Merlin


REGULATION

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 10Ă—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

Merlin and Expanded colocalize in Drosophila tissues and cells, and physically interact through a conserved N-terminal region (CNTR) of Expanded, characteristic of the Protein 4.1 family, and the C-terminal domain of Merlin. Loss of function of both Merlin and expanded in clones reveals that these proteins function to regulate differentiation in addition to proliferation in Drosophila (McCartney, 2000).

What is the functional significance of this interaction? Removal by genetic mutation of either protein does not appear to affect the subcellular localization of the other, indicating that the interaction is not required for proper subcellular localization in tissues. Studies of ERM proteins have shown that they exist in multiple conformations that appear to regulate their ability to interact with transmembrane and other interacting proteins. Thus, the formation of homotypic and heterotypic dimers or oligomers via interactions between the CNTR and C-terminal domains may function to maintain ERM proteins in either an active or inactive state. Recent studies of human Merlin indicate that it can form folded monomers, homotypic dimers and heterotypic dimers with ERM proteins. The results presented here indicate that the CNTR of Expanded interacts directly with the C-terminal domains of Expanded and Merlin, suggesting that Expanded can exist in a similar range of conformations. Based on these results, it is proposed that Merlin and Expanded form a heterodimeric complex that actively suppresses proliferation. According to this model, in the heterodimer, the N-terminal domain of Merlin would be free to interact with other proteins, and therefore would be in an activated state. Consistent with this view, removal of the 35 amino acid C-terminal region of Merlin results in a constitutively active form of the protein that contains all essential Merlin functions. In addition, both Merlin and Expanded must possess growth suppression functions that are independent of each other, because the observed Mer-;ex- double mutant phenotype is much more severe than either mutation alone (McCartney, 2000).

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).

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

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).

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 PP1 phosphatase flapwing regulates the activity of Merlin and Moesin in Drosophila

The signalling activities of Merlin and Moesin, two closely related members of the protein 4.1 Ezrin /Radixin/Moesin family, are regulated by conformational changes. These changes are regulated in turn by phosphorylation. The same sterile 20 kinase-Slik co-regulates Merlin or Moesin activity whereby phosphorylation inactivates Merlin, but activates Moesin. Thus, the corresponding coordinate activation of Merlin and inactivation of Moesin would require coordinated phosphatase activity. Drosophila protein phosphatase type 1 β (Flapwing) fulfils this role, co-regulating dephosphorylation and altered activity of both Merlin and Moesin. Merlin or Moesin are detected in a complex with Flapwing both in-vitro and in-vivo. Directed changes in flapwing expression result in altered phosphorylation of both Merlin and Moesin. These changes in the levels of Merlin and Moesin phosphorylation following reduction of flapwing expression are associated with concomitant defects in epithelial integrity and increase in apoptosis in developing tissues such as wing imaginal discs. Functionally, the defects can be partially recapitulated by overexpression of proteins that mimic constitutively phosphorylated or unphosphorylated Merlin or Moesin. These results suggest that changes in the phosphorylation levels of Merlin and Moesin lead to changes in epithelial organization (Yang, 2012).

The results suggest that Flw would act antagonistically to the kinase Slik during the coordinate regulation of Mer, acting as a tumour suppressor protein, and Moe, required to maintain epithelial integrity. If Flw acts as a coordinate regulatory phosphatase for Mer and/or Moe, it would be expected that Flw is in a protein complex with both Mer and Moe, and this was found to be true. A reproducible increase was found in the ratio of dephosphorylated to phosphorylated Mer isoforms when flw is overexpressed, and a decrease in this ratio was found when flw expression is reduced. In addition, four distinct Mer phosphorylation isoforms were detected. Supporting these observations, the over-expression of flw increases the amount of dephosphorylated Mer signal present as compared to the wild type tissue. Flw also affects the phosphorylation of Moe. The amount of phosphorylated Moesin protein is reduced when flw is over-expressed as compared to when flw expression is reduced. Thus, Flw appears to be a phosphatase specific for both Mer and Moe (Yang, 2012).

Most importantly, using functional assays in whole animals, Flw mediated regulation of Mer and Moe has clear effects on both Mer and Moe protein localization to the plasma membrane and on epithelial organization. There is a higher intensity of staining of both Mer and phosphorylated Moe associated with the plasma membrane upon reduction of flw expression. When the levels of other typical apical domain markers as well as basolateral markers were examined by maximum intensity projection analysis, it was found that maximum projections from larval wing discs show increased brightness of p-ERM, F-actin and anti-Armadillo, within the cells in which flw expression is reduced, whereas the septate junction marker anti-Coracle staining is not changed in intensity over the whole disc. This suggests that as a result of changes in Mer and Moe phosphorylation there are changes in links to the actin cytoskeleton and adherens junctions where both Mer and Moe play roles in wild type cells. Previous studies have demonstrated that phosphorylated Mer is more tightly associated with the plasma membrane. In agreement with data from Drosophila, mammalian cells also show increased plasma membrane association of a phosphomimic form of moesin or the related protein ezrin whereas dephosphorylated ERM proteins are less associated with the plasma membrane. Following flw knockdown in selected cells in the wing epithelium, cells within the boundary between cells with reduced flw expression levels and cells with wild type flw expression levels undergo the greatest amount of change in terms of epithelial integrity. The loss of polarity leads to increased apoptosis in these cells. These effects are observed when flw expression is reduced in only a few cells such as using the ptc Gal4 driver or in the entire dorsal compartment of the wing such as using the apterous Gal4 driver. The cells along the boundary region appear to fold inwards and detach from the rest of epithelium. This is likely the direct result of the difference in adhesion between cells that have reduced flw expression and cells which express wild type levels of Flw protein. As Mer and Moe appear to be direct targets of Flw, and both Mer and Moe have roles in adhesion, the changes in the adhesion of wing epithelium upon reduction of flw are likely a result of changes in Mer and Moe phosphorylation and thus activity. The combination of excess active Moe and excess inactive Mer would affect the balance between maintenance and loss of stabilization of adherens junctions leading to the changes in adhesion and deformation of the wing epithelia that were observed. These adhesion differences could account for the formation of the large folds along the boundary of the ptc expression domain, since cells of similar adhesion are more likely to adhere to themselves (Yang, 2012).

The deformation of the wing imaginal tissue appears to be progressive, since in pre-pupal wing discs (10 h after pupariation) deep holes are observed that extend from the apical surface basally indicating that cells at the apical surface have left the epithelium and are forming balls of cells basally within the disc. In further support of the results, the loss of sds22, a PP1 regulatory subunit, in clonal analysis shows that in large clones in wing discs there is infolding of the mutant tissue with cells being extruded from the epithelium. Cells with loss of function Sds22 also exhibit Moe hyper-phosphorylation. Notably, this is reminiscent of what was observe with reduction of flw expression and overexpression of phosphomimic or nonphsophorylatable Mer or Moe (Yang, 2012).

While a likely cause of some of the changes seen in functional assays are due to changes in Mer and Moe phosphorylation as a result of changes in flw expression, the possibility remains that the level of analysis and resolution of the functional assays in both larval and pupal imaginal wing discs may be insufficient to clearly show subtle differences in the subcellular localization on the membrane of Mer, Moe and apical markers. Thus, it cannot be concluded that the defects associated with flw are due solely to defects in Mer and Moe activity (Yang, 2012).

The ability to partially recapitulate the loss of flw phenotype in ptc expressing cells by the over-expression of either a phosphomimic or nonphsophorylatable Mer or Moe also strongly suggests that this phenotype is, in part, due to the differences in the ratios of active Mer or Moe to inactive Mer or Moe which lead to the corresponding changes in apical epithelial integrity, in third instar discs. This is exemplified by the observation that often with overexpression of either the phosphomimic or nonphosphorylatable Mer or Moe, the formation of a fold is most apparent at the edge of ptc expression at the boundary where the difference in the expression of Mer or Moe within the ptc expressing cells and the neighbouring wild type cells would be greatest. In this way it is not unexpected that the overall effect on the tissue deformation and adhesion is the same with phosphomimic or nonphosphorylatable Mer or Moe, although it is possible that the underlying causes are different due to the predicted opposite activities of the transgenes (Yang, 2012).

