation by Hpo was examined. Unlike wild-type Sav, Sav^shrp6 can not associate with Hpo, suggesting that the coiled-coil domain of Sav is required for Hpo/Sav interaction. Importantly, coexpression of Sav^shrp6 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 Drosophila RASSF homolog antagonizes the Hippo pathway

Correct organ size is determined by the balance between cell death and proliferation. Perturbation of this delicate balance leads to cancer formation. Hippo (Hpo), the Drosophila ortholog of MST1 and MST2 (Mammalian Sterile 20-like 1 and 2) is a key regulator of a signaling pathway that controls both cell death and proliferation. This pathway is so far composed of two Band 4.1 proteins, Expanded (Ex) and Merlin (Mer), two serine/threonine kinases, Hpo and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). It has been proposed that Ex and Mer act upstream of Hpo, which in turn phosphorylates and activates Wts. Wts phosphorylates Yki and thus inhibits its activity and reduces expression of Yki target genes such as the caspase inhibitor DIAP1 and the micro RNA bantam. However, the mechanisms leading to Hpo activation are still poorly understood. In mammalian cells, members of the Ras association family (RASSF) of tumor suppressors have been shown to bind to MST1 and modulate its activity. In this study it is shown that the Drosophila RASSF ortholog (dRASSF) restricts Hpo activity by competing with Sav for binding to Hpo. In addition, dRASSF also possesses a tumor-suppressor function (Polesello, 2006).

The mammalian RASSF family comprises six different loci encoding a variety of splice variants. Most transcripts encode proteins that contain a Ras association domain (RA), an N-terminal C1-type zinc finger, and a C-terminal SARAH (Sav RASSF Hippo) domain. RASSF family members, most notably RASSF1A, are frequently silenced in a variety of solid tumors. Thus, it has been proposed that RASSF genes act as tumor suppressors (Polesello, 2006).

The biological function of these genes is not well understood. RASSF1A and Nore1A have both been shown to interact with MST1 via its SARAH domain. Overexpression of RASSF1A or Nore1A inhibits MST1 activation, but coexpression of these RASSF proteins with Ras enhanced MST1 activity. RASSF1A knockout mice have mildly increased tumor susceptibility, confirming that RASSF genes can act as tumor suppressors. The weakness of the mouse phenotype, which is at odds with the frequency of RASSF1A inactivation in human tumors, can be ascribed to redundancy with other family members (Polesello, 2006).

By contrast, Drosophila melanogaster has a single RASSF family member, which is encoded by the CG4656 gene and which will be referred to as dRASSF. Like its vertebrate counterparts, dRASSF encodes a protein bearing an RA and SARAH domain at its C terminus. It also possesses a LIM domain that shares some similarities with C1 zinc fingers at its N terminus (Polesello, 2006).

Mutant alleles of dRASSF were generated by imprecise excision of two nearby transposons, GE23517 and EY2800. Multiple alleles, which delete up to the fourth intron, including the initiating ATG, were obtained. Some transcript was still detected in dRASSF^X16, dRASSF^X36, but a strong reduction was found in dRASSF^44.2, which lacks the transcription start. However, antibodies raised against the C terminus (amino acids 792–806) and a nonconserved region (amino acids 294–308) of dRASSF showed that full-length dRASSF is absent in lysates from all mutant lines, suggesting the dRASSF mutants are indeed loss-of-function mutations for the locus. All of these alleles were viable and behaved identically in subsequent assays. In addition, dRASSF staining was severely reduced in FLP/FRT-generated dRASSF mutant clones in the eye-imaginal disc, the larval precursor to the adult eye (Polesello, 2006).

Although the dRASSF mutant flies are viable, they present a clear growth defect in comparison to wild-type animals when reared in carefully controlled conditions. dRASSF mutant flies were 15% lighter than their wild-type counterparts, a phenotype which was significantly rescued by introduction of a single copy of a dRASSF rescue construct, although wild-type levels of dRASSF were not fully restored. dRASSF mutant flies were fully fertile and normally proportioned but sensitive to γ-irradiation. Wing surface area was reduced by 8% in dRASSF mutant flies, whereas wing hair density was unaffected. This suggests that the growth defect of dRASSF mutant flies is due to a reduction in cell number and not a defect in cell size (Polesello, 2006).

In mammals, members of the RASSF family are known to interact with MST1 and thus to modulate its pro-apoptotic activity. Therefore whether dRASSF can interact with Hpo was tested. Coimmunoprecipitation (Co-IP) experiments were performed in Drosophila Kc cells with dRASSF antibodies to immunoprecipitate endogenous protein. As expected, dRASSF robustly coimmunoprecipitated with Hpo. The association between Hpo and Sav is mediated by these proteins' shared SARAH domains. Likewise, Hpo's SARAH domain is required for its association with dRASSF, as shown by the fact that a truncated form of Hpo (Hpo^ΔC) lacking this domain fails to bring down dRASSF. Thus, the Hpo SARAH domain can associate with both Sav and dRASSF (Polesello, 2006).

Sav is stabilized by the presence of Hpo. Therefore whether dRASSF levels are modulated by Hpo was tested. dRASSF immunostaining was reduced in clones mutant for a hpo allele that lacks the SARAH domain. In addition, RNAi-mediated depletion of Hpo from Drosophila Kc cells resulted in a reduction of endogenous dRASSF expression, whereas dRASSF transcripts were unaffected. By contrast, dRASSF levels were unaffected in clones mutant for other Hpo-pathway members, such as ex, sav, and wts. These results suggest that direct binding to Hpo through its SARAH domain, rather than signaling through the Hpo pathway, is necessary for dRASSF stability. This is analogous to the situation for Sav, which is also stabilized by a kinase-dead form of Hpo (Polesello, 2006).

Because Hpo, Sav, and dRASSF all contain a SARAH domain, it was speculated that dRASSF might also bind Sav. To test this, whether dRASSF interacts with Sav was investigated by co-IP but no such an interaction was detected. Because the possibility of a ternary complex had been raised by another study, whether the three proteins could be found in the same complex was tested. Hpo, Sav, and dRASSF were co-expressed in cultured Kc cells. As expected, Hpo was able to bind Sav and dRASSF. However, Sav immunoprecipitates only contained Hpo and not dRASSF, and dRASSF immunoprecipitates contained Hpo but not Sav. Identical results were obtained with endogenous IPs by using dRASSF and Sav antibodies. These data support the notion that Sav and dRASSF are not present in the same complex but are in two different Hpo complexes (Polesello, 2006).

Sav has been shown to be a positive regulator of the Hpo pathway, whereas genetic results suggest that dRASSF might antagonize Hpo function. It was therefore of interest to determining whether complexing with Sav or dRASSF might influence Hpo activity. Immunoprecipitates were probed with an phospho-MST1 antibody that recognizes phosphorylated (active) Hpo. Interestingly, although Hpo that was coimmunoprecipitated with dRASSF showed barely detectable levels of phosphorylation, the Sav-associated fraction was highly phosphorylated. Thus, Hpo can exist as two pools, a highly active Sav-associated pool and an inactive dRASSF-associated pool. This correlates with data showing that Nore1 can repress MST1 activity in mammalian cells. This also suggests that Sav can promote Hpo activation and provides the first direct evidence of a function for the Hpo/Sav interaction (Polesello, 2006).

Next, the prediction that dRASSF depletion would promote Hpo activation was tested. Like that of Hpo's mammalian counterparts, phosphorylation of endogenous Hpo can be potently stimulated by the drug Staurosporine (STS) in Kc cells. Although RNAi depletion of dRASSF alone was not able to induce Hpo phosphorylation, dRASSF depletion markedly potentiated STS-induced Hpo activation. Thus, dRASSF restricts Hpo activation in cultured cells (Polesello, 2006).

Given their opposing effects on Hpo activation, the relationship between Sav and dRASSF was investigated. Depletion of dRASSF in Kc cells gives rise to an increase in Sav protein levels. Although dRASSF levels were unaltered in sav mutant clones, overexpression of Sav in the wing disc results in a robust decrease of dRASSF staining. Whether dRASSF and Sav compete to bind Hpo was tested. To address this question, because Sav and dRASSF repress each other's expression and dRASSF has reduced affinity for phosphorylated Hpo, separate Kc cell lysates expressing a kinase-dead form of Hpo (Hpo^KD-Flag), Sav-HA, and HA-dRASSF were mixed and IPs were performed after the proteins were allowed to bind overnight. Both Sav and dRASSF were able to interact with Hpo. In these conditions, increasing the amount of Sav was able to displace the dRASSF fraction bound to Hpo, showing that Sav and dRASSF are competing to bind Hpo. The outcome of the competition probably determines the stability of Sav and dRASSF; both proteins are downregulated when Hpo is depleted by RNAi. Thus, it is suggested that interplay between the inhibitor dRASSF and the activator Sav determines the level of Hpo activation and therefore affects body size (Polesello, 2006).

This model was tested by performing genetic-interaction experiments. A mutant allele of hpo was crossed into the dRASSF mutant background and the adult body mass was measured. The body-mass reduction of dRASSF mutant flies (15% reduction) was substantially rescued by removal of just one copy of Hpo (8% reduction). Flies overexpressing Sav showed a reduction of 10% in weight and 5% in wing area, mimicking dRASSF loss of function. This wing defect was significantly increased in a dRASSF mutant background. In addition, misexpression of dRASSF was able to robustly rescue the rough-eye phenotype elicited by coexpression of Sav and Wts. These data support the notion that dRASSF can antagonize Sav-mediated Hpo activation in vivo (Polesello, 2006).

Though the results are consistent with biochemical data on mammalian RASSF family members, they are at odds with the fact that RASSF genes are commonly silenced in tumor cells. It has been proposed that one RASSF protein, Nore1, possesses a tumor-suppressor function that is independent of MST1 and MST2. Two lines of evidence to support this notion were found. First, in vivo clones were made in the head (by using the eyeless FLP system) that were mutant for two hpo hypomorphic alleles, hpo^42–48 and hpo^KC203, that remove the SARAH domain in a dRASSF mutant background. Interestingly, the overgrowth phenotype elicited by these hpo alleles was strongly enhanced by loss of dRASSF. By contrast, a hpo allele (hpo^42–47) bearing an inactivating deletion in the kinase domain but an intact SARAH domain was barely if at all enhanced by dRASSF loss of function. This suggests that dRASSF may possess a tumor-suppressor function, which may be uncovered when the Hpo function is compromised (Polesello, 2006).

In addition, the relationship between Ras1 and dRASSF was examined because the mammalian RASSF proteins have all been shown to bind Ras proteins. In Drosophila imaginal tissues, Ras1 mutant clones grow poorly and are eliminated by apoptosis. When double-mutant clones for Ras1 and dRASSF were made in the developing eye, a substantial rescue was observed of the growth defect observed in clones mutant for Ras1 alone. This rescue of Ras loss of function was the result of both increased proliferation quantified with phosphorylated Histone 3 staining and a reduction of apoptosis visualized with a cleaved-Caspase 3 antibody. Thus, dRASSF appears to antagonize Ras1 signaling in growth control, which is again suggestive of a “tumour-suppressing” effect distinct from its “oncogenic” role in opposing the Hpo pathway. However, it has been suggest that NORE1 may also have both Ras- and MST-independent functions. Future experiments will therefore be aimed at gaining a better understanding of the RASSFs' growth-restricting functions. The fact that the dRASSF mutations are viable might therefore reflect the facts that its ability to regulate the Hpo pathway may be redundant with other modes of regulation and that loss of dRASSF's tumor-suppressive activity is balanced by loss of its growth-promoting activity. It has been proposed that MST2 may be inactivated by binding to Raf-1. It will be interesting to determine whether this mode of regulation is redundant with RASSF (Polesello, 2006).

In summary, mutant alleles of the sole Drosophila ortholog of the RASSF family of tumor suppressors were generated. Surprisingly, dRASSF mutant flies are smaller than control flies. This growth defect can probably be ascribed in part to dRASSF's ability to antagonize Hpo signaling by competing with Sav for binding to Hpo. In addition, dRASSF also possesses a tumor-suppressor activity, which is uncovered when hpo or Ras1 function is compromised. It will be interesting to investigate whether some mammalian RASSF proteins share these properties (Polesello, 2006).

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

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

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

Despite the presence of mammalian homologs for all the known components of the Drosophila Hippo pathway (Mst1/2 for Hpo, WW45 for Sav, Lats1/2 for Wts, and YAP for Yki), previous studies in mammals have failed to unite these proteins in a physiologically relevant signaling cascade. The conservation of of the S168 phosphorylation site in mammalian YAP provides the first opportunity to functionally link Mst1/2, WW45, and Lats1/2 in a single kinase cascade that culminates in YAP S127 phosphorylation. The mammalian Hippo signaling pathway antagonizes YAP function by promoting its cytoplasmic localization in a S127 phosphorylation-dependent manner. The identification of S168/S127 as Wts/Lats-mediated phosphorylation site in Yki/YAP is rather unexpected given that previous studies have implicated this residue as an Akt phosphorylation site. The observation that both Yki^S168A and YAP^S127A result in a loss-of-Wts rather than a loss-of-Akt phenotype in Drosophila strongly suggests that this site is regulated by the Hippo pathway rather than Akt under normal physiological conditions (Dong, 2007).

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

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

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

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

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

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

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

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

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

The function of Mob proteins has been better studied in yeast, Drosophila and mammalian cells, which revealed a conserved property of Mob proteins as a binding partner as well as a coactivator of protein kinases of the Ndr (nuclear Dbf2-related) family (Hergovich, 2006b). As stated above, Drosophila Mats/dMob1 is required for mediating Hpo signaling by regulating Wts kinase activity in growth inhibition and tumor suppression. All four Drosophila mob genes dMob1-4 genetically interact with trc (tricornered) (He, 2005a), the fly Ndr homolog important for maintaining integrity of epidermal outgrowths and regulating dentritic tiling and branching (Emoto, 2004; He, 2005b). In the budding yeast Saccharomyces cerevisiae, Mob1 binds to and activates Dbf2/Dbf20 protein kinases for controlling mitotic exit and cytokinesis (Komarnitsky, 1998; S. E. 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).

Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and Expanded

The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. This study identified Kibra as another upstream component of the Hippo signaling pathway. This study shows that Kibra functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function and implicate Kibra as a potential tumor suppressor with relevance to neurofibromatosis (Yu, 2010).

In multicellular organisms, cell growth, proliferation, and death must be coordinated in order to attain proper organ size during development and to maintain tissue homeostasis in adult life. Recent studies in Drosophila have led to the discovery of the Hippo signaling pathway as a key mechanism that controls organ size by impinging on cell growth, proliferation, and apoptosis. Central to the Hippo pathway is a kinase cascade comprised of four tumor suppressors, including the Ste20-like kinase Hippo (Hpo) and its regulatory protein Salvador (Sav), the NDR family kinase Warts (Wts) and its regulatory protein Mats. The Hpo-Sav complex phosphorylates and activates the Wts-Mats complex, which in turn phosphorylates and inactivates the oncoprotein Yki, which normally functions as a coactivator for the TEAD/TEF family transcription factor Scalloped (Sd). Recent studies have also implicated the atypical cadherin Fat (Ft) as well as the membrane-associated FERM-domain proteins Expanded (Ex) and Merlin (Mer) as upstream components of the Hippo pathway. How these proteins are biochemically linked to the Hippo kinase cascade remains largely unknown, although Ex can at least partially regulate the Hippo pathway by directly binding and sequestering Yki in the cytoplasm. Ft differs from Ex, Mer, and core components of the Hippo kinase cascade in that, besides tissue growth, Ft also regulates planar cell polarity (PCP), for which it interacts with another cadherin Dachsous (Ds). Most recently, it was shown that a gradient of Ds activity in imaginal discs can modulate Hippo-mediated growth regulation, thus potentially linking PCP to the Hippo kinase cascade, although the biochemical mechanism of this linkage remains to be determined (Yu, 2010).

The physiological function of the Hippo pathway is best understood in Drosophila imaginal discs, where inactivation of the Hippo pathway tumor suppressors, or overexpression of the Yki oncoprotein, results in tissue overgrowth characterized by excessive cell proliferation, diminished apoptosis, and increased transcription of Hippo pathway target genes such as the cell death inhibitor diap1 and the microRNA bantam, as well as ex and mer as part of a negative feedback regulatory loop. Recent studies further implicated the Hippo pathway as a conserved mechanism of organ size control and tissue homeostasis in mammals. Thus, the mammalian homologs of Hpo (Mst1/2), Sav (WW45), Wts (Lats1/2), and Yki (YAP) constitute an analogous kinase cascade, and transgenic overexpression of YAP or inactivation of Mst1/2 led to massive organomegaly and rapid progression to tumorigenesis. Furthermore, NF2, the mammalian homolog of mer, is a well-established tumor suppressor gene whose mutations lead to neurofibromatosis (Yu, 2010 and references therein).

Besides its prominent role in controlling imaginal disc growth, the Hippo pathway is required during Drosophila oogenesis for the proper maturation of posterior follicle cells (PFCs). In the absence of Hippo signaling, the PFCs fail to undergo a Notch-mediated mitotic cycle-endocycle switch and accumulate in extra layers of follicular epithelium. The PFC maturation defects, in turn, lead to a disruption of the anterior-posterior (AP) polarity of the underlying oocyte, which manifests itself as mislocalization of the oocyte nucleus and AP axis determinants such as the RNA-binding protein Staufen (Stau). Interestingly, the oocyte polarity defect is observed in mutants for components of the Hippo kinase cascade as well as ex and mer, but not ft, suggesting that the canonical Hippo pathway may integrate different signals in different developmental contexts (Yu, 2010).

This study identifies Kibra as an upstream component of the Hippo pathway. Loss of kibra leads to oogenesis defects, imaginal disc overgrowth, and aberrant gene expression characteristic of defective Hippo signaling. Kibra functions together with Mer and Ex in an apical protein complex, which, through direct binding to the Hpo-Sav complex, regulates the Hippo kinase cascade and thus Yki phosphorylation. These findings uncover an important missing link in the Hippo signaling pathway and shed light on the molecular mechanism of the Ex and Mer tumor suppressor proteins (Yu, 2010).

