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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Fly (Austin). Author manuscript; available in PMC 2010 July 21.
Published in final edited form as:
Fly (Austin). 2010 April; 4(2): 104–116.
Published online 2010 April 24.
PMCID: PMC2908041

The gang of four gene regulates growth and patterning of the developing Drosophila eye


We report here the identification of a novel complementation group in the fruit fly Drosophila melanogaster named gang of four (gfr). Mutations in gfr disrupt patterns of cell differentiation in the eye and increase eye size through a proliferative mechanism that can be enhanced by a block in apoptosis. gfr mutant cells show several features of deregulated Ras/MAP kinase activity, including reduced expression of the Capicua growth suppressing transcription factor and synthetically lethality with alleles of the Jun N-terminal kinase phosphatase puckered. gfr alleles also upreguate Notch activity in the eye. Thus, gfr alleles appear to elicit growth and patterning phenotypes via effects on multiple signaling pathways. Moreover, the gfr alleles behave as gain-of-function lesions and overexpress the gene, bruno-3 (bru-3), which is located at the genomic region to which gfr lesions map. Genetic reduction of bru-3 suppresses phenotypes caused by gfr alleles, and like gfr alleles, overexpression of bru-3 depresses levels of Cic protein, indicating that overexpression of bru-3 is central to gfr mutant phenotypes.

Keywords: Drosophila, eye, growth, Notch, MAPK, puckered, bru-3


Genetic screens in the fruit fly Drosophila melanogaster have identified many genes that are required to restrict the growth of developing tissues (reviewed in refs. 1 and 2). Some of these genes exclusively control the process of tissue growth by changes in cell number or cell size. However, since a small number of signaling pathways are used reiteratively during metazoan development to control different processes, it is perhaps not surprising that others of these growth mutants exhibit more complex patterning phenotypes indicative of roles in multiple developmental pathways. Indeed, most developing Drosophila organs are patterned by a combination of signals from factors such as Hedgehog, Wingless, Notch, Decapentaplegic (Dpp), and Epidermal Growth Factor (EGF),3 yet it is also well established that mutations that affect these pathways can produce ectopic tissue growth in Drosophila and drive cancers in humans.4,5

Here we report the isolation of a complementation group called gang of four (gfr) that displays effects on multiple cell biological processes, including tissue growth and retinal patterning. gfr alleles were identified in an eyFLP;FRT mosaic screen in the Drosophila eye for mutations that confer a clonal growth advantage relative to wild-type tissue.610 The gfr gene also controls the overall size of the adult head and the specification of certain retinal cell types in the developing eye. Genetic and molecular data argue that this growth advantage arises by an increased rate of growth rather than a decrease in cell death and that the gene acts on Notch and JNK/ERK MAP kinase (MAPK) signaling pathways—pathways that play dual roles in cell fate and cell proliferation control.1113 gfr alleles interact very strongly with alleles of the JNK phosphatase puckered (puc), and gfr mutant cells show several features of deregulated ERK activity, including reduced expression of the Capicua (Cic) growth suppressor.10 A subset of gfr growth and patterning phenotypes may thus arise due to a requirement for the gene as a MAPK regulator. Finally, although multiple alleles of the gfr locus were recovered in the original screen, these alleles do not appear to behave as simple recessive, loss-of-function lesions. Deletions spanning the region to which gfr alleles map fully complement gfr mutant chromosomes. gfr alleles also overexpress the bruno-3 (bru-3) gene, which maps to the same genetic interval as the gfr lesions and encodes an RNA-binding protein that can bind to the EDEN translational repression sequence and drive overproliferation of cells in the hemocyte lineage.14,15 Genetic reduction of bru-3 suppresses phenotypes caused by gfr alleles, and like gfr alleles, overexpression of bru-3 depresses levels of Cic protein. In sum, these data suggest that gfr alleles behave as gain-of-function lesions and that overexpression of bru-3 is central to gfr mutant phenotypes.


gang of four alleles regulate growth in the developing eye

To identify genes that regulate cell growth or cell number during development of the Drosophila melanogaster eye, the eyFLP;FRT system of mitotic recombination was used to screen for mutations that allow homozygous mutant cells to overgrow relative to their wild-type neighbors.610 Flies whose eyes were composed of more mutant than wild-type tissue were retained and placed into complementation groups. In addition to alleles of genes such as Tsc1, salvador, and hippo, clones of which result in overgrowth of the entire eye (reviewed in ref. 1), mutations in genes such as archipelago and capicua, which produce clonal overgrowth without overt organ hyperplasia, were also identified.8,10 Four mutations of the latter type were found to represent a single complementation group that was named gang of four (gfr). A fifth allele, gfrx, was independently isolated by its ability to synergize with a block in cell death to produce clonal overgrowth in the eye (Moberg KH, Gilbert MM, unpublished; see below). Each allele is recessive lethal over itself and in trans to other gfr alleles; some gfr/gfr animals die as L1 larvae, indicating that the gfr alleles lead to late embryonic/early larval death. After extensive meiotic recombination mapping using several P-element insertions (see Materials and Methods), the lethality of the gfr1 allele was mapped on chromosome 3L to 0.39 cM (18 recombinants; 4593 scored) from KG01069 (located at 13.23 Mb; FlyBase) and 0.82 cM (36 recombinants; 4412 scored) from BG00690 (located at 13.48 Mb; reviewed in ref. 16) (Table S1), confirming that gfr mutations are tightly linked to this chromosomal region. Though all gfr alleles are lethal in trans to each other, they are viable with no obvious defects in trans to the molecularly defined Df(3L) ED4502 (13.2–13.95 Mb), which spans the putative gfr region on 3L, and are also viable in trans to all available lethal alleles in this region. As Df(3L)ED4502 fails to complement all other tested lethal alleles in the region (CKB, data not shown), the lack of an interaction between Df(3L)ED4502 and the gfr alleles is not likely due to incomplete coverage of the region but instead to a failure of gfr alleles to behave as simple recessive, loss-of-function lesions (see below).

