Whole-mount immunostaining with anti-Mats antibodies indicated that mats is activated throughout development and ubiquitously expressed at a low level in tissues such as larval eye discs (Lai, 2005).
The directed expression of a dominant negative Trc protein provides a sensitized system for identifying interacting genes (He, 2005a). Deficiencies for each of the fly mob genes enhances the wing hair phenotype that results from driving expression of UAS-trcT453A using either ap-GAL4 or ptc-gal4. The strongest enhancement is seen with deficiencies for mats and Dmob2. These results suggested the possibility that all 4 Dmobs can redundantly interact with Trc, although it is possible that the interactions could be indirect or due to other genes in deleted regions. It is worth noting that such interactions are not common. When the Drosophila deficiency collection was screened for enhancement or suppression of ap-GAL4 UAS-trcT453A, <10% of the Dfs showed an interaction (He, 2005b).
To confirm that the genetic interaction between trc and Df (mats) was due to the reduction in mats dose, two independent alleles were used. One was the null allele described by Lai (2005) (matse235), which is deleted for almost the entire coding region, and the other was a lethal PiggyBac insertion allele of mats [PBac{RB}CG13852e03077] (this allele is referred to as matsPB). Because this later allele has not been well characterized, the insertion was determined to be lethal over a deficiency for the region (Df(3R)Exel6191), and it failed to complement the recessive lethality of matse235 consistent with the lethality being due to the PB insertion. This mutation could be reverted using a source of PiggyBac transposase. It was found that both mats alleles dominantly enhance the trc dominant negative phenotype and this enhancement is lost in the PB revertant. It was also found that over expression of mats from a UAS-mats transgene (Lai, 2005; driven by ptc-Gal4) partially suppresses the multiple hair cell phenotype that results from driving expression of Trc-DN using ptc-Gal4. These dose responses argue that Mats activates Trc. Interestingly, it was found that heterozygosity for a wts mutation also enhances the Trc dominant negative phenotype, although somewhat less strongly (He, 2005b).
Evidence was also obtained for mats functioning with trc and fry using simple loss of function mutations. Wild-type flies or flies heterozygous for either trc, fry, or mats appear normal and only rarely (on fewer than 5% of wings) is even a single multiple hair cell seen. Flies that were heterozygous for two of these genes showed a slightly higher frequency of wings with one or a couple of multiple hair cells (often ~10%) but the increase was not routinely significant. However, almost half of the wings from flies that are heterozygous for all three genes (e.g., fry2 trc1+/+ + matsPB) show a weak multiple hair cell phenotype, a significant increase. This genetic interaction is further support for the hypothesis that trc, fry, and mats function together in regulating wing hair development. In this assay no equivalent interaction with wts3-17 was seen (He, 2005b).
Previous studies established that trc also has a larval denticle phenotype (Geng, 2000). 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 matsPB/matsPB homozygous larvae is also disorganized and contains many split denticles. The number of split denticles is similar in matsPB/Df larvae, suggesting that for this phenotype, matsPB is a strong, near phenotypic null allele. The matse235 also showed a similar denticle phenotype. The phenotype of matsPB homozygotes is slightly less severe than that of trcP/trcP larvae. Notably, trcP matsPB/trcP matsPB 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 (He, 2005b).
Both mats alleles are larval lethals with death typically in the second or early third instar. To examine the phenotype of mats in wing cells, mosaics were generated using FLP/FRT. mats clones on the wing, leg, thorax, and head display two types of phenotype. The most notable is indistinguishable from those produced by clones of wts, suggesting that mats also functions with wts as has been shown by Lai (2005). On the wing, small clones produced bulges that can be seen at low magnification with a stereomicroscope. In mounted wings individual cell outlines are visible in the cuticle and the cells appear to have a bulging apical surface. The hairs are located on an elevated pedestal, a phenotype that is indistinguishable from those seen in wts clones. The hairs were often broader than normal. Particularly in other body regions clones were abnormally pigmented (either darker or lighter than normal, and there were outgrowths of clone tissue. In highly abnormal wing clones, evidence of clustered and split hairs were often seen that were typical of trc mutant clones. Some multiple hair cells were seen in very abnormal wts clones but this phenotype appears less severe (e.g., number of hairs per cell) than that seen with mats or trc. These observations suggest that mats functions with both Trc and Wts (He, 2005b).
matsPB and matse235 clones in pupal wings were examined. Mutant mats cells are able to outcompete their neighbors and end up comprising most of the wing when clones are induced early. As was expected from the morphology of clones in adult wings, the pupal clones produce bulges in the wing and individual cells also often appear bulged. Clone cells stain more brightly for F-actin. This is true both in developing hairs and in the general apical cortex. This phenotype is clear-cut enough that it could be used as a convenient marker of mats mutant cells. These phenotypes were seen with both mats alleles tested. A similar, increase in actin staining was seen in wts clones. A similar, but perhaps less severe increase in staining, is seen in trc clones (He, 2005a). In some, but not all mats clones, large numbers of multiple hair cells can be seen. At later stages a circular pedestal of actin staining could be seen surrounding the base of the hair in mutant cells but not in surrounding wild-type cells. At still later stages the wild-type cells also had a circular pedestal of actin staining, suggesting that the mutant cells might be developmentally more advanced. Consistent with this possibility, in many clones hair initiation and outgrowth appear to be advanced in mats mutant cells compared with neighbors. This is also the case for wts clones, but it is the opposite of trc clones, in which hair development is often delayed (He, 2005a). Cells in mats clones have a smaller cross section so that the array of hairs appears denser, which is also the opposite of what is seen in trc clones, in which there is an increase in cross-sectional area (He, 2005a). Once again the phenotype of the wts clone cells resembles that seen for mats cells. Thus, for several wing phenotypes mats mutant cells resembled wts and not trc cells. Indeed, the mats phenotype is the opposite of trc for both cell area and the timing of hair morphogenesis (He, 2005b).
