A candidate for a Salvador-interacting protein is encoded by the warts (wts; also known as LATS) gene that encodes a serine-threonine kinase. Clones of wts tissue generate outgrowths that resemble tumors. Nine alleles of wts were identified in the screen, and the phenotype of sav3 is similar to that elicited by hypomorphic mutations in wts. Null alleles of wts display a more severe phenotype. Like sav, wts clones in the pupal retina have additional interommatidial cells. Larval imaginal discs containing large wts clones are enlarged and convoluted. Larval eye discs that contain eyFLP-induced wts clones are composed mostly of mutant tissue with small regions of wild-type tissue. Many additional BrdU-incorporating nuclei are observed in mutant clones posterior to the SMW. As observed with sav, the stripe of cyclin E RNA expression is also broadened in these discs. Moreover, the normal cell death that occurs in the pupal retina is almost completely abolished in wts mutant clones. Thus, as for sav, wts mutations generate additional interommatidial cells resulting from both increased cell proliferation posterior to the SMW as well as reduced apoptosis in the pupal retina. In addition, Drice activation induced by GMR-hid is markedly diminished in wts clones (Tapon, 2002).
Overexpression of sav alone using the GMR promoter has no effect, and overexpression of wts generates subtle irregularities in ommatidial architecture. However, combined overexpression of sav and wts results in a smaller eye where the ommatidial pattern is highly irregular. This effect appears to reflect a synergistic increase in cell death in the eye discs of flies that express both transgenes as well as a minor effect on reducing cell proliferation assoiated with the SMW (Tapon, 2002).
Thus, Sav and Wts may function in the same pathway and may bind to each other. Indeed, the Sav protein has a Group I WW domain that is predicted to interact with the PPXY (PY) motif, five of which are found in the Wts protein. To test whether Drosophila Sav and Wts proteins could physically interact, a GST pull-down assay was employed. The region containing the two potential WW domains of Sav was fused to GST and incubated with cell lysates that expressed Myc-tagged Wts protein. Using this assay, Wts was found to interact specifically with the region of Sav that contained the WW domain. Furthermore, a 15 amino acid peptide, designed to mimic one of the PY motifs of Wts, was found to inhibit the interaction between the WW domain region of Sav and Wts. An identical peptide where the tyrosine residue that is required for interaction with type I WW domains had been replaced by an alanine did not prevent this interaction. Thus, at least under the conditions of this experiment, Sav and Wts interact in a WW domain- and PY motif-dependent fashion, suggesting that an analogous interaction could occur in vivo (Tapon, 2002).
Discs containing clones of the wts null allele, wtslatsX1, are much larger than discs containing sav3 clones. If all sav functions were wts dependent, the double mutant phenotype should not be more severe than the wts phenotype. When mutant clones were generated with eyFLP, average disc sizes were 39,669 pixels for sav3, wtslatsX1 double mutant discs and 31,360 pixels for wtslatsX1 discs. Thus, the double mutant discs were significantly larger than the wtslatsX1 discs. Thus, while sav and wts appear to function together in certain ways, they are also likely to have functions that are independent of each other (Tapon, 2002).
Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously. The salvador (sav) gene, which encodes a scaffold protein, restricts cell number by coordinating cell-cycle exit and apoptosis during Drosophila development. Hippo (Hpo), the Drosophila ortholog of the mammalian MST1 and MST2 serine/threonine kinases, is a partner of Sav. Hippo was described in five publications that appeared simutaneously: Pantalacci (2003) identified Hippo in a yeast two-hybrid screen in a search for Salvador interacting proteins, Udan (2003) identifed and positionally cloned hippo in a mutagenesis screen for genes that regulate tissue growth, and Harvey (2003), Jia (2003) and Wu (2003) identified hippo in screens for genes that restrict growth and cell number. Loss of hpo function leads to sav-like phenotypes, whereas gain of hpo function results in the opposite phenotype. Whereas Sav and Hpo normally restrict cellular quantities of the Drosophila inhibitor of apoptosis protein DIAP1 (Thread), overexpression of Hpo destabilizes DIAP1 in cell culture. DIAP1 is phosphorylated in a Hpo-dependent manner in S2 cells and Hpo can phosphorylate DIAP1 in vitro. Thus, Hpo may promote apoptosis by reducing cellular amounts of DIAP1. In addition, Sav is an unstable protein that is stabilized by Hpo. It is proposed that Hpo and Sav function together to restrict tissue growth in vivo (Pantalacci, 2003; Harvey, 2003; Jia, 2003; Udan, 2003 and Wu, 2003).
The dMST mutant phenotypes closely resemble those caused by sav or wts mutations. It has been shown that Sav and Wts physically and genetically interact, suggesting that they may act in common pathways. To determine if dMST could act in the same pathways, coimmunoprecipitation assays with Sav and Wts were performed. S2 cells were transfected with DNA constructs expressing HA-tagged Sav and Flag-tagged full-length dMST (dMSTf), its N-terminal fragment containing the kinase domain (dMSTn), or its C-terminal fragment containing the regulatory domains (dMSTc). Both dMSTf and dMSTc, but not dMSTn, coimmunoprecipitate with Sav, suggesting that dMST binds Sav through its C-terminal regulatory region. The dimerization domain at the C terminus of dMST appears to be essential for interaction as deletion of this domain from dMSTc abolishes its ability to bind Sav (Jia, 2003).
To define the domain in Sav that binds dMST, a series of truncated forms of Sav were generated. Both C-terminal fragments, SavC1 and SavC2, bind dMST. In contrast, all the C-terminally truncated fragments, including SavDeltaC1, SavDeltaC2, and SavDeltaC3, fail to bind dMST, suggesting that dMST binds the C-terminal region of Sav and the coiled-coil domain of Sav is essential (Jia, 2003).
dMST also binds Wts. S2 cells were transfected with DNA constructs expressing Myc-tagged Wts and Flag-tagged dMSTf, dMSTn, or dMSTc. Wts binds dMSTf and dMSTn, but not dMSTc, suggesting that Wts interacts with the N-terminal region of dMST. The interaction between Wts and dMST is not affected by Sav, since coexpression of Sav does not increase the amount of dMSTf coimmunoprecipitated with Wts. However, it remains possible that Sav might regulate dMST/Wts interaction in vivo at physiological concentration. Taken together, these results suggest that dMST, Wts, and Sav form a complex in which Wts and Sav bind the kinase and regulatory domains of dMST, respectively (Jia, 2003).
Sav quantities increase markedly in the presence of Hpo. Furthermore, Sav mobility on acrylamide gels shifts toward higher molecular weights. Sav immunoprecipitated from lysates containing Hpo was treated with phosphatases and it was found that this band shift disappeared, confirming that Sav becomes phosphorylated in the presence of Hpo. Sav is also stabilized by treatment with the proteasome inhibitor LLnL, suggesting that Sav is normally targeted for degradation by the proteasome. Unexpectedly, kinase-dead Hpo also induces stabilization and a mobility shift of Sav, whereas Hpo lacking the Sav-binding domain has little effect (Pantalacci, 2003).
Correct organ size is determined by the balance between cell death and proliferation. Perturbation of this delicate balance leads to cancer formation. Hippo (Hpo), the Drosophila ortholog of MST1 and MST2 (Mammalian Sterile 20-like 1 and 2) is a key regulator of a signaling pathway that controls both cell death and proliferation. This pathway is so far composed of two Band 4.1 proteins, Expanded (Ex) and Merlin (Mer), two serine/threonine kinases, Hpo and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). It has been proposed that Ex and Mer act upstream of Hpo, which in turn phosphorylates and activates Wts. Wts phosphorylates Yki and thus inhibits its activity and reduces expression of Yki target genes such as the caspase inhibitor DIAP1 and the micro RNA bantam. However, the mechanisms leading to Hpo activation are still poorly understood. In mammalian cells, members of the Ras association family (RASSF) of tumor suppressors have been shown to bind to MST1 and modulate its activity. In this study it is shown that the Drosophila RASSF ortholog (dRASSF) restricts Hpo activity by competing with Sav for binding to Hpo. In addition, dRASSF also possesses a tumor-suppressor function (Polesello, 2006).
The mammalian RASSF family comprises six different loci encoding a variety of splice variants. Most transcripts encode proteins that contain a Ras association domain (RA), an N-terminal C1-type zinc finger, and a C-terminal SARAH (Sav RASSF Hippo) domain. RASSF family members, most notably RASSF1A, are frequently silenced in a variety of solid tumors. Thus, it has been proposed that RASSF genes act as tumor suppressors (Polesello, 2006).
The biological function of these genes is not well understood. RASSF1A and Nore1A have both been shown to interact with MST1 via its SARAH domain. Overexpression of RASSF1A or Nore1A inhibits MST1 activation, but coexpression of these RASSF proteins with Ras enhanced MST1 activity. RASSF1A knockout mice have mildly increased tumor susceptibility, confirming that RASSF genes can act as tumor suppressors. The weakness of the mouse phenotype, which is at odds with the frequency of RASSF1A inactivation in human tumors, can be ascribed to redundancy with other family members (Polesello, 2006).
By contrast, Drosophila melanogaster has a single RASSF family member, which is encoded by the CG4656 gene and which will be referred to as dRASSF. Like its vertebrate counterparts, dRASSF encodes a protein bearing an RA and SARAH domain at its C terminus. It also possesses a LIM domain that shares some similarities with C1 zinc fingers at its N terminus (Polesello, 2006).
Mutant alleles of dRASSF were generated by imprecise excision of two nearby transposons, GE23517 and EY2800. Multiple alleles, which delete up to the fourth intron, including the initiating ATG, were obtained. Some transcript was still detected in dRASSFX16, dRASSFX36, but a strong reduction was found in dRASSF44.2, which lacks the transcription start. However, antibodies raised against the C terminus (amino acids 792–806) and a nonconserved region (amino acids 294–308) of dRASSF showed that full-length dRASSF is absent in lysates from all mutant lines, suggesting the dRASSF mutants are indeed loss-of-function mutations for the locus. All of these alleles were viable and behaved identically in subsequent assays. In addition, dRASSF staining was severely reduced in FLP/FRT-generated dRASSF mutant clones in the eye-imaginal disc, the larval precursor to the adult eye (Polesello, 2006).