In a genetic screen for oocyte polarity mutants based on FRT/FLP-induced mitotic clones in follicle cells, four lethal P element insertion lines on chromosome 3R were identified that caused mislocalization of Stau-GFP and Stau to the center of the oocyte when the PFCs were made homozygous mutant for the P element insertions. This polarity defect was observed with variable penetrance depending on the specific P element line analyzed, likely due to their hypomorphic nature. These lethal lines (264/09, 1156/7, f06952, and EP3494) fail to complement each other and all carry a P element insertion near the 5' UTR or within the first intron of CG33967. CG33967 encodes a 1288 amino acid protein that shares 39% identity with KIBRA, a cytoplasmic protein named after its predominant expression in kidney and brain in humans. Both CG33967 and KIBRA contain two N-terminal WW domains and one C-terminal C2 domain. CG33967 is referred to as kibra to distinguish it from its human ortholog KIBRA (Yu, 2010).

This study identifies Kibra as a tumor suppressor and an essential component of the Hippo pathway. A model is proposed in which Kibra functions together with Mer and Ex in an apical protein complex to transduce growth-regulatory signals to the Hpo-Sav complex, which, through the canonical Hippo kinase cascade, controls Yki phosphorylation and target gene transcription. Of note, the findings do not exclude the possibility that Kibra, Ex, or Mer may interact with additional Hippo pathway components besides Hpo-Sav, especially given the recent report that Ex can directly bind Yki. How Kibra, Ex, and Mer function together to integrate upstream signals remains to be determined. One possibility is that these proteins function redundantly in receiving signals from the same upstream regulator(s). Alternatively, each protein may be regulated by distinct upstream regulator(s) (Yu, 2010).

A commonly used assay for Hippo signaling in Drosophila S2 cells involves examining mobility shifts of the Wts protein on SDS-PAGE. Given its large size and that not all protein phosphorylation causes discernable mobility shift on SDS-PAGE, this assay is less sensitive in detecting Wts phosphorylation than the phospho-specific antibody used in the present study. Indeed, overexpression of Ex in S2 cells has no effect on Wts mobility, yet this study demonstrates that Ex induces robust Wts phosphorylation at its hydrophobic motif. The fact that this hydrophobic motif is a well-established direct phosphorylation site by Hpo homologs in mammalian cells further suggests that Ex, as well as Mer and Kibra, regulates Wts through the canonical Hippo kinase cascade. Indeed, it was found that Ex-induced Wts phosphorylation is Hpo dependent. These results are not incompatible with recent report that Ex can also regulate the Hippo pathway in a kinase-independent manner. Using a well-established assay for Yki transcriptional activity, this study found that while Ex, Mer plus Kibra, or Hpo could all suppress the activity of a Yki-Gal4 fusion protein, only Ex was able to suppress the activity of a Yki-Gal4 fusion protein in which all the possible Wts-phosphorylation sites are mutated. These observations are consistent with the view that Ex can regulate the Hippo pathway through both Wts-dependent and -independent mechanisms (Yu, 2010).

A comparison of the loss-of-function phenotypes of mer, ex, and kibra in egg chambers and imaginal discs reveals tissue-specific differences in the relative contribution of each gene to Hippo pathway regulation. For example, loss of ex alone, but not mer or kibra, is sufficient to cause robust diap1 upregulation in imaginal discs, suggesting that ex has a more essential role in diap1 transcriptional regulation. However, the converse is true in the ovary, where loss of mer or kibra results in stronger oocyte polarity and Notch signaling defects than loss of ex. In fact, the severity of mer or kibra mutant phenotypes in oogenesis are comparable to those of core components of the Hippo pathway such as hpo and sav, even though the former display much milder overgrowth than the latter in imaginal discs. Perhaps the most extreme case of tissue-specific requirement is provided by the ft tumor suppressor gene, which is required for Hippo pathway regulation in the imaginal discs but dispensable in developing egg chambers. While the underlying molecular basis remains to be determined, such tissue-specific requirements suggest that the core Hippo kinase cascade may function as a signal integrator of multiple inputs in a dynamic and versatile manner, and that additional cell surface receptors besides Ft may signal to the Hippo pathway (Yu, 2010).

Considerable efforts have been directed at identifying the key signaling pathways regulated by the NF2/Merlin tumor suppressor protein. These investigations have led to the identification of a number of effector mechanisms downstream of NF2/Merlin, such as growth control pathways mediated by Ras, Rac, STAT, or PI3K, contact inhibition mediated by cell surface receptors or adherens junctions, and endocytosis/degradation of various membrane proteins. The recent identification of Mer as an upstream regulator of Hpo in Drosophila provides yet another plausible mechanism through which Mer functions as a tumor suppressor protein. The identification of Kibra as a regulator of the Hippo pathway further strengthens the case for a functional link between NF2/Mer and the Hippo pathway. The observation that NF2/Mer and KIBRA can synergistically stimulate Lats1/2 phosphorylation in mammalian cells not only supports an NF2/Mer-Hippo connection, but further implicates KIBRA as a potential tumor suppressor in humans with relevance to neurofibromatosis (Yu, 2010).

The identification of Kibra as an upstream regulator of the Hippo pathway has implications for understanding memory-related functions of the human KIBRA gene. Besides its well-established roles in growth control, the Hippo pathway is also required for differentiation and morphogenesis of certain postmitotic neurons in Drosophila. It is speculated that modulation of the Hippo pathway may influence the growth or differentiation of memory-related neuronal structures, a hypothesis that can be directly tested by genetic manipulation of Hippo signaling activity in animal models (Yu, 2010).

Tao-1 phosphorylates Hippo/MST kinases to regulate the Hippo-Salvador-Warts tumor suppressor pathway

Recent studies have shown that the Hippo-Salvador-Warts (HSW) pathway restrains tissue growth by phosphorylating and inactivating the oncoprotein Yorkie. How growth-suppressive signals are transduced upstream of Hippo remains unclear. This study shows that the Sterile 20 family kinase, Tao-1, directly phosphorylates T195 in the Hippo activation loop and that, like other HSW pathway genes, Tao-1 functions to restrict cell proliferation in developing imaginal epithelia. This relationship appears to be evolutionarily conserved, because mammalian Tao-1 similarly affects MST kinases. In S2 cells, Tao-1 mediates the effects of the upstream HSW components Merlin and Expanded, consistent with the idea that Tao-1 functions in tissues to regulate Hippo phosphorylation. These results demonstrate that one family of Ste20 kinases can activate another and identify Tao-1 as a component of the regulatory network controlling HSW pathway signaling, and therefore tissue growth, during development (Boggiano, 2011).

During development, organisms must determine the overall size and shape of their individual organs through mechanisms not fully understood. The recent discovery of the evolutionarily conserved Hippo-Salvador-Warts (HSW) signaling pathway has revealed a unique mechanism to regulate proliferation independent of developmental patterning. The core members of the HSW pathway, Hippo (Hpo) and Warts (Wts), together with their scaffolding partners Salvador (Sav) and Mats, phosphorylate and inactivate the transcriptional co-activator Yorkie (Yki). Phosphorylation prevents Yorkie from translocating to the nucleus where it binds to TEAD-family transcription factors and drives the transcription of genes that promote growth and inhibit apoptosis. Loss of HSW pathway function in Drosophila leads to increased cellular proliferation resulting in tumor-like overgrowths in epithelial tissues. Similarly, knockout mouse models of HSW homologs grow tumors, and human HSW homologs have been implicated in cancers. These studies suggest that HSW signaling is a crucial part of an organism's ability to regulate cell proliferation and overall tissue size (Boggiano, 2011 and references therein).

A central, unanswered question regarding HSW function is how the activity of Hpo, the most upstream kinase in the pathway is regulated. The atypical cadherin Fat and its ligand Dachsous can function at the plasma membrane to initiate HSW signaling, however the extracellular cues that trigger signaling and the mechanism by which Fat activates Hpo remain elusive. In addition, genetic evidence strongly suggests that another source of Hpo activation functioning in parallel to Dachsous-Fat activation must exist. At least three different cytoplasmic proteins are believed to act upstream of Hpo to initiate signaling through the pathway, Expanded (Ex), Merlin (Mer), and Kibra. Ex and Mer are members of the Four-point-one, Ezrin, Radixin, Moesin (FERM) family and Kibra is a WW-domain containing protein. Though these three proteins are thought to physically interact with each other in varying complexes, only Ex can form a complex with Hpo and it is unclear how this interaction leads to activation of Hpo. Moreover, there is strong genetic evidence that Ex, Mer, and Kibra act in parallel to each other, implying that other mechanisms for activating Hpo independently of Ex must exist (Boggiano, 2011).

This study sought to identify genes that might function upstream of Hpo to activate the pathway using a candidate gene approach and discovered that the Sterile 20 kinase Tao-1 is a member of this signaling pathway. Tao-1 previously has been shown to destabilize microtubules and has been implicated in apoptosis in the Drosophila germline. This study shows that loss of Tao-1 function results in increased cellular proliferation and upregulation of Yki target gene expression. It was further demonstrated that Tao-1 regulates HSW pathway activity by phosphorylating Hpo at a critical activating residue. Thus, these results identify Tao-1 as a member of the HSW pathway and provide a molecular mechanism for Hpo activation (Boggiano, 2011).

In an effort to identify additional regulators of HSW signaling, this study examined the role of Tao-1 in growth control during development. Tao-1 depletion in either the eye or wing epithelium results in overgrowth phenotypes as well as transcriptional upregulation of HSW targets. Using a combination of genetic epistasis, experiments in cultured S2 cells, and in vitro biochemistry, it was demonstrated that Tao-1 directly phosphorylates the critical T195 regulatory residue in the activation loop of Hpo to promote HSW pathway activation. The observation that a mammalian orthologue of Tao-1, TAOK3, can phosphorylate MST kinases at the same residue further suggests that this regulatory function is conserved in mammals. Taken together, these results implicate Tao-1 as a component of HSW signaling (see A model for Tao-1's function in the HSW pathway) and reveal a mechanism for regulation of Hpo activity (Boggiano, 2011).

While Tao-1 depletion results in overgrowth phenotypes that are similar to mutations in other HSW pathway genes, these phenotypes are less severe than those of core components such as hpo and wts. One likely explanation for this is that the RNAi transgenes that were used in these studies do not completely remove Tao-1 function. It is also possible that there are multiple mechanisms for activating HSW signaling, including, but not limited to, Tao-1 phosphorylation of Hpo. Indeed, previous studies have demonstrated that the upstream components Mer, Ex, and Kibra act, at least in part, in parallel to activate Hpo. Biochemical evidence indicates that two of these proteins, Mer and Ex, function with Tao-1 to activate HSW signaling. While it is probable that Kibra functions upstream of Tao-1, it cannot be ruled out that Kibra functions independently of Mer and Ex to activate HSW signaling in a Tao-1-independent manner. Further genetic analysis using a Tao-1 null allele would be helpful in defining Tao-1's role relative to other HSW components, but unfortunately the deletions associated with the sole existing Tao-1 null allele, Tao-150, also appear to affect an adjacent gene. In addition, Tao-1 maps very close to the most proximal FRT element on the X chromosome, making it difficult to generate recombinant chromosomes for somatic mosaic analysis (Boggiano, 2011).

How do Mer, Ex and Tao-1 cooperate to regulate Hpo phosphorylation? Given that Ex has been shown to interact with Hpo, one possibility is that Mer and Ex function to scaffold Tao-1 together with Hpo, thereby promoting the ability of Tao-1 to phosphorylate and activate Hpo. However, despite repeated attempts it has not been possible to detect Tao-1 in a complex with either Mer or Ex, and knockdown of Mer, ex or kibra does not diminish the ability of Tao-1 to promote Hpo phosphorylation in S2 cells. For these reasons, the possibility is favored that Mer and Ex indirectly affect Tao-1 function, perhaps by interacting with other proteins that in turn directly regulate Tao-1. For example, Tao-1 activity could be directly regulated by an unknown receptor at the cell surface whose localization or activity is controlled by interaction with Mer and Ex. This notion is consistent with the fact that both Mer and Ex have FERM domains, which are known to interact with the cytoplasmic tails of transmembrane proteins. Previous studies have suggested that Ex interacts with the transmembrane protein Crumbs, though the mechanistic significance of this interaction is unclear. It is not currently known whether Drosophila Merlin has transmembrane binding partners (Boggiano, 2011).

Two additional ideas related to Tao-1 function are suggested by the current data. In S2 cells, Tao-1 kinase activity is required for normal levels of Hpo phosphorylation at T195 in the kinase activation loop, suggesting that Tao-1 could function to maintain constant, low levels of pathway activation. In turn, this low level of Hpo activation might be necessary so that other, regulated inputs into HSW activity can quickly transition cells away from actively dividing and into a differentiated state following periods of growth. Alternatively, it is possible that Tao-1 activity itself is dynamically regulated during development, allowing it to rapidly alter levels of HSW pathway activity via its effect on Hpo phosphorylation. In either case, phosphorylation by Tao-1 at T195 is likely to promote Hpo's known ability to undergo autophosphorylation, thus amplifying the effect of even a small change in Tao-1 activity. Further studies will be required to answer these questions and to determine if, and how, Tao-1 activity is regulated (Boggiano, 2011).

An interesting aspect of the discovery that Tao-1 regulates HSW signaling is that Tao-1, and its mammalian orthologues TAOK1-3, have been shown to regulate MT stability. The current results indicate that this effect on MT stability is not mediated through HSW signaling, since mutations in other HSW pathway components do not display similar MT phenotypes. However, it is interesting to speculate that Tao-1's association with MTs might affect its ability to regulate HSW pathway activation. More work will be required to determine whether the function of Drosophila Tao-1 in HSW signaling is entirely independent of its role in microtubule dynamics, though a recent study in mammalian cultured cells found that microtubule disruption did not affect localization of Yap, a mammalian Yki ortholog, suggesting that in mammalian cells these roles might be independent (Boggiano, 2011).

An additional possible mechanistic link between Tao-1 and HSW signaling is suggested by studies in flies and in mammalian cells indicating that Par-1, a polarity protein, is positively regulated by Tao-1. Par-1 has been shown to promote basolateral polarity in the Drosophila follicular epithelium and to regulate the stability and organization of MTs in these cells. Recent studies have implicated components of both apical and basolateral polarity in the regulation of HSW signaling. Conversely, HSW signaling also seems to feed back onto Crumbs, an apical determinant, and perhaps other components to regulate apical-basal polarity. Whether Tao-1 plays a role in the linkage between cell polarity and growth control remains to be established, but the ability to both directly activate Hpo function through phosphorylation and control cytoskeletal organization and cell polarity through microtubule organization potentially places Tao-1 in a unique position to coordinate these important cellular processes (Boggiano, 2011).

The sterile 20-like kinase Tao-1 controls tissue growth by regulating the Salvador-Warts-Hippo pathway

The Salvador-Warts-Hippo (SWH) pathway is a complex signaling network that controls both developmental and regenerative tissue growth. Using a genetic screen in Drosophila melanogaster, the sterile 20-like kinase, Tao-1, was identified as an SWH pathway member. Tao-1 controls various biological phenomena, including microtubule dynamics, animal behavior, and brain development. This study describes a role for Tao-1 as a regulator of epithelial tissue growth that modulates activity of the core SWH pathway kinase cassette. Tao-1 functions together with Hippo to activate Warts-mediated repression of Yorkie. Tao-1's ability to control SWH pathway activity is evolutionarily conserved because human TAO1 can suppress activity of the Yorkie ortholog, YAP. Human TAO1 controls SWH pathway activity by phosphorylating, and activating, the Hippo ortholog, MST2. Given that SWH pathway activity is subverted in many human cancers, these findings identify human TAO kinases as potential tumor suppressor genes (Poon, 2011).

Genetic and biochemical studies have pointed to the existence of unidentified SWH pathway kinases. Among known SWH pathway kinases, Wts phosphorylates and represses Yki, whereas Hpo activates Wts by phosphorylating the other core kinase cassette proteins, Wts, Sav, and Mats. Proteins that activate Hpo are less well defined. Using a Drosophila genetic screen, this study has identified the sterile 20-like kinase Tao-1 as a SWH pathway protein. By modulating Tao-1 expression, a role was uncovered for this kinase as a suppressor of epithelial tissue growth during Drosophila development. Several points of evidence have led to the proposal that Tao-1 regulates tissue growth by activating the core SWH pathway kinase cassette: (1) tissue with reduced Tao-1 activity displayed several phenotypic similarities to tissue mutant for members of the core SWH pathway kinase cassette, including Yki hyperactivation; (2) alterations in tissue size caused by altered SWH pathway activity were modifiable by Tao-1 hemizygosity; and (3) Tao-1 overexpression activated the core SWH pathway kinase cassette (Poon, 2011).

The observation that Tao-1 is necessary for Hpo overexpression to suppress tissue growth suggests that either Hpo functions either upstream of Tao-1 or, alternatively, that Tao-1 was required to activate Hpo, even when Hpo is overexpressed. The latter scenario is favored based on several biochemical results: (1) Tao-1 overexpression requires Hpo to stimulate Wts activity in Drosophila S2 cells, but the inverse relationship was not observed; (2) human TAO1 stimulates phosphorylation of the Hpo ortholog, MST2, in in vitro kinase assays; and (3) TAO1 activates MST2 in human cultured cells. This model is also in keeping with biochemical studies that showed that Hpo/MST1/2 activate Wts/LATS1/2 by phosphorylating these proteins directly, rather than via an intermediary kinase(Poon, 2011).

Tao-1's role as a SWH pathway kinase appears to be conserved throughout evolution because one of its three human homologs, TAO1, represses YAP activity in a manner that is reliant on the SWH pathway core kinase cassette and because TAO1 was found to phosphorylate and activate MST2. This is the first described example of a sterile 20-like kinase family member phosphorylating another kinase from this family. To help address the mechanism by which TAO1 regulates MST2, it will be important to define the MST2 residue(s) that TAO1 phosphorylates (Poon, 2011).

Currently, it is unclear whether Tao-1's ability to control the SWH pathway is regulated or whether it is constitutively active. Several upstream regulatory branches of the SWH pathway have been identified in recent years and appear to regulate the SWH pathway largely by impinging on the core kinase cassette. Ft promotes Wts stability, whereas both Ft and Crb regulate the levels and apical junctional localization of Ex. Lgl and aPKC have been proposed to influence SWH pathway activity by regulating the subcellular localization of Hpo and dRASSF. Kibra, Ex, and Mer can interact with members of the core kinase cassette, recruit Hpo to the plasma membrane, and induce activation of Hpo and Wts. Given that no evidence of physical interactions was found between Tao-1 and several upstream SWH pathway proteins (e.g., Kibra, Ex, and Mer), biochemical studies aimed at identifying Tao-1-interacting proteins are likely to shed light on the question of Tao-1 regulation(Poon, 2011).