It has been found that the accumulation of Fry in wing cells is subject to feedback control that is dependent on Trc activity. Hence, in a trc mutant, increased Fry accumulation is found (He, 2005b). Several of the observations described above suggest the hypothesis that mats functions along with trc and is important for Trc activation. From this it is predicted that Fry accumulation would also be elevated in Dmob1 clones. Increased levels of Fry immunostaining were found in Dmob1 clone cells; this is consistent with the hypothesis. This is also seen in wts mutant clones, although the increase appears less dramatic (He, 2005b).
Tumorous overgrowth phenotypes are a consequence of mutations in a number of Drosophila genes. In several cases, such as lethal giant discs overgrown imaginal discs are found in late third instar larvae. To determine whether that is also the case for mats, mats/Df mutant larvae were examined. These larvae grow slowly and after 5 d of growth, when wild-type larvae begin to pupate, mats/Df larvae are the size of early third instar larvae. These larvae routinely die without growing substantially larger. When 5.5-5-d-old mats larvae were dissected, not evidence of tumors or overgrowth of imaginal or other tissues was found. Rather, the imaginal discs were approximately the size of those seen in 4-d larvae. However, the mats homozygous discs did not appear normal, since they were abnormally shaped and more folded than normal discs of this size (He, 2005b).
In a number of experiments involving mats or wts, what appeared to be spontaneous tumors or clones was observed. This was seen most often in flies that also carried reduced doses of Dmob3 and Dmob4. It is thought that these overgrowths are due to spontaneous mitotic recombination, because when the flies were also mutant for trc or fry, evidence of trc or fry clones was seen. The trc and fry clones were seen less frequently. This could be due to these genes being located more proximally on the chromosome than mats or wts, but it might also be due to the competitive advantage of mats and wts clones, resulting in these clones being larger and easier to detect. The basis for these clones is unclear but suggests genomic instability in mats and/or wts mutants (He, 2005b).
A spontaneous lethal mutation was identified in the Drosophila gene mob as tumor suppressor (mats). From mutant clones generated in mats mosaic flies, large tumors can be induced in many organs including the head, notum, eye, wing, leg, antenna, and halteres. Thus, mats appears to function as a general inhibitor of tissue growth. The tumor cells formed unpatterned tissue with many folds on the surface. Using green fluorescent protein (GFP) to positively label mutant cells, it was apparent that mutant cells overproliferated in comparison to wild-type cells. Mosaic larval eye discs with mats mutant clones were apparently larger and folded in many areas. Overproliferation of mats mutant cells could not be explained by change in cell size, because the size of the mutant cells is not significantly different from that of wild-type cells. To directly examine the cell proliferation phenotype, Bromdeoxyuridine (BrdU) was incubated with eye discs to identify cells in the S phase. In wild-type larval eye discs, BrdU incorporation is evident in cells anterior to the morphogenetic furrow (MF) and in the second mitotic wave (SMW), which is a narrow stripe of dividing cells a few rows posterior to the MF. Consistent with an inhibitory role of mats in regulating cell proliferation, BrdU incorporation was elevated in mats mutant clones in eye discs. This phenotype was more evident in mats clones located in the MF and SMW regions. Moreover, immunostaining with anti-phospho histone H3 (PH3) antibody was carried out to identify mitotic cells in eye discs. In wild-type, PH3-positive cells can be found in the anterior and SMW regions, but not in the MF. They were rarely detected in the posterior region. In mats mosaic eye discs, more mitoses were observed in mats clones compared to neighboring wild-type cells, and mitoses can even sometimes occur in the MF. Excess mitoses were also found in mats clones located in the posterior margin. On the basis of these observations, it is concluded that mats is required to restrict cell proliferation and loss of mats function allows cells to divide at a time when they should exit the cell cycle (Lai, 2005).
To characterize the molecular nature of mats gene, deficiency and meiotic mapping experiments were carried out, and mats was localized in the 94A region on the third chromosome. By using a P transposon-mediated site-specific recombination method, mats was further mapped to a 13 kb region at 94A12. Molecular analysis of a candidate gene in this region, designated CG13852, revealed that a 428 bp Roo transposon sequence was inserted behind codon 84 to cause a premature termination. This first mutant allele of CG13852 is named roo in this study. A second mutant allele of this gene, referred to as e235, was generated by mobilizing a P transposon EP(3)3303 inserted approximately 2 kb downstream of CG13852. Like roo, e235 causes homozygous lethality at early second larval stage and induces tumors in somatic clones of mosaic flies. Based on the larval lethal phenotype, e235 failed to complement roo as well as deficiency chromosomes with the 94A12 region deleted. Sequence analysis of e235 has indicated that the second and third exons of CG13852 were deleted. Although the first exon is still intact, it encodes only the first four amino acids of CG13852. Thus, e235 is a null allele of CG13852 (Lai, 2005).