Although the dRASSF mutant flies are viable, they present a clear growth defect in comparison to wild-type animals when reared in carefully controlled conditions. dRASSF mutant flies were 15% lighter than their wild-type counterparts, a phenotype which was significantly rescued by introduction of a single copy of a dRASSF rescue construct, although wild-type levels of dRASSF were not fully restored. dRASSF mutant flies were fully fertile and normally proportioned but sensitive to γ-irradiation. Wing surface area was reduced by 8% in dRASSF mutant flies, whereas wing hair density was unaffected. This suggests that the growth defect of dRASSF mutant flies is due to a reduction in cell number and not a defect in cell size (Polesello, 2006).
In mammals, members of the RASSF family are known to interact with MST1 and thus to modulate its pro-apoptotic activity. Therefore whether dRASSF can interact with Hpo was tested. Coimmunoprecipitation (Co-IP) experiments were performed in Drosophila Kc cells with dRASSF antibodies to immunoprecipitate endogenous protein. As expected, dRASSF robustly coimmunoprecipitated with Hpo. The association between Hpo and Sav is mediated by these proteins' shared SARAH domains. Likewise, Hpo's SARAH domain is required for its association with dRASSF, as shown by the fact that a truncated form of Hpo (HpoΔC) lacking this domain fails to bring down dRASSF. Thus, the Hpo SARAH domain can associate with both Sav and dRASSF (Polesello, 2006).
Sav is stabilized by the presence of Hpo. Therefore whether dRASSF levels are modulated by Hpo was tested. dRASSF immunostaining was reduced in clones mutant for a hpo allele that lacks the SARAH domain. In addition, RNAi-mediated depletion of Hpo from Drosophila Kc cells resulted in a reduction of endogenous dRASSF expression, whereas dRASSF transcripts were unaffected. By contrast, dRASSF levels were unaffected in clones mutant for other Hpo-pathway members, such as ex, sav, and wts. These results suggest that direct binding to Hpo through its SARAH domain, rather than signaling through the Hpo pathway, is necessary for dRASSF stability. This is analogous to the situation for Sav, which is also stabilized by a kinase-dead form of Hpo (Polesello, 2006).
Because Hpo, Sav, and dRASSF all contain a SARAH domain, it was speculated that dRASSF might also bind Sav. To test this, whether dRASSF interacts with Sav was investigated by co-IP but no such an interaction was detected. Because the possibility of a ternary complex had been raised by another study, whether the three proteins could be found in the same complex was tested. Hpo, Sav, and dRASSF were co-expressed in cultured Kc cells. As expected, Hpo was able to bind Sav and dRASSF. However, Sav immunoprecipitates only contained Hpo and not dRASSF, and dRASSF immunoprecipitates contained Hpo but not Sav. Identical results were obtained with endogenous IPs by using dRASSF and Sav antibodies. These data support the notion that Sav and dRASSF are not present in the same complex but are in two different Hpo complexes (Polesello, 2006).
Sav has been shown to be a positive regulator of the Hpo pathway, whereas genetic results suggest that dRASSF might antagonize Hpo function. It was therefore of interest to determining whether complexing with Sav or dRASSF might influence Hpo activity. Immunoprecipitates were probed with an phospho-MST1 antibody that recognizes phosphorylated (active) Hpo. Interestingly, although Hpo that was coimmunoprecipitated with dRASSF showed barely detectable levels of phosphorylation, the Sav-associated fraction was highly phosphorylated. Thus, Hpo can exist as two pools, a highly active Sav-associated pool and an inactive dRASSF-associated pool. This correlates with data showing that Nore1 can repress MST1 activity in mammalian cells. This also suggests that Sav can promote Hpo activation and provides the first direct evidence of a function for the Hpo/Sav interaction (Polesello, 2006).
Next, the prediction that dRASSF depletion would promote Hpo activation was tested. Like that of Hpo's mammalian counterparts, phosphorylation of endogenous Hpo can be potently stimulated by the drug Staurosporine (STS) in Kc cells. Although RNAi depletion of dRASSF alone was not able to induce Hpo phosphorylation, dRASSF depletion markedly potentiated STS-induced Hpo activation. Thus, dRASSF restricts Hpo activation in cultured cells (Polesello, 2006).
Given their opposing effects on Hpo activation, the relationship between Sav and dRASSF was investigated. Depletion of dRASSF in Kc cells gives rise to an increase in Sav protein levels. Although dRASSF levels were unaltered in sav mutant clones, overexpression of Sav in the wing disc results in a robust decrease of dRASSF staining. Whether dRASSF and Sav compete to bind Hpo was tested. To address this question, because Sav and dRASSF repress each other's expression and dRASSF has reduced affinity for phosphorylated Hpo, separate Kc cell lysates expressing a kinase-dead form of Hpo (HpoKD-Flag), Sav-HA, and HA-dRASSF were mixed and IPs were performed after the proteins were allowed to bind overnight. Both Sav and dRASSF were able to interact with Hpo. In these conditions, increasing the amount of Sav was able to displace the dRASSF fraction bound to Hpo, showing that Sav and dRASSF are competing to bind Hpo. The outcome of the competition probably determines the stability of Sav and dRASSF; both proteins are downregulated when Hpo is depleted by RNAi. Thus, it is suggested that interplay between the inhibitor dRASSF and the activator Sav determines the level of Hpo activation and therefore affects body size (Polesello, 2006).
This model was tested by performing genetic-interaction experiments. A mutant allele of hpo was crossed into the dRASSF mutant background and the adult body mass was measured. The body-mass reduction of dRASSF mutant flies (15% reduction) was substantially rescued by removal of just one copy of Hpo (8% reduction). Flies overexpressing Sav showed a reduction of 10% in weight and 5% in wing area, mimicking dRASSF loss of function. This wing defect was significantly increased in a dRASSF mutant background. In addition, misexpression of dRASSF was able to robustly rescue the rough-eye phenotype elicited by coexpression of Sav and Wts. These data support the notion that dRASSF can antagonize Sav-mediated Hpo activation in vivo (Polesello, 2006).
Though the results are consistent with biochemical data on mammalian RASSF family members, they are at odds with the fact that RASSF genes are commonly silenced in tumor cells. It has been proposed that one RASSF protein, Nore1, possesses a tumor-suppressor function that is independent of MST1 and MST2. Two lines of evidence to support this notion were found. First, in vivo clones were made in the head (by using the eyeless FLP system) that were mutant for two hpo hypomorphic alleles, hpo42–48 and hpoKC203, that remove the SARAH domain in a dRASSF mutant background. Interestingly, the overgrowth phenotype elicited by these hpo alleles was strongly enhanced by loss of dRASSF. By contrast, a hpo allele (hpo42–47) bearing an inactivating deletion in the kinase domain but an intact SARAH domain was barely if at all enhanced by dRASSF loss of function. This suggests that dRASSF may possess a tumor-suppressor function, which may be uncovered when the Hpo function is compromised (Polesello, 2006).
In addition, the relationship between Ras1 and dRASSF was examined because the mammalian RASSF proteins have all been shown to bind Ras proteins. In Drosophila imaginal tissues, Ras1 mutant clones grow poorly and are eliminated by apoptosis. When double-mutant clones for Ras1 and dRASSF were made in the developing eye, a substantial rescue was observed of the growth defect observed in clones mutant for Ras1 alone. This rescue of Ras loss of function was the result of both increased proliferation quantified with phosphorylated Histone 3 staining and a reduction of apoptosis visualized with a cleaved-Caspase 3 antibody. Thus, dRASSF appears to antagonize Ras1 signaling in growth control, which is again suggestive of a “tumour-suppressing” effect distinct from its “oncogenic” role in opposing the Hpo pathway. However, it has been suggest that NORE1 may also have both Ras- and MST-independent functions. Future experiments will therefore be aimed at gaining a better understanding of the RASSFs' growth-restricting functions. The fact that the dRASSF mutations are viable might therefore reflect the facts that its ability to regulate the Hpo pathway may be redundant with other modes of regulation and that loss of dRASSF's tumor-suppressive activity is balanced by loss of its growth-promoting activity. It has been proposed that MST2 may be inactivated by binding to Raf-1. It will be interesting to determine whether this mode of regulation is redundant with RASSF (Polesello, 2006).
In summary, mutant alleles of the sole Drosophila ortholog of the RASSF family of tumor suppressors were generated. Surprisingly, dRASSF mutant flies are smaller than control flies. This growth defect can probably be ascribed in part to dRASSF's ability to antagonize Hpo signaling by competing with Sav for binding to Hpo. In addition, dRASSF also possesses a tumor-suppressor activity, which is uncovered when hpo or Ras1 function is compromised. It will be interesting to investigate whether some mammalian RASSF proteins share these properties (Polesello, 2006).
The Hippo (Hpo) signaling pathway controls tissue growth and organ size in species ranging from Drosophila to mammals and is deregulated in a wide range of human cancers. The core pathway consists of the Hpo/Warts (Wts) kinase cassette that phosphorylates and inactivates the transcriptional coactivator Yorkie (Yki). This study reports that Echinoid (Ed), an immunoglobulin domain-containing cell adhesion molecule, acts as an upstream regulator of the Hpo pathway (see Ed regulates organ size through the Hpo-Yki pathway). Loss of Ed compromises Yki phosphorylation, resulting in elevated Yki activity that increases Hpo target gene expression and drives tissue overgrowth. Ed physically interacts with and stabilizes the Hpo-binding partner Salvador (Sav) at adherens junctions. Ed/Sav interaction is promoted by cell-cell contact and requires dimerization of Ed cytoplasmic domain. Overexpression of Sav or dimerized Ed cytoplasmic domain suppressed loss-of-Ed phenotypes. It is proposed that Ed may link cell-cell contact to Hpo signaling through binding and stabilizing Sav, thus modulating the Hpo kinase activity (Yue, 2012).
The Hpo signaling pathway has emerged as a conserved regulatory pathway that controls tissue growth and organ size. Although the core pathway components (i.e., the Hpo/Sav/ Wts/Mats kinase cassette and its effector Yki/Yap), have been well defined, the upstream regulators, especially the membrane receptors that link cell-cell communication to Hpo signaling, remain poorly defined. This study provides both genetic and biochemical evidence that the transmembrane cell adhesion molecule Ed functions as a upstream regulator of the Hpo pathway. Evidence is provided that Ed physically interacts with Sav/Hpo and regulates the abundance of Sav at adherens junctions. Loss of Ed compromises the ability of Hpo/Wts kinase cassette to phosphorylate Yki, leading to elevated levels of nuclear Yki that drive tissue overgrowth. Ed/Sav association is facilitated by cell-cell contact, raising an interesting possibility that Ed may serve as a mechanism that links Hpo signaling to cell contact inhibition (Yue, 2012).