Interestingly, Tao-1 has been shown to regulate microtubule polymerization at the cell cortex in both Drosophila and mammalian cells (Liu, 2010; Timm, 2003). At present there is no evidence to suggest that Tao-1's abilities to regulate SWH pathway-dependent tissue growth and microtubule stability are in any way linked. However, it is interesting to note that mechanical tension has been shown to regulate microtubule polymerization at the cell cortex, and has been hypothesized to regulate tissue growth and the SWH pathway. Therefore, it is tempting to speculate that Tao-1/TAO1 kinases could act as a point of convergence between mechanical tension, the SWH pathway, and control of tissue growth (Poon, 2011).

This study has shown that Tao-1's SWH pathway regulatory function is conserved in mammalian cells. It will be important to extend these findings to determine whether the TAO1, 2, and 3 kinases control the growth of mammalian tissues. In addition, given that SWH pathway activity is subverted at a high frequency in many human tumor types, it raises the possibility that TAO1, 2, and 3 function as tumor suppressor genes. According to the COSMIC and Tumorscape databases, TAO1, 2, and 3 do not appear to be mutated or deleted at a high frequency in the tumors that were surveyed. However, based on the current finding that Tao-1 controls SWH pathway-dependent tissue growth, a closer examination of the potential tumor suppressor function of human TAO kinases is warranted, particularly in cancers that are known to exhibit enhanced YAP activity (Poon, 2011).

Par-1 regulates tissue growth by influencing hippo phosphorylation status and hippo-salvador association

The evolutionarily conserved Hippo (Hpo) signaling pathway plays a pivotal role in organ size control by balancing cell proliferation and cell death. This study reports the identification of Par-1 as a regulator of the Hpo signaling pathway using a gain-of-function EP screen in Drosophila melanogaster. Overexpression of Par-1 elevates Yorkie activity, resulting in increased Hpo target gene expression and tissue overgrowth, while loss of Par-1 diminishes Hpo target gene expression and reduces organ size. par-1 functions downstream of fat and expanded and upstream of hpo and salvador (sav). In addition, it was also found that Par-1 physically interacts with Hpo and Sav and regulates the phosphorylation of Hpo at Ser30 to restrict its activity. Par-1 also inhibits the association of Hpo and Sav, resulting in Sav dephosphorylation and destabilization. Furthermore, evidence is provided that Par-1-induced Hpo regulation is conserved in mammalian cells. Taken together, these findings identified Par-1 as a novel component of the Hpo signaling network (Huang, 2013).

The Hpo signaling pathway has emerged as a conserved pathway that controls tissue growth and balances tissue homeostasis via the regulation of the downstream Sd-Yki transcription complex. Despite the importance of this pathway in development and carcinogenesis, many unknown regulators of the Hpo pathway remain to be identified. This study identified Par-1 as one such Hpo pathway regulator via a genetic overexpression screen using Drosophila EP lines. This study demonstrated that Par-1 was essential for the restriction of Hpo signaling. It was also demonstrated that overexpression of Par-1 promotes tissue growth via the inhibition of the Hpo pathway, whereas loss of Par-1 promotes Hpo signaling to suppress growth and induce apoptosis. Using the Drosophila eye and wing imaginal discs as well as cultured cells, this study provides the first genetic and biochemical evidence for a function of Par-1 in the Hpo pathway (Huang, 2013).

Although the conserved function of Hpo has been well studied, the regulatory mechanism of its kinase activity is still largely obscure. Currently, the regulatory mechanism of Hpo kinase activity is believed to mainly be dependent on autophosphorylation by altering the phosphorylation status of the Thr195. However, whether the uncharacterized phosphorylation events of Hpo, which have been identified in several recent proteome-wide phosphorylation studies, contribute to the regulation of Hpo activity is still unknown. By studying the mechanism underlying Par-1 function in Hpo signaling, this study demonstrated that Par-1 induces Hpo phosphorylation at Ser30 and this leads to the regulation of Hpo kinase activity (Huang, 2013).

In recent proteome-wide phosphorylation studies using Drosophila embryos, it was suggested that Hpo was phosphorylated at Ser30 in vivo, indicating an important role for the Ser30 site in the regulation of Hpo activity. To determine the biological significance of Hpo phosphorylation at Ser30 induced by Par-1, whether Ser30 phosphorylation state affects Hpo phosphorylation at Thr195, which is important for Hpo activation, was tested. Par-1, but not Par-1-KD, was shown to significantly inhibit Hpo phosphorylation levels at Thr195, whereas this inhibitory effect was abolished when the Ser30 site was mutated. More importantly, phosphorylation at Thr195 was slightly elevated when Ser30 was mutated into an alanine. These findings suggested that Par-1 regulates Hpo activity via antagonizing phosphorylation at the Thr195 site by regulating Ser30 phosphorylation. It has been reported that the Hpo Thr195 site is not only auto-phosphorylated but also phosphorylated by Tao-1 (Boggiano, 2011; Poon, 2011), a partner of Par-1 in the regulation of microtubule dynamics. Thus, it was asked whether Par-1-induced phosphorylation at Ser30 also affects Tao-1-mediated phosphorylation at Thr195. Par-1 was shown to suppress Tao-1-mediated phosphorylation at Thr195. The antagonistic effect of Par-1 and Tao-1 on Hpo phosphorylation at Thr195 motivated the examination of the interrelationship of Par-1 and Tao-1 in the Hpo pathway. It was found that Tao-1 disrupted Par-1-induced phosphorylation mobility shift of Hpo-KD, suggesting that the function of Par-1 in the Hpo pathway was modulated by upstream signaling (Huang, 2013).

Several unresolved questions remain. The interaction between Par-1 and Hpo/Sav may be tightly regulated because full-length Par-1 only weakly interacts with Hpo/Sav, unlike the interaction with the N-terminal fragment of Par-1. However, the triggering signal for Par-1 to interact with Hpo/Sav is still unknown. It has been reported that Par-1 is activated by Tao-1 and LKB1. This study established that Par-1 antagonized Tao-1 in Hpo signaling: in Drosophila, the antagonistic relationship between Par-1 and Tao-1 in microtubule regulation has been previously reported (Liu, 2010; King, 2011; Wang, 2007). Thus, it is unlikely that Tao-1 functions as the trigger. Whether LKB1 functions as an activator of Par-1 in Hpo signaling was investigated by expressing the LKB1 transgene in different organs. Unlike Par-1, ectopic LKB1 expression limits both wing and eye growth, indicating that LKB1 is also not the trigger (Huang, 2013).

This study has shown that Par-1 and Tao-1 exhibit opposing effects on Hpo signaling. Given that Tao-1 and Par-1 are partners that regulated microtubule dynamics via the phosphorylation of Tau, Tau may have a function in Hpo signaling. To investigate this hypothesis, genetic and biochemical studies were employed, and it was found that Tau RNAi failed to suppress the expression of Hpo pathway-responsive genes. In addition, Tau did not trigger Hpo phosphorylation and Sav dissociation in vitro, indicating that Par-1 regulates Hpo signaling independent of Tau. Interestingly, it has been previously suggested that Par-1 does not regulate Tau activity in Drosophila, indicating an evolutionary difference between Par-1 and Tau-1 function (Huang, 2013).

This study has provided evidence that Par-1 regulates Hpo signaling via the phosphorylation of Hpo or the destruction of the Hpo/Sav complex. Because Par-1 is a well-known polarity regulator and polarity components, such as Crumb and Lgl, have been shown to be involved in the Hpo signaling pathway, it is possible that Par-1 may regulate Hpo signaling via a polarity complex, or its activity might be regulated via a polarity complex. Indeed, the localization of Crumb and Patj were affected by Par-1 expression. Thus, further studies on polarity complexes and Hpo signaling will help elucidate this problem (Huang, 2013).

Modulating F-actin organization induces organ growth by affecting the Hippo pathway

The Hippo tumour suppressor pathway is a conserved signalling pathway that controls organ size. The core of the Hpo pathway is a kinase cascade, which in Drosophila involves the Hpo and Warts kinases that negatively regulate the activity of the transcriptional coactivator Yorkie. Although several additional components of the Hippo pathway have been discovered, the inputs that regulate Hippo signalling are not fully understood. This study reports that induction of extra F-actin formation, by loss of Capping proteins A or B, or caused by overexpression of an activated version of the formin Diaphanous, induces strong overgrowth in Drosophila imaginal discs through modulating the activity of the Hippo pathway. Importantly, loss of Capping proteins and Diaphanous overexpression does not significantly affect cell polarity and other signalling pathways, including Hedgehog and Decapentaplegic signalling. The interaction between F-actin and Hpo signalling is evolutionarily conserved, as the activity of the mammalian Yorkie-orthologue Yap is modulated by changes in F-actin. Thus, regulators of F-actin, and in particular Capping proteins, are essential for proper growth control by affecting Hippo signalling (Sansores-Garcia, 2011).

This study investigated a role of actin Capping proteins and changes in actin organization on tissue growth. Changing the organization of the actin cytoskeleton affects growth by modulating the activity of the Hpo pathway. Several observations support this conclusion. First, loss of Capping proteins, or induction of extra F-actin by overexpression of Dia^CA, induced strong overgrowth of Drosophila imaginal discs. Second, changes in actin organization lead to the upregulation of Hpo pathway target genes, which depended on normal Yki activity. Third, the effects of Dia^CA or loss of Capping proteins on Hpo signalling are specific downstream effects and not the cause of general defects in cellular organization and signalling. Fourth, actin dynamics and the Hpo pathway interact with each other in evolutionary distant species. Therefore, F-actin regulates growth in different species through effects on the Hpo pathway (Sansores-Garcia, 2011). p>Several observations were striking. First, the data suggest that the effects on Hpo signalling are specific effects of F-actin accumulation. Given the crucial role for F-actin in numerous cellular processes, it might have been expected that imbalances in F-actin organization lead to defects in many different signalling pathways. Surprisingly, however, while changing F-actin organization had strong effects on Hpo signalling, it did not significantly affect epithelial cell polarity, or Hh and Dpp signalling, indicating a specific molecular effect. Second, given the pleiotropic functions of F-actin, it might have been expected that knockdown of Capping proteins would lead to reduced growth. On the contrary, loss of Capping proteins or higher levels of F-actin induced by Dia^CA lead to increased proliferation and overgrowth, although mutant regions showed some dying cells. It is therefore concluded that Capping proteins act as tumour suppressors that affect growth through the Hpo pathway (Sansores-Garcia, 2011).

The observations that both loss of Cpa and Cpb, as well as overexpression of activated Dia-induced overgrowth indicate that their effects on growth are due to F-actin accumulation. It is currently not known whether the observed effects involve a specific pool of F-actin or whether any increase in F-actin induces growth. A screen in S2 cells identified several other genes involved in F-actin formation that modulated Yki activity. It remains to be seen whether these also modulate Hpo signalling in vivo (Sansores-Garcia, 2011).

The effect of modulating F-actin organization on the Hpo pathway may be evolutionary conserved as strong effects on Yap localization and activity is seen in mammalian cells. Therefore, proteins that restrict F-actin formation may be tumour suppressors in humans and associated with cancer. Indeed, one example of an inhibitor of F-actin polymerization that is downregulated in several cancers is Gelsolin. Gelsolin is known to sever F-actin filaments and to cap them, which inhibits F-actin polymerization. Thus, modulators of the F-actin cytoskeleton affect cell proliferation in mammals and may be involved in the development of cancer (Sansores-Garcia, 2011).

To gain insight into the mechanism by which F-actin affects the Hpo pathway, the localization of different Hpo pathway components was analyzed. Mer and Ex, which contain FERM (4.1 protein-ezrin-radixin-moesin) domains and are known to bind F-actin, localized normally in cells that lost Cpa function, and similarly Hpo localization was unaffected. However, Yki localization was affected such that more Yki protein localized to the nuclei in cells that lost Capping protein function. Therefore, the F-actin status affects growth upstream of Yki, but might not affect growth by regulating the localization of upstream components in the Hpo pathway. The in vivo data show that overexpression of Ex or Hpo did not significantly rescue Dia^CA-induced phenotypes in contrast to their ability to rescue fat and ex;mer mutant phenotypes. Overexpression of Wts, however, significantly suppressed Dia^CA-induced overgrowth and Hpo pathway target gene expression. Interestingly, ex mutant cells have increased levels of F-actin, although not as much as cells depleted for Capping proteins. Thus, Ex could regulate Hpo signalling indirectly through its effect on F-actin. However, two observations argue against this possibility. First, overexpression of Hpo can rescue ex mutant phenotypes, but not those caused by Dia^CA. Second, Ex and Mer directly interact with the Hpo cofactor Sav. Altogether, these data suggest that F-actin affects growth in parallel to Ex and Hpo but upstream of Yki (Sansores-Garcia, 2011).

Recent work showed that a small fraction of the mammalian homologues of Hpo, MST1, and MST2, localize to apical actin filaments. Upon disruption of the actin filaments, MST1/2 were activated, although it is not known whether this involves a relocalization of MST1/2. Consistent with MST activation, it was found that under similar conditions in which sever F-actin bundles are severed, Yap is exported from the nucleus and its activity is downregulated. It is not known whether the same or different mechanisms are engaged to regulate Hpo signalling in response to severing or inducing actin filaments, but elucidation of the molecular mechanisms involved will answer this question (Sansores-Garcia, 2011).

The data reveal an interaction between F-actin organization and the Hpo pathway in the regulation of growth. A possible connection between F-actin and growth may involve the sensing of mechanical forces. In vitro, cells change their rate of proliferation in response to external mechanical forces, which requires an intact actin cytoskeleton. In vivo, the actin cytoskeleton might act as a sensor to couple mechanical forces to growth control. While it is not clear whether these effects depend on the Hpo pathway, it is an exciting possibility to be tested in the future (Sansores-Garcia, 2011).

Regulation of Drosophila glial cell proliferation by Merlin-Hippo signaling

Glia perform diverse and essential roles in the nervous system, but the mechanisms that regulate glial cell numbers are not well understood. This study identified and characterize a requirement for the Hippo pathway and its transcriptional co-activator Yorkie in controlling Drosophila glial proliferation. Yorkie was found to be both necessary for normal glial cell numbers and, when activated, sufficient to drive glial over-proliferation. Yorkie activity in glial cells is controlled by a Merlin-Hippo signaling pathway, whereas the upstream Hippo pathway regulators Fat, Expanded, Crumbs and Lethal giant larvae have no detectable role. Functional characterization of Merlin-Hippo signaling was extended by showing that Merlin and Hippo can be physically linked by the Salvador tumor suppressor. Yorkie promotes expression of the microRNA gene bantam in glia, and bantam promotes expression of Myc, which is required for Yorkie and bantam-induced glial proliferation. These results provide new insights into the control of glial growth, and establish glia as a model for Merlin-specific Hippo signaling. Moreover, as several of these genes have been linked to human gliomas, the results suggest that this linkage could reflect their organization into a conserved pathway for the control of glial cell proliferation (Reddy, 2011).

Merlin was first identified as the product of a human tumor suppressor gene, NF2, loss of which in peripheral glial cells results in benign tumors. Merlin has also been identified as an inhibitor of gliomas. The current observations indicate that the role of Merlin as a negative regulator of glial cell proliferation is conserved from humans to Drosophila and, thus, that Drosophila can serve as a model for understanding Merlin-dependent regulation of glial growth (Reddy, 2011).

Studies in Drosophila imaginal discs first linked Merlin to Hippo signaling, and Merlin was subsequently linked to Hippo signaling in mammalian cells, including its role in meningioma. However, the tumor suppressor activity of Merlin has also been linked to other downstream effectors in mammals, including Erb2, Src, ras, rac, TORC1 (CRTC1 -- Human Gene Nomenclature Database; see Drosophila CRTC) and CRL4 (IL17RB -- Human Gene Nomenclature Database), creating some uncertainty regarding the general importance of the linkage of Merlin to Hippo in growth control. This study found that depletion of Merlin, depletion of other tumor suppressors in the Hippo pathway, or expression of an activated form of Yki, all result in similar glial overgrowth phenotypes. Moreover, depletion of Merlin increased nuclear localization of Yki, and depletion of Yki suppressed the overgrowth phenotype of Merlin. Together, these observations clearly establish that the glial overgrowth phenotype associated with Merlin depletion in Drosophila is mediated through the Hippo signaling pathway (Reddy, 2011).

A noteworthy feature of Hippo signaling in Drosophila glial cells is that Merlin appears to be uniquely required as an upstream regulator of Hippo signaling, as the Fat-dependent, Ex-dependent and Lgl-dependent branches have no detectable role. Glia might, thus, provide an ideal model for mechanistic investigations of the Merlin branch of Hippo signaling. Fat-Hippo signaling employs Fat as a transmembrane receptor and Dachsous as its transmembrane ligand, whereas Ex-Hippo signaling appears to employ Crumbs as a transmembrane receptor and ligand. By contrast, Drosophila transmembrane proteins that mediate extracellular signaling and interact with Merlin have not yet been identified. Distinct mechanisms might also be involved in signal transduction downstream of Merlin. Although there is evidence that Ex and Merlin can both influence Hippo activity, Ex, but not Mer, can directly associate with Hpo. Conversely, Merlin, but not Ex, can interact directly with Salvador, and Merlin, Salvador and Hippo can form a trimeric complex. Moreover, the kibra loss-of-function phenotype is weaker than expanded in imaginal discs, but comparable to Merlin, and it was found that depletion of kibra also has a significant effect on glial cell proliferation. Kibra is highly expressed in mammalian brain, and alleles of KIBRA (WWC1 -- Human Gene Nomenclature Database) have been linked to human memory performance. The role of kibra in regulating glial cell numbers in Drosophila thus raise the possibility that the influence of KIBRA on human memory might reflect a role in glial cells (Reddy, 2011).

Finally, it is noted that although Hippo signaling has been investigated in several different organs in Drosophila, including imaginal discs, ovarian follicle cells, neuroepithelial cells and intestinal cells, these all involve roles in epithelial cells, in which upstream regulators of the pathway (e.g. Fat, Ex, Mer) all have a distinctive localization near adherens junctions. The identification of a requirement for Hippo signaling in glia is the first time in Drosophila that a role for the pathway has been identified in non-epithelial cells. Indeed, in previous studies it was found that Hippo signaling influences proliferation of neuroepithelial cells, but other neuronal cell types, including neuroblasts, ganglion mother cells and neurons, are insensitive to Yki (Reddy, 2011).