Within or directly beside the edge of the ptc expression domain in wing imaginal discs, significantly more cells stain positively for activated Caspase 3. This suggests that cells in these affected domains are undergoing increased levels of apoptosis. These phenotypes are again reminiscent of what is observed in loss of function clones of Sds22, which exhibit an increase in the number of apoptotic cells in the wing discs (Yang, 2012).

It was also demonstrated that Flw binds to the scaffold protein Sip1. It functions with the kinase Slik to regulate Moe activity to maintain epithelial cell integrity. Therefore, the findings suggest that Mer, Moe, Flw, and Sip1 function within a protein complex. This coordinate incorporation within a regulated protein complex is necessary to coordinate cellular response to changing epithelial integrity. This might also explain why the overexpression of flw does not have a strong effect on epithelial integrity. If Mer and Moe need to be part of a complex with Flw and Sip1 in order to regulate epithelial integrity and proliferation, then expression of excess phosphatase outside the complex would have no effect on tissue morphology and growth. In contrast, loss of the phosphatase would have a direct effect since there would be reduced levels of functional protein complex (Yang, 2012).

Future studies are required to determine additional members of this regulatory complex, such as the likely candidates Sds22 and MYPT-75D. The similarity in phenotypes between Sds22 mutant cells and the results of knockdown of flw function would also support a role of Sds22 to interact with Flw in regulating Moe function (Yang, 2012).

This study has shown that the Mer and Moe proteins are direct targets of the catalytic subunit of the PP1 phosphatase Flw. This identifies another important player in the regulation of both Mer and Moe in Drosophila. This is the first identification of a phosphatase coordinately regulating both Mer and Moe activity in vivo. What remains to be determined is how Flw is targeted to regulate Mer and Moe function and what downstream pathways may be affected by these interactions (Yang, 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).

Regulation of cell proliferation and adhesion via a novel region of Drosophila Merlin interacting with Sip1

The tumour suppressor protein Merlin is thought to regulate cell proliferation and cell adhesion through interaction with protein partners. Loss of Merlin is associated with Neurofibromatosis Type 2 (NF2) tumours. NHERF1 or EBP50 is a scaffolding protein that functions in apical organization of polarized cells. Merlin and NHERF1 have been shown to interact in vitro in vertebrates. This study investigated how the Drosophila NHERF1 orthologue, Sip1, and Merlin function to regulate cell proliferation and adhesion. Two conserved arginine residues (R325 and R335) were identified in Merlin that, in addition to the FERM domain, are required for interaction with Sip1. Mutation of the arginine residues result in reduced Sip1 binding to Merlin and loss of Merlin growth suppressor function. Over-expression of MerlinR325A and/or MerlinR335L in Drosophila wings result in increased proliferation in the adult wing (increase in size), which is rescued by co-over-expression of constitutively active Merlin protein. Reduced Sip1 binding to Merlin also produces defects in adhesion in follicle epithelial cells. It is concluded that Sip1 facilitates the activation of Merlin as a tumour suppressor protein. Thus, this work provides insight into how Merlin functions as a tumour suppressor and in adhesion and this provides insight into the mechanism of NF2 pathogenesis (Abeysundara, 2014).


DEVELOPMENTAL BIOLOGY

Embryonic

In contrast to the largely complementary pattern observed for Merlin and Moesin-like during oogenesis, the two appear to be coexpressed in most cells during embryogenesis. Merlin and Moesin-like are present from cellularization throughout embryonic development. Merlin expression is found to be enhanced in the early mesoderm of the germband extended embryo, whereas Moesin-like is expressed uniformly throughout the embryo at this stage. Late in embryogenesis, both proteins are expressed ubiquitously throughout the tissues of the embryo, including the epidermis, salivary glands, foregut, midgut, hindgut, and the embryonic nervous system. At this stage Merlin expression is enhanced in the midgut (McCartney, 1996).

In the embryonic central nervous system (stage 15), Merlin and Moesin-like staining is detected in the neuropil, a structure composed of the developing axonal bundles of the ventral nerve cord and in the developing brain. Merlin and Moesin-like localization is also observed in the neuronal cell bodies of the CNS, with Moesin-like expression enhanced in these tissues. During stage 11 of embryogenesis, the neurons of the embryonic peripheral nervous system develop from specialized regions within the epidermis termed the proneural clusters. The cell bodies of the differentially bipolar sensory neurons can be observed in a regular pattern within the epidermis of a stage 17 embryo. At this stage, Moesin-like is enriched at the membranes of these cells, whereas Merlin is found to localize to an intensely staining spot within the cell body (McCartney, 1996)

Larval

In the developing eye Moesin-like is localized at the membranes of the cone cells, secondary and tertiary pigment cells, and in the bristle precursor cells. In contrast, Merlin is localized primarily in the cytoplasm of the secondary and tertiary pigment cells, and is greatly enhanced in the bristle precursor cells, which are interspersed between outer pigment cells. Both Merlin and Moesin-like are more intensely expressed in the center of each ommatidium. This corresponds to the region of the rhabdomeres, the photosensitive microvilli of the photoreceptors, which project into the center of each ommatidium (McCartney, 1996).

Adult

During oogenesis, Merlin and Moesin-like display strikingly different tissue distributions: this distinction is clearly observed as early as the germarium, the location of the germline stem cells. Merlin is expressed predominantly in the germline, while Moesin-like is expressed at greater levels in the follicle cells. In addition, Merlin expression becomes enhanced in the developing oocyte at approximately stage 6 of development and persists until the end of oogenesis. Lower levels of Merlin expression are also detected at the apical ends of the follicle cells at stage 10, late in oogenesis. In contrast, Moesin expression is found at the apical and basolateral ends of the follicular epithelium, although some expression is detected in the germline of early egg chambers and in the nurse cells at stage 10. Fully developed oocytes (stage 14) clearly display membrane associated Merlin, while no Moesin-like expression is detected at this stage (McCartney, 1996).

Effects of Mutation or Deletion

Neurofibromatosis-2 is an inherited disorder characterized by the development of benign schwannomas and other Schwann-cell-derived tumors associated with the central nervous system. The Neurofibromatosis-2 tumor suppressor gene encodes Merlin, a member of the Protein 4.1 superfamily most closely related to Ezrin, Radixin and Moesin. This discovery suggested a novel function for Protein 4.1 family members in the regulation of cell proliferation; proteins in this family were previously thought to function primarily to link transmembrane proteins to underlying cortical actin. Loss of Merlin function in Drosophila results in hyperplasia of the affected tissue without significant disruptions in differentiation. Similar phenotypes have been observed for mutations in expanded, another Protein 4.1 superfamily member in Drosophila. Because of the phenotypic and structural similarities between Merlin and expanded, it was asked whether Merlin and Expanded function together to regulate cell proliferation. Recessive loss of function of either Merlin or expanded can dominantly enhance the phenotypes associated with mutations in the other gene. Consistent with this genetic interaction, Merlin and Expanded colocalize in Drosophila tissues and cells, and physically interact through a conserved N-terminal region (CNTR) of Expanded, characteristic of the Protein 4.1 family, and the C-terminal domain of Merlin. Loss of function of both Merlin and expanded in clones reveals that these proteins function to regulate differentiation in addition to proliferation in Drosophila. Further genetic analyses suggest a role for Merlin and Expanded specifically in Decapentaplegic-mediated differentiation events. These results indicate that Merlin and Expanded function together to regulate proliferation and differentiation, and have implications for understanding the functions of other Protein 4.1 superfamily members (McCartney, 2000).