CG13852 encodes a 219-amino acid polypeptide, which is approximately 25 kDa in size. Due to CG13852's apparent homology to the Mob superfamily, it is renamed here Mats (Mob as tumor suppressor). As a member of the Mob superfamily, Mats has no significant homology with any other previously characterized protein domains. To test if CG13852 can rescue defects induced by mats mutations, an in vivo assay was established to allow CG13852 transgene expression only in mutant cells by using the MARCM system. It was found that expression of a full-length CG13852 cDNA in mutant cells suppresses tumor growth and pupal lethality associated with the mosaic flies. Moreover, ubiquitous expression of UAS-CG13852 driven by arm-Gal4 rescues larval lethality of mats homozygous mutants. These results further demonstrated that CG13852 corresponds to mats (Lai, 2005).
Cyclin E, a key regulator for the G1-S transition, is normally expressed in the second mitotic wave (SMW) of larval eye discs. In mats mosaic eye discs, Cyclin E levels are elevated in mutant clones located in the morphogenetic furrow (MF) and SMW regions. Moderate upregulation of Cyclin A and Cyclin B expression is also observed. Thus, an important mechanism for mats to control cell proliferation is to negatively regulate expression of key cell cycle regulators such as Cyclins. Interestingly, Cyclin E expression in mutant cells immediately anterior to the MF is much less elevated than that immediately posterior to the MF, suggesting that mats may use a different mechanism to restrict cell proliferation in cells anterior to the MF. The cell proliferation defects observed in mats mutants are similar to those caused by sav, wts, and hpo mutations (Lai, 2005).
Whether mats plays a role in regulating cellular differentiation in the developing eye was investigated. In larval eye discs, photoreceptors (R) and cone cells in mats clones appear to be specified normally. However, they fail to fully differentiate to generate ommatidia in adult. Defective retinal differentiation occurs at least at mid-pupal stages. Thus, mats is required for cellular differentiation during eye development. In contrast, hpo, sav, and wts do not significantly affect differentiation of retinal cells during eye development (Lai, 2005).
Apoptosis provides an important mechanism for the control of cell number and organ size. To test if mats plays a role in cell death control, expression of DIAP1 in eye discs was examined. DIAP1 is a caspase inhibitor essential for cell survival. Through immunostaining of mats mosaic eye discs, it was found that the level of DIAP1 protein is increased in mats clones. To examine if mats regulates diap1 at the transcriptional level, an enhancer trap line thj5C8 was used, in which a lacZ reporter gene is inserted in diap1 and expression pattern of diap1-lacZ reflect that of the endogenous diap1 gene. It was found that expression of diap1-lacZ was elevated. Thus, mats is required to negatively regulate DIAP1 expression. To directly test the idea that mats promotes apoptosis, mats mutant clones were induced in larval eye discs that overexpress an apoptosis-promoting gene head involution defective (hid) in all cells behind the MF. As expected, expression of hid in a wild-type background increased apoptosis to cause a reduced eye phenotype. Notably, removal of mats function blocks hid-induced cell death and significantly suppresses the small eye phenotype. In these same tissues, developmental cell death is observed in regions anterior to the MF, where expression of the hid transgene is not induced. In these cases, apoptosis occurs only in wild-type tissues but not in mats mutant clones. Thus, mats is also required for developmentally programmed apoptosis. All together, these findings support a model that mats is required to facilitate cell death, and loss of mats’ apoptosis-promoting activity may contribute to tumor development (Lai, 2005).
Through phylogenetic analysis, four major groups of Mob proteins within the Mob superfamily were found. CG13852/Mats and its orthologs from animals and plants form the Mats gene family. Mats proteins are highly conserved. For instance, fly Mats and human Mats1 (also named Mob1A) are 87% identical. Even plant Mats orthologs are over 64% identical to fly Mats. In comparison, fly Mats shares no more than 40%-50% sequence identity with all other non-Mats Mob proteins from species such as yeast, fly, and humans. Such high levels of sequence conservation suggest that function of Mats proteins is evolutionarily conserved. To test functional homology, human Mats1 was introduced into Drosophila; it can effectively suppress tumor growth and rescue pupal lethality of mats mosaic flies. Thus, the growth inhibitory function of Mats has been conserved from insects to humans (Lai, 2005).