Considerable attention has been paid to genes for which mutation or inappropriate activation can cause over-proliferation of glial cells, resulting in glial tumors. However, less is known about the mechanisms required for normal glial growth. Through loss-of-function studies, several genes essential for normal glial cell numbers were identified, including yki, sd, ban, mad and myc. The requirement for yki, mad and sd, together with epistasis studies, identifies a requirement for active Yki in glial growth. This in turn implies that downregulation of Hippo signaling is important for normal glial growth. Understanding how this is achieved will provide further insights into the regulation of glial cell numbers (Reddy, 2011).

A requirement for Mad, together with its upstream regulator Thickveins (Tkv), in promoting retinal glial cell proliferation was has been established in previous studies. Current studies of glial cells, together with recent work in imaginal discs, emphasize that in mediating the growth-regulating activity of Hippo signaling, Yki utilizes multiple DNA-binding partners (i.e. Mad and Sd) in the same cells at the same time to regulate distinct downstream target genes required for tissue growth (Reddy, 2011).

Although Yki activity influenced glial cell numbers throughout the nervous system, direct analysis of cell proliferation by EdU labeling revealed that retinal glia were more sensitive to Yki activation at late third instar than central brain glia, and significant induction of central brain glial cell proliferation was only observed when Yki activation was combined with Myc over-expression. Further studies will be required to define the basis for this differential sensitivity, but the implication that the proliferative response to Yki is modulated by developmental stage and/or glial cell type has important implications for diseases associated with both excess and deficits of glial cells (Reddy, 2011).

These studies in Drosophila delineate functional relationships among genes involved in the control of glial cell proliferation. Mammalian homologs of Merlin, Yki and Myc have been implicated in glioma. Although a mammalian homolog of ban has not been described, other miRNAs have also been linked to glioma. These observations imply that these genes can be placed into a pathway, in which Merlin, through Hippo signaling, regulates Yki, Yki regulates ban, and ban regulates Myc. However, as expression of Myc alone did not lead to substantial overgrowth of glia, Yki and ban must also have other downstream targets important for the promotion of glial cell proliferation. Moreover, the current observations indicate that a Yki-Sd complex is also required for glial growth. In addition to the well characterized downstream target Diap1, Yki-Sd complexes in glial cells might regulate Myc directly, as suggested by studies in imaginal discs, and might regulate cell cycle genes in conjunction with E2F1 (Reddy, 2011).

The influence of activated-Yki on a ban-GFP sensor, together with the observations that yki is not required for ban-mediated overgrowth, whereas ban is required for Yki-mediated overgrowth, position ban downstream of Yki. This is consistent with studies of Hippo signaling in imaginal discs, in which ban has also been identified as a target of Yki for growth regulation. The placement of Myc downstream of Yki and ban is supported by the observation that Myc levels can be increased by expression of ban or activated-Yki, and by genetic tests that indicate that Myc is required for Yki- and ban-promoted glial overgrowth. A mechanism by which ban can regulate Myc levels, involving downregulation of a ubiquitin ligase that negatively regulates Myc, was identified recently in imaginal discs, and might also function in glial cells. Myc has been reported to downregulate Yki expression in imaginal discs and, although this study has not investigated whether a similar negative-feedback loop exists in glial cells, the synergistic enhancement of glial cell proliferation observed when Yki and Myc were co-expressed is consistent with this possibility, as the expression of both genes under heterologous promoters could bypass negative regulation of Yki by Myc (Reddy, 2011).

The Myc proto-oncogene is de-regulated or amplified in several human cancers, including gliomas. The sensitivity of Yki/ban-induced overgrowth to reduced Myc levels parallels studies of glioma models involving other signaling pathways. For example, Myc is upregulated by EGFR, and is limiting for EGFR-PI3K-induced glial cell overgrowth in a Drosophila glioma model, and p53 and Pten-driven glioma in mouse models is also Myc dependent. Considering the evidence linking Merlin and Yap to glial growth in mammals, and the identification of Myc as a downstream target of Yap in cultured cells, it is likely that Yap could also influence glial growth in mammals, in part, through regulation of Myc (Reddy, 2011).

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

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

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

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

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

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

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

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

Dimerization and cytoplasmic localization regulate Hippo kinase signaling activity in organ size control

The Hippo (Hpo) signaling pathway controls organ size by regulating the balance between cell proliferation and apoptosis. Although the Hpo function is conserved, little is known about the mechanism of how its kinase activity is regulated. Based on structural information, mutation-function analysis was performed and in vitro and in vivo evidence is provided that Hpo activation requires proper dimerization of its N-terminal kinase domain as well as the C-terminal SARAH domain. Hpo carrying point mutation M242E can still dimerize, yet the dimers formed between intermolecular kinase domains were altered in conformation. As a result, autophosphorylation of Hpo at Thr-195 was blocked, and its kinase activity was abolished. In contrast, Hpo carrying I634D, a single mutation introduced in the Hpo C-terminal SARAH (for Sav/Rassf/Hpo) domain, disrupted the dimerization of the SARAH domain, leading to reduced Hippo activity. It was also found that the Hpo C-terminal half contains two nuclear export signals that promote cytoplasmic localization and activity of Hpo. Taken together, these results suggest that dimerization and nucleocytoplasmic translocation of Hpo are crucial for its biological function and indicate that a proper dimer conformation of the kinase domain is essential for Hpo autophosphorylation and kinase activity (Jin, 2012).

Previous studies have revealed that Hpo signaling is essential for tissue growth and organ size control in both Drosophila and vertebrate. However, despite the emerging significance of this signaling transduction pathway, the mechanism by which the Hpo kinase is regulated is poorly understood. Based on a combination of structural and functional analyses, the current results demonstrate that the autophosphorylation and kinase activation of Hpo depends on its homodimerization, which is mediated by two distinct functional domains. Dimerization of the N-terminal kinase domain, which is mediated predominantly by hydrophobic interactions, is essential for Hpo autophosphorylation and its kinase activation, whereas the coiled coil-mediated dimerization of the C-terminal SARAH domain is probably necessary to modulate Hpo function (Jin, 2012).

The dimerization of the Hpo SARAH domain is not essential for its kinase activity. Disruption of the dimerization mediated by the SARAH domain only led to a partial decrease for its autophosphorylation and kinase activity. It is important to emphasize that although the Hpo SARAH domain seems to be non-essential for autophosphorylation, it may be crucial for regulatory functions based on existing studies of Sav and RASSF in both Drosophila and mammalian cells. Indeed, wild type Hpo always shows higher activity than just the kinase domain in vivo because GMR-Hpo results in 100% lethality at the early pupal stage, but expressing the GMR-Hpo-kinase domain alone results in a weaker phenotype. Misexpressed HpoI634D in Drosophila wing results in a discernible change compared with the wild type Hpo. It is likely that the strong kinase activity caused by overexpression may conceal the regulatory function of the SARAH domain. FRET analysis showed that a higher basal level of interaction between Hpo C termini is detected, compared with that between the Hpo N termini. However, when activated by the upstream signaling, the FRET change between C termini is not as dramatic as that observed for that between the N termini, suggesting that the SARAH domain may constitutively form dimers, whereas the conformation of the N-terminal dimer is inducible. At this point, it is speculated that one possible role of the Hpo C-terminal dimerization is to function as a platform to facilitate the intermolecular association between the N-terminal kinase domains. In addition, it seems that the dimerization of SARAH domain may help to stabilize Hpo because it was repeatedly detected that the I634D is less stable than the wild type Hpo when expressed in S2 cells. The immunoprecipitation data support this point by showing that the FLAG-Hpo-C-I634D barely pulled down the endogenous Hpo. Furthermore, the activity of Hpo could be regulated by the dimerization partners, which are recognized by the SARAH domain in the upstream or downstream branches of the Hpo signaling pathway, such as dRASSF and Sav. Consistent with this finding, this study showed that Hpo and Sav/dRASSF heterodimerize via the conserved C-terminal SARAH domains of both proteins (Jin, 2012).

Hpo SARAH domain plays a dual role in regulating Hpo activity. Besides the functional regulation of Hpo through homodimerization as well as through heterodimerization with other SARAH-containing partners, the SARAH domain of Hpo participates in the regulation of nucleocytoplasmic distribution of Hpo via a CRM1-dependent nuclear export mechanism. Two functional NESs were identified that are both located in the Hpo C-terminal region, including the one located in the SARAH domain. The nucleus-cytoplasm shuttling of Hpo is regulated by the nuclear export pathway, and the translocation of Hpo is critical for its functional regulation because the Hpo^NES1/2 lost most of its signaling activity in vitro and in vivo. These findings will bring novel insights to the present functional studies and help to answer questions regarding the importance in regulating nuclear-cytoplasmic translocation of Hpo and the function of Hpo in the nucleus (Jin, 2012).

The Hpo kinase domain (Hpo-N) shows a diffused localization when the Hpo C-terminal part is deleted, and its pathway activity is much lower than the activity of wild type Hpo, which localizes mainly in the cytoplasm. However, the mammalian Hpo homolog Mst1 is activated by autophosphorylation and the caspase-dependent cleavage, which releases the active 36-kDa N-terminal kinase domain. The active truncated kinase translocates into the nucleus and promotes apoptosis by phosphorylating nuclear substrates. Because Hpo does not have the conserved caspase cleavage site, the observation suggested that Hpo may exhibit the growth-suppressive activity mainly in the cytoplasm; therefore, a different mechanism from the mammalian MST needs to be determined. Mammalian Mst1/2 protein was localized predominantly or entirely in the cytoplasm in an LMB-dependent manner, and the function of nucleocytoplasmic shuttling of MST1/2 needs to be further studied (Jin, 2012).

Although considerable progress has been made in investigating the mechanisms of regulation and function of the SARAH domain in the Hpo signaling pathway, much less is known about the activating mechanisms of the N-terminal kinase domain, especially in vivo. A structure-function analysis of Drosophila Hpo was performed based on the modeled three-dimensional structure of Hpo kinase domain. As a result, Thr-195 was identfied as the autophosphorylation site and critical for Hpo kinase activity. More importantly, it was demonstrated that the N-terminal kinase domain of Hpo mediates homodimerization independently of the C-terminal SARAH domain and that the dimeric conformation of the kinase domain is essential for intermolecular autophosphorylation. A point mutation, M242E, which is predicted to interfere with the N-terminal dimeric conformation, resulted in the loss of autophosphorylation and kinase activity but not self-interaction. All of the genetic analyses and genetic interaction data in Drosophila were consistent with the biochemistry results and supported the findings. Moreover, overexpression of the deletion form Δ238-246 induces bigger organ size using different drivers in Drosophila. This is consistent with the prediction that Δ238-246 interferes with the dimeric conformation to an even larger extent and therefore functionally imposes a stronger dominant-negative effect compared with the point mutation M242E. Hpo KD and T195A behaved in a dominant-negative manner because they may nonproductively associate with endogenous Hpo or Sav and block signal activation, whereas overexpressed M242E and Δ238-246 may interfere with endogenous Hpo homodimer, and exhibited similar dominant effects. In addition, evidence is provided that the intermolecular autophosphorylation of Thr-195 is only achieved when its kinase domains are properly dimerized. It is proposed that the kinase activity of Hpo depends on Thr-195 phosphorylation, whereas proper dimerization of the kinase domain is the prerequisite of its phosphorylation. Thus, once the autophosphorylation happens, the dimerization may not be that critical anymore. The hybrid form T195E-Δ still possesses pathway activity (Jin, 2012).

In summary, these biochemical and genetic results demonstrate, for the first time, that homodimerization and nucleocytoplasmic shuttling regulate the biological function of Hpo. The N-terminal dimeric conformation of Hpo is essential for its intermolecular autophosphorylation and kinase activation and organ size control (Jin, 2012).

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).

CYLD negatively regulates Hippo signaling by limiting Hpo phosphorylation in Drosophila

Cylindromatosis (CYLD), a deubiquitinase and regulator of microtubule dynamics, has important roles in the regulation of inflammation, immune response, apoptosis, mitosis, cell migration and tumorigenesis. Although great progress has been made in the biochemical and cellular functions of CYLD, its role in animal development remains elusive. This study identified Drosophila CYLD (dCYLD) as a negative regulator of the Hippo pathway in vivo. dCYLD associates and colocalizes with Hpo , a core component of the Hippo pathway, in the cytoplasm, and decreases Hpo activity through limiting its phosphorylation at T195. dCYLD also limits Hippo signal transduction as evidenced by decreasing phosphorylation and thereby increasing activity of Yki, the key downstream effector of the Hippo pathway. These findings uncover dCYLD as a negative regulator of the Hippo pathway and provide new insights into the physiological function of dCYLD in animal development (Chen, 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).

Salvador has an extended SARAH domain that mediates binding to Hippo kinase

The Hippo pathway controls cell proliferation and differentiation through the precisely tuned activity of a core kinase cassette. The activity of Hippo kinase is modulated by interactions between its C-terminal coiled-coil, termed the SARAH domain, and the SARAH domains of either dRassF or Salvador. This study examined the molecular basis of SARAH domain mediated interactions and their influence on Hippo kinase activity. Focused was placed on Salvador, a positive effector of Hippo activity and the least well characterized SARAH domain-containing protein. The crystal structure was determined of a complex between Salvador and Hippo SARAH domains from Drosophila. This structure provided insight into the organization of the Salvador SARAH domain including a folded N-terminal extension that expands the binding interface with Hippo SARAH domain. This extension was found to improve the solubility of Salvador SARAH domain, enhances binding to Hippo, and is unique to Salvador. It is therefore suggested that the definition of the Salvador SARAH domain be expanded to include this extended region. The heterodimeric assembly observed in the crystal was confirmed by crosslinked mass spectrometry and provided a structural basis for the mutually exclusive interactions of Hippo with either dRassF or Salvador. Salvador influenced the kinase activity of Mst2, the mammalian Hippo homolog. In co-transfected HEK293T cells, human Salvador increased the levels of Mst2 autophosphorylation and Mst2-mediated phosphorylation of select substrates, whereas Salvador SARAH domain inhibited Mst2 autophosphorylation in vitro. These results suggest Salvador enhances the effects of Hippo kinase activity at multiple points in the Hippo pathway (Cairns, 2018).

Homeostatic control of Hpo/MST kinase activity through autophosphorylation-dependent recruitment of the STRIPAK PP2A phosphatase complex

The Hippo pathway controls organ size and tissue homeostasis through a kinase cascade leading from the Ste20-like kinase Hpo (MST1/2 in mammals) to the transcriptional coactivator Yki (YAP/TAZ in mammals). Whereas previous studies have uncovered positive and negative regulators of Hpo/MST, how they are integrated to maintain signaling homeostasis remains poorly understood. This study identifies a self-restricting mechanism whereby autophosphorylation of an unstructured linker in Hpo/MST creates docking sites for the STRIPAK PP2A phosphatase complex to inactivate Hpo/MST. Mutation of the phospho-dependent docking sites in Hpo/MST or deletion of Slmap, the STRIPAK subunit recognizing these docking sites, results in constitutive activation of Hpo/MST in both Drosophila and mammalian cells. In contrast, autophosphorylation of the Hpo/MST linker at distinct sites is known to recruit Mats/MOB1 to facilitate Hippo signaling. Thus, multisite autophosphorylation of Hpo/MST linker provides an evolutionarily conserved built-in molecular platform to maintain signaling homeostasis by coupling antagonistic signaling activities (Zheng, 2017).

DEVELOPMENTAL BIOLOGY

Larval

Imaginal discs from third-instar larvae were probed with either an antisense hpo probe. hpo is expressed in an unpatterned fashion throughout all imaginal discs including the eye imaginal disc (Harvey, 2003).

Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo

Neutral competition, an emerging feature of stem cell homeostasis, posits that individual stem cells can be lost and replaced by their neighbors stochastically, resulting in chance dominance of a clone at the niche. A single stem cell with an oncogenic mutation could bias this process and clonally spread the mutation throughout the stem cell pool. The Drosophila testis provides an ideal system for testing this model. The niche supports two stem cell populations that compete for niche occupancy. This study shows that cyst stem cells (CySCs) conform to the paradigm of neutral competition and that clonal deregulation of either the Hedgehog (Hh) or Hippo (Hpo) pathway allows a single CySC to colonize the niche. The driving force behind such behavior is accelerated proliferation. These results demonstrate that a single stem cell colonizes its niche through oncogenic mutation by co-opting an underlying homeostatic process (Amoyel, 2014).

This study characterized the behavior of somatic CySCs in the Drosophila testis and explored the molecular mechanisms that regulate their ability to compete with their neighbors for limited space at the niche. Single stem cell clones were found to bias stem cell replacement dynamics in their favor, leading to non-neutral competition, when they had increases in Hh signaling, Yki activity or in the rate of proliferation, but not when JAK/STAT signaling or adhesion were dys-regulated. Furthermore, it was found that the dynamics of CySCs were well-described by a model in which they were continually and stochastically lost and replaced, leading to neutral drift dynamics and a consolidation of clonal diversity (Amoyel, 2014).

This observation contrasts with the dynamics of GSC offspring fate choices, where oriented divisions and mother centromere retention determine which cells remain as stem cells and which are thrust out of the niche to differentiate. However, careful analysis of GSC dynamics has suggested that they also undergo neutral competition, albeit at a slower loss/replacement rate than CySCs. Thus, within the same stem cell niche, two markedly different strategies for self-renewal are in use, exemplified by the requirement for yki in CySC self-renewal, but not in GSC self-renewal. This is particularly surprising as the two stem cell populations are by necessity linked, in that they need to produce offspring in the correct ratio, as well as the fact that CySCs support GSC self-renewal through BMP production. It has been hypothesized that the careful choice of stem cell retention in the GSC pool is a requirement of their role in preserving the genetic integrity of the species. CySCs are under no such constraint, and moreover, need to proliferate twice as fast in order to produce two cyst cells for every germ cyst. Thus it may be that the functional imperatives of the tissue (e.g., careful replication of DNA versus rapid production of offspring) determine which type of self-renewal strategy a stem cell adopts (Amoyel, 2014).

