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Genetics, Vol. 177, 1667-1677, November 2007, Copyright © 2007
doi:10.1534/genetics.107.078360
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Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
1 Corresponding author: Department of Molecular and Cell Biology, 142 LSA 3200, University of California, Berkeley, CA 94720-3200.
E-mail: bilder{at}berkeley.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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1000-fold to approach their final size. The size of the imaginal disc at the initiation of pupation is a major determinant of the size of the adult organ following metamorphosis. This size is highly regular, reflecting the importance for appropriate physiology and functioning of, for example, the complex optics of the compound eye or the aerodynamics of the wing and haltere flight organs. Thus, tight developmental controls must exist to permit sufficient but not excessive growth of the imaginal discs. Classic and contemporary manipulative studies have been performed to define the general parameters controlling growth of discs. These studies indicate that growth control is largely intrinsic to the disc (BRYANT and SIMPSON 1984). While the bulk of imaginal disc growth takes place in the larvae prior to pupation, transplanted discs can proliferate in other growth-permissive environments, such as adult abdomens. In this context, discs cease proliferation at approximately the appropriate final size, indicating that the mechanisms that terminate growth are disc autonomous and do not require specific systemic or hormonal cues such as those associated with metamorphosis. Consistent with this, artificially extending the larval period by delaying metamorphosis does not in most cases lead to disc overgrowth. Interestingly, some evidence suggests that proliferating disc tissue can itself influence the onset of pupariation in Drosophila as well as in other insects (SIMPSON et al. 1980; BRYANT and SIMPSON 1984). However, details of a potential interaction between disc growth and the timing of metamorphosis remain unclear.
Insight into the mechanisms that act in the disc itself to control organ size has come from the identification of mutations that act cell autonomously to cause increased growth of imaginal tissue. These mutations disrupt a subset of Drosophila tumor suppressor genes (TSGs) and are generally divided into two categories: "hyperplastic" and "neoplastic" TSGs (HARIHARAN and BILDER 2006). Mutations in hyperplastic TSGs lead to larger discs with relatively little effect on epithelial structure and, often, differentiation of the tissue. By contrast, mutations in neoplastic TSGs cause imaginal cells to lose epithelial polarity and usually also block terminal differentiation.
A relatively large number of hyperplastic TSGs have been identified, and studies have assigned many of these into several signaling pathways whose mechanisms of action on cell growth, cell death, and cell cycle regulation are becoming increasingly clear. By contrast, only seven neoplastic TSGs have been reported to date. Three of these—scrib, dlg, and lgl—encode cytoplasmic proteins with various protein–protein interaction domains (JACOB et al. 1987; WOODS and BRYANT 1991; BILDER and PERRIMON 2000). Four others—avl, Rab5, tsg101, and vps25—encode components of the endocytic machinery (LU and BILDER 2005; MOBERG et al. 2005; THOMPSON et al. 2005; VACCARI and BILDER 2005; HERZ et al. 2006). The relationship between the cytoplasmic scaffolds and the endocytic regulators, the mechanism of action of these proteins in cell polarity, and in particular how they each act to restrain cell proliferation remains mysterious. Identification of additional neoplastic TSGs in the Drosophila genome may shed light on these mechanisms.
There has not yet been a systematic attempt to isolate neoplastic TSGs. Any such attempt must cope with the significant maternal contribution of known neoplastic TSGs, which prevents detection of mutant phenotypes in zygotically mutant embryos. While the phenotypes of neoplastic TSGs are dramatic in the larval imaginal discs, only scrib, dlg, and lgl have sufficient maternally provided transcript to enable zygotically homozygous animals to survive to L3, when they grow to be distinctively "giant" larvae (GATEFF and SCHNEIDERMAN 1967; STEWART et al. 1972; PERRIMON 1988; BILDER et al. 2000). Instead, avl, Rab5, tsg101, and vps25 homozygotes die as L1 larvae without obvious phenotypes. The neoplastic phenotypes of these latter genes were detected either in labor-intensive follicle cell screens or in assays dependent on a nonautonomous growth phenotype that is associated with tsg101 and vps25 but not shared by other known neoplastic TSGs. These limitations mean that the question of how many genes in Drosophila act to prevent neoplastic growth remains unaddressed.