To further test the hypothesis that mammalian Mats functions as a tumor suppressor, 89 human and 8 mouse tumor-derived Mats cDNA sequences reported as expressed sequence tags (EST) in GenBank were examined and 2 Mats1 ESTs with disruptions in the coding region were identified, which were subsequently verified by sequencing analysis. In the first case, three nucleotides were deleted in a Mats1 cDNA (hMats1ΔS6/7) derived from a human skin melanoma, which caused deletion of the sixth (or seventh) codon for Ser. This mutation greatly destabilized hMats1 because no hMats1 protein was detectable from hMats1ΔS6/7-transfected human 293T cells. Another Mats1 cDNA (mMats1Δ6-216) derived from a mouse mammary gland carcinoma contains a 38 bp insertion immediately downstream of the fifth codon that causes a premature termination. As expected, no mMats1 protein product was detected. These findings are exciting as they support a model that Mats1 may function as a tumor suppressor in mammals (Lai, 2005).
Recent studies in Drosophila of the protocadherins Dachsous and Fat suggest that they act as ligand and receptor, respectively, for an intercellular signaling pathway that influences tissue polarity, growth and gene expression, but the basis for signaling downstream of Fat has remained unclear. This study characterizes functional relationships among Drosophila tumor suppressors and identifies the kinases Discs overgrown and Warts as components of a Fat signaling pathway. fat, discs overgrown and warts regulate a common set of downstream genes in multiple tissues. Genetic experiments position the action of discs overgrown (dco) upstream of the Fat pathway component dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs and discs overgrown in vivo, consistent with its placement as a downstream component of the pathway. The tumor suppressors Merlin, expanded (ex), hippo, salvador (sav) and mob as tumor suppressor (mats) also share multiple Fat pathway phenotypes but regulate Warts activity independently. These results functionally link what had been four disparate groups of Drosophila tumor suppressors, establish a basic framework for Fat signaling from receptor to transcription factor and implicate Warts as an integrator of multiple growth control signals (Cho, 2006).
Since Dachs is required for loss of Wts protein in fat mutants, and Dachs encodes a large Myosin protein, a model was considered in which Dachs acts as a scaffold to link Wts to proteins that promote Wts proteolysis, analogous to the roles of Costal2 in Hedgehog signaling, or APC in Wnt signaling. This model predicts that Dachs should be able to bind to Wts. To evaluate this possibility, tagged forms of Dachs and Wts were coexpressed in cultured cells and assayed for coimmunoprecipitation. These experiments identified a specific and reproducible interaction between Dachs and Wts (Cho, 2006).
Recent studies have identified the transcriptional coactivator Yorkie (Yki) as a downstream component of the Hippo pathway and a substrate of Wts kinase activity. Phosphorylation of Yki by Wts inactivates Yki, and overexpression of Yki phenocopies wts mutation. The determination that the Fat tumor suppressor pathway acts through modulation of Wts thus predicts that Yki should also be involved in Fat signaling. When the influence of Yki overexpression was examined on Fat target genes, expression of Wg in the proximal wing, Ser in the proximal leg and fj in the wing and eye were each upregulated by Yki overexpression, consistent with the inference that Fat tumor suppressor pathway signaling acts through Yki (Cho, 2006).
In order to identify additional components of the Fat tumor suppressor pathway, advantage was taken of the observation that loss of fat in clones of cells is associated with an induction of Wingless (Wg) expression in cells just proximal to the normal ring of Wg expression in the proximal wing, reflective of its role in distal-to-proximal wing signaling. It was reasoned that this influence on Wg expression could be used to screen other Drosophila tumor suppressors for their potential to contribute to Fat signaling. Analysis of mutant clones in the proximal wing identified dco, ex, mats, sav, hpo and wts as candidate components of the Fat tumor suppressor pathway. As for fat, mutation of each of these genes is associated with induction of Wg expression specifically in the proximal wing, whereas Wg expression is not affected in more distal or more proximal wing cells. Although Wg expression often seems slightly elevated within its normal domain, the effect of these mutations is most obvious in the broadening of the Wg expression ring. The induction of Wg expression does not seem to be a nonspecific consequence of the altered growth or cell affinity associated with these mutations, since Wg expression is unaffected by expression of the growth-promoting microRNA gene bantam or by expression of genes that alter cell affinity in the proximal wing (Cho, 2006).
dco encodes D. melanogaster casein kinase I delta/epsilon. The overgrowth phenotype that gave the gene its name is observed in allelic combinations that include a hypomorphic allele, dco3, and it is this allele that is associated with induction of Wg. Null mutations of dco actually result in an 'opposite' phenotype: discs fail to grow, and clones of cells mutant for null alleles fail to proliferate. This is likely to reflect requirements for dco in multiple, distinct processes, as casein kinase I proteins phosphorylate many different substrates, and dco has been implicated in circadian rhythms, Wnt signaling and Hedgehog signaling (Cho, 2006).
Mer and ex encode two structurally related FERM domain-containing proteins. ex was first identified as a Drosophila tumor suppressor, whereas Drosophila Mer was first identified based on its structural similarity to human Merlin. Mutation of Mer alone causes only mild effects on imaginal disc growth, but Mer and ex are partially redundant, and double mutants show more severe overgrowth phenotypes than either single mutant. Consistent with this, elevation of Wg expression was observed in ex mutant clones (7/10 proximal wing clones induced Wg) and not in Mer mutant clones (0/8 clones), whereas Mer ex double mutant clones showed even more severe effects on Wg than ex single mutant clones. Because of the partial redundancy between Mer and ex, when possible, focus was placed for subsequent analysis on Mer ex double mutant clones (Cho, 2006).