Hypomorphic alleles of either Mer (Mer3) or ex (ex697) result in very similar adult wing and eye phenotypes. In both cases, mutant adults displayed enlarged wings due to an increase in cell number rather than an increase in cell size. In fact, the cell size in the mutants appears to be slightly decreased. Furthermore, this expansion in wing area is often accompanied by the disruption or complete absence of the posterior cross vein. In addition, the anterior cross vein was sometimes disrupted in ex697 adults. Mer and ex mutants have smaller, weakly roughened eyes. Histological sections of Mer3 eyes reveal only minor perturbations in interommatidial organization and no obvious disruptions in ommatidial polarity. Concomitant with the reduction in eye size is an apparent expansion of the ventral peripheral head cuticle and the development of ectopic vibrissae. Although stronger alleles of both Mer and ex (exe1, a null allele) result in lethality, Mer mutant larvae do not develop the hyperplastic discs characteristic of ex mutant larvae (McCartney, 2000).

Dose-sensitive genetic interactions have been shown to be a reliable indicator of functional interactions between genes. Reduction of ex function in the Mer3 hemizygous background with either ex697 or exe1 results in an enhancement of the Mer3 head phenotypes; the adult eye is reduced in size, more ectopic head cuticle and vibrissae are observed, and the bristles normally found between ommatidia are often duplicated and disorganized. Those ommatidia that form contain the normal complement of photoreceptors, however. In addition, Mer3 wing area and the frequency of posterior cross vein disruptions increase when ex function is reduced. Consistent with these observations, reduction of Mer function in ex697 mutants causes an increase in ex697 wing size (McCartney, 2000).

Previous studies indicate that loss of function of either Mer or ex in clones results in a 2- to 3-fold overproliferation of the mutant tissue compared with the wild-type twin spot. In the wing, loss of either Mer or ex alone in clones has no apparent effect on the differentiation and morphology of the affected tissue. Similarly, in the eye, loss of function of Mer results in overproliferation without obvious changes in the underlying morphology. In contrast, loss of ex function in the eye results in defects in planar polarization, in addition to proliferation defects (Blaumueller and Mlodzik, 2000, in press, cited by McCartney, 2000). Because the loss of function of either Mer or ex results in overproliferation, the consequences of loss of function of both genes were examined using somatic mosaic analysis. In the adult wing, both vein and intervein cells differentiate in mutant tissue. In contrast, clones that intersect the position of the posterior cross vein disrupt its development, consistent with the variably penetrant disruption of the posterior cross vein observed in hypomorphic alleles of Mer or ex. Clones in the position of the anterior cross vein differentiate normally. Within the mutant intervein and vein clones, apparent defects were observed in proliferation control. In the proximal region of the wing, clonal vein tissue forms a raised protrusion. In other regions of the wing, bulges in the veins are also observed, although more frequently vein clones are merely broadened when compared with the surrounding vein. In the intervein regions, the clonal tissue appears to bulge and crinkle within the confines of the normal tissue, suggesting overproliferation. Similar cuticular bulges or protrusions have been reported for mutant clones of genes that have tumor suppressor phenotypes, such as warts. Thus this phenotype is interpreted to indicate that the Mer;ex double mutant clones in the wing proliferate at a greater rate than the single mutant clones, though this could not be confirmed directly. Based on general morphology, the cells within the clone appear to differentiate as intervein cells, however, the cuticle deposited at the base of each wing hair within the mutant clone appears to be thickened and is distinct from cuticle produced by either the heterozygous intervein or vein cells. Clones that develop within the eye appear either as small scars with associated clusters of bristles, or as elongated scars and associated indentations running from within the eye field toward the anterior margin. Although these clones do not differentiate ommatidia, the position of the twin spot was used to indicate the position of the mutant clone. Mutant clones are often associated with overproliferated head cuticle (McCartney, 2000).

Reduction of dpp function in the eye imaginal disc (dppblk) results in reduction of the eye along the dorsoventral axis such that the ventral portion of the eye is replaced by head cuticle. A similar, yet less severe, phenotype is observed in Mer3 hemizygotes and ex697 homozygotes. To ask whether dpp expression is disrupted in Mer mutants, a dpp-lacZ transgene was ovexpressed in the Mer3 background. In the wild-type eye-antennal complex, dpp is expressed at the lateral margins and in the morphogenetic furrow of the eye disc and in a ventral wedge of tissue in the antennal disc. In the Mer3 mutants, the ventral portion of the disc is significantly enlarged. The expression pattern of dpp is disrupted such that the cells expressing dpp at the margin are displaced to the outer tip of the overproliferated tissue. In some cases, this dpp staining is associated with an ectopic furrow and developing photoreceptors. To better understand the functional relationship between dpp, Mer and ex, genetic interactions were examined between these genes. Reduction of dpp dose in Mer3 hemizygotes enhances the severity of the Mer3 eye phenotype, resulting in a smaller, more roughened eye and expansion of the head cuticle. Similar reduction of dpp function in ex697 homozygotes results in enhanced eye phenotypes and variably penetrant truncated leg phenotypes, reminiscent of those observed in pharate adults null for ex function. Although it is appealing to think that the effects of Mer and ex on patterning and proliferation are both mediated through the DPP pathway, this seems unlikely given that loss of either gene seems to negatively affect DPP patterning functions, but simultaneously causes overproliferation of mutant cells. It therefore seems more likely that the proliferation phenotypes of Mer and ex loss-of-function mutations are mediated through effects on one or more other pathways that regulate proliferative events (McCartney, 2000).

Mammalian Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor-suppressor gene, is a member of the protein 4.1 superfamily that is most closely related to ezrin, radixin, and moesin (ERM). As is the case for NF2 tumors in vertebrates, Drosophila cells lacking Merlin function will overproliferate, relative to their neighbors. Using in vitro mutagenesis, functional domains within Merlin have been defined that are required for proper subcellular localization and for genetic rescue of lethal Merlin alleles. Remarkably, the results of these experiments demonstrate that all essential genetic functions reside in the plasma membrane-associated NH2-terminal 350 amino acids of Merlin. Removal of a seven-amino acid conserved sequence within this domain results in a dominant-negative form of Merlin that is stably associated with the plasma membrane and causes overproliferation when expressed ectopically in the wing. Evidence is provided that the COOH-terminal region of Merlin has a negative regulatory role, as has also been shown for ERM proteins. These results provide insights into the functions and functional organization of a novel tumor suppressor gene (LaJeunesse, 1998).

Reverse genetic analysis in Drosophila has been greatly aided by a growing collection of lethal P transposable element insertions that provide molecular tags for the identification of essential genetic loci. However, because the screens performed to date primarily have generated autosomal P-element insertions, this collection has not been as useful for performing reverse genetic analysis of X-linked genes. A reverse genetic screen has been designed that takes advantage of the hemizygosity of the X chromosome in males together with a cosmid-based transgene that serves as an autosomally linked duplication of a small region of the X chromosome. The efficacy and efficiency of this method is demonstrated by the isolation of mutations in Drosophila homologs of two well-studied genes, the human Neurofibromatosis 2 tumor suppressor and the yeast CDC42 gene. The method describe should be of general utility for the isolation of mutations in other X-linked genes, and should also provide an efficient method for the isolation of new alleles of existing X-linked or autosomal mutations in Drosophila (Fehon, 1997).

Merlin, the Drosophila homologue of neurofibromatosis-2, is specifically required in posterior follicle cells for axis formation in the oocyte

In Drosophila, the formation of the embryonic axes is initiated by three signals -- one from Gurken, a transforming growth factor; another signal from the oocyte to the posterior follicle cells, and an unknown polarizing signal back to the oocyte. Drosophila Merlin is specifically required only within the posterior follicle cells to initiate axis formation. Merlin mutants show defects in nuclear migration and mRNA localization in the oocyte. Merlin is not required to specify posterior follicle cell identity in response to the Gurken signal from the oocyte, but is required for the unknown polarizing signal back to the oocyte. Merlin is also required non-autonomously, only in follicle cells that have received the Gurken signal, to maintain cell polarity and limit proliferation, but is not required in embryos and larvae. These results are consistent with the fact that human Merlin is encoded by the gene for the tumor suppressor neurofibromatosis-2 and is a member of the Ezrin-Radixin-Moesin family of proteins that link actin to transmembrane proteins. It is proposed that Merlin acts in response to the Gurken signal by apically targeting the signal that initiates axis specification in the oocyte (MacDougall, 2001).