The atypical cadherin Ft functions as a receptor for the Hpo pathway; however, Ft mainly acts through Dachs to control the stability of Wts. Genetic study indicated that Ed does not act through Ft-Dachs to regulate Yki activity because inactivation of Dachs did not block Yki activation induced by loss of Ed. Furthermore, loss of Ed synergized with loss of Ds to induce the expression of Hpo-responsive genes, supporting a model in which Ed acts in parallel with Ds/Ft in the Hpo pathway. Several lines of evidence suggest that Ed regulates Hpo signaling, at least in part, through Sav. (1) Using coimmunoprecipitation, colocalization, and FRET assays, it was demonstrated that Ed physically interacts with Sav. (2) Deleting the Sav-interacting domain in Ed compromises its in vivo activity. (3) Ed regulates the abundance and subcellular localization of Sav both in vitro and in vivo. (4) Overexpression of Sav suppresses tissue overgrowth induced by loss of Ed. Sav is a binding partner and activator of Hpo. Hence, Ed could influence the Hpo kinase activity by regulating the abundance and subcellular location of the Sav/Hpo complex. How Ed regulates Sav stability is currently unknown; however, it was found that Sav is degraded in a proteasome-dependent manner. It is possible that binding of Ed to Sav leads to some modifications of Sav and prevents it from ubiquitin/proteasome-mediated degradation (Yue, 2012).
Sav binds Ed and Hpo through its N- and C-terminal regions, respectively, and thus may function as a bridge to bring Hpo to Ed. Indeed, enhanced Ed/Hpo association was observed in the presence of cotransfected Sav. It has been suggested that apical membrane recruitment of Hpo promotes phosphorylation of Wts. Thus, it is conceivable that Ed may regulate the Hpo kinase by recruiting Sav/Hpo complex to the apical membrane. It was found that Ed/Sav interaction requires membrane association and dimerization/oligomerization of Ed intracellular domain. As Sav also forms a dimer/oligomer, dimerization/oligomerization of Ed intracellular domain may enhance binding to Sav through multimeric interactions. It is also possible that membrane association and dimerization/oligomerization could lead to a modification of Ed intracellular domain, resulting in increased binding affinity toward Sav (Yue, 2012).
It has been shown that the Hpo pathway can mediate cell contact inhibition in mammalian cultured cells, although the underlying mechanism has remained poorly defined. Interestingly, this study found that cell-cell contact can facilitate the recruitment of Sav to Ed at the contact site. Cell-cell contact may facilitate homophilic interaction in trans and clustering of Ed intracellular domain or induce posttranslational modification of Ed C-tail at the contact site, leading to enhanced Sav association. It is proposed that regulation of Ed/Sav association may provide a mechanism for cell-cell contact to modulate Hpo signaling and tissue growth (Yue, 2012).
The mechanism by which Ed regulates Hpo signaling is likely to be more complex than simply regulating Sav/Hpo. For example, it was also observed that Ed interacts with Ex/Mer/Kibra as well as Yki. It has been proposed that enrichment of Hpo pathway components to the apical membrane domain may facilitate the activation of the kinase cassette and increase the accessibility of Yki to its kinase (Genevet, 2011). The finding that Ed facilitates the apical localization of Sav lends further support to this notion. Through interacting with multiple components of the Hpo pathway, Ed could function as a molecular scaffold to facilitate Hpo activation and Yki phosphorylation. Loss of Ed did not alter the apical membrane localization of Ex and Mer in wing discs even though overexpression of Ed in S2 cells facilitates membrane recruitment of Ex. The apical localization of Ex and Mer is likely to be mediated by other upstream components such as Ft and Crb in the absence of Ed. Indeed, Crb physically interacts with Ex, and both loss and gain of function of Crb caused mislocalization of a fraction of Ex to the basal region. It has been shown that Ex physically interacts with Yki, which may sequester Yki in the cytoplasm independent of Yki phosphorylation. The finding that Ed interacts with Yki through a domain distinct from those mediating its binding to the upstream Hpo pathway components raises a possibility that Ed may also directly sequester Yki in the cytoplasm in addition to regulating its subcellular localization through phosphorylation (Yue, 2012).
It is interesting to note that Ed is related to TSLC1, a tumor suppressor implicated in human non-small-cell lung cancer and other cancers including liver, pancreatic, and prostate cancers. Like Ed, TSLC1 also mediates cell-cell adhesion through homophilic interactions. TSLC1 interacts with MPP3, a human homolog of Drosophila tumor suppressor Discs large (Dlg) that has been implicated in the Hpo pathway, as well as DAL-1, a FERM-domain containing tumor suppressor related to Ex/Mer. Therefore, it would be interesting to determine whether TSLC1 inhibits tumor formation through the Hpo pathway (Yue, 2012).
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).
EGFR and Hippo signaling pathways both control growth and, when dysregulated, contribute to tumorigenesis. This study found that EGFR activates the Hippo pathway transcription factor Yorkie and demonstrates that Yorkie is required for the influence of EGFR on cell proliferation in Drosophila. EGFR regulates Yorkie through the influence of its Ras-MAPK branch on the Ajuba LIM protein Jub. Jub is epistatic to EGFR and Ras for Yorkie regulation, Jub is subject to MAPK-dependent phosphorylation, and EGFR-Ras-MAPK signaling enhances Jub binding to the Yorkie kinase Warts and the adaptor protein Salvador. An EGFR-Hippo pathway link is conserved in mammals, as activation of EGFR or RAS activates the Yorkie homolog YAP, and EGFR-RAS-MAPK signaling promotes phosphorylation of the Ajuba family protein WTIP and also enhances WTIP binding to the Warts and Salvador homologs LATS and WW45. These observations implicate the Hippo pathway in EGFR-mediated tumorigenesis and identify a molecular link between these pathways (Reddy, 2013).
The evolutionarily conserved Hippo (Hpo) signaling pathway plays a pivotal role in organ size control by balancing cell proliferation and cell death. This study reports the identification of Par-1 as a regulator of the Hpo signaling pathway using a gain-of-function EP screen in Drosophila melanogaster. Overexpression of Par-1 elevates Yorkie activity, resulting in increased Hpo target gene expression and tissue overgrowth, while loss of Par-1 diminishes Hpo target gene expression and reduces organ size. par-1 functions downstream of fat and expanded and upstream of hpo and salvador (sav). In addition, it was also found that Par-1 physically interacts with Hpo and Sav and regulates the phosphorylation of Hpo at Ser30 to restrict its activity. Par-1 also inhibits the association of Hpo and Sav, resulting in Sav dephosphorylation and destabilization. Furthermore, evidence is provided that Par-1-induced Hpo regulation is conserved in mammalian cells. Taken together, these findings identified Par-1 as a novel component of the Hpo signaling network (Huang, 2013).
The Hpo signaling pathway has emerged as a conserved pathway that controls tissue growth and balances tissue homeostasis via the regulation of the downstream Sd-Yki transcription complex. Despite the importance of this pathway in development and carcinogenesis, many unknown regulators of the Hpo pathway remain to be identified. This study identified Par-1 as one such Hpo pathway regulator via a genetic overexpression screen using Drosophila EP lines. This study demonstrated that Par-1 was essential for the restriction of Hpo signaling. It was also demonstrated that overexpression of Par-1 promotes tissue growth via the inhibition of the Hpo pathway, whereas loss of Par-1 promotes Hpo signaling to suppress growth and induce apoptosis. Using the Drosophila eye and wing imaginal discs as well as cultured cells, this study provides the first genetic and biochemical evidence for a function of Par-1 in the Hpo pathway (Huang, 2013).
Although the conserved function of Hpo has been well studied, the regulatory mechanism of its kinase activity is still largely obscure. Currently, the regulatory mechanism of Hpo kinase activity is believed to mainly be dependent on autophosphorylation by altering the phosphorylation status of the Thr195. However, whether the uncharacterized phosphorylation events of Hpo, which have been identified in several recent proteome-wide phosphorylation studies, contribute to the regulation of Hpo activity is still unknown. By studying the mechanism underlying Par-1 function in Hpo signaling, this study demonstrated that Par-1 induces Hpo phosphorylation at Ser30 and this leads to the regulation of Hpo kinase activity (Huang, 2013).
In recent proteome-wide phosphorylation studies using Drosophila embryos, it was suggested that Hpo was phosphorylated at Ser30 in vivo, indicating an important role for the Ser30 site in the regulation of Hpo activity. To determine the biological significance of Hpo phosphorylation at Ser30 induced by Par-1, whether Ser30 phosphorylation state affects Hpo phosphorylation at Thr195, which is important for Hpo activation, was tested. Par-1, but not Par-1-KD, was shown to significantly inhibit Hpo phosphorylation levels at Thr195, whereas this inhibitory effect was abolished when the Ser30 site was mutated. More importantly, phosphorylation at Thr195 was slightly elevated when Ser30 was mutated into an alanine. These findings suggested that Par-1 regulates Hpo activity via antagonizing phosphorylation at the Thr195 site by regulating Ser30 phosphorylation. It has been reported that the Hpo Thr195 site is not only auto-phosphorylated but also phosphorylated by Tao-1, a partner of Par-1 in the regulation of microtubule dynamics. Thus, it was asked whether Par-1-induced phosphorylation at Ser30 also affects Tao-1-mediated phosphorylation at Thr195. Par-1 was shown to suppress Tao-1-mediated phosphorylation at Thr195. The antagonistic effect of Par-1 and Tao-1 on Hpo phosphorylation at Thr195 motivated the examination of the interrelationship of Par-1 and Tao-1 in the Hpo pathway. It was found that Tao-1 disrupted Par-1-induced phosphorylation mobility shift of Hpo-KD, suggesting that the function of Par-1 in the Hpo pathway was modulated by upstream signaling (Huang, 2013).
Several unresolved questions remain. The interaction between Par-1 and Hpo/Sav may be tightly regulated because full-length Par-1 only weakly interacts with Hpo/Sav, unlike the interaction with the N-terminal fragment of Par-1. However, the triggering signal for Par-1 to interact with Hpo/Sav is still unknown. It has been reported that Par-1 is activated by Tao-1 and LKB1. This study established that Par-1 antagonized Tao-1 in Hpo signaling: in Drosophila, the antagonistic relationship between Par-1 and Tao-1 in microtubule regulation has been previously reported. Thus, it is unlikely that Tao-1 functions as the trigger. Whether LKB1 functions as an activator of Par-1 in Hpo signaling was investigated by expressing the LKB1 transgene in different organs. Unlike Par-1, ectopic LKB1 expression limits both wing and eye growth, indicating that LKB1 is also not the trigger (Huang, 2013).