We have therefore developed a novel and efficient screen for identifying new neoplastic TSGs, based on analysis of genetic mosaic larvae. Strikingly, while eye discs are not themselves required for viability, our data demonstrate that disruption of known neoplastic TSGs in the eye discs alone induces nonautonomous defects that result in death prior to adult eclosion. These defects are associated with delayed or defective pupation and are consistent with a requirement for diminished disc proliferation prior to the onset of metamorphosis. We exploit this pupation-defect phenotype to carry out a genetic mosaic screen for new neoplastic TSGs. Screening of two chromosome arms using this strategy resulted in the identification of at least seven new neoplastic TSGs and illustrates that many unidentified neoplastic TSGs exist in the Drosophila genome.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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EMS mutagenesis:
Male flies carrying an isogenized FRT chromosome were starved for 8 hr and subsequently fed a 25-mM EMS solution overnight at room temperature. To screen the 2L chromosome arm, mutagenized FRT40 males were mated en masse to Gla/CyO TwiGal4 UAS-GFP females. Single F1 males of the genotype *FRT40/Cyo TwiGal4 UAS-GFP were each crossed to three females of the genotype eyFLP cl GMR-hid FRT40/CyO TwiGal4 UAS-GFP. Absence of non-CyO adults in the F2 progeny indicated a positive MENE phenotype. Mutant chromosomes were then recovered by crossing F2 males of the genotype *FRT40/Cyo TwiGal4 UAS-GFP to Gla/Cyo TwiGal4 UAS-GFP females.
To screen the 3R chromosome arm, mutagenized FRT82 males were mated en masse to females of the genotype Lyra/TM6B. Single F1 males of the genotype *FRT82/TM6B were each crossed to three females of the genotype eyFLP cl GMR-hid FRT 82/TM6C. Absence of non-tubby and nonhumeral F2 adults indicated a positive MENE phenotype. Mutant chromosomes were recovered by crossing F2 males of the genotype * FRT82/TM6C to females of the genotype TM3 hshid/TM6B.
Immunohistochemistry and microscopy:
All dissections were from wandering third instar larvae taken at the onset of pupariation. Fixations were done at room temperature for 20 min in a methanol-free, 4% paraformaldehyde solution. F-actin stains were done using tetramethylrhodamine isothiocyanate-conjugated phalloidin 1:500 (Sigma, St. Louis). Prior to antibody staining, samples were incubated in a blocking solution of 5% normal goat serum. Primary antibody stains were done at 4° overnight, and secondary antibody stains were done at room temperature for 4 hr. Primary antibodies used were rat anti-Elav 1:50 Developmental Studies Hybridoma Bank (DSHB), rabbit anti-aPKC (Santa Cruz) 1:1000, mouse anti-Dlg (DSHB) 1:100, and mouse anti-Mmp1 1:100 (DSHB) (ZHANG et al. 2006). Secondary antibodies from Molecular Probes (Eugene, OR) were anti-rat Alexa488, anti-rabbit Alexa488, and anti-mouse Alexa647, all used at 1:200. All images are single confocal sections taken with a TCS microscope (Leica) using x16/NA 0.5, x40/NA 1.25, or x63/NA 1.4 oil lenses. Images were edited with Adobe Photoshop CS and were assembled with Adobe Illustrator 10.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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We therefore adopted an experimental context in which cell competition was eliminated. Such a context can be found using the eyFLP-cell lethal system, in which a recessive cell-lethal mutation distal to an FRT recombination site is used to eliminate the genotypically wild-type cells that result as the reciprocal recombination product of homozygous mutant cells (STOWERS and SCHWARZ 1999; NEWSOME et al. 2000). We will refer to these discs, which are predominantly composed of homozygous mutant tissue, as "mutant eye discs" to distinguish them from "mosaic eye discs," in which a significant portion of wild-type tissue remains. We compared the phenotypes of mutant eye discs for null alleles of six of the neoplastic TSGs (dlg was not included) to each other and to wild-type eye discs. In neoplastic TSG mutant eye discs, the mutant tissue survives, loses apicobasal polarity, and fails to undergo terminal differentiation (Figure 1, A–G). All of these characteristics are seen in other epithelia mutant for null alleles of neoplastic TSGs, such as the wing imaginal disc and follicle cells, confirming that the use of the eyFLP-cell lethal system confers a strong mutant phenotype (GATEFF and SCHNEIDERMAN 1969; GOODE and PERRIMON 1997; WOODS et al. 1997; BILDER et al. 2000). Interestingly, the amount of overgrowth seen prior to pupariation consistently differed among different genotypes. Eye discs mutant for tsg101 showed the largest overgrowth, whereas eye discs mutant for three different null alleles of scrib were in fact smaller than wild type. Nevertheless, the epithelial disorganization and impaired differentiation was consistent for all of the neoplastic TSG mutations, confirming this as a reliable assay for neoplastic TSG detection.