Wts, Mats, Sav and Hpo interact biochemically, show similar overgrowth phenotypes and regulate common target genes. Mats, Sav and Hpo are all thought to act by regulating the phosphorylation state and thereby the activity of Wts. Mutation of any one of these genes is associated with upregulation of Wg in the proximal wing. The effects of sav (47/84 clones in the proximal wing induced Wg) and hpo (23/31 clones) were weaker than those of mats (19/19 clones) and wts (92/97 clones), but this might result from differences in perdurance or allele strength. Because sav, hpo and mats all act through Wts, focus for most of the subsequent analysis was placed on wts (Cho, 2006).
The observation that mutation of dco, Mer, ex, mats, sav, hpo or wts all share the distinctive upregulation of Wg expression in the proximal wing observed in fat mutants suggests that the functions of these genes are closely linked. To further investigate this, the effects of these tumor suppressors were characterized on other transcriptional targets of Fat signaling. Expression of the Notch ligand Ser is upregulated unevenly within fat mutant cells in the proximal region of the leg disc. A very similar upregulation occurred in dco3, Mer ex, and wts mutant clones. fj is a target of Fat signaling in both wing and eye imaginal discs, and fj expression was also upregulated in dco3, Mer ex, or wts mutant clones. The observation that these genes share multiple transcriptional targets in different Drosophila tissues implies that they act together in a common process (Cho, 2006).
The hypothesis that Fat pathway genes and Hippo pathway genes are linked predicts that not only should Fat target genes be regulated by Hippo pathway genes, but Hippo pathway target genes should also be regulated by Fat pathway genes. The cell cycle regulator CycE and the inhibitor of apoptosis Diap1 (encoded by thread) have been widely used as diagnostic downstream targets to assign genes to the Hippo pathway. Notably, then, clones of cells mutant for fat showed upregulation of both Diap1 and CycE protein expression. Genes whose expression is upregulated within fat mutant cells (such as wg, Ser and fj) have been shown previously to be induced along the borders of cells expressing either fj or dachsous (ds), and Diap1 is also upregulated around the borders of ds- or fj-expressing clones. That thread is affected by fat at a transcriptional level was confirmed by examining a thread-lacZ enhancer trap line. The regulation of Diap1 by the Hippo pathway is thought to be responsible for a characteristic eye phenotype in which an excess of interommatidial cells results from their failure to undergo apoptosis; an increase was also observed in interommatidial cells in fat mutant clones. Upregulation of both Diap1 and CycE is also observed in Mer ex double mutant clones. In dco3 mutant clones, consistent upregulation was detected only for Diap1, and CycE was upregulated only weakly and inconsistently. dco3 also has weaker effects on Wg and fj expression; the weaker effects of dco3 could result from its hypomorphic nature. ex has recently been characterized as another Hippo pathway target, and an ex-lacZ enhancer trap that is upregulated in wts or Mer ex mutant clones is also upregulated in fat or dco3 mutant clones. Analysis of ex transcription by in situ hybridization also indicated that ex is regulated by fat. Altogether, this analysis of Hippo pathway targets further supports the conclusion that the functions of the Fat pathway, the Hippo pathway and the tumor suppressors Mer, ex and dco are linked (Cho, 2006).
Genetic epistasis experiments provide a critical framework for evaluating the functional relationships among genes that act in a common pathway. The relationships was evaluated between each of the tumor suppressors linked to the Fat pathway and dachs, using both wing disc growth and proximal Wg expression as phenotypic assays. dachs is the only previously identified downstream component of the Fat tumor suppressor pathway. It acts oppositely to fat and is epistatic to fat in terms of both growth and gene expression phenotypes (Cho, 2006).
dachs is also epistatic to dco3 for overall wing disc growth and for proximal Wg expression. The epistasis of dachs to dco3 implies that the overgrowth phenotype of dco3 is specifically related to its influence on Fat signaling, as opposed to participation of dco in other pathways. By contrast to the epistasis of dachs to dco3, both wts and ex are epistatic to dachs for disc overgrowth phenotypes, and wts and Mer ex are epistatic to dachs in their influence on proximal Wg expression. Together, these epistasis experiments suggest that dco acts upstream of dachs, whereas Mer ex and wts act downstream of dachs (Cho, 2006).
Because wts and Mer ex have similar phenotypes, their epistatic relationship cannot be determined using loss-of-function alleles. However, overexpression of ex inhibits growth and promotes apoptosis, which suggests that ex overexpression affects ex gain-of-function. Clones of cells overexpressing ex are normally composed of only a few cells, and over time most are lost, but coexpression with the baculovirus apoptosis inhibitor p35 enabled recovery of ex-expressing clones. These ex- and p35-expressing clones were associated with repression of proximal Wg expression during early- to mid-third instar, as has been described for dachs2, consistent with ex overexpression acting as a gain-of-function allele in terms of its influence on Fat signaling. In epistasis experiments using overexpressed ex and mutation of wts, wts was epistatic; Wg was induced in the proximal wing. Additionally, when wts is mutant, coexpression with p35 was no longer needed to ensure the viability and growth of ex-expressing clones, indicating that wts is also epistatic to ex for growth and survival. Consistent with this conclusion, others have recently described phenotypic similarities between Mer ex and hpo pathway mutants and have reported that hpo is epistatic to Mer ex (Cho, 2006).