To identify new genes required for axis specification, a screen was carried out of a collection of X-linked ts lethal mutations generated by selecting for male lethality at 29°C and viability at 21°C. Homozygous female progeny were collected at 21°C from 73 viable ts lethal lines, shifted to 29°C for 3 days and GRK RNA in situ hybridization on ovaries was performed. In wild type at 29°C or in all strains at 21°C, the oocyte nucleus migrates correctly to the antero-dorsal corner of the oocyte with GRK mRNA localiz ing between the nucleus and the overlying future dorsal follicle cells. In one mutant line, l(1)ts594, identified as a mutation in Merlin (termed here Merts1), 55% of oocyte nuclei fail to migrate and GRK mRNA localizes at the posterior after stage 8. The remaining 45% of cases were similar to wild type (MacDougall, 2001).

To test whether the defects in the oocyte are primarily due to a defect in MT organization, MT polarity was examined. Kin:betagal, a plus end-directed MT motor fusion that leads to betagal accumulation at the posterior of the oocyte, was used. Nod:betagal, a MT motor fusion that leads to betagal accumulation at the anterior, where the minus ends of MTs are thought to localize, was also used. The betagal motor fusions indicate that prior to stage 7, there is an MTOC at the posterior. In wild-type oocytes after stage 7, the posterior MTOC disassembles, a diffuse MTOC appears at the anterior with MT plus ends at the posterior. Prior to stage 7, Merts1 mutant oocytes show a similar MT organization to wild type, but after stage 7, the MTOC fails to disassemble at the posterior and a second diffuse MTOC forms at the anterior. This leads to a symmetric organization of MTs, with their plus ends at the center of the oocyte and minus ends at the anterior and posterior. The overall distribution of MTs was examined using a maternally expressed TauGFP line showing the highest concentration of MTs at the anterior cortex of wild-type oocytes. A similar Tau-GFP distribution was observed in Merts1 oocytes at 21°C. In Merts1 at 29°C Tau-GFP shows an abnormally high level at the posterior, consistent with a failure to disassemble the posterior MTOC. It is concluded that the mislocalization of mRNA and failure of the oocyte nucleus to relocate in Merts1 oocytes are due primarily to defects in MT organization (MacDougall, 2001).

Merlin protein is expressed in the oocyte and in posterior follicle cells. To determine where Merlin functions in egg chambers, homozygous Merts1 germline clones were generated using X-rays in females raised at the restrictive temperature (29°C). Ten Merts1 oocytes surrounded by Merts1/+ follicle cells were examined, and they all showed normal mRNA localization and led to normal eggs. It is concluded that Merlin is not required in the germline derived nurse cells or oocyte (MacDougall, 2001).

To test whether Merlin is required within the somatically derived posterior follicle cells to receive the Grk signal from the oocyte, the expression of different follicle cell markers were examined in Mer egg chambers. The results show that Merts1 posterior follicle cells receive the Grk signal correctly; they express posterior and not anterior markers. It is concluded that Merlin is not required for any aspect of Grk signaling or its reception in the posterior follicle cells. Merlin is also not required for Notch signaling among the posterior follicle cells, which is required to specify the correct number of posterior cells (MacDougall, 2001).

The posterior follicle cells in fixed and living Mer egg chambers often have a slightly disrupted morphology. To study these defects in more detail, actin and DNA were covisualized to highlight each cell and its boundaries. Posterior follicle cells in controls have a uniform columnar appearance characteristic of epithelial sheets. However, after stage 6, Merts1 egg chambers have a double layer of follicle cells only at the posterior where follicle cells are in contact with the oocyte. To determine whether the double layer of posterior follicle cells is due to overproliferation, the number of cells was counted using three-dimensional microscopy and a twofold increase in the number of posterior follicle cells was found, but no changes in other follicle cells. To determine whether the overproliferation of posterior follicle cells is accompanied by polarity defects, MT polarity was studied by covisualizing DNA, the nuclear envelope and centrosomes. In control egg chambers, most centrosomes lie on the apical side of each nucleus, where the minus ends of MTs are found. In contrast, Merts1 posterior follicle cells mostly lose the apical-basal polarity of their MTs (MacDougall, 2001).

To investigate whether other aspects of the apical-basal polarity of the posterior follicle cells are also disrupted, the distribution of beta-spectrin heavy chain (betaH-spectrin) was examined in Mer mutants. betaH-spectrin is normally restricted to the apical side of follicle cells within a Spectrin-based membrane skeleton. In Merts1 mutants, betaH-spectrin is apically localized in the cells adjacent to the oocyte, but not detected in the second layer of follicle cells. These results suggest that in Mer mutants, the apical surface of posterior follicle cells contacts the oocyte correctly, and is probably competent to send and receive signals to the oocyte (MacDougall, 2001).

To determine whether the defects in cell proliferation and polarity in Mer egg chambers are dependent on receiving the Grk signal, the follicle cells of Mer;grk double mutants was examined. Even a hypomorphic allele of grk suppresses the Mer posterior follicle cell phenotype entirely. It is concluded that Merlin is required only in cells that receive the Grk signal and is not a constitutive factor required for cell polarity and proliferation. Additional experiments show that Merlin is required only in posterior follicle cells. Also, Merlin has been shown to acts cell non-autonomously among the posterior follicle cells (MacDougall, 2001).

These results show that Merlin is required for two distinct processes involving signalling, but whether the two processes depend on a single signal or two distinct signals cannot be distinguished. The restriction of posterior follicle cell proliferation could require the same unknown signal that initiates MT repolarization in the oocyte. Both processes could depend on the same signal secreted into the space between the follicle cells and oocyte. Indeed, it is intriguing that Merlin egg chambers have MT polarity defects in both the oocyte and the posterior follicle cells. However, further progress awaits the identification of the signal or signals involved (MacDougall, 2001).

The identity of the polarizing signal is unknown, but some genes are known to be required for the signal, including PKA, Mago and Laminin A. Merlin is unlikely to be required for PKA and Mago functions because they are required in the oocyte. In contrast, Laminin A is expressed and required in posterior follicle cells as a component of the extracellular matrix (Deng, 2000). It is tempting to speculate that Merlin and Laminin A could be functionally linked as specialized structural components required specifically in the posterior follicle cells for the transduction of the polarizing signal (MacDougall, 2001).

A systematic screen for dominant second-site modifiers of Merlin/NF2 phenotypes reveals an interaction with blistered/DSRF and scribbler

Merlin, the Drosophila homolog of the human tumor suppressor gene Neurofibromatosis 2 (NF2), is required for the regulation of cell proliferation and differentiation. To better understand the cellular functions of Merlin, recent work has concentrated on identifying proteins with which Merlin interacts either physically or functionally. Genetic screens designed to isolate second-site modifiers of Merlin phenotypes are described from which five multiallelic complementation groups have been identified that modify both loss-of-function and dominant-negative Merlin phenotypes. Three of these groups, Group IIa/scribbler (also known as brakeless), Group IIc/blistered, and Group IId/net, are known genes, while two appear to be novel. In addition, two genes, Group IIa/scribbler and Group IIc/blistered, alter Merlin subcellular localization in epithelial and neuronal tissues, suggesting that they regulate Merlin trafficking or function. Mutations in scribbler and blistered display second-site noncomplementation with one another. These results suggest that Merlin, blistered, and scribbler function together in a common pathway to regulate Drosophila wing epithelial development (LaJeunesse, 2001).

In this screen seven new alleles of sbb were identified; allelism was based on noncomplementation with a null sbb allele and the presence of nonsense mutations in two sbb alleles identified. Null and strong hypomorphic mutations in scribbler result in aberrant axon guidance and behavioral phenotypes. However, none of the sbb alleles identified in this screen display either of these phenotypes. In addition, none of the previously identified P-element insertional mutations in sbb modify Merlin phenotypes, although the null sbb4 allele and Df(2R)PC4 do enhance Merlin phenotypes. These data suggest that sbb has two distinct functions, one in axon guidance of photoreceptor cells and the other in regulation of proliferation in epithelial cells, and that these functions are independent. Consistent with this model, previous studies showed that sbb encodes two novel proteins of unknown function, SBB-A and SBB-B. Although it was shown that the two SBB isoforms are functionally redundant in axon guidance, the presence of a zinc finger domain and a novel Region B in the larger SBB-B isoform suggests that it may have functions distinct from SBB-A. Sequence analysis indicates that two of the alleles isolated as Merlin modifiers correlate with nonsense mutations that affect the SBB-B product but leave the BSKA product intact (LaJeunesse, 2001).

