warts

REGULATION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Protein Interactions

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

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

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

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

Discs containing clones of the wts null allele, wts^latsX1, 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, wts^latsX1 double mutant discs and 31,360 pixels for wts^latsX1 discs. Thus, the double mutant discs were significantly larger than the wts^latsX1 discs. Thus, while sav and wts appear to function together in certain ways, they are also likely to have functions that are independent of each other (Tapon, 2002).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Individual Mob family proteins also interact with Tricornered (Trc), the Drosophila Ndr (Nuclear Dbf2-related) serine/threonine protein kinase that is required for the normal morphogenesis of a variety of polarized outgrowths including epidermal hairs, bristles, arista laterals, and dendrites. In yeast the Trc homolog Cbk1 needs to bind Mob2 to activate the RAM pathway. Genetic and biochemical data is provided that Drosophila Trc interacts with and is activated by Drosophila Dmob proteins, specifically Mats and Dmob2 (FlyBase terms the gene Dmob2 Mob1). Evidence is provided that Drosophila Mob proteins also interact with the related Warts/Lats kinase, which functions as a tumor suppressor in flies and mammals. In trc mutants the overall pattern of denticles is partly disorganized and many denticles are split. Split denticles are infrequent in wild-type larvae. The denticle pattern of mats mutant larvae is also disorganized and contains many split denticles. Interestingly, the overgrowth tumor phenotype that results from mutations in Dmob1 (mats) is only seen in genetic mosaics and not when the entire animal is mutant. Unlike in yeast, in Drosophila individual Mob proteins interact with multiple kinases and individual NDR family kinases interact with multiple Mob proteins; in particular, Mats interacts physically and genetically with trc and mats phenotype resembles that of trc. Notably, trc:mats double mutant larvae do not have a more severe phenotype than the single mutants. This lack of additivity argues that trc and mats function in a common pathway during denticle development. These observations also suggest that mats functions with both Trc and Wts (He, 2005).

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

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

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

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

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

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

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

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

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

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

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

DEVELOPMENTAL BIOLOGY

Embryonic

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

Effects of Mutation or Deletion

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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date revised: 20 April 2007

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