This study revealed an unexpected ratio of CySCs to GSCs, close to 1:1 and different from the 2:1 ratio described by a previous study. However it is noted that both studies find the same number of CySCs (approximately 13), and that the difference resides in the number of GSCs. Indeed, the previous study find a ratio of 1.3 CySCs:1 GSC in larval testes which increases to 1.8:1 in young adults, due entirely to a drop in the number of GSCs. This may be a function of the genetic background used previously, as this study established the 1:1 ratio through three different experiments in distinct genetic stocks. Although the analysis of the data is consistent with neutral competition between 13 equipotent CySCs, by the nature of the neutral competition model, the possibility cannot be ruled out that the stem cell compartment is heterogeneous with cells moving reversibly between states in which they become primed for duplication or loss, as recently defined in the mouse intestinal crypt. In this case, the effective number of CySCs may be smaller than the observed figure of N = 13, while the true loss/replacement rate, λ, might be proportionately adjusted to a lower value such that the ratio N²/λ remains constant (Amoyel, 2014).

The results also show that the predominant force driving niche colonization by CySCs is proliferation. How proliferation causes stem cells to replace neighbors more efficiently is not established by this study. However, it is hypothesized that in such a competitive situation, the rate of stem cell loss is not altered but the overproliferating mutants simply produce more offspring, which are in the right place to fill a vacant seat at the niche. It remains possible that a mechanism of active displacement is involved in CySC dominance (i.e., the colonizing stem cells crowd out the wild-type ones), and live-imaging of competing clones might distinguish between passive replacement and active displacement (Amoyel, 2014).

A related issue is how CySCs outcompete GSCs. GSC loss is only observed after most of the CySC pool is comprised of colonizing mutant CySCs. The model is therefore favored that competition among CySCs for niche space precedes that between CySCs and GSCs. It is unclear whether the numerous offspring of the competitive CySCs are passively replacing GSCs that have spontaneously left a vacancy at the niche, or whether colonizing CySCs actively push the GSCs out of the niche. The latter scenario is reminiscent of competition among GSCs in the Drosophila ovary, where the contact area between the GSC and niche depended on DE-cadherin. GSCs that elevated cadherins adhered better to the niche and caused the physical displacement of neighbors. This study explored the contribution of integrin- and cadherin-based adhesion and found that neither affected the competitiveness of CySCs. Moreover, this study found that integrin binding was entirely dispensable for CySC self-renewal, unlike cadherin. Importantly, clonal gain of integrin or cadherin did not lead to niche colonization, indicating that they are not instructive for CySC maintenance. Moreover, no role was found for JAK/STAT signaling in inducing competition at the niche. The fact that neither Stat92E nor integrin was causal to colonization in clonal assays is surprising because both were ascribed critical roles in CySC-dependent niche competition. The reasons for the difference in results by this group and a previous study are not entirely clear. However, it is noted that gain of Stat92E activity in CySCs in an otherwise wild-type background leads to expansion (not loss) of GSCs because JAK/STAT signaling in CySCs enables their extended niche function to support GSC self-renewal. The latter niche role is specific to JAK/STAT signaling in CySCs and cannot be fulfilled by Hh signaling, another CySC self-renewal pathway. Moreover, the clonal assays (as opposed to lineage mis-expression) are able to recapitulate the constant jostling for space at the niche that normally occurs. Regardless, the findings establish that competition and self-renewal are two facets of the same homeostatic process (i.e., proliferation) and that colonizing stem cells have not acquired a new cellular property, but are simply better at self-renewing (Amoyel, 2014).

This study exemplifies how corrupting the naturally occurring process of neutral competition endows a stem cell with greater competitiveness, enabling it to gain dominance within a tissue. Such behavior may be relevant to the early steps of oncogenesis driven by tumor-initiating cells, which have stem cell-like properties, as in the case of carcinoma, glioma and leukemia caused by sustained Hh signaling . The process described in this study of biasing neutral drift by stem cells harboring oncogenic mutations and the mechanism underlying it appear to be conserved. Taken together, these findings may explain observations such as field cancerization, in which a molecular lesion spreads through a tissue, causing multiple foci of the primary tumor (Vanharanta & Massague, 2012) (Amoyel, 2014).

EFFECTS OF MUTATION

Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway

Proliferation and apoptosis must be precisely regulated to form organs with appropriate cell numbers and to avoid tumor growth. Hippo (Hpo), the Drosophila homolog of the mammalian Ste20-like kinases (Dan, 2001), MST1/2, promotes proper termination of cell proliferation and stimulates apoptosis during development. hpo mutant tissues are larger than normal because mutant cells continue to proliferate beyond normal tissue size and are resistant to apoptotic stimuli that usually eliminate extra cells. Hpo negatively regulates expression of Cyclin E to restrict cell proliferation, downregulates the Drosophila inhibitor of apoptosis protein DIAP1, and induces the proapoptotic gene head involution defective (hid) to promote apoptosis. The mutant phenotypes of hpo are similar to those of warts (wts), which encodes a serine/threonine kinase of the myotonic dystrophy protein kinase family, and salvador (sav), which encodes a WW domain protein that binds to Wts. Sav binds to a regulatory domain of Hpo that is essential for its function, indicating that Hpo acts together with Sav and Wts in a signalling module that coordinately regulates cell proliferation and apoptosis (Udan, 2003).

To identify genes that regulate tissue growth, a mutagenesis screen was performed by using the eyFLP system and three complementation groups were isolated that showed similar overgrowth phenotypes. These were sav (also known as shar-pei), wts and a previously uncharacterized locus that has been named 'hippo' (hpo) because of the dark, folded and overgrown cuticle of the mutant heads. Four alleles of hpo (hpo^KC202, hpo^KC203, hpo^KC221 and hpo^KC249) were isolated that all show similar overgrowth phenotypes. Adult mutant heads are enlarged, with larger eyes and expanded and folded cuticle on head and antennae. hpo^KC202 mutant cells show overgrowths on head cuticle, halteres, thorax, wings and legs. Notably, the positions of pattern elements such as ommatidia, bristles and ocelli are relatively normal, and overgrown structures differentiated proper cell types. It is therefore concluded that Hpo is a general growth regulator required to restrict cell number and tissue size in adult structures. Analysis of hpo^KC202 mutant clones in adult eyes by thin sections show that there is a considerable increase in spacing between photoreceptor clusters in mutant regions as compared with wild-type regions. This is due to a large increase in numbers of interommatidial cells, which are evident in hpo^KC202 mutant mid-pupal retinae. These extra cells eventually differentiate into extra bristles and pigment cells. In contrast to the interommatidial cells, most hpo^KC202 mutant ommatidia have normal numbers of photoreceptor, cone and primary pigment cells. Notably, the sizes of all cell types are normal in mutant eyes, although the morphology of photoreceptors are often abnormal. Both the expression of Elav, a marker for differentiating photoreceptor cells, and the R8 marker Senseless (Sens) were also analyzed to test whether hpo affects pattern formation during development. The initial spacing of R8 cells and the number of photoreceptor cells per ommatidium are normal in hpo^KC202 mutant clones. Thus, mutations in hpo primarily affect tissue size but not patterning or differentiation (Udan, 2003).

The effects of loss of Hpo function on cell proliferation were tested by analysing the pattern of bromodeoxyuridine (BrdU) incorporation, which marks cells in S phase. In wild-type eye discs, BrdU-incorporating cells are randomly distributed anterior to the morphogenetic furrow, arrested in G1 in the furrow, and start either to differentiate into photoreceptor cells or to undergo an additional round of cell division referred to as the second mitotic wave posterior to the furrow. After the second mitotic wave, cells cease proliferation and differentiate into the remaining photoreceptor, cone, pigment and bristle cells. Mutant cells in eye discs that were almost completely mutant for hpo^KC202 (subsequently referred to as eyFLP hpo^KC202 eye discs because the clones were induced by eyFLP) properly synchronize their cell cycles in the furrow and progress through the second mitotic wave. In contrast to wild-type, however, cells in eyFLP hpo^KC202 eye discs show ectopic incorporation of BrdU after the second mitotic wave. DNA synthesis was followed by cell division, as assessed by the ectopic expression of phosphorylated histone H3 (PH3), which marks mitotic chromosomes. Thus, Hpo mutant cells fail to arrest in G1 and continue to proliferate. This phenotype is cell autonomous and ectopic proliferation is restricted to uncommitted precursor cells and not observed in differentiating photoreceptor cells. Furthermore, Cyclin E, which is limiting for S-phase initiation in imaginal disc cells, is cell autonomously upregulated in hpo^KC202 mutant clones. Likewise, Cyclin E messenger RNA is upregulated in and around the second mitotic wave in eyFLP hpo^KC202 eye discs. Thus, the transcriptional repression of Cyclin E expression is probably an important downstream effect of Hpo to promote proliferation arrest of uncommitted precursor cells (Udan, 2003).

Because extra interommatidial cells are normally removed by developmentally induced apoptosis, the phenotype of hpo^KC202 mutant retinae indicates that Hpo is required for apoptosis in addition to cell proliferation arrest. Indeed, in contrast to wild-type, clones of hpo^KC202 mutant cells lack apoptotic cells, showing that hpo is required for developmentally regulated apoptosis in the retina. Whether hpo mutant cells are protected from ectopically induced cell death was tested. Overexpression of Hid in the developing eye induces apoptosis and results in loss of eye structures. hpo^KC202 loss-of-function partially rescues cell death in eye discs that ectopically express Hid (Udan, 2003).

The proapoptotic genes hid, reaper, grim, sickle (skl) and Jafrac2 induce apoptosis by activating caspase cascades through direct targeting of DIAP1 for auto-ubiquitination and degradation, which in turn liberates caspases from DIAP1-mediated inhibition. hpo^KC202 mutant clones show an increase in DIAP1 protein and mRNA. Regulation of DIAP1 might include transcriptional mechanisms because DIAP1 mRNA is upregulated just behind the furrow. The increase in DIAP1 might protect cells from apoptosis induced both in normal development and by ectopic expression of Hid. Notably, sav and wts mutants show similar defects in proliferation arrest and apoptosis (Udan, 2003).

Overexpression of several kinases including members of the Ste20 family has been used successfully in yeast, cell culture and Drosophila to activate signalling pathways (Dan, 2001). Thus, overexpression of human MST1/2 in cell culture activates caspases and triggers apoptosis. Ectopic expression of wild-type Hpo during imaginal disc development results in loss of eye, head and wing tissues through the arrest of cell proliferation and the induction of apoptosis. By contrast, overexpression of a kinase-dead variant of Hpo, HpoK71R, does not result in smaller structures, but partially rescues the phenotypes caused by overexpressing wild-type Hpo. Overexpression of Hpoauto, a hyperactive form of Hpo, produces phenotypes similar to those caused by overexpressing wild-type Hpo. The Hpoauto construct carries a deletion that is analogous to a deletion in human MST1 that causes an increase in kinase activity in vitro. Thus, kinase activity is required for Hpo action and HpoK71R is acting as a dominant-negative form of Hpo, suggesting that overexpression of Hpo activates its pathway. GMR-driven Hpo overexpression in the eye disc caused large amounts of apoptosis, similar to GMR-driven Hid expression. Hpo and Hid expression induce DNA fragmentation, activate Drice caspase, downregulate DIAP1 and induce nuclear condensation, which are all hallmarks of apoptosis. The small head and wing phenotypes are partially suppressed by coexpression of DIAP1 and of p35, a general caspase inhibitor. Expression is observed of the proapoptotic gene hid, which is required for developmentally regulated apoptosis observed during wild-type eye development (Udan, 2003).

GMR-driven Hpo expression upregulates hid mRNA and protein but does not effect grim or reaper mRNA. The small wing and eye phenotypes caused by Hpo overexpression are partially rescued by heterozygosity for hid, indicating that Hid is crucial for Hpo to exert its effects. Similarly, heterozygosity for wts suppresses the ectopic Hpo phenotype, suggesting that Hpo requires wts for its action. However, heterozygosity for Tsc1, a gene that functions in the target of rapamycin (TOR) signalling pathway that regulates cell growth, does not affect the Hpo overexpression phenotype. Hpo thus seems to regulate apoptosis at several levels. In one case, Hpo negatively regulates DIAP1 to promote apoptosis. The increase in DIAP1 in hpo mutant clones protect cells from Hid-induced apoptosis, indicating that Hpo effects cell death downstream of Hid. In addition, Hpo seems to act upstream of Hid, because overexpression of Hpo upregulates Hid expression and causes Hid-dependent cell death. In naturally occurring cell death pathways that require Hpo activity, such as the killing of interommatidial cells, a death-inducing signal may activate Hpo, which in turn may induce expression and activation of Hid, as well as downregulation of DIAP1 to trigger apoptosis. It has been shown that the activity of Hid is regulated at the transcriptional and posttranslational level and that hid is required for apoptosis in the retina. MAPK, for example, promotes cell survival by downregulating Hid activity through direct phosphorylation and through transcriptional mechanisms that depend on the downstream transcription factor Pointed. Although no significant changes of MAPK phosphorylation has been detected either in Hpo mutant clones or by Hpo overexpression, subtle effects of Hpo on MAPK activity may contribute to the effect of Hpo on Hid activity and apoptosis. Determining whether hid induction by Hpo is a direct effect or a secondary consequence of Hpo-induced cell death, however, requires further investigation (Udan, 2003).

Because Hpo is expressed ubiquitously, its activity is probably regulated by posttranscriptional mechanisms such as phosphorylation. Indeed, the activities of the mammalian homologs of Hpo, MST1/2, are regulated by phosphorylation during stress-induced cell death. The Hpo/Sav/Wts signalling pathway is unique in that it regulates apoptosis and proliferation. In fact, the hpo mutant overgrowth phenotypes are the outcome of deregulated cell proliferation, which generates extra cells, along with a lack of apoptosis, which allows extra cells to survive and to contribute to adult structures. Hpo, Wts and Sav mutants specifically affect cell numbers but not cell size, in contrast to other growth regulators such as InR signalling, Ras and Myc, which typically affect both cell size and cell number. In addition, the function of Hpo/Sav/Wts signalling is distinct from mechanisms directing cell-cycle exit during terminal differentiation because Hpo, Wts and Sav are required for cell proliferation arrest of uncommitted precursor cells but not differentiating cells. Because of this specificity for precursor cells, only the last cell types to differentiate in the retina, namely the interommatidial pigment and bristle cells, are produced in excess, whereas the number of photoreceptor and cone cells is normal in mutant ommatidia. Wts negatively regulates the activity of Cdc2/Cyclin A33, and Sav, Wts and Hpo negatively regulate Cyclin E. Thus, excessive activity of Cdc2/Cyclin A and Cyclin E may contribute to drive cell proliferation of mutant cells. The promotion of cell-cycle progression alone, however, cannot account for the excess growth (mass accumulation) of mutant tissues. Thus, Hpo/Sav/Wts signalling must regulate additional targets to restrict cell growth and cell proliferation (Udan, 2003).

Hpo, Wts and Sav are highly conserved in vertebrates. As in Drosophila, the mammalian homologs of Hpo, MST1/2, have been implicated in regulating apoptosis. LATS acts as a tumor suppressor gene in vertebrates and the human ortholog of Sav (hWW45) is mutated in cancer cell lines. It will be interesting to determine whether MST1/2 signalling is downregulated in cancer cells (Udan, 2003).

hippo, restricts growth and cell proliferation and promotes apoptosis

Establishing and maintaining homeostasis is critical to the well-being of an organism and is determined by the balance of cell proliferation and death. Two genes that function together to regulate growth, proliferation, and apoptosis in Drosophila are warts, encoding a serine/threonine kinase, and salvador, encoding a WW domain containing Wts-interacting protein. However, the mechanisms by which sav and wts regulate growth and apoptosis are not well understood. Mutations are described in hippo, which encodes a protein kinase most related to mammalian Mst1 and Mst2. Like wts and sav, hpo mutations result in increased tissue growth and impaired apoptosis characterized by elevated levels of the cell cycle regulator cyclin E and apoptosis inhibitor DIAP1. Hpo, Sav, and Wts interact physically and functionally, and regulate DIAP1 levels, likely by Hpo-mediated phosphorylation and subsequent degradation. Thus, Hpo links Sav and Wts to a key regulator of apoptosis (Harvey, 2003).

Three alleles of hippo are lethal either when homozygous or in trans to another allele. Eyes containing hpo mutant clones and wild-type clones have an overrepresentation of mutant tissue when compared to eyes containing clones of the wild-type parental chromosome suggesting that the mutant tissue may have a relative growth advantage. Mutant ommatidial facets are slightly larger than wild-type facets and sometimes contain extra interommatidial bristles. When homozygous clones of hpo were generated in other imaginal discs using hsFLP, outgrowths of tissue were observed and portions of wings containing large hpo clones were larger than the corresponding portion of a wild-type wing, indicating a role for hpo in regulating organ size in tissues other than the eye. Retinal sections of adult eyes containing hpo clones reveal that mutant ommatidia appear to have the normal complement and arrangement of photoreceptor cells. However, hpo mutant ommatidia appear to have significantly more tissue between adjacent ommatidia. Cell outlines are visualized more readily in the pupal retina. In contrast to the single layer of interommatidial cells observed in wild-type retinas, mutant ommatidia have several additional interommatidial cells. These phenotypic abnormalities are very similar to those observed in sav or wts mutant clones (Harvey, 2003).

sav or wts mutant cells in the eye imaginal disc fail to exit from the cell cycle at the appropriate time. The additional rounds of cell division generate an excess of interommatidial cells. Elevated levels of cyclin E protein detected in mutant cells may underlie the delayed cell cycle exit (Harvey, 2003).

In a disc mosaic for the wild-type parent chromosome, BrdU-incorporation was evident in the anterior portion of the disc and in a narrow stripe, the second mitotic wave (SMW), but not in the morphogenetic furrow (MF) or posterior to the SMW where cells arrest in the G1 phase of the cell cycle. In hpo mosaic eye discs, the pattern of S phases was normal in the anterior portion of the disc and in the SMW but in mutant portions of the disc, BrdU-incorporating nuclei were observed posterior to the SMW and also in the MF. Thus, hpo cells continue to cycle when surrounding wild-type cells are arrested in G1, indicating that hpo function is essential for timely cell cycle exit. In discs containing clones of the wild-type parental chromosome, mitoses, visualized with anti-phospho histone H3, were observed in the anterior portion of the eye disc and in several rows of developing ommatidia immediately posterior to the SMW. In discs mosaic for hpo however, extra mitoses were seen in mutant clones many ommatidial rows posterior to the SMW, indicating that at least a subset of hpo mutant cells continue to divide when wild-type cells are mitotically quiescent. These abnormalities are similar to, though less severe than, those found in sav and wts imaginal discs (Harvey, 2003).