The identification of SBB-B mutations that specifically modify Merlin phenotypes but do not affect photoreceptor axon guidance supports a model where SBB-B has distinct functions in the proliferation and differentiation of wing tissue. Both SBB isoforms are reported to be nuclear proteins and the presence of a zinc finger in SBB-B suggests that it may be involved in transcriptional regulation. How SBB proteins interact with Merlin, a membrane-associated cytoplasmic protein, is unclear. The observation that Merlin subcellular localization is disrupted in sbb mutant cells makes this question particularly intriguing and suggests that sbb may play a role in a cellular pathway that regulates Merlin function. The identity of this pathway is currently unknown (LaJeunesse, 2001).

While sbb encodes novel proteins with unknown function, the bs gene product, also known as the Drosophila serum response factor (BS/DSRF), is a well-characterized transcription factor. bs is required for formation of terminal tracheal branches and differentiation of the adult wing. BS/DSRF activity, like that of its mammalian homolog, is regulated by the epidermal growth factor receptor (Egfr) signaling pathway. During development of the wing imaginal disc, cells can adopt one of two fates; most cells form wing blade (intervein tissue), while a subset form the characteristic longitudinal veins. BS/DSRF is believed to promote the intervein cell fate -- loss-of-function bs mutations result in wings in which all cells develop as vein tissue. Activity of the Egfr pathway is believed to promote the vein cell fate by downregulating BS/DSRF function in the vein primordia and promoting the expression of vein-specific genes. Thus interactions between the Egfr pathway and BS/DSRF play a crucial role in wing development (LaJeunesse, 2001 and references therein).

The identification of bs as a dominant modifier of Merlin phenotypes suggests that Merlin, like Blistered, is involved in Egfr signaling. Specifically, the observation that bs mutations enhance Merlin dominant-negative and loss-of-function phenotypes suggests that Merlin may function antagonistically to Egfr pathway function. Although this hypothesis should be considered as tentative, several lines of evidence support this notion: (1) developing wing cells that have lost both Merlin and expanded, which appear to function redundantly, produce abundant ectopic vein material adjacent to endogenous veins; (2) net, which was also identified as a Merlin modifier, has been shown to modify phenotypes of components of Egfr signaling in the wing; (3) a role for Merlin in negatively regulating Egfr function is consistent with the observation that Merlin mutations result in overproliferation phenotypes and (4) a hypermorphic Egfr mutation called Ellipse enhances phenotypes expressed by dominant-negative and hypomorphic Merlin alleles. However, despite these intriguing indications that Merlin may function to regulate Egfr pathway activity, it should be noted that Merlin does not interact genetically with several other known pathway members (Star, asteroid, and rhomboid), nor does it interact with hypomorphic Egfr mutations. In addition, because other signaling pathways, including dpp, wingless, and Notch, are involved in vein specification, it is possible that Merlin functions to regulate one or more of these either instead of or in addition to the Egfr pathway. In support of this notion, Merlin and expanded have both been shown to genetically interact with dpp. Further experiments are required to determine the significance of these genetic interactions. Nonetheless, the identification of Merlin modifiers suggests testable hypotheses regarding Merlin cellular functions and opens new avenues for further investigation of the molecular basis of the NF2 disorder (LaJeunesse, 2001).

The tumor suppressors Merlin and Expanded function cooperatively to modulate receptor endocytosis and signaling

The precise coordination of signals that control proliferation is a key feature of growth regulation in developing tissues. While much has been learned about the basic components of signal transduction pathways, less is known about how receptor localization, compartmentalization, and trafficking affect signaling in developing tissues. This paper examines the mechanism by which the Drosophila Neurofibromatosis 2 (NF2) tumor suppressor ortholog Merlin (Mer) and the related tumor suppressor expanded (ex) regulate proliferation and differentiation in imaginal epithelia. Merlin and Expanded are members of the FERM (Four-point one, Ezrin, Radixin, Moesin) domain superfamily, which consists of membrane-associated cytoplasmic proteins that interact with transmembrane proteins and may function as adapters that link to protein complexes and/or the cytoskeleton. Merlin and Expanded function to regulate the steady-state levels of signaling and adhesion receptors, and loss of these proteins can cause hyperactivation of associated signaling pathways. In addition, pulse-chase labeling of Notch in living tissues indicates that receptor levels are upregulated at the plasma membrane in Mer; ex double mutant cells due to a defect in receptor clearance from the cell surface. It is proposed that these proteins control proliferation by regulating the abundance, localization, and turnover of cell-surface receptors and that misregulation of these processes may be a key component of tumorigenesis (Maitra, 2006).

Merlin's tumor suppressor function is conserved from humans to flies, but the cellular basis for this function remains unclear. Genetic studies in Drosophila suggest that Mer regulates signaling pathways that control proliferation, and cell biological experiments indicate that Merlin may play a role in endocytic processes. In addition, Merlin physically interacts with Expanded, a distantly related member of the FERM superfamily, and these proteins colocalize in the apical junctional region of epithelial cells. Furthermore, genetic studies have shown that while mutations of each gene produce modest overproliferation phenotypes in the eye and wing, double mutant Mer; ex cells display severe overgrowth and differentiation defects that are not seen in either mutation alone. Thus, Mer and ex are partially redundant in regulating proliferation and differentiation (Maitra, 2006).

Given these observations, it was reasoned that the difficulty in identifying precise cellular functions for Merlin might stem from its redundancy with Expanded and that this difficulty could be overcome by examining tissues from double mutant animals and double mutant cell clones generated by somatic recombination. Overproliferation of Mer; ex wing imaginal discs is more extreme than that observed with either mutation alone. Surprisingly, however, Mer4; ex697 eye-antennal imaginal discs have severely reduced eye primordia with a substantial reduction in or total absence of photoreceptors, although the antennal portion is normal or slightly larger than normal and occasionally is duplicated. Apoptosis does not appear to be enhanced in double mutant eye-antennal discs, suggesting that loss of the eye primordium is not due to cell death. Thus, loss of Mer and ex function has a tissue-specific defect in the developing eye that is very different from its effects on proliferation in the wing imaginal disc (Maitra, 2006).

Why does the combined loss of two tumor suppressors cause reduction rather than hypertrophy of eye tissue? Previous studies have shown that initiation of the morphogenetic furrow, which organizes development of the eye, is regulated by a complex network of signals at the posterior and lateral margins of the eye-antennal disc. Mutations that affect these signals not only block furrow initiation, but also may significantly reduce the size of the eye field and disrupt photoreceptor differentiation. For example, ectopic Wingless expression either at the posterior and lateral margins or throughout the eye primordium results in dramatic losses of eye tissue that closely resemble the Mer; ex phenotype just described. Similar effects are seen from reduction in Decapentaplegic (DPP) or Hedgehog signaling in the same cells (Maitra, 2006).

If Merlin and Expanded affect initiation of the morphogenetic furrow rather than differentiation of photoreceptors, then Mer; ex double mutant somatic clones should block ommatidial development only when present at the posterior or lateral margins of the eye field. Indeed, Mer; ex clones could differentiate photoreceptors, but only when located in the middle of the eye field. In contrast, clones in contact with the posterior or lateral margin of the eye fail to produce photoreceptors. It is inferred from these observations that one or more of the signaling pathways that control initiation of the morphogenetic furrow are likely disrupted in Mer; ex double mutant cells (Maitra, 2006).