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The MENE phenotype results from effects on imaginal discs:
To test whether the MENE phenotype was specifically induced by neoplastic growth in the eye disc, we employed an alternative FLP recombinase driver that is expressed in other imaginal discs. UbxFLP drives recombination in a portion of the wing, haltere, and leg discs at early larval stages, with little recombination evident in eye discs (HUTTERER and KNOBLICH 2005) (supplemental Figure S1G at http://www.genetics.org/supplemental/; data not shown). We built UbxFLP-cell lethal stocks and assessed the degree of recombination in wing discs by crossing to chromosomes carrying a distinct set of cell-lethal mutations, reasoning that recombinant tissue homozygous for either cell-lethal mutation would be eliminated. The wing discs in the resultant larvae were partially reduced (Figure 2C) and the overall morphology was retained, indicating that, unlike in the eyFLP-cell lethal system (Figure 2B), recombinant cells do not occupy most of the disc and therefore significant wild-type tissue remains. We then crossed UbxFLP-cell lethal flies to flies carrying known neoplastic TSG mutations. While eye discs from the resultant larvae were normal (data not shown), wing discs contained regions of neoplastic growth (Figure 2, E–G). Since UbxFLP-driven recombination does not occur in the entire wing disc, homozygous mutant cells are still subject to cell competition, and accordingly the disc phenotype was less extreme than that seen with eyFLP-cell lethal. Nevertheless, in all cases no adult eclosers were seen, indicating that recombination of neoplastic TSG mutations driven by UbxFLP as well as eyFLP can block pupation.
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Not all MENE mutants cause overgrowth:
Having established that neoplastic TSG mutants can cause the MENE phenotype, we wondered whether other classes of mutations could do so as well. As an initial assessment of this possibility, we analyzed animals containing eye discs mutant for several genes essential for cell viability (see MATERIALS AND METHODS) or that act as hyperplastic TSGs (pten, salvador). In both cases, adults eclosed, albeit at sub-Mendelian rates, with eyes that were reduced or overgrown, respectively. We therefore anticipated that known hyperplastic TSGs and cell-lethal mutations were unlikely to cause significant background of pre-eclosion lethality in a MENE screen.
We first conducted a pilot screen for MENE phenotypes among a collection of transposon-induced mutations that have been placed on FRT chromosomes (CHEN et al. 2005). We crossed 635 of these lines to eyFLP-cell lethal stocks and identified 44 lines that displayed the MENE phenotype (supplemental Table S1 at http://www.genetics.org/supplemental/). Among this collection we found alleles of Rab5 as well as lgl that caused neoplastic growth of eye imaginal discs, validating the screen design. Surprisingly, the majority of the other lines showed either wild-type discs or smaller discs, and, unlike Rab5 and lgl, did not prevent eclosion when crossed to UbxFLP-cell lethal flies. Inspection of the transposon insertion sites in these lines suggested that a diverse set of genes can be disrupted to cause the MENE phenotype without inducing overgrowth. This pilot screen established that, while loss of growth control is one of multiple pathways that can induce the MENE phenotype, the strategy can indeed be used in a forward genetic screen to detect neoplastic imaginal disc phenotypes.