This study has shown that Par-1 and Tao-1 exhibit opposing effects on Hpo signaling. Given that Tao-1 and Par-1 are partners that regulated microtubule dynamics via the phosphorylation of Tau, Tau may have a function in Hpo signaling. To investigate this hypothesis, genetic and biochemical studies were employed, and it was found that Tau RNAi failed to suppress the expression of Hpo pathway-responsive genes. In addition, Tau did not trigger Hpo phosphorylation and Sav dissociation in vitro, indicating that Par-1 regulates Hpo signaling independent of Tau. Interestingly, it has been previously suggested that Par-1 does not regulate Tau activity in Drosophila, indicating an evolutionary difference between Par-1 and Tau-1 function (Huang, 2013).
This study has provided evidence that Par-1 regulates Hpo signaling via the phosphorylation of Hpo or the destruction of the Hpo/Sav complex. Because Par-1 is a well-known polarity regulator and polarity components, such as Crumb and Lgl, have been shown to be involved in the Hpo signaling pathway, it is possible that Par-1 may regulate Hpo signaling via a polarity complex, or its activity might be regulated via a polarity complex. Indeed, the localization of Crumb and Patj were affected by Par-1 expression. Thus, further studies on polarity complexes and Hpo signaling will help elucidate this problem (Huang, 2013).
The specification of tissue size during development involves the coordinated action of many signalling pathways responding to organ-intrinsic signals, such as morphogen gradients, and systemic cues, such as nutrient status. The conserved Hippo (Hpo) pathway, which promotes both cell-cycle exit and apoptosis, is a major determinant of size control. The pathway core is a kinase cassette, comprising the kinases Hpo and Warts (Wts) and the scaffold proteins Salvador (Sav) and Mats, which inactivates the pro-growth transcriptional co-activator Yorkie (Yki). A split-TEV-based genome-wide RNAi screen was performed for modulators of Hpo signalling. In the TEV screen, inactive fragments of the NIa protease from the tobacco etch virus (TEV protease) regain activity only when coexpressed as fusion constructs with interacting proteins. The Drosophila salt-inducible kinases (Sik2 and Sik3) were characterized as negative regulators of Hpo signalling. Activated Sik kinases increase Yki target expression and promote tissue overgrowth through phosphorylation of Sav at Ser 413. As Sik kinases have been implicated in nutrient sensing, this suggests a link between the Hpo pathway and systemic growth control (Wehr, 2013).
This study combined the protein-protein interaction detection method split TEV and RNAi screening in Drosophila cell culture to identify Hpo pathway modulators. Split TEV was first developed in mammalian cells, and subsequently shown to be a valuable tool to monitor phosphorylation-dependent interactions of proteins. This study applied split TEV in Drosophila cell culture, both for constitutive (Hpo dimerization) and regulated (Yki/14.3.3) interactions. The success of the screening approach suggests that split TEV will prove invaluable in mapping signalling pathways by providing functional readouts that can be combined with RNAi or pharmacological approaches (Wehr, 2013).
The results identify Sik kinases as Hpo upstream regulators. In particular, activated Sik2 can induce overgrowth and activation of Yki transcriptional activity, whereas depletion of Sik2/3 in the wing leads to undergrowth. Interestingly, Sik3 can also antagonize Hpo signalling, but in an isoform-specific manner. The relative contribution of Sik2 and Sik3 to growth in various tissues needs to be addressed to appreciate the possible redundancy between these two kinases, and potentially other AMPK family kinases. A recent report shows that LKB1, which regulates all AMPK family members, may inhibit YAP activity independently of the core Hpo cassette, suggesting a complex interplay between LKB1/AMPKs and the Hpo pathway (Wehr, 2013).
The effect of Sik2/3 on Hpo signalling is mediated, at least in part, by Sav phosphorylation on Ser 413. The data suggest that Sav phosphorylation by Sik reduces its ability to efficiently scaffold the Hpo/Wts core kinase complex, thereby reducing Yki inhibitory phosphorylation. In agreement with this notion, Sav-S413A exhibited an enhanced ability to reduce the level of growth in vivo. Siks play a major role in inhibiting gluconeogenesis in the liver in response to high glucose levels through inhibitory phosphorylation of the transcriptional co-activator CRTC2 (CREB-regulated transcription coactivator 2)/TORC2, and activatory phosphorylation of the histone deacetylase HDAC4, a function that seems to be conserved in Drosophila (Wehr, 2013).
The Drosophila Siks have mostly been studied in the context of energy balance in the brain and fat body (the Drosophila equivalent of the liver and adipose tissue), where they prevent the mobilization of fat and glycogen stores by antagonizing CRTC and Foxo-activated transcription. The Siks are under hormonal control by insulin receptor (InR) signalling through the downstream kinase Akt, which phosphorylates and activates Sik2/3, and the glucagon homologue adipokinetic hormone (AKH), which signals through a G-protein-coupled receptor and PKA, which phosphorylates and inhibits Siks. Under fasting conditions, low insulin and high AKH activity combine to shut down Sik3, thereby promoting gluconeogenesis and inducing mobilization of fat-body lipid stores to restore circulating glucose levels. The current study suggests a role for Sik2/3 in growth control during development. Analogously to InR signalling in Drosophila, which promotes both nutrient storage in the fat body and developmental growth of peripheral tissues, the Sik kinases might couple Hpo pathway activity to nutrient or energy availability, ensuring that Yki is able to drive tissue growth only under favourable conditions. Recent work has shown that Drosophila InR signalling can promote cell proliferation through Yki. Furthermore, mammalian liver cells stimulated with glucagon or the PKA activator forskolin exhibit reduced levels of phosphorylated YAP and increased YAP activity, an effect that may conceivably be mediated by the SIKs (Wehr, 2013).
Recent work has established the SIK kinases as candidate oncogenes in ovarian and lung cancer. This study has shown that SIK2 promotes YAP-dependent transcription in human cells. However, because Sav-S413 is not conserved, the molecular mechanism by which SIK2 regulates the mammalian Hpo pathway may differ and should be investigated further. Interestingly, YAP, the Yki orthologue, has also been reported to function as an ovarian cancer oncogene. SIK2 inhibitors may therefore prove attractive candidates to boost Hpo pathway activity in ovarian tumour cells, although this strategy may be less effective in tumours harbouring YAP amplifications (Wehr, 2013).
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).
Hippo signalling controls organ growth and cell fate by regulating the activity of the kinase Warts. Multiple Hippo pathway components localize to apical junctions in epithelial cells, but the spatial and functional relationships among components have not been clarified, nor is it known where Warts activation occurs. This study reports that Hippo pathway components in Drosophila wing imaginal discs are organized into distinct junctional complexes, including separate distributions for Salvador, Expanded, Warts and Hippo. These complexes are reorganized on Hippo pathway activation, when Warts shifts from associating with its inhibitor Ajuba LIM protein (Jub) to its activator Expanded, and Hippo concentrates at Salvador sites. This study identify mechanisms promoting Warts relocalization, and using a phospho-specific antisera and genetic manipulations, where Warts activation occurs was identified: at apical junctions where Expanded, Salvador, Hippo and Warts overlap. These observations define spatial relationships among Hippo signalling components and establish the functional importance of their localization to Warts activation (Sun, 2015).
Wts is a key control point within the Hippo pathway, where multiple upstream regulatory processes converge. A fundamental gap in understanding of Hippo signal transduction has been the cellular location of Wts activation. This study established that Wts activation in wing disc epithelial cells occurs at sub-apical junctions where Hpo, Sav, Ex and Wts overlap. Co-recruitment of Hpo and Wts kinases to a common scaffold is implicated as a central feature of Hippo pathway activation, and this helps to explain why genes required for apical junctions and apical-basal polarity promote Hippo signalling and can act as tumour suppressors (Sun, 2015).
These studies indicate that a key step in Wts activation in disc epithelia is its relocalization from Jub to Ex. No special mechanism is needed to transport Wts from Jub to Ex, as Wts localization could simply be governed by equilibrium binding with a limited cytoplasmic pool. That is, if Wts normally binds relatively strongly to Jub, and relatively weakly to Ex, it could, depending on its concentration, accumulate at Jub sites but not at Ex sites. Expression of activated Yki induced a robust relocalization of Wts from Jub to Ex, and these studies identify three factors that contribute to the visible accumulation of Wts at Ex sites under these conditions. First, Yki activation appears to increase Hpo activity. It was also found that hpo RNAi suppresses the relocalization of Wts from Jub to Ex, and that increased Hpo activity promotes Ex-Wts binding, as assayed by co-immunoprecipitation experiments. These observations are consistent with the hypothesis that Wts shifts from Jub sites towards Ex sites due to an increased Ex-Wts binding affinity induced by Hpo. Second, Yki activation increases levels of Ex, which under equilibrium binding would also increase the recruitment of Wts to Ex sites. The relocalization of Wts back to adherens junctions in the absence of Ex indicates that the shift in Wts localization is Ex dependent, and implies that Jub and Ex can compete for association with Wts. A third factor that contributes to detection of Wts-Ex co-localization is the increase in Wts protein levels induced by activated Yki, which could lead to Wts concentrations high enough to bind even lower-affinity Ex sites, and indeed it was observed that simply overexpressing Wts was sufficient to induce Wts-Ex overlap, without removing Wts from adherens junctions where it co-localizes with Jub. It is suggested that an additional consequence of increased Wts levels that enables detection of Wts and pWts overlapping Ex could be a saturation of pWts removal. While at present this remains speculative, all signal transduction pathways require mechanisms to turn off after they have been activated, so there should exist mechanisms that either degrade or dephosphorylate pWts. Relatively low levels of pWts due to rapid turnover could also help explain why pWts was undetectable in wild-type wing discs (Sun, 2015).
The discovery of Ex-Wts binding, together with earlier studies that identified Ex-Hpo binding, implicate Ex as a scaffold that could promote Wts activation by co-localizing it with Hpo, and thus define a role for Ex distinct from previous suggestions that it functions as an activator of Hpo. Similarly, recent studies in cultured cell models showed that activated forms of Mer could bind Wts, and suggested a model in which Mer promotes Wts activation by recruiting it to membranes where it could be activated by Hpo. This suggests that in tissues where Mer, rather than Ex, plays key roles in Wts activation, such as glia, Mer, which can also associate with Hpo, through Sav, could play an analogous role in assembling a Wts activation complex. It is thus noteworthy that the best characterized upstream branches of Hippo signalling characterized in Drosophila (Fat, Ex and Mer) can all now be said to act principally at the levels of Wts regulation rather than Hpo regulation. Moreover, it is noted that Kibra, which has been suggested to act at a similar point in the Hippo pathway as Mer and Ex, has also been reported to be able to physically interact with both Hpo and Wts, and thus might also act principally as a scaffold that links them together rather than as a promoter of Hpo activation (Sun, 2015).