Given that Merlin is associated with the plasma membrane and may function in endocytic processes, it was asked if Merlin and Expanded play a role in regulating localization and/or abundance of transmembrane receptors that function in eye development. For these studies, Mer; ex somatic mosaic cell clones were examined to allow side-by-side comparisons of wild-type and mutant cells in the wing and eye imaginal discs. Immunofluorescence staining with specific antibodies then allowed comparison of the steady-state levels of receptors between adjacent wild-type and mutant cells. Intriguingly, Notch, the EGF receptor, Patched, and Smoothened all displayed increased antibody staining in double mutant cells relative to their wild-type neighbors. Notch, which is primarily localized to the apical junctional domain in wild-type cells, showed not only increased junctional staining in mutant cells, but also more diffuse staining. Similarly, preparations with anti-EGFR display more abundant membrane-associated and cytoplasmic staining in mutant than in wild-type cells. Patched staining, which is less obviously junctional than Notch or EGFR, appeared more punctate in Mer; ex cells. Thus, simultaneous loss of Merlin and Expanded results in increased abundance of receptors for multiple signaling pathways, though the precise localization defect seems to be specific to each receptor. Two adhesion-related receptors, E-cadherin and Fat, a cadherin superfamily member, were examined; both are similarly upregulated in Mer; ex cells. However, Coracle, a membrane-associated cytoplasmic protein, is not affected. In addition, the localization of markers for apical-basal polarity, including DLG, PATJ, and aPKC, was unaffected in the double mutant cells, indicating that epithelial polarity is not disrupted. In contrast to the double mutant cells, clones lacking just Merlin show no apparent difference in receptor localization or abundance, and exe1 cells display only a slight increase in staining. Taken together, these results indicate that Merlin and Expanded are required to reduce the steady-state abundance of a variety of signaling and adhesion receptors in developing epithelia (Maitra, 2006).

Membrane trafficking was examined in Mer; ex double mutant cells. Antibodies were used against the extracellular domain of Notch (anti-ECN) to label protein on the surface of living cells in imaginal discs bearing somatic mosaic clones. Side-by-side comparisons of wild-type and Mer; ex mutant cells show increased cell-surface Notch labeling, consistent with what was observed with fixed tissue and indicating that there are increased levels of receptor at the plasma membrane in mutant cells. In addition, in double mutant cells, the junctional band of Notch staining is broader, indicating that Notch localization to the junctional region also may be affected. Similar differences in junctional staining were observed with the same antibody on fixed and permeabilized tissues, indicating that surface labeling of live cells does not affect Notch localization (Maitra, 2006).

To ask if the increased abundance is due to a defect in turnover, a pulse-chase approach was used to label Notch receptor at the plasma membrane and then its removal from the cell surface was followed. To restrict analysis to Notch that remains at the cell surface, tissues were fixed but not permeabilized at the end of the chase period. A progressive loss was observed of Notch staining at the cell surface during the chase period that appeared more rapid in wild-type than in mutant cells, suggesting a defect in trafficking off the plasma membrane. Quantitative fluorescence analysis was used to determine the relative quantities of Notch on wild-type and mutant cells at the various chase time points. The results indicate that the ratio of cell-surface Notch fluorescence in mutant versus wild-type cells increases significantly between 0 and 10, 30, or 60 min postlabeling. Therefore, Notch protein is cleared more rapidly from the surface of wild-type than mutant cells (Maitra, 2006).

It is worth noting that current models for Notch receptor activation require cleavage and release of its extracellular domain in response to ligand binding. Because an antibody was used that recognizes this domain, it follows that these studies examined only ligand-independent trafficking of the receptor. In support of this inference, the pattern of Notch internalization in pulse-chase experiments was unaffected in Delta clones. These observations suggest that Merlin and Expanded function in steady-state, ligand-independent clearance of receptors from the plasma membrane, rather than internalization and degradation that occurs in response to ligand binding (Maitra, 2006).

Increased receptor abundance may be expected to result in increased signaling output, if receptor quantity is a limiting factor. In addition, even if overall receptor quantity is not limiting, alterations in subcellular localization or the dynamics of receptor trafficking may have dramatic effects on receptor function. To ask if loss of Merlin and Expanded result in increased output from signaling pathways that regulate eye development and cell proliferation, markers specific for downstream activation of the EGFR, Wingless, and Notch signaling pathways were used. First, double mutant clones were stained with an antibody that recognizes the phosphorylated, activated form of MAP kinase (anti-dpERK), a downstream effector of the EGFR pathway. In addition to the normal anti-dpERK pattern in the wing imaginal disc, increased staining was observed in Mer; ex clones relative to their wild-type neighbors, suggesting upregulation of EGFR pathway activity. Similarly, output from the Wingless pathway was monitored by looking at expression of Distalless, a target of Wingless signaling and it was found to be dramatically higher in the double mutant wing clones. In contrast, similar experiments with the mAb323 antibody to E(spl) bHLH proteins, a marker for Notch pathway activity, did not show upregulation of Notch signaling. This result is consistent with the observation that overexpression of Notch in a wild-type genetic background has little or no phenotype. To examine this further, a genetic context was analyzed in which Notch receptor quantities are known to be limiting, that is, in animals that are heterozygous for a null Notch mutation. Such animals display a dominant, haploinsufficient phenotype characterized by notching along the wing margin. To ask if reduction in Merlin and Expanded in this context can cause upregulation of Notch pathway output, animals triply heterozygous for Notch, Merlin, and expanded were generated and it was found that the characteristic Notch wing phenotype was strongly suppressed (Maitra, 2006).

Taken together, these results are consistent with the observation that the steady-state level of multiple receptors is elevated in Mer; ex cells and indicate that, depending on the precise developmental or genetic context, loss of Merlin and Expanded can result in increased output from the corresponding signaling pathways. In Mer; ex eyes, upregulation of Wingless signaling may be a primary contributor to the observed defect in ommatidial development. Previous studies have shown that ectopic Wingless signaling produces remarkably similar eye phenotypes, and preliminary data suggest that inhibiting Wingless signaling partially suppresses the Mer; ex eye phenotype. In the wing, the dramatic overproliferation of Mer; ex cells may be the combined result of upregulation of several pathways, including EGFR and Wingless (Maitra, 2006).

Merlin and Expanded are associated with the apical junctional region in imaginal epithelia and with endocytic vesicles in cultured cells. Results shown in this study indicate that loss of these proteins affects abundance, cell-surface localization, and endocytic trafficking of Notch, EGFR, and other signaling and adhesion receptors in epithelial cells. Recent studies of endocytic trafficking in receptor/ligand regulation suggest aspects of endocytosis that could relate to Merlin and Expanded function. For example, it is possible that Merlin and Expanded function at the plasma membrane to recruit or anchor transmembrane proteins at sites on the membrane from which they are endocytosed or in the sorting between recycling endosomes and lysosomal degradation by promoting receptor degradation. Both possibilities are consistent with observations of increased receptor levels at the plasma membrane in Mer; ex mutant cells and colocalization of Merlin and Expanded with Notch in punctate structures at the plasma membrane. In addition, a partial colocalization was observed of Merlin and Expanded with Rab 11, a marker for recycling endosomes, and with EEA-1, which labels early endosomes. Intriguingly, it has been suggested that the closely related ERM protein Ezrin functions to promote recycling rather than degradation of the β2-adrenergic receptor via its interactions with filamentous actin. Understanding the exact relationship of Merlin and Expanded to endocytosis and recycling of receptors, as well as their possible relationship to ERM proteins in this process, will require further analysis (Maitra, 2006).

A recent study has proposed that Merlin and Expanded function upstream of Hippo in the Warts signaling pathway, which regulates proliferation. Merlin and expanded mutants display similar phenotypes to those seen in hippo mutants. However, there are significant phenotypic differences between Mer; ex and hippo mutations, most notable of which is that hippo mutations have not been reported to block induction of eye morphogenesis. In addition, there is no evidence to suggest that the Hippo pathway regulates output of the EGFR, Wingless, or Notch signaling pathways. Thus, the relationship of Merlin and Expanded to the Hippo pathway may be more complicated than the linear pathway proposed. One possibility is that Hippo activation is a downstream consequence of Merlin and Expanded's effects on output of multiple signaling pathways (Maitra, 2006).