When Fat was overexpressed, a slight reduction was detected in Wg expression during early- to mid-third instar, suggesting that overexpression can result in a weak gain-of-function phenotype. Clones of cells overexpressing Fat but mutant for dco3 still showed reduced Wg levels, whereas clones of cells overexpressing Fat but mutant for warts showed increased Wg levels. Although experiments in which the epistatic mutation is not a null allele cannot be regarded as definitive, these results are consistent with the conclusion that wts acts downstream of fat and suggest that dco might act upstream of fat (Cho, 2006).
The epistasis results described above suggest an order of action for Fat tumor suppressor pathway genes in which dco acts upstream of fat, fat acts upstream of dachs, dachs acts upstream of Mer and ex, and Mer and ex act upstream of wts. However, the determination that one gene is epistatic to another does not prove that the epistatic gene is biochemically downstream, as it is also possible that they act in parallel but converge upon a common target. Thus, to better define the functional and hierarchical relationships among these genes, experiments were initiated to investigate the possibility that genetically upstream components influence the phosphorylation, stability or localization of genetically downstream (that is, epistatic) components. Focus in this study was placed on the most downstream of these components, Wts. As available antibodies did not specifically recognize Wts in imaginal discs, advantage was taken of the existence of functional, Myc-tagged Wts-expressing transgenes (Myc:Wts) to investigate potential influences of upstream Fat pathway genes on Wts protein. In wing imaginal discs, Myc:Wts staining outlines cells, suggesting that it is preferentially localized near the plasma membrane, and it was confirmed that expression of Myc:Wts under tub-Gal4 control can rescue wts mutation. Notably, mutation of fat results in a reduction of Myc:Wts staining. As Myc:Wts is expressed under the control of a heterologous promoter in these experiments, this must reflect a post-transcriptional influence on Wts protein. fat does not exert a general influence on the levels of Hippo pathway components; fat mutant clones had no detectable influence on the expression of hemagglutinin epitope-tagged Sav (HA:Sav) (Cho, 2006).
The decrease in Wts protein associated with mutation of fat contrasts with studies of the regulation of Wts activity by the Hippo pathway, which have identified changes in Wts activity due to changes in its phosphorylation state. To directly compare regulation of Wts by Fat with regulation of Wts by other upstream genes, Myc:Wts staining was examined in ex, sav and mats mutant clones. In each of these experiments, the levels and localization of Myc:Wts in mutant cells was indistinguishable from that in neighboring wild-type cells (Cho, 2006).
Since Myc:Wts appears preferentially localized near the plasma membrane, it was conceivable that the apparent decrease in staining reflected delocalization of Wts, rather than destabilization. To investigate this possibility, Wts levels were examined by protein blotting. Antisera against endogenous Wts recognized a band of the expected mobility in lysates of wing imaginal discs or cultured cells, and this band was enhanced when Wts was overexpressed. The intensity of this band was reproducibly diminished in fat or dco3 homozygous mutant animals but was not diminished in fat or dco3 heterozygotes or in ex mutants. Conversely, levels of Hpo, Sav, Mer or Mats were not noticeably affected by fat mutation (Cho, 2006).
The determination that Wts is affected by Fat, together with the genetic studies described above, place Wts within the Fat signaling pathway, as opposed to a parallel pathway that converges on common transcriptional targets. Indeed, given that even hypomorphic alleles of wts result in disc overgrowth, the evident reduction in Wts levels might suffice to explain the overgrowth of fat mutants. As a further test of this possibility, Wts levels were examined in fat dachs double mutants. As the influence of Fat on gene expression and growth is absolutely dependent upon Dachs, if Fat influences growth through modulation of Wts, its influence on Wts levels should be reversed by mutation of dachs. Examination of Myc:Wts staining in fat dachs clones and of Wts protein levels in fat dachs mutant discs confirmed this prediction (Cho, 2006).
Prior observations, including the influences of fat and ds on gene expression, and the ability of the Fat intracellular domain to rescue fat phenotypes, suggested that Fat functions as a signal-transducing receptor. By identifying kinases that act both upstream (Dco) and downstream (Wts) of the Fat effector Dachs and by linking Fat to the transcriptional coactivator Yki, these results have provided additional support for the conclusion that Fat functions as a component of a signaling pathway and have delineated core elements of this pathway from receptor to transcription factor. Fat activity is regulated, in ways yet to be defined, by Ds and Fj. The influences of Fat on gene expression, growth, and cell affinity, as well as on Wts stability, are completely dependent on Dachs, indicating that Dachs is a critical effector of Fat signaling. Since Dachs can associate with Wts or a Wts-containing complex, it is suggested that Dachs might act as a scaffold to assemble a Wts degradation complex. The observations that Fat, Ds and Fj modulate the subcellular localization of Dachs, that Wts is preferentially localized near the membrane and that Dachs accumulates at the membrane in the absence of Fat, suggest a simple model whereby Fat signaling regulates Wts stability by modulating the accumulation of Dachs at the membrane and thereby its access to Wts. The working model is that dco3 is defective in the phosphorylation of a substrate in the Fat pathway, but the recessive nature of dco3, the genetic epistasis experiments, and biochemical experiments argue that this substrate is not Wts, and further work is required to define the biochemical role of Dco in Fat signaling (Cho, 2006).