Indeed, external signals that impinge directly on Hpo activity have not yet been identified. The current discovery that Hpo localization to Sav is greatly increased by Yki activation reveals that regulators of Hpo localization exist, and implies that they are subject to negative feedback regulation downstream of Yki. As Hpo kinase activity can be promoted by Hpo dimerization, it is proposed that the increased recruitment of Hpo to Sav could elevate Hpo activity by increasing its local concentration, and thereby its dimerization. Relocalization of Hpo might also affect its interactions with kinases that can modulate Hpo activity. Recruitment of Hpo to Sav also concentrates Hpo near Ex, where it would more efficiently phosphorylate Ex-bound Wts. However, since most junctional Wts in disc epithelia is normally complexed with Jub rather than Ex, a mechanism-based solely on Hpo recruitment to apical junctions would not be expected to induce robust Wts activation. Importantly, then, these studies revealed that Hpo can increase Ex-Wts binding, possibly by phosphorylating Ex. Increased Ex-Wts binding would help recruit Wts to Ex, where it could then be phosphorylated by Hpo. Thus, it is now possible to suggest a sequential model for Hippo pathway activation in which Hpo is first recruited to membranes and activated, activated Hpo then phosphorylates Ex to recruit Wts and finally Hpo phosphorylates and activates Wts complexed with Ex. While further studies will be required to validate this model, it provides a framework that could guide future investigations, and these current studies clearly emphasize the importance of determining the in vivo localization of endogenous pathway components (Sun, 2015).
In the eye disc, sav is expressed in a stripe in the MF, and expression decreases in the region of the second mitotic wave (SMW). Expression increases once again posterior to the SMW. Thus, to a first approximation, sav expression coincides with regions of temporary or permanent cell cycle arrest and supports the notion that sav functions in promoting exit from the cell cycle (Tapon, 2002).
In sav1 clones in the adult retina, almost all the ommatidia contain the normal complement of eight photoreceptor cells. However, there is increased spacing between adjacent ommatidia. In contrast to wild-type retinas from late pupae that contain a single layer of interommatidial cells, mutant clones contain many additional interommatidial cells. Generation of sav1 mutant clones in a white+ background indicates that most of these additional interommatidial cells contain pigment. Thus, these cells can undergo terminal differentiation. The more disorganized retinas of the sav3 allele display all of these phenotypic abnormalities. In addition, almost half of the ommatidia in sav3 clones lack one or more photoreceptor cells (Tapon, 2002).
In wild-type imaginal discs, S phases, as visualized by BrdU incorporation, are observed anterior to the morphogenetic furrow (MF) and as a single stripe of incorporation posterior to the furrow referred to as the second mitotic wave (SMW). In sav clones, many BrdU-incorporating nuclei are observed posterior to the SMW. Clones spanning the MF have some BrdU-incorporating nuclei in the anterior half of the MF, a region that is normally composed of cells arrested in G1. Using the anti-phosphohistone H3 antibody, additional cells in mitosis are also visualized in sav mutant clones posterior to the MF, suggesting that at least some of these cells are completing additional cell cycles. BrdU incorporation persists in mutant clones during the first 12 hr after puparium formation (APF) but has ceased by 24 hr APF. Thus, sav mutant cells continue to proliferate for 12–24 hr after wild-type cells stop dividing but are eventually able to exit from the cell cycle and undergo terminal differentiation (Tapon, 2002).
In cycling cells in the anterior portion of the eye imaginal disc, the distribution of mutant cells in the cell cycle, as assessed by flow cytometry, is extremely similar to that of wild-type cells. The mutant cells are very slightly smaller than their wild-type counterparts. Posterior to the MF, mutant populations have an increased proportion of cells in S and G2, indicating that mutant cells continue to cycle in this portion of the disc. Mutant cells are of normal size. The population doubling times of clones of mutant cells and wild-type cells generated in the wing imaginal disc during the proliferative phase of development did not differ significantly. Thus, when they are proliferating, mutant cells behave like wild-type cells. However, exit from the cell cycle is delayed in sav cells (Tapon, 2002).
Elevated levels of Cyclin E protein are found in the basal nuclei of sav clones posterior to the MF. These are the nuclei of the undifferentiated cells that continue to proliferate in sav clones. Such discs were examined for levels of cyclin E RNA. When sav clones are generated using eyFLP, a large proportion of cells in third instar discs are mutant, and these discs contain large patches of mutant tissue. In wild-type discs, cyclin E RNA is expressed in a narrow stripe immediately posterior to the morphogenetic furrow. In discs containing sav clones, the stripe of expression is broader and more intense, indicating that cyclin E RNA levels are elevated in these discs. Thus, the increased level of Cyclin E protein is likely to result, at least in part, from an increase in cyclin E RNA levels (Tapon, 2002).
In wild-type eyes, excessive interommatidial cells are eliminated by a wave of apoptosis that is evident in 38 hr pupal retinas. Even in sav mutant clones, cell proliferation, as assessed by BrdU incorporation, has ceased within 24 hr APF. When mosaic retinas were examined 38 hr APF, cell death is mostly confined to the wild-type portions of the retina. Thus, the apoptotic cell deaths that are part of normal retinal development appear to require sav function (Tapon, 2002).
Apoptosis in the pupal retina requires hid function, since hid mutants display additional interommatidial cells. Hid is thought to induce caspase activation by binding to the DIAP1/Thread protein and preventing it from inhibiting caspase function. Overexpression of hid using the eye-specific GMR promoter generates a small eye. The induction of cell death by hid is severely impaired in sav mutant clones. As a consequence, eyes derived from GMR-hid-expressing discs that contain sav mutant clones are larger than those derived from wild-type discs that express GMR-hid. Since sav function is required for hid-induced cell death, sav is likely to function either downstream of hid or in a parallel pathway (Tapon, 2002).
Several studies have shown that Hid and Rpr activate caspases by another mechanism in which they induce the autoubiquitination of DIAP1 and target it for degradation by the proteasome. DIAP1 levels are markedly elevated in sav clones in the larval eye disc and remain elevated in the interommatidial cells in mutant clones in the pupal eye disc. Thus, increased levels of DIAP1 in sav cells may be able to overcome the effect of many proapoptotic signals (Tapon, 2002).
To examine DIAP1 RNA levels, in situ hybridization was used to examine 20 wild-type discs and 20 mutant discs. The presence of sav (GFP-) clones in the mutant discs was confirmed by examining the discs by fluorescence microscopy prior to hybridization. There is a modest level of DIAP1 RNA expression posterior to the furrow in both populations of discs and no evidence of increased DIAP1 RNA in the discs containing sav clones. Thus, at least at this level of detection, the increased DIAP1 expression in sav cells does not appear to result from increased transcription (Tapon, 2002).
In wild-type eye discs, DIAP1 protein is expressed at higher levels posterior to the morphogenetic furrow. DIAP1 protein levels are downregulated by GMR-rpr or, to a lesser extent, by GMR-hid expression. In sav mutant clones expressing GMR-rpr, DIAP1 protein levels remain elevated. Similar results are observed with GMR-hid. Thus, neither GMR-rpr nor GMR-hid appears capable of downregulating the elevated levels of DIAP1 sufficiently in sav clones to activate caspases (Tapon, 2002).
Expression of hid or reaper (rpr) in the eye imaginal disc results in activation of the effector caspase Drice. An antibody that recognizes the cleaved (activated) form of Drice was used to stain eye discs expressing GMR-hid or GMR-rpr. In wild-type cells, Drice is activated by GMR-hid or GMR-rpr. However, in clones of sav tissue, Drice activation by either GMR-hid or GMR-rpr is almost completely blocked. These experiments indicate that sav blocks activation of Drice by both rpr and hid (Tapon, 2002).
A mutant form of Hid (Hid-Ala5) is resistant to inactivation by MAP kinase phosphorylation. GMR-hid-Ala5 is a more potent inducer of cell death than is GMR-hid, as assessed by the extent of Drice activation in the eye disc. Cell death induced by GMR-hid-Ala5 is only partially blocked in sav clones, indicating that the increased potency of Hid-Ala-5 may be able to overcome increased DIAP1 levels (Tapon, 2002).
The mutations in sav1, sav2, and sav4 result in stop codons in positions 289, 231, and 160, respectively, that would truncate the protein N-terminal to the WW domains. As expected, the more N-terminally located sav4 mutation has a more severe phenotype than sav1 or sav2. Surprisingly, the sav3 mutation, which elicits the most severe phenotype, maps 3' to those found in sav1 and sav2. The sav3 mutation causes a frameshift and generates a protein consisting of 406 sav-encoded amino acids and a C-terminal portion of 84 amino acids derived from the use of an alternate open reading frame that has no sequence similarity to any protein in the database. It is possible that sav1, sav2, and sav4 proteins may have some residual activity despite the absence of the WW domains and that sav3 is a null allele. The sav3 allele may have a more severe phenotype because the novel C-terminal sequences may further impair its stability or function. Alternatively, the novel C terminus of the sav3 protein may confer some neomorphic properties. Any such properties, if present, are not apparent in the presence of the wild-type protein, since sav3/+ flies display no overt phenotypic abnormalities. In different transheterozygous combinations, sav3 is similar in strength to a deletion. In four independent experiments, sav1/sav3 animals and sav1/Df(3R)EB6 animals have hatching rates of 85.5% (SD 2.5%) and 83.3% (SD 3.2%), respectively, and 90%–95% of the animals of each genotype subsequently failed to grow and died as first instar larvae. Thus, at least by this criterion, sav3 behaves like a null mutation. Importantly, the abnormalities in cell proliferation and apoptosis were analyzed using at least two different sav alleles and only quantitative differences were observed between sav3 and the weaker alleles (Tapon, 2002).
During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. Mutations have been identified in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. This phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei, identified by Nolan (2002) and termed salvador in that study, encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved (Kango-Singh, 2002).