More than a decade after its molecular characterization, the precise cellular functions of Merlin in regulating cell proliferation remain unclear. Based on the current studies, it is proposed that Merlin's tumor suppressor phenotype results from defects in endocytic trafficking of signaling receptors and accompanying hyperactivation of associated signaling pathways. Recent studies highlight the importance of endocytosis in regulation of signaling pathways. Based on the results presented in this study, it is suggested that proper regulation of membrane trafficking also may have important implications for understanding the cellular basis of tumor suppression in flies and mammals (Maitra, 2006).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

This study has analyzed the requirement of the SWH pathway during oogenesis. Several of the components of this pathway, but not ft, are required in the PFCs to induce the axis specification in the germline. The defects in oocyte polarity, however, are probably due to a lack of proper differentiation of the PFCs in SWH mutant egg chambers. In addition, the pathway is required in the terminal cells to control their proliferation. It has already been shown that terminal follicle cells are different from lateral follicle cells. The distinct spatial requirement of the SWH pathway for differentiation and proliferation is another feature that distinguishes the terminal from the lateral FCs, and the posterior from the anterior FCs. These results point out that this dual function of the SWH pathway might be achieved by modulation of the Notch and EGFR signals. In conclusion, the SWH pathway lies at the intersection of two signaling pathways and is permissive for the signal that is sent from the follicle cells to repolarize the oocyte (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 (hpoJM1) or a truncating (hpoBF33) allele of hpo. These two alleles behave similarly in all subsequent experiments (Polesello, 2007).

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

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

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

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

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

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

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

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

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

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

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

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

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

The Hippo tumor-suppressor pathway regulates apical-domain size in parallel to tissue growth

The Hippo tumor-suppressor pathway controls tissue growth in Drosophila and mammals by regulating cell proliferation and apoptosis. The Hippo pathway includes the Fat cadherin, a transmembrane protein, which acts upstream of several other components that form a kinase cascade that culminates in the regulation of gene expression through the transcriptional coactivator Yorkie (Yki). Work in Drosophila has indicated indicated that Merlin (Mer) and Expanded (Ex) are members of the Hippo pathway and act upstream of the Hippo kinase. In contrast to this model, it was suggested that Mer and Ex primarily regulate membrane dynamics and receptor trafficking, thereby affecting Hippo pathway activity only indirectly. This study examined the effects of Mer, Ex and the Hippo pathway on the size of the apical membrane and on apical-basal polarity complexes. It was found that mer;ex double mutant imaginal disc cells have significantly increased levels of apical membrane determinants, such as Crb, aPKC and Patj. These phenotypes were shared with mutations in other Hippo pathway components and required Yki, indicating that Mer and Ex signal through the Hippo pathway. Interestingly, however, whereas Crb was required for the accumulation of other apical proteins and for the expansion of the apical domain observed in Hippo pathway mutants, its elimination did not significantly reverse the overgrowth phenotype of warts mutant cells. Therefore, Hippo signaling regulates cell polarity complexes in addition to and independently of its growth control function in imaginal disc cells (Hamaratoglu, 2009).

The results show that the Hippo pathway regulates the amount of apical protein complexes and thereby the size of the apical domain and that this effect is independent of its growth control function. Importantly, the regulation of apical complexes is a specific effect of the Hippo pathway, since other growth control pathways do not regulate apical complexes. In addition, this effect of the Hippo pathway is a general effect, since upregulation of apical complexes was observed in multiple tissues and cell types. Although overexpression of Crb and aPKC are sufficient to drive extra growth, the results show that the upregulation of apical complexes is not required for the overgrowth phenotype and for the induction of Hippo target genes in wts mutant cells. It is thus concluded that the Hippo pathway regulates the amount of apical complexes in Drosophila imaginal disc cells in addition to and independently of its growth control function (Hamaratoglu, 2009).

It has been suggested that Mer and Ex regulate the levels of membrane receptors independently of the Hippo pathway. However, the current results show that the upregulation of DER, Ft and apical complexes was similar in hpo and wts mutant cells and mer;ex double mutant cells, and that this effect requires Yki. These results thus indicate that Mer and Ex act through the Hippo pathway to exert their effect and that they are bona fide members of the Hippo pathway. Similar conclusions have been drawn based on observations that overexpression of wts suppresses the lethality and overgrowth phenotypes of ex mutants (Hamaratoglu, 2009 and references therein).

How does the Hippo pathway regulate the size of the apical domain and the amount of the apical complexes? The observation that Yki is required and sufficient for the effect on the apical domain indicates that this effect of the Hippo pathway is mediated by transcriptional regulation. However, although the upregulation of Crb is necessary and sufficient for the expansion of the apical domain and for the accumulation of the other apical polarity complex proteins, it is not required for the upregulation of DER and Ft, which still accumulate in wts,crb double mutant cells. Thus a model is favored in which the Hippo pathway regulates the turnover of several apical membrane components, for example through regulation of endocytosis. Notably mer;ex mutant cells in wing imaginal discs have defects in Notch (N) endocytosis, which leads to accumulation of N. Moreover, the endosomal protein Hrs accumulates in hpo mutant follicle cells in Drosophila ovaries, and this study observed a similar accumulation of Hrs in wts mutant clones in imaginal discs. These observations thus support the hypothesis that Hippo signaling regulates the amount of endocytosis and membrane turnover, thereby affecting the amount of apical membrane proteins. The target of Yki that mediates these effects, however, is currently not known (Hamaratoglu, 2009).

Several other studies also demonstrated roles for the Hippo pathway beyond its function in growth control. For example, the Hippo pathway is required for the proper selection of photoreceptor subtypes in the Drosophila eye, and it is required in follicle cells to generate a signal that polarizes the underlying oocyte. For both of these functions, Hippo signals through Yki, but Yki may regulate different sets of target genes, since the phenotypic effects are different. In addition, the Hippo pathway regulates cellular behavior through pathways that may not require Yki and thus may not involve the regulation of gene expression. For example, Yki-independent functions of the Hippo pathway may regulate dendritic tiling of larval neurons and the death of salivary gland cells during metamorphosis. The finding that Hippo regulates apical polarity complexes in addition to and independently of its growth control function in imaginal discs cells thus further reveals the complex function of this pathway in the regulation of cellular behavior (Hamaratoglu, 2009).

Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration

An amputated cricket leg regenerates all missing parts with normal size and shape, indicating that regenerating blastemal cells are aware of both their position and the normal size of the leg. However, the molecular mechanisms regulating this process remain elusive. This study used a cricket model (the two-spotted cricket, Gryllus bimaculatus) to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration. Knockdown of ft or ds transcripts by regeneration-dependent RNA interference (rdRNAi) suppressed proliferation of the regenerating cells along the proximodistal (PD) axis concomitantly with remodelling of the pre-existing stump, making the regenerated legs shorter than normal. By contrast, knockdown of the expanded (ex) or Merlin (Mer) transcripts induced over-proliferation of the regenerating cells, making the regenerated legs longer. These results are consistent with those obtained using rdRNAi during intercalary regeneration induced by leg transplantation. A model is presented to explain these results in which the steepness of the Ds/Ft gradient controls growth along the PD axis of the regenerating leg (Bando, 2009).

Regeneration-dependent RNAi is a type of RNAi that occurs specifically after leg amputation in cricket nymphs that have been injected with double-stranded RNA (dsRNA) for a target gene (Nakamura, 2008a). In this system, when the metathoracic (T3) tibia of the third-instar nymph is amputated, it takes approximately 40 days (six ecdyses) to restore the adult leg. The process begins with the covering of the amputated region by newly formed cuticle. A ligand of Epidermal growth factor receptor (Gb'Egfr) is then induced by Decapentaplegic (Gb'Dpp) and Wingless (Gb'Wg) in a blastema composed of epithelial stem cells, which begins to undergo rapid proliferation to restore the lost portion in the fourth instar (Mito, 2002; Nakamura, 2008b). In the fifth instar, the tibiae, tibial spurs, tarsi and tarsal claws are restored in miniature. In the seventh instar, the amputated legs restore the missing portion to regain a nearly normal appearance. As no leg regeneration was observed after amputation in the case of rdRNAi against Gb'armadillo (Gb'arm), the canonical Wnt pathway should be involved in the initiation of the regeneration (Nakamura, 2007; Bando, 2009 and references therein).