Elevated levels of cyclin E were found in hpo mutant clones immediately anterior to the MF, in the SMW, and posterior to the SMW. Cyclin E expression appeared normal in hpo clones in the most anterior portions of the third instar larval eye-antennal disc. In contrast, cyclins A, B, and D were expressed at normal levels throughout the disc. In wild-type discs cyclin E RNA is expressed in a narrow stripe corresponding to the SMW. In discs mosaic for hpo, the level of cyclin E RNA was elevated and the expression domain of cyclin E was broader. Additionally, an increase in cyclin E mRNA was detected by semiquantitative RT-PCR performed on eye imaginal discs composed almost entirely of hpo mutant tissue. This indicates that, at least in part, hpo regulates cyclin E at the level of transcription or RNA stability but additional posttranscriptional regulation is also possible. In experiments where hpo function was reduced in S2 cells by RNAi, the levels of cyclin E protein were increased without an obvious change in RNA levels as assessed by Northern blotting. Thus, hpo may be capable of regulating cyclin E levels at both transcriptional and posttranscriptional levels (Harvey, 2003).

The cycling properties of hpo mutant cells were analyzed by generating clones of mutant cells in third instar larval wing discs. Larvae were heat shocked 48 hr after egg deposition (AED) and wing discs were dissected 120 hr AED. Following dissociation with trypsin and staining with Hoechst, cells were subjected to flow cytometry. Cell size, as gauged by forward scatter, and DNA content were measured and found to be almost indistinguishable between wild-type and hpo mutant cells (Harvey, 2003).

The number of cells in hpo mutant clones is consistently larger than in wild-type sister clones. The median population doubling time in hpo clones was 13.1 hr, which is significantly shorter than that of the GFP-bearing tester chromosome, which was 14.7 hr. By comparison, clones derived from the parent FRT42D chromosome had a population doubling time that was not significantly different from the same tester chromosome. It is unlikely that the increased cell numbers in hpo clones can be explained by a block in apoptosis, since overexpression of the caspase inhibitor p35 in wing disc cells at this stage of development does not appreciably alter the population doubling time. Thus, cells appear to divide faster in hpo clones. Since cell size is essentially unchanged, this would imply that hpo cells have an increased rate of growth (mass accumulation) and a commensurate increase in the rate of cell division (Harvey, 2003).

In wild-type pupal retinas, excess interommatidial cells are eliminated in a wave of apoptosis during the midpupal stage. There is a defect in apoptosis in sav and wts mutant tissue. As a result, the additional cells generated by excess cell division in sav and wts tissue are not eliminated and account for the increased number of interommatidial cells (Harvey, 2003).

Drosophila Ste20 family kinase dMST functions as a tumor suppressor by restricting cell proliferation and promoting apoptosis

In a genetic screen for mutations that restrict cell growth and organ size, a new tumor suppressor gene, dMST (CG11228), was identified that encodes the Drosophila homolog of the mammalian Ste20 kinase family members MST1 and MST2. Loss-of-function mutations in dMST result in overgrown tissues containing more cells of normal size. dMST mutant cells exhibit elevated levels of Cyclin E and DIAP1, increased cell growth and proliferation, and impaired apoptosis. dMST forms a complex with Salvador (Sav) and Warts (Wts)/Large tumors (Lats), a WW domain-containing protein and a Ser/Thr kinase resepectively, two tumor suppressors also implicated in regulating both cell proliferation and apoptosis, suggesting that they act in common pathways (Jia, 2003).

To identify novel tumor suppressor genes, the Drosophila genome was systematically screened for mutations that cause tissue overgrowth phenotypes. From this screen, three alleles of a gene named dMST (Drosophila homolog of MST1 and MST2) were identified. dMST^BF33 was used for most analyses described in this study; the molecular nature of dMST^BF33 suggests that it is likely to be a null allele (Jia, 2003).

Compared with wild-type eyes, dMST mosaic eyes are significantly larger and often protrude out in folds. Tumorous outgrowths are also observed when dMST clones are induced in other places, including the thorax, wing, and haltere. Hence, dMST is generally required for restricting tissue growth and organ size (Jia, 2003).

To determine how dMST controls organ size, labeled dMST clones were generated in wing discs and cell size and clone size were compared between mutant clones and twin spots. dMST mutant cells do not exhibit discernible changes in cell size. Moreover, dMST mutant cells differentiate into wing margin bristles of normal size. However, dMST clones occupy significantly larger areas and contain more cells than do wild-type twin spots. Since mutant clones and twin spots are derived from mitotic sister cells born at the same developmental stages, the increase in cell numbers and tissue mass of dMST mutant clones over twin spots suggests that dMST mutant cells grow and proliferate faster than do wild-type cells. Hence, the increase in size of dMST mosaic organs is caused by an increase in cell number but not cell size (Jia, 2003).

To further explore cell proliferation defects caused by dMST mutations, focus was placed on eye development. In wild-type eye discs, a single stripe of cells, referred to as the second mitotic wave (SMW), enters S phase synchronously posterior to the morphogenetic furrow(MF), and little bromodeoxyuridine (BrdU) labeling is present posterior to the SMW. In contrast, dMST mosaic discs exhibit extensive BrdU incorporation posterior to the SMW. To determine if extra mitosis also occurs in dMST mutant eye discs, the anti-phosphohistone H3 (pH3) antibody was used to label cells in M phase. In wild-type eye discs, few cells posterior to the SMW exhibit pH3 staining. In contrast, dMST mutant discs contain increased number of cells in M phase posterior to the SMW, suggesting that dMST mutations increase cell proliferation (Jia, 2003).

Cyclin E is an important regulator of S-phase initiation and progression in imaginal disc development. In wild-type discs, Cyclin E is up-regulated at the SMW. dMST mutant clones accumulate high levels of Cyclin E in a cell-autonomous fashion, which is consistent with the increased cell proliferation in dMST mutant discs (Jia, 2003).

In wild-type eyes, excessive cells between differentiated ommatidias are eliminated by a wave of apoptosis at early pupal stage so that a single layer of cells exists between two adjacent ommatidias. In contrast, the dMST mutant disc contains multiple layers of interommatidial cells. The persistence of excessive interommatidial cells in dMST mutant discs implies that apoptosis could be compromised. To test this, cell death was examined in wild-type and dMST mosaic pupal retina 38 h after pupa formation (APF); apoptosis was found to be diminished in dMST mutant cells. Hence, dMST is required for apoptosis during development (Jia, 2003).

In Drosophila, apoptosis is triggered by death inducers, including head involution defective (hid). Expressing GMR-hid induces precocious cell death in larval eye discs, which is blocked in dMST mutant cells. As a consequence, adult eyes derived from GMR-hid-expressing discs that also contain dMST mutant clones are larger than those derived from wild-type discs expressing GMR-hid. In Drosophila, death promoters induce apoptosis in part by down-regulating the levels of the death inhibitor DIAP1. dMST mutant cells exhibit higher levels of DIAP1 than do wild-type cells, suggesting that dMST promotes cell death at least in part by down-regulating the levels of DIAP1. The expression of a diap1-lacZ reporter gene is elevated in dMST mutant cells, indicating that dMST inhibits the transcription of diap1 (Jia, 2003).

The dMST mutations were mapped by high-resolution meiotic recombination. Several candidate genes were sequences for molecular lesions present in mutagenized chromosomes containing dMST alleles. Both dMST^JM1 and dMST^BF33 introduce point mutations in the open reading frame (ORF) of annotated gene CG11228, which encodes the Drosophila homolog of mammalian Ste20 kinase family members MST1 and MST2. A full-length cDNA corresponding to CG11228 was placed under the control of a tubulinalpha1 promoter to generate tub-dMSTf. Both dMST^JM1 and dMST^BF33 homozygotes expressing one copy of tub-dMSTf are viable, morphologically normal, and fertile, demonstrating that CG11228 is dMST. dMST^JM1 introduced a single amino acid substitution at a conserved residue (G181E), whereas dMST^BF33 introduced a stop codon at amino acid 174, suggesting that dMST^BF33 is likely to be a null allele (Jia, 2003).

When overexpressed in larval eye discs by using the GMR-gal4 driver, dMSTn, but not its kinase dead form (dMSTnK>R), induced ectopic apoptosis, leading to the formation of small rough eyes. Although expressing GMR-Wts alone does not cause any significant defect, coexpressing GMR-Wts with dMSTn enhances the defects caused by expressing dMSTn alone. For example, although flies expressing GMR-Gal4/dMSTn are viable, coexpressing GMR-Wts with GMR-gal4/dMSTn causes flies to die as pharate adults that have smaller eyes than those of flies expressing GMR-gal4/dMSTn alone. Similar phenotypic enhancement was obtained when two copies of UAS-dMSTn were expressed under GMR-gal4. In contrast, the defects caused by dMSTn are not enhanced by GMR-Sav. These genetic interactions are consistent with the findings that dMSTn binds Wts but not Sav (Jia, 2003).

To determine the genetic epistasis, dMSTn was overexpressed with GMR-gal4 in eye discs that contain wts or sav mutant clones. dMSTn induces ectopic Drice activation in sav mutant cells, as in the case of wild-type cells. In contrast, the ectopic Drice activation induced by dMSTn is diminished in wts mutant cells. Consistently, dMSTn blocks the up-regulation of DIAP1 in sav, but not in wts, mutant cells. In addition, dMSTn blocks the up-regulation of Cyclin E in sav but not in wts mutant cells. These results suggest that dMST acts downstream of sav but upstream of or in parallel with wts to restrict cell proliferation and promote apoptosis (Jia, 2003).

The MST subfamily of Ser/Thr kinases is classified as putative mitogen-activated protein kinase kinase kinase kinase (MAP4K). Although numerous studies have been carried out to address their biochemical properties and regulations in cultured cells, their physiological roles remain elusive. dMST is shown to play a pivotal role in controlling cell number and organ size in Drosophila development. dMST fulfills such a role both by restricting cell growth and proliferation and by promoting cell death. The cell-autonomous elevation of Cyclin E and DIAP1 levels in dMST mutant clones suggest that dMST regulates cell proliferation and cell death cell autonomously. Biochemical evidence is provided that dMST, Sav, and Wts form a complex. Genetic epitasis study suggests sav acts upstream of dMST whereas wts acts downstream of or in parallel with dMST. Sav could regulate the formation or activity of dMST/Wts kinase complex. Alternatively, Sav could act to bridge the dMST/Wts kinase complex to its substrates, a function that can be bypassed by excess amounts of dMSTn (Jia, 2003).

It should be noted that sav, dMST, and wts may not simply act in a linear pathway, because wts mutant phenotypes appear to be more severe than those caused by sav or dMST mutations. In addition, sav wts double-mutant phenotypes are stronger than sav or wts single-mutant phenotypes. Hence, although the results suggest that dMST, Sav, and Wts act in common pathways, they may exist in multiple complexes and have independent functions (Jia, 2003).

It has been shown that human Sav is mutated in several cancer cell lines, and mice lacking a homolog of wts/lats develop hyperplasia and tumors in several tissues, raising the possibility that mammalian MST1 and MST2 may also function as tumor suppressors. In support of this notion, MST1 and MST2 have been implicated in promoting cell death in cultured mammalian cells. It remains to be determined whether MST1 and MST2 also regulate cell proliferation in mammals. Given the functional conservation between many insect and mammalian pathways, it is speculated that the mammalian MST/Sav/Lats pathway may also participate in restricting cell number and organ size during normal development, and its malfunction may lead to cancers (Jia, 2003).

Autophagy regulates tissue overgrowth in a context-dependent manner

Autophagy is a catabolic process that has been implicated both as a tumor suppressor and in tumor progression. This study investigated this dichotomy in cancer biology by studying the influence of altered autophagy in Drosophila models of tissue overgrowth. The impact of altered autophagy was found to depend on both genotype and cell type. As previously observed in mammals, decreased autophagy suppresses Ras-induced eye epithelial overgrowth. In contrast, autophagy restricts epithelial overgrowth in a Notch-dependent eye model. Even though decreased autophagy did not influence Hippo pathway-triggered overgrowth, activation of autophagy strongly suppresses this eye epithelial overgrowth. Surprisingly, activation of autophagy enhances Hippo pathway-driven overgrowth in glia cells. These results indicate that autophagy has different influences on tissue growth in distinct contexts, and highlight the importance of understanding the influence of autophagy on growth to augment a rationale therapeutic strategy (Perez, 2014).

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, 2005).

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, 2005).

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, 2005).

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, 2005).

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, 2005).

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, 2005).

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, 2005).

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).

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 (Meignin, 2007).

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 (hpo^JM1) or a truncating (hpo^BF33) 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).

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 WW domain protein Kibra acts upstream of Hippo in Drosophila

The conserved Hippo kinase pathway plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. Whereas the function of the core kinase cascade, consisting of the serine/threonine kinases Hippo and Warts, in phosphorylating and thereby inactivating the transcriptional coactivator Yorkie is well established, much less is known about the upstream events that regulate Hippo signaling activity. The FERM domain proteins Expanded and Merlin appear to represent two different signaling branches that feed into the Hippo pathway. Signaling by the atypical cadherin Fat may act via Expanded, but how Merlin is regulated has remained elusive. This study shows that the WW domain protein Kibra is a Hippo signaling component upstream of Hippo and Merlin. Kibra acts synergistically with Expanded, and it physically interacts with Merlin. Thus, Kibra predominantly acts in the Merlin branch upstream of the core kinase cascade to regulate Hippo signaling (Baumgartner, 2010).

Overexpression of Drosophila Kibra in the developing eye has been shown to decrease the size of the adult organ. Four different loss-of-function alleles of Kibra were generated to define its function in growth control. Deletion of the first exon (harboring the translational start site) by imprecise excision of a P element resulted in the alleles Kibra¹ and Kibra². Kibra³, a mutation in the initiating ATG, was generated by means of an EMS reversion mutagenesis of the EP-mediated Kibra overexpression phenotype. Finally, the entire Kibra locus was removed by the hybrid element insertion (HEI) technique. All alleles were lethal when homozygous and failed to complement each other but were complemented by the precise P element excision used as a control throughout this study. All mutants displayed the same growth phenotypes, and homozygous mutant animals died as first-instar larvae. It is concluded that all Kibra alleles are genetically null (Baumgartner, 2010).

Kibra mutant heads were enlarged in comparison to controls. Similarly, wings containing posterior compartments largely mutant for Kibra were larger than control wings. The presence of a UAS-Kibra overexpression construct, without any Gal4 driver, rescued the lethality of Kibra homozygous mutant flies as well as the size defects of Kibra mutant organs, proving that the growth alterations are caused by the loss of Kibra function. Thus, Kibra is a general regulator of growth that is required to restrict organ size (Baumgartner, 2010).

To determine the cause of the Kibra mutant overgrowth phenotypes, a clonal analysis in wing imaginal discs was performed. Clones of Kibra mutant cells were larger than their corresponding wild-type sister clones. The number of cells per clone was increased in Kibra mutant clones compared to wild-type clones but not to the same extent as the clone size. However, FACS analysis revealed that cell size was unchanged in Kibra mutant cells, suggesting a change in cellular architecture in cells devoid of Kibra function. It is concluded that Kibra mutant clones in the wing imaginal disc were enlarged because Kibra mutant cells exhibit a proliferative advantage over wild-type cells (Baumgartner, 2010).

Tangential sections were analyzed of mosaic compound eyes consisting of Kibra mutant cells surrounded by heterozygous cells. The mutant ommatidia were normally structured and the different cell types properly differentiated, but the interommatidial regions were enlarged compared to the control. The increased distance between mutant ommatidia was due to more cells, because clones of Kibra mutant cells in the pupal retina displayed an increase in the number of interommatidial cells. Supernumerary interommatidial cells are a hallmark of inactivation of the Hippo pathway. Whereas a complete loss of Hippo signaling causes a pronounced excess of interommatidial cells, a mild extra interommatidial cell phenotype is observed in mutants that reduce but do not abrogate Hippo signaling, such as ex or Mer (Baumgartner, 2010).

A reduction in Hippo signaling activity results in extra interommatidial cells because the developmental apoptosis in pupal retinae is largely eliminated. Conversely, overexpression of hpo or ex induces apoptosis in third instar eye discs. Overexpression of Kibra in clones in the wing imaginal disc reduced clone size. Kibra-overexpressing clones contained fewer cells than control clones. To investigate whether overexpression of Kibra induces apoptosis, Kibra overexpression clones were generated in the third instar eye disc by using the Gene-Switch system. Indeed, the Kibra-overexpressing clones located anterior to the morphogenetic furrow (MF) showed an increase in programmed cell death as judged by staining for cleaved Caspase-3 and TUNEL staining, suggesting that overexpression of Kibra induces inappropriate apoptosis of proliferating cells. Consistently, co-overexpression of Diap1, a direct Yorkie transcriptional target, partially rescued the small eye phenotype associated with Kibra overexpression. Co-overexpression of CycE, another target of the Hippo pathway, also resulted in a partial rescue of the small eye. The size of Kibra-overexpressing eyes was further restored by concomitant overexpression of Diap1 and CycE. These results suggest that the effects elicited by Kibra overexpression are at least partly due to a reduction in the expression of the Hippo pathway target genes Diap1 and CycE (Baumgartner, 2010).

The striking similarities of the Kibra, ex, and Mer phenotypes prompted a genetic test of whether Kibra restricts tissue size via Hippo signaling. Interaction studies were started at the level of the transcriptional coactivator yki, which induces target genes promoting cell proliferation and cell survival and is inactivated by Hippo signaling. Three lines of evidence suggest that Kibra acts via inactivation of Yki. First, the coexpression of Kibra and yki during eye development suppressed the eye size reduction caused by Kibra and resulted in the same overgrowth phenotype as observed in eyes overexpressing yki alone. Second, the growth advantage of Kibra mutant cells was completely abolished by the concomitant loss of yki function. Third, a pupal lethal hypomorphic combination of Kibra alleles was rescued to viability by removal of a single copy of yki (Baumgartner, 2010).

To determine whether (and at which level) Kibra acts in the Hippo pathway to inactivate Yki, a series of epistasis tests were performed. It was found that the loss-of-function phenotypes of hpo, sav, and wts were epistatic to the Kibra overexpression phenotype, indicating that Kibra acts upstream of Hpo (Baumgartner, 2010).