In addition to identifying core components of the Fat pathway, the results establish close functional links between the Fat pathway, the Hippo pathway and the FERM-domain tumor suppressors Mer and Ex. The common phenotypes observed among these tumor suppressors can be explained by their common ability to influence Wts. However, they seem to do this in distinct ways, acting in parallel pathways that converge on Wts rather than a single signal transduction pathway. The Fat pathway modulates levels of Wts, apparently by influencing Wts stability. By contrast, the Hippo pathway seems to regulate the activity of Wts by modulating its phosphorylation state. Thus, Wts seems to act as an integrator of distinct growth signals, which can be transmitted by both the Fat pathway and the Hippo pathway. It has been suggested that Mer and Ex also act through the Hippo pathway, although present experiments cannot exclude the possibility that Mer and Ex act in parallel to Hpo. Moreover, it should be noted that Mats might regulate Wts independently of Hpo and Sav and hence function within a distinct, parallel pathway. Although it is simplest to think of parallel pathways, there is also evidence for cross-talk. fj and ex are both components and targets of these pathways. Thus, they can be regarded as feedback targets within their respective pathways, but their regulation also constitutes a point of cross-talk between pathways. Another possible point of cross-talk is suggested by the observation that levels of Fat are elevated within Mer ex mutant clones. Although the potential for cross-talk complicates assessments of the relationships between tumor suppressors, the observations that fat, dco3 and dachs affect Warts protein levels in vivo, whereas ex, hippo, sav and mats do not, argues that there are at least two distinct pathways that converge on Warts. This conclusion is also consistent with the observations that ex, hippo, sav and mats can influence Wts phosphorylation in cultured cell assays, but Fat, Dachs and Dco do not (Cho, 2006).
Although the Fat and Hippo pathways converge on Wts, Hippo pathway mutants seem more severe. Thus, hpo, wts or mats mutant clones show a distinctive disorganization and outgrowth of epithelial tissues that is not observed in fat mutant clones, and they show a greater increase in interommatidial cells. This difference presumably accounts for the previous failure to recognize the tight functional link between Fat and Hippo signaling, and it can be explained by the finding that Wts levels are reduced but not completely absent in fat mutant cells. Thus, fat would be expected to resemble a hypomorphic allele of wts rather than a null allele, and consistent with this, a hypomorphic allele, wtsP2, results in strong overgrowth phenotypes. The effects of Yki overexpression on growth and target gene expression can be even stronger than those of fat or wts mutations, which suggests that Yki levels become limiting when upstream tumor suppressors are mutant (Cho, 2006).
fat encodes a protocadherin, which in the past has led to speculation that its influences on growth and cell affinity might result from Fat acting as a cell adhesion molecule. However, all of the effects of fat on growth and affinity require dachs, which is also required for the effects of fat on transcription. Additionally, targets of Fat signaling include genes that can influence growth and affinity; recent studies identified an influence of fat on E-cadherin expression, and as describe in this study, Fat influences CycE and Diap1 expression. Thus, one can account for the influence of fat on growth and affinity by its ability to regulate gene expression. fat interacts genetically with other signaling pathways, including EGFR and Wnt, and in some cells Fat signaling also influences the expression of ligands (such as Wg and Ser) for other signaling pathways. Regulation of these ligands contributes to fat overgrowth phenotypes, but since clonal analysis indicates that fat is autonomously required for growth control in most imaginal cells, the principal mechanism by which fat influences growth presumably involves the regulation of general targets (Cho, 2006).
Normal tissue growth and patterning depend on a relatively small number of highly conserved intercellular signaling pathways. The Fat pathway is essential for the normal regulation of growth and PCP in most or all of the external tissues of the fly and also participates in local cell fate decisions. In this regard, its importance to fly development can be considered comparable to that of other major signaling pathways. Although the biological roles and even the existence of a Fat pathway in mammals remain to be demonstrated, there is clear evidence that the mammalian Warts homologs Lats1 and Lats2 act as tumor suppressors and that a mammalian Yorkie homolog, YAP, can act as an oncogene. Moreover, other genes in the Drosophila Fat pathway have apparent structural homologs in mammals. Thus, it is likely that mammals also have a Fat tumor suppressor pathway that functions in growth control (Cho, 2006).