To identify novel components of growth control pathways, a genetic screen was performed in adult Drosophila to isolate mutants in which tissue size but not tissue patterning is affected. Because genes involved in growth control may have ubiquitous functions, it was anticipated that animals homozygous for mutations in these genes might die during embryogenesis. Therefore randomly mutagenized chromosome arms that were made homozygous only in the head using an eyeless enhancer driven Flipase transgene (eyFLP) were screened. Mutations in several genes were isolated that resulted in enlarged heads but did not affect patterning. These include mutations in the Drosophila homologs of PTEN and TSC1/2 tumor suppressor genes, which act in cell growth control pathways that affect cell number as well as cell size, mutations in warts/lats, a previously described tumor suppressor gene encoding a Ser/Thr kinase that affect cell number but not cell size and mutations in a previously undescribed gene. This gene was named 'shar-pei' because of its folded cuticle phenotype in the head, which resembles the folded skin of Shar-pei dogs (Kango-Singh, 2002).
Mutations in shrp were isolated on chromosome arm 3R using chemical mutagenesis. Complementation tests showed that six mutations (shrp1-6) that caused a head overgrowth phenotype fail to complement each other. All mutations showed a very similar phenotype and caused early larval lethality. Given the nature of the molecular lesions it is likely that they are either null alleles or very severe loss-of-function alleles. All experiments involving cell clones were performed with at least three independent alleles (Kango-Singh, 2002).
The heads of flies in which over 90% of cells are homozygous mutant for shrp1 are proportionally larger than other structures but have a normal overall pattern, including bristles, ocelli and ommatidia. All mutant fly heads have folded head cuticle and eye tissue and over 15% of flies are severely affected. Smaller clones generated by heat-shock induced Flipase expression do not exhibit this folding phenotype. Folding may therefore be a secondary consequence of limited space within the pupal case, which does not allow overgrown tissue to fully expand. In addition to producing structures that are too big, shrp mutant cells appear to out-compete wild-type cells. The phenotype suggests that shrp mutant cells proliferate more rapidly than wild-type cells (Kango-Singh, 2002).
To test whether shrp affects cell proliferation in tissues other than the head, random clones were generated by heat-shock induced Flipase expression. Such mutant clones resulted in overgrowths on thorax, wings, halteres and legs. As observed for the eye and head, these structures differentiated the correct tissue-specific cell types. It is concluded that shrp is generally required to restrict the size of imaginal disc-derived adult structures, whereas tissue-specific cell-type specification and differentiation remain unaffected in shrp mutant cells (Kango-Singh, 2002).
To define the developmental basis for the enlarged tissue phenotypes, a focus was placed on patterning and cell proliferation in the developing eye because the eye has a precise pattern of cell types and highly regulated cell proliferation. The pattern of differentiated photoreceptor cells was analyzed in adult shrp mutant clones in 1 µm sections. Eight photoreceptors per ommatidium with a normal trapezoidal arrangement are observed, indicating that this aspect of pattern formation is not affected. However, spacing between individual photoreceptor clusters is significantly increased in shrp5 clones when compared with wild-type areas. To test whether the increased space is due to an excess of interommatidial cells, wild-type and mutant midpupal retinas were stained with an antibody against Discs-large (Dlg), a protein that localizes to apical junctions and hence reveals cell outlines. It was found that shrp4 mutant clones exhibit a dramatic increase in the numbers of interommatidial cells when compared with wild type. These extra interommatidial cells differentiate into pigment cells that produce normal pigmentation when clones are induced in a w+ background. These data indicate that Shrp regulates cell number but not differentiation in the retina (Kango-Singh, 2002).
The extra interommatidial cells could be due to excess cell proliferation, increased spacing of photoreceptor clusters during patterning, lack of apoptosis or a combination thereof. In wild-type flies, interommatidial cells are initially produced in excess but the extra cells are later eliminated by apoptosis during pupal development in a process that requires cell-cell signaling. This system generates a very precise retinal lattice. To determine whether shrp mutant cells initiate the apoptotic program, shrp mosaic pupal retinas were stained with an antibody that detects the activated form of Drice, a caspase that triggers the apoptotic program and specifically marks cells undergoing apoptosis. Many Drice-positive cells were detected in wild-type retinal tissue, but none were found in shrp3 mutant territories. Importantly, all Drice-positive cells were wild type. This suggests that the apoptotic pathway is blocked in shrp mutant cells and that this block occurs upstream of Drice activation. It is concluded that shrp mutant cells do not receive or are resistant to signals that induce apoptosis (Kango-Singh, 2002).
To test directly whether lack of apoptosis is sufficient to produce the shrp mutant phenotype, the phenotype of shrp mutant retinas was compared with that of wild-type retinas in which apoptosis was blocked by expressing the apoptosis inhibitor p35. Ectopic expression of p35 eliminates most, if not all, normally occurring cell death in the retina and results in extra interommatidial cells. However, the number of additional cells is significantly less than that observed in shrp4 mutant clones. Therefore, while lack of apoptosis allows additional cells to survive, it is not sufficient to explain the amount of extra interommatidial cells generated in shrp mutants (Kango-Singh, 2002).
To test directly whether shrp affects cell proliferation, the distribution of cell cycle progression was monitored in mutant third larval eye discs by bromodeoxyuridine (BrdU) incorporation. In wild-type discs, BrdU-incorporating cells are randomly distributed in front of the morphogenetic furrow. In the furrow, cells synchronously arrest in G1 and do not incorporate BrdU. Posterior to the furrow, cells go through a synchronous S phase in the second mitotic wave, revealed as a band of cells incorporating BrdU. Few BrdU-positive cells are found posterior to the second mitotic wave. shrp1 mutant cells also synchronize their cell cycles in the furrow and progress normally through the second mitotic wave. However, in contrast to wild-type cells, shrp1 mutant cells still display BrdU incorporation after the second mitotic wave. The extra DNA synthesis is followed by cell division, as revealed by ectopic expression of phosphorylated histone H3 (PH3), which marks chromosomes during mitosis. This phenotype of shrp is cell autonomous, because only mutant cells undergo extra rounds of cell proliferation, and all territories of mutant cells show the excess interommatidial cell phenotype in pupal retinas, whereas non-mutant tissue appears wild type. Extra cell proliferation continues into the pupal stage but ceases by 24 hours after pupariation. Double labeling with BrdU and antibodies against Elav to detect differentiating photoreceptor cells revealed that only Elav-negative cells incorporate BrdU. Therefore, shrp is required to arrest cell proliferation in developmentally uncommitted cells after the second mitotic wave, but is not required for cell cycle arrest of differentiating photoreceptor cells. The ectopic proliferation produces extra interommatidial cells, which together with the lack of apoptosis, are sufficient to explain the overgrowth phenotypes observed in pupal and adult retinas (Kango-Singh, 2002).
Cyclin E is limiting for S-phase initiation and progression during imaginal disc development and several tumor suppressor genes negatively regulate its activity or levels. Cyclin E levels are upregulated in shrp1 mutant cells in the second mitotic wave and posterior to it. Elevated levels were also observed just anterior to the second mitotic wave, although this effect was not as pronounced. The effect on Cyclin E is cell autonomous and observed in most or all mutant cells, even though only a fraction of them are actively progressing through S phase. Thus, the effect of Shrp on cell proliferation arrest may be mediated by regulating the levels of Cyclin E (Kango-Singh, 2002).
The data show that although shrp mutant cells are able to exit the cell cycle during cell differentiation, they are delayed in arresting cell proliferation at the end of eye imaginal disc growth. To determine whether shrp has a function in uncommitted cells anterior to the morphogenetic furrow, it was necessary to measure whether mutant eye discs are already larger than wild-type before ommatidial clusters are specified. Because initial spacing of photoreceptor clusters is normal in shrp mutant eye discs, the final number of ommatidia provides a measure of the number of cells present in mutant eye discs before R8 cells are specified in the morphogenetic furrow. The numbers of ommatidia were determined in wild-type and mutant retinas by counting clusters of photoreceptor cells revealed by Elav-Gal4 driven GFP expression. Mutant retinas contained an average of 913 ommatidial clusters, whereas wild-type retinas had an average of 776 photoreceptor clusters. It is concluded that shrp mutant eye discs are already larger than normal at the time when the positions of ommatidia are specified in the morphogenetic furrow. Shrp thus functions in uncommitted cells anterior to the morphogenetic furrow (Kango-Singh, 2002).
To test whether shrp affects the rate of cell proliferation during the growth phase of imaginal discs, cell numbers were compared in mutant clones and their associated twin clones in third instar wing discs. To reduce variability in the proliferation rate of wild-type twin clones, isogenized FRT 82B ubi-GFP chromosomes were used to generate mitotic clones. Cell numbers in shrp3 mutant clones were almost always larger than their twin clones, and the difference in cell numbers was significant when assessed using a t-test. The same experiment with a second allele, shrp4, showed similar differences. By contrast, cell numbers in clones of the isogenized wild-type chromosome on which the shrp mutations were induced during the mutagenesis screen were similar and not significantly different from the corresponding ubi-GFP/ubi-GFP twin clones. Based on these cell counts and assuming exponential proliferation, the cell division rate of shrp mutant cells is 1.10 times faster than that of wild-type cells. These data thus indicate that shrp mutant cells proliferated more. This phenotype is also manifest in mosaic adult eyes, where shrp1 mutant cells out-compete wild-type cells. Determination of the distribution of cell cycle phases in third instar wing discs using FACS analysis shows that the population of shrp4 mutant cells has the same distribution of cell cycle phases as wild-type cells. Thus, shrp mutant cells do not accelerate a particular step in the cell cycle. Rather, mutant cells show an even acceleration of cell cycle progression (Kango-Singh, 2002).
Manipulating the activity of cell growth regulators such as components of the insulin receptor signaling pathway results in larger organs because of more and larger cells. To determine whether shrp also affects cell size, mosaic wing discs were stained with antibodies against Dlg to detect apical cell outlines. Cells in shrp3 mutant clones show normal cell sizes, as judged by cell outlines and have normal height as judged by the thickness of the wing disc epithelium in the mutant clones. In addition, rhabdomeres of mutant photoreceptor cells were of normal diameter, and shrp mutant cone and pigment cells are of normal size at the pupal stage. Furthermore, forward light scatter (FSC) data, a measurement of cell size collected by FACS analysis confirms that mutant cells have normal size. Therefore, Shrp does not regulate cell size. Rather, extra proliferation of shrp mutant cells is induced by stimulation of cell growth and cell cycle progression, resulting in balanced growth and extra cells that are of normal size (Kango-Singh, 2002).