A genetic screen for pre-eclosion lethality in mosaic larvae:
The MENE phenotype, characterized by penetrant pre-eclosion lethality, is seen with all neoplastic TSG mutations in the eyFLP-cell lethal system. This finding provided an opportunity to use this easily assayed phenotype as a proxy to identify new neoplastic TSGs. To screen for new mutations that caused the MENE phenotype (Figure 3), we first used EMS to mutagenize male flies that carry an isogenized chromosome with an FRT recombination site near the centromere. These males were mated en masse to virgin females carrying a balancer for the mutagenized chromosome; this balancer carried a dominant marker (either Tubby or a GFP transgene) such that its presence could be assayed in larval stages. Individual balanced male F1 progeny of this cross, carrying independent mutations on the FRT chromosome, were then crossed in vials to virgin females of a tester line. The tester carries the eyFLP recombinase driver and an autosome with both a matched FRT site and a recessive cell-lethal mutation, all in trans to a dominantly marked balancer chromosome. Each vial was ultimately scored for the presence of nonbalanced F2 progeny. Vials lacking such progeny were considered a "hit" and evaluated, using the markers present on the balancer chromosomes to determine whether nonbalanced pupae, puparia, or larvae were present. Although the animals with mutant eye discs in "hit" vials were dead, the mutant chromosome was recovered in balanced siblings and used to establish a stock.
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The distribution within the classes was as follows: 55% gave no consistent defect (class IV), 29% showed a small disc phenotype (class III), and 6% gave a consistent phenotype that did not alter disc size (class II), while 6% gave apparent hyperplastic overgrowth (class Ia) and 4% gave apparent neoplastic overgrowth (class Ib). Examples of the class Ia (hyperplastic) mutants are shown in Figure 4, A and B. Phenotypes of the class II mutants included discs that showed general defects in epithelial organization (Figure 4, C and D), severely impaired photoreceptor differentiation (Figure 4, E and F), excess axonal fibers (Figure 4, G and H), and increased actin polymerization (Figure 4, I and J). Examples of the class III (small disc) mutants are shown in Figure 4, K and L. Distinctive phenotypes allowed us to identify three alleles of capulet among the elevated actin mutants (Figure 4J) and two alleles of kuzbanian among the small disc mutants (Figure 4K). As in the pilot screen, the majority of mutations that give a MENE phenotype do not cause the production of larger imaginal discs, confirming that loss of growth control is only one of multiple pathways that can induce the MENE phenotype.
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Initial categorization of MENE mutants in the secondary screen was based on actin staining of imaginal discs, which provides only a general sense of tissue architecture and differentiation. To assess whether the class Ib mutations truly showed neoplastic phenotypes, we examined three characteristics more closely. First, we tested for the presence of Elav immunoreactivity, which reflects differentiation into a neuronal fate and found that Elav was absent in the majority of disc tissue mutant for all eight complementation groups (Figure 6, A–C). Second, we examined the distribution of markers for the polarized apical (atypical protein kinase C) and basolateral (large disc) membrane domains and found that seven of the complementation groups showed an expansion of apical markers that were intermixed with the basolateral domain (Figure 6, F and G). Finally, we asked whether the mutant discs expressed matrix metalloprotease 1 (Mmp1), which is upregulated in tissue mutant for known neoplastic tumor suppressors (PAGE-MCCAW et al. 2003; UHLIROVA and BOHMANN 2006; BEAUCHER et al. 2007; SRIVASTAVA et al. 2007; our unpublished results), and found upregulation of Mmp1 in tissue mutant for seven of the complementation groups (Figure 6, K and L). Expanded apical domains, loss of terminal differentiation, and upregulation of Mmp1, shown for a MENE(3R)-A allele in Figure 6, are all characteristic phenotypes caused by known neoplastic TSG mutations. We therefore conclude that seven of the complementation groups represent bona fide neoplastic TSGs that have not been previously identified.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The MENE screen reported here, which covered 40% of the genome, identified at least seven clear complementation groups that represent new nTSGs. While not saturating, the isolation of multiple alleles of known neoplastic TSGs as well as most new complementation groups suggests that we have identified many of the genes on 2L and 3R that can mutate to this phenotype. All of the new mutations show an expansion of apical membrane domains in the overgrowing disc tissue, providing further evidence for the close coupling between polarity and proliferation control in Drosophila. The seven complementation groups identified here now double the number of known neoplastic TSG loci, which had previously been identified using various alternative strategies. Notably, lethal-phase analysis indicates that homozygotes for all seven new complementation groups die during early larval stages and would not have been identified in zygotic "giant larvae" screens. Moreover, in preliminary experiments we have been unable to recover follicle clones for MENE(3R)-A and -E; these alleles at least would have been missed in follicle cell-based screens. Phenotypic and molecular analysis of the mutants is ongoing and will reveal whether the affected genes act through mechanisms similar to the junctional scaffold proteins, the endocytic regulators, or reveal additional pathways that shed light on why normal epithelial structure is required for disc size control.