Studies in Drosophila have defined a new growth inhibitory pathway mediated by Fat (Ft), Merlin (Mer), Expanded (Ex), Hippo (Hpo), Salvador (Sav)/Shar-pei, Warts (Wts)/Large tumor suppressor (Lats), and Mob as tumor suppressor (Mats), which are all evolutionarily conserved in vertebrate animals. The Mob family protein Mats functions as a coactivator of Wts kinase. This study shows that mats is essential for early development and is required for proper chromosomal segregation in developing embryos. Mats is expressed at low levels ubiquitously, which is consistent with the role of Mats as a general growth regulator. Like mammalian Mats, Drosophila Mats colocalizes with Wts/Lats kinase and cyclin E proteins at the centrosome. This raises the possibility that Mats may function together with Wts/Lats to regulate cyclin E activity in the centrosome for mitotic control. While Hpo/Wts signaling has been implicated in the control of cyclin E and diap1 expression, this study found that it also modulates the expression of cyclin A and cyclin B. Although mats depletion leads to aberrant mitoses, this does not seem to be due to compromised mitotic spindle checkpoint function (Shimizu, 2008).
Mats is essential for normal development; mats mutants stop their growth at the second instar larval stage and eventually die. In fact, this growth retardation phenotype facilitated identification of matsroo and matse235 mutant larvae for DNA sequence analysis. Using matse235 allele and the P-element-induced allele matsPB, it has been shown that mats homozygotes and hemizygotes grow slowly and their imaginal discs are much smaller than that of wild-type larvae at the same age. mats mutant cells in mosaic tissues acquire growth advantage likely through comparison and competition with neighboring wild-type cells. In contrast, the absence of wild-type cells in homozygous mats mutant animals renders no competitive growth advantage to mutant cells. The mechanism by which mats mutants acquire growth advantage in the context of mosaic tissue still needs to be investigated. mats mutant embryos missing both maternal and zygotic mats functions failed to hatch, indicating that mats is essential for embryonic development. By analyzing mitotic cells, it was found that maternally mats-depleted embryos show aberrant DNA segregation such that uneven amounts of DNA are segregated toward opposing centrosomes. However, this does not appear to be due to the compromised function of mitotic spindle checkpoint, since mats mutant tissue still accumulate M-phase cells in response to inhibition of mitotic spindle formation by colcemid treatment. Thus, mats is not required for mitotic spindle checkpoint, unlike mps1 (Shimizu, 2008).
Cyclin E is a critical cell cycle regulator. Through a Cdk2-dependent mechanism, cyclin E-Cdk2 plays a critical role in accelerating G1-S transition in the cell cycle. As a general rule, cyclin E is tightly regulated during the cell cycle by Cdk2 and GSK-mediated phosphorylation and subsequent degradation. A nondegradable cyclin E mutant can cause extra rounds of DNA synthesis and polyploidy, and overexpression of cyclin E is frequently detected in tumor cells exhibiting polyploidy. Intriguingly, cyclin E is a centrosomal protein that functions to promote S-phase entry and DNA synthesis in a Cdk2-independent manner (Matsumoto, 2004). Loss of cyclin E expression in the centrosome inhibits DNA synthesis, whereas ectopic expression of cyclin E in the centrosome accelerates S-phase entry. Thus, the centrosome is an important subcellular organelle for cyclin E to regulate cell proliferation, and the level and activity of cyclin E in centrosomes must be tightly controlled. The fact that Mats and Wts colocalize with cyclin E at the centrosome raises the possibility that Mats may function together with Wts kinase to regulate cyclin E function in the centrosome for mitotic control. In support of this hypothesis, loss-of-function mutations in mats increase the levels of cyclin E protein and both gain- and loss-of-function mutant alleles of cyclin E modulate the eye phenotypes caused by Wts overexpression. Although Mats/Wts-mediated inhibition of cyclin E could occur through Yki to regulate cyclin E transcription, a direct control of cyclin E at the protein level would allow a rapid response to an upstream signal (Shimizu, 2008).
The fact that both Mats and Wts show a intracellular localization pattern very similar to that of their respective yeast relatives Mob1 and Dbf2 suggests that their function is conserved. This conservation may extend to mammals; human LATS1, LATS2, and MOB1A (MATS2) also localize at the centrosome. In addition, localization at the bud neck/midbody appears to be conserved in humans. Interestingly, such centrosomal localization of Mats and Wts does not seem to rely on Wts kinase activity as kinase-inactive Wts and Mats can be still localized at the centrosome. To examine whether endogenous Mats protein localizes at the centrosome, embryo immunostaining was done with Mats antibodies. As in larval tissues, expression of Mats protein in developing embryos does not exhibit any obvious pattern and Mats expression level is low and ubiquitous. Although centrosomal localization of endogenous Mats protein has not been shown, likely due to some technical problems, Mats (CG13852/Mob4) has been recently reported to be a centrosomal protein (Shimizu, 2008 and references therein).
Both loss- and gain-of-function analysis supports a model in which cyclin E and diap1 are critical downstream targets of Hpo/Wts signaling. Evidence in this report suggests that Hpo/Wts signaling may also target cyclin A and cyclin B. Consistent with this notion, elevated levels of cyclin B were found in ex mutant cells. In addition, wts has been shown to be required for a negative control of cyclin A but not cyclin B expression. In humans, LATS1 was shown to be a negative regulator of Cdc2/cyclin A and to function at the G2/M-phase transition, while LATS2 affects cyclin E/Cdk2 activity and regulates G1/S phase passage. Thus, the ability of Hpo/Wts signaling to target cyclin genes important for cell cycle progression appears to be evolutionarily conserved (Shimizu, 2008).
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date revised: 10 August 2009
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