It is proposed that the arrest of cell proliferation during imaginal disc development is controlled by several genetically separable mechanisms. (1) Cells stop proliferating when imaginal discs have reached their correct sizes. This process requires Shrp function. (2) Cells permanently exit the cell cycle during terminal differentiation. Because terminal cell cycle exit is part of cell differentiation, it is directly regulated by patterning mechanisms that determine the identity and position of each individual cell. This regulation is governed by tissue-and cell-type specific enhancers of cell cycle regulators such as dacapo, cyclin E and string, which all have complex cis- regulatory regions. Similarly, patterned regulation of cell cycle progression may occur before terminal differentiation, as is observed in the second mitotic wave in the eye and in the zone of non-proliferation along the presumptive wing margin in the wing disc. None of these processes are affected in shrp mutants. Thus, the direct control of cell cycle progression by patterning mechanisms acts epistatically to the control of cell proliferation observed during organ growth and can impose cell cycle arrest on cells that otherwise may continue to proliferate. Therefore, shrp mutations do not deregulate cell proliferation of terminally arrested cells, and cells differentiate normally. In summary, Shrp identifies a molecular mechanism that is required to arrest cell proliferation during organ growth and that appears to be distinct from the ones used to arrest cells during terminal cell differentiation (Kango-Singh, 2002).
What are the downstream effectors of Shrp that are deregulated in shrp mutants and induce cell proliferation? shrp mutant clones behind the second mitotic wave in eye discs show elevated levels of Cyclin E. Notably, Cyclin E was elevated in all developmentally uncommitted cells of the clones, apparently irrespective of the phase of the cell cycle. The effect on Cyclin E levels may thus be direct and not just a reflection of the ectopic cell proliferation observed in mutant clones. Precise regulation of Cyclin E expression and activity is crucial because ectopic expression of Cyclin E induces entry into S phase and limited cell proliferation in imaginal discs and embryos. Several other negative regulators of cell proliferation directly regulate the levels of Cyclin E activity. Dap directly inhibits Cyclin E/Cdk2 complexes, and Archipelago is required for degradation of Cyclin E. The regulation of Cyclin E is thus likely to be an important downstream effect of Shrp function (Kango-Singh, 2002).
However, ectopic expression of Cyclin E alone is not sufficient to generate the number of extra cells observed in shrp mutant tissues. Artificial acceleration of the cell cycle by ectopic expression of specific cell cycle regulators such as E2F accelerates cell division, but does not stimulate cell growth rates, and cells divide when they are smaller. This results in an increase in cell number and a concomitant decrease in cell size, yet does not affect the overall tissue size. Thus, cell cycle progression is not sufficient to drive cell and organ growth. Conversely, stimulating cell growth alone produces larger organs, but also affects cell size. For example, artificially stimulating the activities of Ras, Myc or insulin receptor signaling produces more and bigger cells and thus bigger but otherwise normal flies. Thus, cell proliferation during organ growth requires coordinate stimulation of cell cycle progression and cell growth to produce normal sized cells. Because shrp mutant cells maintain normal size, Shrp appears to be required to regulate cell growth and cell cycle progression coordinately. Thus, Shrp probably regulates other targets driving cell cycle and cell growth in addition to Cyclin E (Kango-Singh, 2002 and references therein).
Several other mutations have been described that fail to arrest imaginal disc growth and were thus classified as tumor suppressor genes. These include discs-large (dlg), lethal giant larva (lgl) and scribble (scrib), encoding proteins that form an architectural complex localized to septate junctions. Mutations in these genes disrupt septate junctions and apical-basal polarity of epithelial cells and result in neoplastic overgrowth of imaginal discs. Mutations in a second group of Drosophila tumor suppressor genes cause hyperplastic overgrowth of imaginal discs that retain their single layered epithelial structure. These include warts/lats, which encodes a kinase that regulates the activity of Cdc2; fat, a large Cadherin; hyperplastic discs, a E3 ubiquitin ligase, and discs overgrown, a Drosophila homolog of Casein kinase IDelta/Epsilon. The imaginal disc overgrowth in mutants of both groups occurs during an extended larval period, and embryonic requirements for these genes appear to be provided by maternal contributions. By contrast, homozygous shrp mutant animals die as first/second instar larvae, which do not show gross morphological defects. Thus, zygotic expression of shrp is required for early larval viability, whereas that of other tumor suppressor genes is not. Cells homozygous mutant for neoplastic or hyperplastic tumor suppressor genes generally differentiate abnormally and show defects in cell morphology and/or pattern formation. These phenotypes are thus different from those of shrp mutant cell clones, which overproliferate but differentiate with normal cell morphology and patterning. In addition to these differences, clones of cells homozygous mutant for shrp proliferate more rapidly and have reduced apoptosis, while cells mutant for most other tumor suppressor genes have reduced viability and a decreased rate of cell proliferation. Only fat and warts/lats mutant cell clones proliferate faster, similar to shrp mutant cells. However, the phenotypes of shrp, fat and warts/lats differ, because fat and warts/lats affect the morphology and pattern of adult tissues in addition to cell number. Therefore, shrp affects cell number more specifically than these other mutants, and future work will have to establish whether and how Shrp interacts with other tumor suppressor gene products to control tissue size (Kango-Singh, 2002).
In summary, these studies provide evidence that Shrp functions in the decision of imaginal disc cells to terminate proliferation and to exit the cell cycle once the correct disc size is attained. The determination of the effectors of Shrp action should reveal mechanisms by which cell growth and cell cycle progression are coordinately regulated during organ growth and how cells arrest proliferation once organs have reached their correct size. The presence of Shrp homologs in mouse and human suggest the existence of a conserved organ size control mechanism in mammals. The characterization of Shrp function should therefore provide valuable insights into the mechanisms that underlie tissue size regulation and cause disproportionate growth and tumorigenesis when defective (Kango-Singh, 2002).
Color vision in Drosophila relies on the comparison between two color-sensitive photoreceptors, R7 and R8. Two types of ommatidia in which R7 and R8 contain different rhodopsins are distributed stochastically in the retina and appear to discriminate short (p-subset) or long wavelengths (y-subset). The choice between p and y fates is made in R7, which then instructs R8 to follow the corresponding fate, thus leading to a tight coupling between rhodopsins expressed in R7 and R8. warts, encoding large tumor suppressor (Lats) and melted encoding a PH-domain protein, play opposite roles in defining the yR8 or pR8 fates. By interacting antagonistically at the transcriptional level, they form a bistable loop that insures a robust commitment of R8 to a single fate, without allowing ambiguity. This represents an unexpected postmitotic role for genes controlling cell proliferation (warts and its partner hippo and salvador) and cell growth (melted) (Mikeladze-Dvali, 2005).
The fly eye provides a powerful system to study cell-fate decisions: it develops from a flat epithelium into a complex three-dimensional structure of multiple cell types in less than a week. The adult eye allows the fly to perform various visual tasks, ranging from motion detection and the discrimination of colors to measuring the orientation of polarized light for navigation (Mikeladze-Dvali, 2005).
In the fly compound eye, each of the 800 ommatidia is a single optical unit that contains 8 photoreceptor cells (PRs). The 8 PRs form widely expanded membrane structures, the rhabdomeres, which contain the photosensitive Rhodopsins (Rh). The rhabdomeres of the six outer PRs (R1-R6) form a trapezoid. R1-R6 all express the broad spectrum rhodopsin1 (rh1 or ninaE) and are morphologically and functionally invariant in all ~800 ommatidia (Mikeladze-Dvali, 2005).
The center of the trapezoid is occupied by the two inner PRs, R7 and R8. The rhabdomeres of R7 are positioned on top of R8, so that they share the same optic path. Inner PRs are involved in color vision and can be viewed as equivalent to vertebrate cones. Each R7 and R8 expresses only one of the four rhodopsins, rh3, rh4, rh5, or rh6 in a highly regulated manner, defining three different subtypes of ommatidia: 'yellow' (y), 'pale' (p) (for their appearance under UV illumination), and the 'dorsal rim area' (DRA). Ommatidia in the DRA express rh3 in both R7 and R8 and are specified in a very restricted region by the gene homothorax. They are believed to function as polarized light detectors (Mikeladze-Dvali, 2005).
In contrast, color vision depends on the y and p ommatidial subtypes that are randomly distributed through the main part of the retina, with a bias of y (~70%) over p subtype (~30%). In the p subtype, R7 expresses the UV-sensitive Rh3 and R8 the blue-sensitive Rh5. In the y subtype, R7 expresses a distinct UV-sensitive Rh4 while R8 expresses the green-sensitive Rh6. As in many other sensory systems, expression of a given Rhodopsin excludes all others to prevent sensory overlap. While the p subtype is better suited to discriminate among shorter wavelengths, the y subtype should discriminate amongst longer wavelengths (Mikeladze-Dvali, 2005).
The choice between the p and y fate is first made in R7: once an R7 commits to the p fate and expresses rh3, it sends an instructive signal to the underlying R8, which then also commits to the p fate and expresses rh5. In the absence of the R7 signal (i.e., when R7 expresses rh4 or in a sevenless mutant), R8 commits to the y fate and expresses rh6. The stochastic choice appears to be made by each R7 independently of its neighbors, resulting in the biased random distribution of p and y ommatidia throughout the main part of the retina (for review see Mikeladze-Dvali, 2005).
Four genes required in R8 cells for ensuring the correct choice of y versus p cell fate have been identified. The warts (wts) gene, which encodes the Drosophila large tumor suppressor (also known as lats) and melted (melt) play a critical role in the specification of p and y R8 cells, without affecting the R7 choice. wts encodes a Ser/Thr kinase, while melt encodes a Pleckstrin Homology (PH) domain protein. wts is necessary and sufficient for R8 to adopt the y fate, while melt plays the opposite role and specifically induces the p fate in R8. wts and melt are expressed in a complementary manner in the yR8 and pR8 subsets, respectively. Evidence is presented that the two genes repress each other's transcription to form a bistable loop. melt seems to respond to the R7 signal, while wts appears to regulate the output of the loop. The tumor-suppressor genes hippo (hpo) and salvador (sav), which encode the two molecular partners of Wts/Lats, have phenotypes identical to wts. Interestingly, melt has been reported to regulate growth and fat metabolism in Drosophila. Thus, genes known to regulate both cell growth (melt) and proliferation (wts, sav, hpo) interact antagonistically during retinal patterning (Mikeladze-Dvali, 2005).