Imaginal disc proliferation and metamorphosis:
In this article, we describe a genetic screening strategy that uses defective pupation as a proxy phenotype to detect mutations that cause imaginal disc overgrowth. Previous work has suggested that the presence of dividing disc cells in an L3 larva can interfere with pupation (SIMPSON et al. 1980). An associated mechanism has not been uncovered but presumably would involve a humoral factor that acts on the neuroendocrine axis to inhibit production of metamorphosis-promoting hormones. In this manner, the autonomous growth control mechanism of the imaginal disc could be coupled to the timing and coordination of metamorphosis throughout the animal. Thus, attainment of proper disc size, as assessed by cessation of most disc cell proliferation, could be used as a "checkpoint" to be cleared before initiating the irreversible process of pupation. In the MENE screen, we suggest that the presence of excess cell division or continually dividing cells ectopically activates this checkpoint to delay or disrupt the normal process of pupation.
The formation of giant L3 larvae along with delayed or defective pupation is a well-known phenotype of animals homozygous for many TSG mutations, including members of both the neoplastic and the hyperplastic classes (GATEFF and SCHNEIDERMAN 1967; STEWART et al. 1972; TAO et al. 1999; BILDER et al. 2000; STEWART et al. 2003; READ et al. 2004). In such animals, all tissue is mutant, and overgrowth often occurs in the brain as well as in the eight pairs of imaginal discs. Our results with the eyFLP-cell lethal system show that a failure to cease proliferation in the pair of eye discs alone can impair pupation in the entire organism, and in some cases induce a giant larva phenotype resembling that of zygotically homozygous mutant animals. We also observe a correlation between the amount of overgrowing tissue and the stage of defective pupation, in which large overgrowths block pupariation whereas small overgrowths do not visibly alter pupation until pharate stages. These results recall those seen with experiments examining regeneration of imaginal discs in irradiated larvae, where the degree of pupation delay correlates with the amount of regenerating tissue (SIMPSON et al. 1980). Interestingly, those experiments also pointed to a threshold amount of proliferation required to induce pupation delay; that threshold—one pair of discs—is met by our screening strategy. Our results thus support a model in which a signal emanating from proliferating disc cells can repress pupation and cessation of proliferation releases this checkpoint (SIMPSON et al. 1980; ZITNAN et al. 1993).
Although the MENE screen was effective in isolating genes controlling imaginal disc growth, the majority of MENE mutations did not cause oversized discs. What occurs in these instances to induce pupation defects? While many cases may involve unrelated pathways, it is interesting to speculate that some cases may nevertheless involve cell proliferation. One possibility is that certain mutations that make small discs, or discs with altered epithelial organization, contain both dying as well as continually proliferating cells, and it is the presence of these proliferating cells that activates the pupation checkpoint. An alternative possibility is that certain mutations disrupt communication between growing imaginal discs and the hormonal system that controls metamorphosis. If proliferating disc cells secrete a signal that inhibits pupation, then mutations in negative regulators of such a signal would fail to release this checkpoint. Interestingly, several mutations isolated in the screen produce normal imaginal discs but still block pupation in both the eyFLP-cell lethal and UbxFLP-cell lethal systems. Further characterization of the MENE mutants may shed light on the mechanisms by which imaginal tissue and the neuroendocrine axis that controls pupation regulate each other.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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