Next, interaction with the upstream components Ex and Mer was tested. Overexpression of ex in a Kibra mutant background resulted in an intermediate phenotype. Vice versa, overexpression of Kibra also yielded an additive effect in an ex mutant head. Conversely, Kibra overexpression failed to reduce organ size in a Mer mutant head, indicating that Kibra requires Mer to exert its function. The eyFlp/FRT recombination system (without cell lethal) was used to generate mosaic animals with heads largely homozygous for ex and Mer mutations, as well as ex Kibra and Mer Kibra double mutations, respectively. Both ex and Mer mosaic heads showed only mild overgrowth. Strikingly, pupae with mosaic heads doubly mutant for ex and Kibra did not eclose, and normal head structures were displaced by overgrown tissue. In contrast, flies with Mer Kibra mosaic heads were viable. However, Mer Kibra double mutant clones showed stronger overgrowth than Mer clones. Reducing ex function during eye development by the expression of a hairpin RNAi construct did not alter the wild-type eye size but resulted in a severe enhancement of the Kibra loss-of-function phenotype, and the resulting eyes resembled those of hpo mutants. Reducing Mer function caused subtle overgrowth but enhanced the Kibra mutant phenotype much less (Baumgartner, 2010).

Whereas single mutants for ex and Mer cause a mild overgrowth phenotype, ex Mer double mutants display strong synergistic effects, suggesting that the two FERM domain proteins act in separate branches to activate Hippo signaling. These findings suggest that Kibra acts primarily upstream of Mer. However, since Mer Kibra double mutant clones show stronger overgrowth than Mer mutant clones and a reduction of Mer function enhances the Kibra loss-of-function phenotype, Kibra also contributes to Mer-independent regulation of Yki activity (Baumgartner, 2010).

To confirm that Kibra acts via Hippo signaling, whether Kibra mutant clones upregulated the expression of a Diap1 enhancer element (diap1-GFP4.3) that had been published to be a minimal Hippo responsive element (HRE) was tested. A pronounced upregulation of diap1-GFP4.3 was evident in clones of hpo mutant cells posterior and, to a weaker extent, anterior to the MF in eye imaginal discs. Cells lacking Kibra function also upregulated diap1-GFP4.3 expression, although to a lesser degree and with restriction to the differentiating tissue posterior to the MF. Clones of ex mutant cells, in resemblance to hpo clones, upregulated diap1-GFP4.3 strongly behind and somewhat weaker before the MF, whereas Mer mutant cells, like Kibra mutant cells, upregulated diap1-GFP4.3 expression weakly and solely posterior to the MF. Thus, the loss of Kibra results in an upregulation of a Hippo signaling reporter gene. The similar response of diap1-GFP4.3 to loss of Kibra or Mer suggests that Kibra and Mer act in the same way on Hippo signaling to regulate the HRE (Baumgartner, 2010).

This study provides genetic and biochemical evidence that the WW domain protein Kibra is a Hippo signaling component. Several lines of evidence indicate that Kibra acts predominantly in the Mer branch. First, the mild overgrowth phenotype caused by loss of Kibra function is akin to the Mer phenotype. Second, genetic epistasis experiments place Kibra upstream of Mer. Third, the effects of Kibra and Mer loss-of-function on a reporter for Hippo signaling activity are very similar. Fourth, Kibra and Mer synergise with ex in a similar fashion. Fifth, Kibra physically interacts with Mer. However, since the genetic analysis of Kibra also revealed a synergism with Mer, Kibra also acts on Yki activity in a Mer-independent manner (Baumgartner, 2010).

FERM domain proteins, such as Mer, have been suggested to connect membrane proteins with the underlying cortical cytoskeleton in order to integrate signals from the membrane and initiate intracellular signaling cascades. Thus, it is conceivable that Mer, together with as yet unknown proteins, assembles downstream cytoplasmic components of the Hippo pathway at the membrane and that controlled assembly and stabilization of such multiprotein complexes regulates the activity of the Hippo kinase cascade. In such a scenario, adaptor proteins providing multiple protein-protein interaction domains are of special interest (Baumgartner, 2010).

The WW domain protein Kibra binds Mer and could enable signaling events at the membrane/cytoskeleton interface that activate the Hpo kinase cascade. Since a truncated Kibra protein lacking the WW domains interacts more fiercely with Mer, it is likely that the physical association of Kibra and Mer is modulated by binding of other factors to the WW domains of Kibra (Baumgartner, 2010).

Interestingly, the effects caused by the concomitant loss of ex and Kibra functions are more severe than those elicited by mutated Hippo signaling core components. In addition to massively overgrowing, clones of ex Kibra double mutant cells round up, a behavior that was never observed in clones of hpo mutant cells. Furthermore, the diap1-GFP4.3 reporter indicates higher Yki activity in proliferating ex Kibra mutant eye imaginal disc cells as compared to hpo mutant cells. It thus appears that Yki activity is unleashed in cells lacking both ex and Kibra functions. Since Ex has been shown to directly bind Yki, it is tempting to speculate that Kibra participates in a distinct (Mer-independent) mechanism to prevent nuclear Yki localization (Baumgartner, 2010).

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).

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).

Identification of Happyhour/MAP4K as alternative Hpo/Mst-like kinases in the Hippo kinase cascade

In Drosophila and mammals, the canonical Hippo kinase cascade is mediated by Hpo/Mst acting through the intermediary kinase Wts/Lats to phosphorylate the transcriptional coactivator Yki/YAP/TAZ. Despite recent reports linking Yki/YAP/TAZ activity to the actin cytoskeleton, the underlying mechanisms are poorly understood and/or controversial. Using Drosophila imaginal discs as an in vivo model, this study shows that Wts, but not Hpo, is genetically indispensable for cytoskeleton-mediated subcellular localization of Yki. Through a systematic screen, the Ste-20 kinase Happyhour (Hppy) and its mammalian counterpart MAP4K1/2/3/5 were identified as an alternative kinase that phosphorylates the hydrophobic motif of Wts/Lats in a similar manner as Hpo/Mst. Consistent with their redundant function as activating kinases of Wts/Lats, combined loss of Hpo/Mst and Hppy/MAP4K abolishes cytoskeleton-mediated regulation of Yki/YAP subcellular localization, as well as YAP cytoplasmic translocation induced by contact inhibition. These Hpo/Mst-like kinases provide an expanded view of the Hippo kinase cascade in development and physiology (Zheng, 2015).

Understanding of the core kinase cascade of the Hippo pathway has been aided by multiple lines of investigation. First, genetic screens for tumor suppressors using mosaic flies have identified Hpo, Sav, Wts, and Mats as main constituents of the core kinase cassette. Second, biochemical studies of the activation mechanism of NDR family kinases, which include Lats1/2 and NDR1/2, demonstrate the importance of regulatory phosphorylation sites on the activation loop and the hydrophobic motif. The realization that the hydrophobic motif of Wts is phosphorylated by Hpo provides a fitting molecular explanation for the linear genetic pathway uncovered by in vivo studies. The simplicity of this linear pathway begun to be challenged based on the observation that Mst1/2 null cells still showed high levels of Lats phosphorylation on the hydrophobic motif. Indeed, in many subsequent reports, various signals have been reported to still regulate YAP/TAZ activity in Mst1/2 null cells. While semantically these observations were implied to support the existence of 'Mst1/2-independent' mechanisms, the molecular underpinning of this phenomenon has been elusive. It was also unclear whether this represents a mammalian-specific phenomenon as there has been no evidence to date that a similar mechanism operates in Drosophila (Zheng, 2015).

The current study addresses these issues in several significant ways. Definitive evidence is provided supporting an alternative Hpo-independent mechanism of Hippo pathway activation in Drosophila by demonstrating the genetic requirement of Wts, but not Hpo, in LatB-induced nuclear exclusion of Yki. Through a systematic screen, the Ste-20 family kinase Hppy/MAP4K was identified as a plausible molecular explanation for Mst1/2-independent regulation of Hippo signaling. Not only does Hppy/MAP4K directly phosphorylate the hydrophobic motif of Wts/Lats in vitro and in cell cultures, but loss of Hppy/MAP4K also abolishes LatB-induced Yki/YAP cytoplasmic translocation in Hpo/Mst null cells in both Drosophila tissues and mammalian cell cultures. These findings support the view that Hpo/Mst and Hppy/MAP4K act as redundant kinases targeting the hydrophobic motif of Wts/Lats. It is also noted that analysis of Hpo/Mst and Hppy/MAP4K in F-actin-mediated Hippo signaling was largely based on LatB treatment. Thus, it remains to be determined how these kinases cooperate with each other in a more physiological setting of cytoskeleton modulation. Nevertheless, the fact that MAP4K mediates Mst-independent regulation of YAP target gene expression and contact inhibition of YAP nuclear localization suggests that these kinases co-regulate Wts/Lats in multiple contexts beyond LatB-induced F-actin disruption. Since the hydrophobic motif of NDR1/2 can be phosphorylated by both Mst1 and Mst3, it is suggested that phosphorylation of hydrophobic motif by multiple Ste-20 kinases may be a common feature of the NDR family kinases. It is noted that two other kinases, CK2 and MSN/MAP4K4, were recently reported to promote Wts/Lats activity toward Yki. However, neither kinase was shown to directly phosphorylate the hydrophobic motif of Wts/Lats. Furthermore, although MSN was shown to promote Yki phosphorylation when Wts was co-expressed in S2 cells, MSN alone did not affect Yki phosphorylation, as was observed in this study. Thus, the mechanisms by which these kinases promote Hippo signaling remain to be determined (Zheng, 2015).

Recent studies have implicated cellular mechanical force as a regulator of Yki/YAP/TAZ activity. Reorganization of F-actin cytoskeleton has been suggested as the common mediator of mechanical forces arising from cell-cell and cell-matrix interactions. However, the underlying mechanism by which F-actin controls Yki/YAP/TAZ activity remains poorly understood and/or controversial. While some studies suggested that cytoskeleton-mediated regulation of YAP/TAZ is independent of the Hippo kinase cascade, others suggested that it requires the Hippo kinase cascade. The observation that LatB-induced Yki cytoplasmic localization is Wts dependent is more consistent with a Hippo signaling-dependent mechanism. An important modification brought by the current study is that the canonical Hippo kinase cascade should be expanded to include Hppy/MAP4K at the level of Hpo. This expanded Hippo kinase cascade may also include NDR1/2 at the level of Lats1/2, given the recent report of NDR1/2 as Lats1/2-like kinases capable of phosphorylating YAP (Zheng, 2015).

Finally, it is suggested that the Hippo-signaling-dependent and -independent regulation of YAP/TAZ by F-actin may be potentially reconciled with each other. A major discrepancy between the two models came from the analysis of mutant YAP/TAZ that lacks all the Lats phosphorylation sites (YAP5SA or TAZ4SA). It is noted, however, that a different readout was used to assay the regulation of these YAP/TAZ mutants in the different studies. A luciferase reporter assay was used to show that the transcriptional activity of YAP5SA or TAZ4SA still responded to F-actin reorganization, whereas subcellular localization was used to show that YAP5SA no longer responded to cytoplasmic localization of YAP induced by F-actin disruption. These results may reflect the functionality of different subcellular pools of F-actin; inasmuch as YAP/TAZ localization is regulated by F-actin through the Hippo pathway, F-actin may also play a separate role in regulating the transcriptional activity of YAP/TAZ in the nucleus, especially given the increasing appreciation of a more direct role of nuclear F-actin in transcriptional regulation (Zheng, 2015).

The Hippo signalling pathway maintains quiescence in Drosophila neural stem cells

Stem cells control their mitotic activity to decide whether to proliferate or to stay in quiescence. Drosophila neural stem cells (NSCs) are quiescent at early larval stages, when they are reactivated in response to metabolic changes. This study reports that cell-contact inhibition of growth through the canonical Hippo signalling pathway maintains NSC quiescence. Loss of the core kinases hippo or warts leads to premature nuclear localization of the transcriptional co-activator Yorkie and initiation of growth and proliferation in NSCs. Yorkie is necessary and sufficient for NSC reactivation, growth and proliferation. The Hippo pathway activity is modulated via inter-cellular transmembrane proteins Crumbs and Echinoid that are both expressed in a nutrient-dependent way in niche glial cells and NSCs. Loss of crumbs or echinoid in the niche only is sufficient to reactivate NSCs. Finally, the Hippo pathway activity discriminates quiescent from non-quiescent NSCs in the Drosophila nervous system (Ding, 2016).

Loss-of-function mutation in Hippo suppressed enlargement of lysosomes and neurodegeneration caused by dFIG4 knockdown

Charcot-Marie-Tooth disease (CMT) is the most common hereditary neuropathy, and more than 80 CMT-causing genes have been identified to date. CMT4J is caused by a loss-of-function mutation in the Factor-Induced-Gene 4 (FIG4) gene, the product of which plays important roles in endosome-lysosome homeostasis. It was hypothesized that Mammalian sterile 20-like kinase (MST) 1 and 2, tumor-suppressor genes, are candidate modifiers of CMT4J. The interactions were examined between dFIG4 and Hippo (hpo), Drosophila counterparts of FIG4 and MSTs, respectively, using the Drosophila CMT4J model with the knockdown of dFIG4. The loss-of-function allele of hpo improved the rough eye morphology, locomotive dysfunction accompanied by structural defects in the presynaptic terminals of motoneurons, and the enlargement of lysosomes caused by the knockdown of dFIG4. Therefore, this study identified hpo as a modifier of phenotypes induced by the knockdown of dFIG4. These results in Drosophila may provide an insight into the pathogenesis of CMT4J and contribute toward the development of disease-modifying therapy for CMT. The regulation of endosome-lysosome homeostasis was also identified as a novel probable function of Hippo/MST (Kushimura, 2018).

Hippo, Drosophila MST, is a novel modifier of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS

Mutations in the Fused in Sarcoma (FUS) gene have been identified in familial ALS in human. Drosophila contains a single ortholog of human FUS called Cabeza (Caz). Drosophila models of ALS have been established targeted to Caz, which developed the locomotive dysfunction and caused anatomical defects in presynaptic terminals of motoneurons. Accumulating evidence suggests that ALS and cancer share defects in many cellular processes. The Hippo pathway was originally discovered in Drosophila and plays a role as a tumor suppressor in mammals. Whether Hippo pathway genes modify the ALS phenotype was determined using Caz knockdown flies. A genetic link was found between Caz and Hippo (hpo), the Drosophila ortholog of human Mammalian sterile 20-like kinase (MST) 1 and 2. Loss-of-function mutations of hpo rescued Caz knockdown-induced eye- and neuron-specific defects. The decreased Caz levels in nuclei induced by Caz knockdown were also rescued by loss of function mutations of hpo. Moreover, hpo mRNA level was dramatically increased in Caz knockdown larvae, indicating that Caz negatively regulated hpo. The results demonstrate that hpo, Drosophila MST, is a novel modifier of Drosophila FUS. Therapeutic targets that inhibit the function of MST could modify the pathogenic processes of ALS (Azuma, 2018).

EVOLUTIONARY HOMOLOGS

Structure of mammalian Mst1 and Mst2

The human serine/threonine protein kinases, Mst1 and Mst2, share considerable homology to Ste20 and p21-activated kinase (Pak) throughout their catalytic domains. However, outside the catalytic domains there are no significant homologies to previously described Ste20-like kinases or other proteins. To understand the role of the nonhomologous regions, a structure/function analysis of Mst1 was performed. A series of COOH-terminal and internal deletions indicates that there is an element within a central 63-amino acid region of the molecule that inhibits kinase activity. Removal of this domain increases kinase activity approximately 9-fold. Coimmunoprecipitation assays, the yeast two-hybrid procedure, and in vitro cross-linking analysis indicate that Mst1 homodimerizes and that the extreme COOH-terminal 57 amino acids are required for self-association. Size exclusion chromatography indicates that Mst1 is associated with a high molecular weight complex in cells, suggesting that other proteins may also oligomerize with this kinase. While loss of dimerization alone does not affect kinase activity, a molecule lacking both the dimerization and inhibitory domains is not as active as one that lacks only the inhibitory domain. Comparison of Mst1 and Mst2 indicates that both functional domains lie in regions conserved between the two molecules (Creasy, 1996).

Initiation of Hippo signaling is linked to polarity rather than to cell position in the pre-implantation mouse embryo

In the mouse embryo, asymmetric divisions during the 8-16 cell division generate two cell types, polar and apolar cells, that are allocated to outer and inner positions, respectively. This outer/inner configuration is the first sign of the formation of the first two cell lineages: trophectoderm (TE) and inner cell mass (ICM). Outer polar cells become TE and give rise to the placenta, whereas inner apolar cells become ICM and give rise to the embryo proper and yolk sac. This study analyzed the frequency of asymmetric divisions during the 8-16 cell division and assessed the relationships between cell polarity, cell and nuclear position, and Hippo signaling activation, the pathway that initiates lineage-specific gene expression in 16-cell embryos. Although the frequency of asymmetric divisions varied in each embryo, it was found that more than six blastomeres divided asymmetrically in most embryos. Interestingly, many apolar cells in 16-cell embryos were located at outer positions, whereas only one or two apolar cells were located at inner positions. Live imaging analysis showed that outer apolar cells were eventually internalized by surrounding polar cells. Using isolated 8-cell blastomeres, the internalization process of apolar cells was carefully analyzed, and indications were found of higher cortical tension in apolar cells than in polar cells. Last, apolar cells were found to activate Hippo signaling prior to taking inner positions. These results suggest that polar and apolar cells have intrinsic differences that establish outer/inner configuration and differentially regulate Hippo signaling to activate lineage-specific gene expression programs (Anani, 2014).

Phosphorylation of MST proteins, activation, and cleavage by caspase and apoptosis

Mst1 is a ubiquitously expressed serine-threonine kinase, homologous to the budding yeast Ste20, whose physiological regulation and cellular function are unknown. Mst1 is specifically cleaved by a caspase 3-like activity during apoptosis induced by either cross-linking CD95/Fas or by staurosporine treatment. CD95/Fas-induced cleavage of Mst1 was blocked by the cysteine protease inhibitor ZVAD-fmk, the more selective caspase inhibitor DEVD-CHO and by the viral serpin CrmA. Caspase-mediated cleavage of Mst1 removes the C-terminal regulatory domain and correlates with an increase in Mst1 activity in vivo, consistent with caspase-mediated cleavage activating Mst1. Overexpression of either wild-type Mst1 or a truncated mutant induces morphological changes characteristic of apoptosis. Furthermore, exogenously expressed Mst1 is cleaved, indicating that Mst1 can activate caspases that result in its cleavage. Kinase-dead Mst1 did not induce morphological alterations and was not cleaved upon overexpression, indicating that Mst1 must be catalytically active in order to mediate these effects. Mst1 activates MKK6, p38 MAPK, MKK7 and SAPK in co-transfection assays, suggesting that Mst1 may activate these pathways. These findings suggest the existence of a positive feedback loop involving Mst1, and possibly the SAPK and p38 MAPK pathways, which serves to amplify the apoptotic response (Graves, 1998).