Using an RNAi knockdown approach against 23 candidate genes, this study identified 15 components of the Ds/Ft signalling pathway that are involved in cricket leg regeneration. Based on additional data from Gryllus and Drosophila, a model signalling cascade was proposed for the regulation of leg regeneration by the Ds/Ft signalling pathway. As the main components of the Ds/Ft signalling pathways are conserved in vertebrates, this signalling cascade may also be involved in vertebrate leg regeneration (Bando, 2009).

The most typical phenotypes were the short and thick legs induced by rdRNAi against Gb'ft or Gb'ds. It is known that the size of each leg segment normally scales with overall body size, a phenomenon known as allometry. Surprisingly, the size of the regenerated legs in the phenotypes that were observed did not scale with overall body size. Furthermore, the size of the regenerated legs depended upon the site of tibial amputation. It is noteworthy that, although the expression patterns of Gb'ft and Gb'ds were different, their short and thick phenotypes were similar. This is consistent with the fact that Drosophila mutant phenotypes of both ft and ds in adult legs are short and thick, despite the fact that ft and ds have distinct expression patterns in Drosophila imaginal discs. Thus, it is concluded that the activity of Ds/Ft signalling regulates leg segment size and shape during regeneration. Furthermore, it was shown that the Ds/Ft signalling pathway may regulate leg size during regeneration through the Hpo signalling pathway. This is also supported by the fact that the Hpo signalling pathway is involved in an intrinsic mechanism that restricts organ size and that the Ds/Ft signalling system defines a cell-to-cell signalling mechanism that regulates the Hpo pathway, thereby contributing to the control of organ size (Bando, 2009).

Meinhardt pointed out that two processes operate during leg regeneration. One, which operates during the restoration of distal structures, is instructed by a morphogen epidermal growth factor (Egf), which is itself induced by two morphogens, Dpp and Wg, at the amputated surface (Meinhardt, 1982; Mito, 2002; Nakamura, 2008a). The other, operating in intercalary regeneration, is directly controlled by neighbouring cells at the junction between host and graft, but not by long-range morphogens (Meinhardt, 2007; Nakamura, 2008a). It is likely that the Ds/Ft signalling pathway participates in both mechanisms, because rdRNAi against Gb'ft or Gb'ds affected leg regeneration after either distal amputation or intercalary transplantation. In the case of distal amputation, as the Gryllus tarsi and claws were not restored after tibial amputation in the Gb'Egfr rdRNAi nymphs (Nakamura, 2008a), it has been speculated that Gb'Egf functions as a morphogen in the leg regeneration, as found in Drosophila leg imaginal discs. Recently, it has been demonstrated in the Drosophila wing disc that the Fat signalling pathway links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth. Thus, it is speculated that the Ds/Ft system links the Egf-mediated establishment of gradients of positional values across regenerating blastemal cells to the regulation of regenerate growth (Bando, 2009).

As Gb'd rdRNAi legs exhibited the short-leg phenotypes, but not thick ones, and Gb'd (decapentaplegic) is epistatic of Gb'ft and Gb'ds, Gb'D may mediate the components of Ds/Ft signalling controlling leg size. The enlarged phenotype of Gb'wts RNAi nymphs was suppressed by RNAi against Gb'd in Gryllus, indicating that Gb'd is downstream of Gb'wts. This result differs from Drosophila data. A genetic analysis is necessary to confirm the difference, because the epistatic allele is not null in RNAi experiments. As the phenotype of rdRNAi treatment against Gb'ds was weaker than that against Gb'ft, as reported in the corresponding Drosophila mutants, Gb'Ft may interact with factors that are as yet unidentified. Although the effect of rdRNAi against Gb'fj on leg size was very mild, the possible involvement of Gb'fj in allometric growth cannot be excluded. The short phenotypes were observed in the Gb'sd RNAi nymphal legs, so it remains a possibility that Gb'sd is involved in allometric leg growth. However, the apparent contribution of the Hpo-Sav-Mats complex is as yet uncertain (Bando, 2009).

Regenerated legs of Gb'ex and Gb'Mer RNAi adults become longer than normal control legs, and Gb'ex and Gb'Mer regulate cell proliferation induced by the presence of positional disparity. These results suggest that Gb'ex and Gb'Mer are also involved in allometric growth of the leg segment. In Drosophila, Ex and Mer negatively regulate cell growth and proliferation through the Hpo/Wts pathway. In mammalian cells, Nf2 (merlin) is known to be a crucial regulator of contact-dependent inhibition of proliferation (Curto, 2008). Thus, it is concluded that activities of Ex and Mer may regulate contact-dependent inhibition of proliferation via the Wts signalling pathway to restore the proper leg segment size during regeneration (Bando, 2009).

A widely accepted model for leg regeneration is the intercalation model, based on positional information. This model is based on the intercalation of new structures so as to re-establish continuity of positional values during regeneration. However, on the basis of this model, it is difficult to explain the changes in leg size that were observed in the present study. Thus, the model need to be extended to include the control of growth and tissue size during regeneration. Several models have been proposed to explain how organ size is regulated. Lawrence (2008) proposed a model, which is referred to here as the Ds/Ft steepness model, to explain the mechanisms underlying the determination of organ size and PCP, including the Warts/Hippo pathway as the mechanism for controlling growth. In this model, it was hypothesized that: (1) the morphogens responsible for the overall pattern of an organ establish and orient the Ds/Ft system, which then forms a linear Ds/Ft gradient. The nature of the Ds/Ft gradient is unknown, although the number of Ds/Ft trans heterodimers is the key variable; (2) the steepness of the Ds/Ft gradient regulates Hpo target expression and cell proliferation, and its direction provides information used to establish the correct cellular polarity; (3) growth would be expected to cease when the slope of the gradient declines below a certain threshold value; and (4) the maximum and minimum limits of the system are conserved, while recently divided cells take up intermediate scalar values from their neighbours (Bando, 2009).

Using this model, a modified Ds/Ft steepness model is proposed for leg regeneration acting as follows. The results indicate that nymphal leg regeneration depends on two major processes: (1) proliferation and differentiation of blastemal cells and (2) growth of the pre-existing stump. In each of these processes, new positional identities are specified in relation to new segment boundaries. According to the Ds/Ft steepness model, in normal regeneration, a very steep gradient should be formed in the regenerating blastema. The regenerate may grow so as to restore the normal pre-existing steepness. Reassignment of positional identities after amputation will correlate with a similar re-setting of the minimum Ds/Ft scalar value, and the results are consistent with the steepness hypothesis (Bando, 2009).

Growth of the pre-existing stump is a normal component of leg growth, in which the pre-existing stump cells proliferate according to some allometric signals, which may be related to the maximum scalar value and a slope of the gradient, keeping their original positional information. This was observed in the truncated leg of Gb'arm rdRNAi adults (Nakamura, 2007; Bando, 2009).

In the absence of the proliferation and differentiation of blastemal cells, as observed in the Gb'ft rdRNAi leg, the minimum scalar value, which is the most distal positional value, would be established at the site of amputation, and the Ds/Ft gradient would be expected, in turn, to shift down with the same slope as the pre-existing one. The Ds/Ft steepness model provides an explanation for the observation that the final leg size depends on the amputated position, if it is assumed that the gradient shifts down with the same slope as that where cells at an amputated position have the minimum scalar value. Thus, the observed re-specification of regeneration legs induced in the legs treated with rdRNAi against Gb'ft or Gb'ds is as would be predicted by the Ds/Ft steepness model. Thus, it is likely that the Ds/Ft gradient functions to link positional and allometric information to the regulation of leg segment growth. Furthermore, if it is assumed that the activity of Ex/Mer is related to a threshold value of the slope of the gradient that determines when growth ceases, all rdRNAi phenotypes in the present study can be interpreted consistently with the Ds/Ft steepness model for regeneration (Bando, 2009).


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Merlin: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation

date revised: 26 December 2014

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