Precise patterning of dendritic fields is essential for neuronal circuit formation and function, but how neurons establish and maintain their dendritic fields during development is poorly understood. In Drosophila class IV dendritic arborization neurons, dendritic tiling, which allows for the complete but non-overlapping coverage of the dendritic fields, is established through a 'like-repels-like' behaviour of dendrites mediated by Tricornered (Trc), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila. The other NDR family kinase, the tumour suppressor Warts/Lats (Wts), regulates the maintenance of dendrites; in wts mutants, dendrites initially tile the body wall normally, but progressively lose branches at later larval stages, whereas the axon shows no obvious defects. Biochemical and genetic evidence is provided for the tumour suppressor kinase Hippo (Hpo) as an upstream regulator of Wts and Trc for dendrite maintenance and tiling, respectively, thereby revealing important functions of tumour suppressor genes of the Hpo signalling pathway in dendrite morphogenesis (Emoto, 2006).
Dendritic arborization patterns are critical to a neuron's ability to receive and process impinging signals. Whereas neurons normally maintain the gross morphology of their dendrites, cortical neurons of Down's syndrome patients gradually lose dendritic branches after initially forming normal dendritic fields. Thus, neurons appear to have separate mechanisms for establishment and maintenance of their dendritic fields (Emoto, 2006).
Dendritic tiling is an evolutionarily conserved mechanism for neurons of the same type to ensure complete but non-redundant coverage of dendritic fields. In the mammalian visual system, for instance, dendrites of each retinal ganglion cell type cover the entire retina with little overlap, like tiles on a floor. In Drosophila, the dendritic arborization sensory neurons can be divided into four classes (I-IV) based on their dendrite morphology, and the dendritic field of class IV dendritic arborization neurons is shaped, in part, through a like-repels-like tiling behaviour of dendrite terminals. The NDR family kinase Trc and its activator Furry (Fry) has been identified as essential regulators of dendritic tiling and branching of class IV dendritic arborization neurons. These proteins are evolutionarily conserved and probably serve similar functions in neurons of different organisms (Emoto, 2006).
In addition to Trc, Drosophila has one other NDR family kinase, Wts, which is a tumour suppressor protein that functions in the coordination of cell proliferation and cell death in flies. To uncover the cell-autonomous functions of Wts in neurons, MARCM (mosaic analysis with a repressive cell marker) was ised to generate mCD8-GFP-labelled wts clones in a heterozygous background. Wild-type class IV neurons elaborate highly branched dendrites that cover essentially the entire body wall. Compared to wild-type ddaC (dorsal dendrite arborization neuron C) neurons, wts clones showed a severe and highly penetrant simplification of dendritic trees, with significantly reduced number (wild type, 575.1; wts, 255.6) and length (wild type, 1,457.0; wts, 590.4) of dendritic branches, and hence a greatly reduced dendritic field (Emoto, 2006).
In contrast to the severe dendritic defects caused by loss of Wts function, wts mutant ddaC axons entered the ventral nerve cord at the appropriate position and showed arborization patterns very similar to wild-type controls, with their axons terminating on the innermost fascicle and sending ipsilateral branches anteriorly and posteriorly and sometimes also a collateral branch towards the midline. Thus, Wts seems to have a crucial role in dendrite-specific morphogenesis in post-mitotic neurons (Emoto, 2006).
In proliferating cells, Wts is part of a signalling complex for tumour suppression that includes the adaptor protein Salvador (Sav) and the serine/threonine kinase Hpo. sav mutant ddaC MARCM clones were examined and dendritic defects were observed similar to wts MARCM clones. In severely affected clones (3 of 15 clones), most of the high-order branches were missing, whereas moderately affected clones (12 of 15 clones) exhibited a partial loss of their fine branches and major branches (Emoto, 2006).
To confirm that Wts and Sav function in the same pathway, genetic interaction between wts and sav in regulating dendrite morphogenesis was tested. Whereas heterozygous wts or sav mutants had no obvious dendritic phenotype, trans-heterozygous combinations of wts and sav alleles resulted in simplified dendrites similar to moderately affected sav clones. Furthermore, sav wts double mutant clones showed a severe dendrite defect comparable to wts mutant clones. Thus, Wts and Sav most probably function together in class IV neurons to regulate dendrite morphogenesis (Emoto, 2006).
The dendritic phenotypes of wts mutants and sav mutants might result from defects in branch formation and/or elongation, or loss of normally formed dendrites. Therefore ddaC dendrites were examined at different time points of larval development using the pickpocket-EGFP reporter, which is specifically expressed in class IV dendritic arborization neurons. Wild-type ddaC neurons elaborated primary and secondary dendritic branches by 24-28 h after egg laying, but large regions of the body wall were not yet covered by dendrites. By 48-52 h after egg laying, the major branches reached the dorsal midline, and the open spaces between major branches were filled with fine branches, resulting in complete dendritic coverage of the body wall. This tiling of dendrites persisted throughout the rest of larval development. In wts and sav mutants, ddaC dendrites were indistinguishable from those of wild-type controls at 24-28 h after egg laying. By 48-52 h after egg laying, wts and sav dendrites tiled the body wall as in wild type. During the next 24 h, however, dendrites of wts and sav mutants no longer tiled the body wall. Therefore, wts and sav seem to be required for maintenance of the already established tiling of dendrites (Emoto, 2006).
The loss of dendrites was further documented in live mutant larvae imaged for 30 h starting in early second instar larvae (48-50 h after egg laying). In wild-type larvae, ddaC dendrites grew steadily; the number of terminal branches increased by 23.0 over this time period. By contrast, dendrites of wts and sav mutants gradually lost their fine branches (decrease of 27.5 and 31.5, respectively) as well as some of the major branches by 78-80 h after egg laying (Emoto, 2006).
Class-IV-neuron-specific expression of wts and sav largely rescued the dendritic phenotype of wts and sav mutants, respectively, confirming that Wts and Sav act cell autonomously in class IV neurons. Furthermore, no detectable defect in patterning of the epidermis (anti-Armadillo antibody) or muscle (Tropomyosin::GFP reporter) was observed in wts or sav mutant third instar larvae. Taken together, these results indicate that the Wts/Sav signalling pathway functions in class IV neurons to maintain dendritic arborizations (Emoto, 2006).
Wts kinase activity is regulated, at least in part, by the Ste20-like serine/threonine kinase Hpo. Indeed, ddaC clones mutant for hpo exhibited simplified dendritic trees in third instar larvae, similar to wts or sav mutant clones, but showed more extensive dendritic arborizations in earlier larval stages (second to early third instar), consistent with the involvement of Hpo in the maintenance of dendrites. Notably, in hpo mutant clones at earlier developmental stages, dendritic branches were often found to overlap. Both the dendritic tiling and maintenance phenotypes were rescued by hpo expression in MARCM clones, consistent with the cell-autonomous function of Hpo in class IV neurons. Because this tiling defect in hpo mutant clones was similar to the tiling defects of trc mutant clones, whether hpo could genetically interact with trc to regulate dendritic tiling was tested. Compared with wild-type controls, trans-heterozygous combinations of trc and hpo exhibited obvious iso-neuronal as well as hetero-neuronal tiling defects, whereas wts and hpo trans-heterozygotes displayed simplified dendrites similar to wts mutants. These dendritic defects were consistently observed in multiple allelic combinations between hpo and trc or wts. In contrast, trans-heterozygous combinations of trc and wts showed no significant dendritic phenotypes. Furthermore, overexpression of wild-type Trc, but not Wts, in hpo MARCM clones partially rescued the dendritic tiling defects in class IV neurons. Thus, Hpo acts through Trc and Wts to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).
Not only did Hpo interact genetically with Trc and Wts, its physical association with these NDR kinases could be detected in vivo. When Flag-tagged Trc was expressed using a nervous-system-specific Gal4 driver, anti-Flag antibodies immunoprecipitated Trc together with Hpo. Similarly, Myc-tagged Wts co-immunoprecipitated with Hpo expressed in embryonic nervous systems. Hpo co-immunoprecipitation appeared to be specific, because Misshapen, another Ste20-like kinase protein present in neurons, was not co-immunoprecipitated by anti-Flag or anti-Myc antibodies in similar experiments. These results suggest that Hpo associates with Trc and Wts in the Drosophila nervous system (Emoto, 2006).
To examine further the physical interaction between Trc and Hpo, analogous experiments were carried out in Drosophila S2 cells co-transfected with a haemagglutinin (HA)-tagged Trc construct and a Flag-tagged Hpo construct containing the full open reading frame, an amino-terminal fragment containing the kinase domain, or a carboxy-terminal fragment containing the regulatory domain. Full-length Hpo and the C-terminal portion of Hpo, but not the N-terminal fragment, were co-immunoprecipitated with Trc, suggesting that the C-terminal domain of Hpo is sufficient for Trc-Hpo complex formation (Emoto, 2006).
Hpo physically interacts with Wts and promotes Wts phosphorylation at multiple serine/threonine sites, including two sites, S920 and T1083 of Drosophila Wts, that appear to be necessary for Wts kinase activation. Indeed, Wts protein with mutations in the S920 and T1083 residues was unable to rescue the wts mutant dendritic phenotypes. Given that the corresponding phosphorylation sites in Trc are critical for Trc activation as well as control of dendritic tiling and branching, it was of interest to know whether Hpo may promote Trc phosphorylation at the critical serine and/or threonine residue. Wild-type Hpo, but not catalytically inactive Hpo or the Misshapen kinase, led to substantial incorporation of 32P-labelled phosphate into recombinant Trc or Trc with a mutation at the S292 site (S292A), but not the T449A mutant form of Trc. Analogous results were obtained with Wts. These results support a model in which Hpo associates with and phosphorylates Trc and Wts at a critical threonine residue to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).
Both genetic and biochemical evidence reveals that Hpo regulates complementary aspects of dendrite development through two distinct downstream signalling pathways: the Trc kinase pathway for tiling and the Wts kinase pathway for maintenance. These studies of class IV dendritic arborization neurons, together with the recent report that Wts signalling is required for cell fate specification of photoreceptor cells in Drosophila retina, demonstrate that the Wts signalling pathway is important for post-mitotic neurons. In proliferating cells, Wts phosphorylates Yorkie (Yki), a transcriptional co-activator, to regulate cell cycle and apoptosis in growing cells. However, Yki is dispensable for Hpo/Wts-mediated dendrite maintenance. Hpo probably functions as an upstream kinase for Trc, as well as Wts, in neurons by phosphorylating a functionally essential threonine, which may also be regulated by MST3, a Ste20-like kinase closely related to Hpo. Given the evolutionary conservation of known components in the Trc and Wts signalling pathways, it will be important to identify their relevant downstream targets and explore mechanisms that coordinate the establishment and maintenance of dendritic fields, and to determine the role of Trc and Wts signalling in the mammalian nervous system (Emoto, 2006).
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date revised: 20 October 2013
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