The Fas system has been extensively investigated as a model of apoptosis and the caspase cascade has been shown to be a characteristic mechanism of signaling of apoptosis. A kinase has been identified and purified that is activated after the stimulation of Fas on human thymoma-derived HPB-ALL cells. Partial amino acid sequencing of the purified kinase revealed it to be MST/Krs, member of the yeast STE20 family of protein kinases. MST/Krs was activated by proteolytic cleavage and proteolytic activation was blocked by the caspase inhibitor, Z-VAD-FK. A mutant MST with Asp-->Asn replacement at a putative caspase cleavage site is resistant to either the proteolytic cleavage or the activation of the kinase activity. These findings suggest that proteolytic activation is one activation mechanism of MST and plays a role in apoptosis (Lee, 1998).

The serine/threonine kinase Mst1, a mammalian homolog of the budding yeast Ste20 kinase, is cleaved by caspase-mediated proteolysis in response to apoptotic stimuli such as ligation of CD95/Fas or treatment with staurosporine. Furthermore, overexpression of Mst1 induces morphological changes characteristic of apoptosis in human B lymphoma cells. Mst1 may therefore represent an important target for caspases during cell death, which serves to amplify the apoptotic response. Mst1 has two caspase cleavage sites, and evidence is presented indicating that cleavage may occur in an ordered fashion and be mediated by distinct caspases. Caspase-mediated cleavage alone is insufficient to activate Mst1, suggesting that full activation of Mst1 during apoptosis requires both phosphorylation and proteolysis. Another role of phosphorylation may be to influence the susceptibility of Mst1 to proteolysis. Autophosphorylation of Mst1 on a serine residue close to one of the caspase sites inhibited caspase-mediated cleavage in vitro. Finally, Mst1 appears to function upstream of the protein kinase MEKK1 in the SAPK pathway. In conclusion, Mst1 activity is regulated by both phosphorylation and proteolysis, suggesting that protein kinase and caspase pathways work in concert to regulate cell death (Graves, 2001).

Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH(2) terminus, whereas there is a regulatory domain at its COOH terminus. IEndogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, the caspase cleavage site, AETD(313), was mapped: it is at the junction of the NH(2)-terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis (Huang, 2002).

The human serine/threonine kinase, mammalian STE20-like kinase (MST), is considerably homologous to the budding yeast kinases, SPS1 and STE20, throughout their kinase domains. The cellular function and physiological activation mechanism of MST is unknown except for the proteolytic cleavage-induced activation in apoptosis. MST1 and MST2 are direct substrates of caspase-3 both in vivo and in vitro. cDNA cloning of MST homologs in mouse and nematode shows that caspase-cleaved sequences are evolutionarily conserved. Human MST1 has two caspase-cleavable sites, which generate biochemically distinct catalytic fragments. Staurosporine activates MST either caspase-dependently or independently, whereas Fas ligation activates it only caspase-dependently. Immunohistochemical analysis reveals that MST is localized in the cytoplasm. During Fas-mediated apoptosis, cleaved MST translocates into the nucleus before nuclear fragmentation is initiated, suggesting it functions in the nucleus. Transiently expressed MST1 induces striking morphological changes characteristic of apoptosis in both nucleus and cytoplasm, which is independent of caspase activation. Furthermore, when stably expressed in HeLa cells, MST highly sensitizes the cells to death receptor-mediated apoptosis by accelerating caspase-3 activation. These findings suggest that MST1 and MST2 play a role in apoptosis both upstream and downstream of caspase activation (Lee, 2001).

MST1 is an upstream kinase of the JNK and p38 MAPK pathways whose expression induces apoptotic morphological changes such as nuclear condensation. During apoptosis, caspase cleavage of MST1 removes a C-terminal regulatory domain, increasing the kinase activity of the MST1 N-terminal domain. Downstream pathways of MST1 in the induction of apoptosis remain to be clarified. The expression of MST1 results in caspase-3 activation. Therefore, MST1 is not only a target of caspases but also an activator of caspases. This caspase activation and apoptotic changes occur through JNK, since the co-expression of a dominant-negative mutant of JNK inhibits MST1-induced morphological changes as well as caspase activation. In contrast, neither a dominant-negative p38 nor the p38 inhibitor SB203580 inhibit the cellular changes. MST1 induces nucleosomal DNA fragmentation, which is suppressed by caspase inhibitors or ICAD (Inhibitor of Caspase-Activated DNase). Surprisingly, however, other changes such as membrane blebbing and chromatin condensation are not inhibited by caspase inhibitors. These results suggest that MST1 most likely promotes two events through JNK activation: (1) MST1 induces the activation of caspases, resulting in CAD-mediated DNA fragmentation, and (2) MST1 induces chromatin condensation and membrane blebbing without utilizing downstream caspases (Ura, 2001a).

MST1 is a member of the Sterile-20 family of cytoskeletal, stress, and apoptotic kinases. MST1 is activated by phosphorylation at previously unidentified sites. This study examines the role of phosphorylation at several sites and effects on kinase activation. Thr(183) in subdomain VIII is defined as a primary site of phosphoactivation. Thr(187) is also critical for kinase activity. Phosphorylation of MST1 in subdomain VIII is catalyzed by active MST1 via intermolecular autophosphorylation, enhanced by homodimerization. Active MST1 (wild-type or T183E), but not inactive Thr(183)/Thr(187) mutants, is also highly autophosphorylated at the newly identified Thr(177) and Thr(387) residues. Cells expressing active MST1 are mostly detached, whereas with inactive MST1, adhesion is normal. Active MKK4, JNK, caspase-3, and caspase-9 were detected in the detached cells. These cells also contain all autophosphorylated and essentially all caspase-cleaved MST1. Similar phenotypes were elicited by a caspase-insensitive D326N mutant, suggesting that kinase activity, but not cleavage of MST1, is required. Interestingly, an S327E mutant mimicking Ser(327) autophosphorylation was also caspase-insensitive, but only when expressed in caspase-3-deficient cells. Together, these data suggest a model whereby MST1 activation is induced by existing, active MST kinase, which phosphorylates Thr(183) and possibly Thr(187). Dimerization promotes greater phosphorylation. This leads to induction of the JNK signaling pathway, caspase activation, and apoptosis. Further activation of MST1 by caspase cleavage is best promoted by caspase-3, although this appears to be unnecessary for signaling and morphological responses (Glantschnig, 2002).

Mammalian STE20-like kinase 2 (MST2), a member of the STE20-like kinase family, has been shown in previous studies to undergo proteolytic activation by caspase-3 during cell apoptosis. A few studies have also implicated protein phosphorylation reactions in MST2 regulation. This study examined the mechanism of MST2 regulation with an emphasis on the relationship between caspase-3 cleavage and protein phosphorylation. Both the full-length MST2 and the caspase-3-truncated form of MST2 overexpressed in 293T cells exist in a phosphorylated state. However, the endogenous full-length MST2 from rat thymus or from proliferating cells is mainly unphosphorylated whereas the caspase-3-truncated endogenous MST2 from apoptotic cells is highly phosphorylated. Cell transfection studies using mutant MST2 constructs indicate that MST2 depends on the autophosphorylation of a unique threonine residue, Thr(180), for kinase activity. The autophosphorylation reaction shows strong dependence on MST2 concentration, suggesting that it is an intermolecular reaction. While both the full-length MST2 and the caspase-3-truncated form of MST2 undergo autophosphorylation, the two forms of the phosphorylated MST2 display marked differences in susceptibility to protein phosphatases. The full-length phospho-MST2 is rapidly dephosphorylated by protein phosphatase 1 or protein phosphatase 2A whereas the truncated MST2 is remarkably resistant to the dephosphorylation. Based on the present results, a novel molecular mechanism for MST2 regulation in apoptotic cells is postulated. In normal cells, because of the low concentration and the ready reversal of the autophosphorylation by protein phosphatases, MST2 is present mainly in the unphosphorylated and inactive state. During cell apoptosis, MST2 is cleaved by caspase-3 and undergoes irreversible autophosphorylation, thus resulting in the accumulation of active MST2 (Deng, 2003).

MST1 complex is a Ras effector unit that mediates the apoptotic effect of Ki-RasG12V

The Ras-GTPase controls cell fate decisions through the binding of an array of effector molecules, such as Raf and PI 3-kinase, in a GTP-dependent manner. NORE1, a noncatalytic polypeptide, binds specifically to Ras-GTP and to several other Ras-like GTPases. NORE is homologous to the putative tumor suppressor RASSF1 and to the Caenorhabditis elegans polypeptide T24F1.3. All three NORE-related polypeptides bind selectively to the proapoptotic protein kinase MST1, a member of the Group II GC kinases. Endogenous NORE and MST1 occur in a constitutive complex in vivo that associates with endogenous Ras after serum stimulation. Targeting recombinant MST1 to the membrane, either through NORE or myristoylation, augments the apoptotic efficacy of MST1. Overexpression of constitutively active Ki-RasG12V promotes apoptosis in a variety of cell lines; Ha-RasG12V is a much less potent proapoptotic agent; however, a Ha-RasG12V effector loop mutant (E37G) that binds NORE, but not Raf or PI 3-kinase, exhibits proapoptotic efficacy approaching that of Ki-RasG12V. The apoptotic action of both Ki-RasG12V and Ha-RasG12V, E37G is suppressed by overexpression of the MST1 carboxy-terminal noncatalytic segment or by the NORE segment that binds MST1. It is concluded that MST1 is a phylogenetically conserved partner of the NORE/RASSF polypeptide family, and the NORE-MST1 complex is a novel Ras effector unit that mediates the apoptotic effect of Ki-RasG12V (Khokhlatchev, 2002).

Nuclear transport of MST1

MST1, mammalian STE20-like kinase 1, is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. MST1 is capable of inducing apoptotic morphological changes such as chromatin condensation upon overexpression. MST1 contains two functional nuclear export signals (NESs) in the C-terminal domain, which is released from the N-terminal kinase domain upon caspase-mediated cleavage. Full-length MST1 is excluded from the nucleus and localized to the cytoplasm. However, either truncation of the C-terminal domain, point mutation of the two putative NESs, or treatment with leptomycin B, an inhibitor of the NES receptor, results in nuclear localization of MST1. Staurosporine treatment induces chromatin condensation, MST1 cleavage, and nuclear translocation. Staurosporine-induced chromatin condensation is partially inhibited by expressing a kinase-negative mutant of MST1, suggesting an important role of MST1 in this process. Significantly, MST1 is more efficient at inducing chromatin condensation when it is constitutively localized to the nucleus by mutation of its NESs. Moreover, inhibition of MST1 nuclear translocation by mutation of its cleavage sites reduces its ability to induce chromatin condensation. Taken together, these results suggest that truncation of the C-terminal domain of MST1 by caspases may result in translocation of MST1 into the nucleus, where it promotes chromatin condensation (Ura, 2001b).

Death-associated protein 4 binds MST1 and augments MST1-induced apoptosis

The protein kinase MST1 is proapoptotic when overexpressed in an active form, however, its physiologic regulation and cellular targets are unknown. An overexpressed inactive MST1 mutant associates in COS-7 cells with an endogenous 761-amino acid polypeptide known as 'death-associated protein 4' (DAP4). The DAPs are a functionally heterogeneous array of polypeptides isolated in a screen for elements involved in the interferon gamma-induced apoptosis of HeLa cells. DAP4, which is encoded by a member of a vertebrate-only gene family, contains no identifiable domains, but is identical over its amino-terminal 488 amino acids to p52(rIPK), a putative modulator of protein kinase R. DAP4 is a widely expressed, constitutively nuclear polypeptide that homodimerizes through its amino terminus and binds MST1 through its carboxyl-terminal segment. MST1 is predominantly cytoplasmic, but cycles continuously through the nucleus, as evidenced by its rapid accumulation in the nucleus after addition of the Crm1 inhibitor, leptomycin B. Overexpression of DAP4 does not cause apoptosis, however, coexpression of DAP4 with a submaximal amount of MST1 enhances MST1-induced apoptosis in a dose-dependent fashion. DAP4 is not significantly phosphorylated by MST1 nor does it alter MST1 kinase activity in vivo or in vitro. MST1-induced apoptosis is suppressed by a dominant interfering mutant of p53. MST1 is unable to directly phosphorylate p53, however, DAP4 binds endogenous and recombinant p53. DAP4 may promote MST1-induced apoptosis by enabling colocalization of MST with p53 (Lin, 2002).

Mst1 phosphorylates histone H2B in cells undergoing apoptosis

DNA in eukaryotic cells is associated with histone proteins; hence, hallmark properties of apoptosis, such as chromatin condensation, may be regulated by posttranslational histone modifications. Phosphorylation of histone H2B at serine 14 (S14) correlates with cells undergoing programmed cell death in vertebrates. A 34 kDa apoptosis-induced H2B kinase has been identified as caspase-cleaved Mst1 (mammalian sterile twenty) kinase. Mst1 can phosphorylate H2B at S14 in vitro and in vivo, and the onset of H2B S14 phosphorylation is dependent upon cleavage of Mst1 by caspase-3. These data reveal a histone modification that is uniquely associated with apoptotic chromatin in species ranging from frogs to humans and provides insights into a previously unrecognized physiological substrate for Mst1 kinase. The data provide evidence for a potential apoptotic 'histone code' (Cheung, 2003).

The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1

Originally identified in Drosophila, the Warts(Wts)/Lats protein kinase has been proposed to function with two other Drosophila proteins, Hippo (Hpo) and Salvador (Sav), in the regulation of cell cycle exit and apoptosis. In mammals, two candidate Warts/Lats homologs, termed Lats1 and Lats2, have been described, and the targeted disruption of LATS1 in mice increases tumor formation. Little, however, is known about the function and regulation of human Lats kinases. Human Mst2, a STE20-family member and purported Hpo ortholog, phosphorylates and activates both Lats1 and Lats2. Deletion analysis reveals that regulation of Lats1 occurs through the C-terminal, catalytic domain. Within this domain, two regulatory phosphorylation sites were identified by mass spectrometry. These sites, S909 in the activation loop and T1079 within a hydrophobic motif, have been highly conserved during evolution. Moreover, a direct interaction is observed between Mst2 and hWW45, a putative ortholog of Drosophila Sav. These results indicate that Mst2-like kinases regulate Lats kinase activities in an evolutionarily conserved regulatory pathway. Although the function of this pathway remains poorly understood in mammals, it is intriguing that, in Drosophila, it has been linked to development and tissue homeostasis ( Chan, 2005 ).

Mst1 and Mst2 protein kinases restrain intestinal stem cell proliferation and colonic tumorigenesis by inhibition of Yes-associated protein (Yap) overabundance

Ablation of the kinases Mst1 and Mst2, orthologs of the Drosophila antiproliferative kinase Hippo, from mouse intestinal epithelium caused marked expansion of an undifferentiated stem cell compartment and loss of secretory cells throughout the small and large intestine. Although median survival of mice lacking intestinal Mst1/Mst2 is 13 wk, adenomas of the distal colon are common by this age. Diminished phosphorylation, enhanced abundance, and nuclear localization of the transcriptional coactivator Yes-associated protein 1 (Yap1) is evident in Mst1/Mst2-deficient intestinal epithelium, as is strong activation of β-catenin and Notch signaling. Although biallelic deletion of Yap1 from intestinal epithelium has little effect on intestinal development, inactivation of a single Yap1 allele reduces Yap1 polypeptide abundance to nearly wild-type levels and, despite the continued Yap hypophosphorylation and preferential nuclear localization, normalizes epithelial structure. Thus, supraphysiologic Yap polypeptide levels are necessary to drive intestinal stem cell proliferation. Yap is overexpressed in 68 of 71 human colon cancers and in at least 30 of 36 colon cancer-derived cell lines. In colon-derived cell lines where Yap is overabundant, its depletion strongly reduces β-catenin and Notch signaling and inhibits proliferation and survival. These findings demonstrate that Mst1 and Mst2 actively suppress Yap1 abundance and action in normal intestinal epithelium, an antiproliferative function that frequently is overcome in colon cancer through Yap1 polypeptide overabundance. The dispensability of Yap1 in normal intestinal homeostasis and its potent proliferative and prosurvival actions when overexpressed in colon cancer make it an attractive therapeutic target (Zhou, 2011).

Hippo kinases MST1 and MST2 control the differentiation of the epididymal initial segment via the MEK-ERK pathway

Although the roles of the Hippo pathway in organogenesis and tumorigenesis have been well studied in multiple organs, its role in sperm maturation and male fertility has not been investigated. The initial segment (IS) of the epididymis plays a critical role in sperm maturation. IS differentiation is governed by ERK1/2 (see Drosophila Rolled), but the mechanisms of ERK1/2 activation in IS are not fully understood. This study shows that double knockout (dKO) of mammalian sterile 20-like kinases 1 and 2 (Mst1 and Mst2), homologs of Hippo in Drosophila, in the epididymal epithelium led to male infertility in mice. Sperm in the cauda epididymides of mutant mice were immotile with flagellar angulation and severely disorganized structures. Loss of Mst1/2 activated YAP and increased proliferation and cell death in all the segments of epididymis. The mutant mice showed substantially suppressed MEK/ERK signaling in the IS and failed IS differentiation. Deletion of Yap (see Drosophila Yorkie) restored the reduced MEK/ERK signaling, and partially rescued the defective IS differentiation and fertility in Mst1/2 dKO mice. These results demonstrate that YAP inhibits the MEK/ERK pathway in IS epithelial cells, and MST1/2 control IS differentiation and fertility at least partially by repressing YAP. Taken together, the Hippo pathway is essential for sperm maturation and male fertility (Meng, 2020).

REFERENCES

Search PubMed for articles about Drosophila hippo

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Fernández, B. G., et al. (2011). Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila. Development 138(11): 2337-46. PubMed Citation: 21525075

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date revised: 25 October 2023

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