Adult gfr mosaic eyes generated using the eyFLP;FRT technique show increased representation of gfr mutant tissue (white) over control tissue (red) (Fig. 1A and B), and ommatidia within these clones can contain either multiplication or loss of interommatidial bristles (Fig. 1C). To examine how gfr mutations might increase clonal growth in the eye, patterns of expression of the major mitotic cyclins, Cyclins A, B, E and D, were analyzed in gfr mosaic larval eye discs. Compared with surrounding wild-type tissue, levels of the mitotic regulator Cyclin A are increased in gfr clones in the asynchronously dividing cells anterior to the morphogenetic furrow (MF); Cyclin A also perdures past the point of wild-type expression in mutant clones posterior to the MF (Fig. 2A–A″ and B–B″). A similar, although more mild, version of this effect is observed with Cyclin B (Fig. 2C–C″). Expression of the S-phase inducer Cyclin E is elevated in gfr clones within the MF and also perdures just posterior to the MF; clones anterior to the MF contain a higher density of Cyclin E-positive cells compared with surrounding wild-type tissue (Fig. 2D–D″). Patterns of Cyclin D were not substantially altered in gfr clones (data not shown). Compared with the parental chromosome (Fig. S1A and D), gfr mutant clones show relatively normal patterns of S phase as visualized by BrdU incorporation in the larval and 24-hour pupal eye (Fig. S1B–B′ and C–C″) and of mitosis as assessed by staining with anti-phospho-Histone H3 (Fig. S1E–E′). Thus, despite their effects on cyclin expression, gfr alleles have relatively little effect on patterns of replication and mitosis. This is confirmed by flow cytometric data showing that the size and cell cycle phasing of gfr mutant cells are very similar to that of wild-type cells at this stage (data not shown).

Figure 1
gfr mutations confer a clonal growth advantage in the adult eye. Light microscopic images of adult eyes mosaic for the parental FRT chromosome (A) or gfr (B). Red pigment marks wild-type cells; white tissue is homozygous for FRT (A) or gfr (B and C). ...
Figure 2
gfr mutant tissue has increased levels of Cyclins A, B and E. Merged confocal sections of gfr1 clones marked by the absence of GFP (green) and stained for Cyclin A (A–A″, B–B″), Cyclin B (C–C″), or Cyclin ...

gang of four alleles affect patterning of the developing eye

The adult bristle defects in gfr mutant eye clones indicate that gfr mutations affect the generation or survival of cells that make up the mature bristle complex. The four cells of the mature bristle complex are clonally derived from a single precursor and are specified by a Notch-dependent process during early pupal development (reviewed in ref. 17). Analysis of Senseless, which marks sensory organ precursor cells,18 at 24 hours after pupariation formation (APF) and BarH1, which marks primary pigment cells and a subset of bristle cells,19 at 48 hours APF, shows a disruption of the normal organization of bristle complexes in gfr clones: in wild-type tissue, bristle precursors are arranged into a linear array, whereas in gfr mutant tissue, this pattern is disrupted by changes in both the number and location of Sens- and BarH1-positive cells (Fig. 3A–A″ and B–B″). Visualizing cell outlines in the mid-pupal eye with anti-Discs large (Dlg) reveals that the bristle complexes and tertiary pigment cells are no longer located at alternating vertices, and the hexagonal lattice created by secondary pigment cells is disrupted (Fig. 3B–B′). Visualizing BarH1 more apically also reveals a disruption in both ommatidial rotation and the number of primary pigment cells: in wild-type tissue, primary pigment cells are found at the top and bottom of each ommatidium creating a linear pattern over the disc, whereas the primary pigment cells in gfr mutant ommatidia are often missing or in inappropriate locations around the ommatidium (Fig. 3C and C″). Moreover, many of the gfr mutant ommatidia have fewer than the wild-type complement of four cone cells (Fig. 3C and C′). Analysis of both Sens and the cone-cell marker Cut in third-instar larval eye discs reveals no changes in the number or pattern of Sens-positive or Cut-positive cells in gfr mutant tissue (data not shown), suggesting that gfr-mediated defects in retinal patterning occur after the third-instar stage of eye development.

Figure 3
Patterning of the pupal retina is disrupted in gfr clones. (A–C″) Merged confocal sections of gfr1 clones marked by the absence of GFP (green). (A–A″) 24-hour mosaic pupal eye disc stained for Sensless (red). (B–B″) ...

The absence of cone cells in gfr clones could be an indirect consequence of the apoptotic cell death of properly specified cell types or a more direct effect of gfr mutations on the specification or recruitment of these cells into the retina during the pupal stage. To distinguish between these two possibilities, cell outlines and bristle cell complexes were visualized in gfr mutant clones 48 hours APF in a background in which cell death is blocked by simultaneous loss of the Df(3L)H99 genomic deletion, which removes the pro-apoptotic genes rpr, grm and hid.20 If gfr mutations lead to the death of properly specified cells, then the cell fate and patterning defects observed in gfr clones should be rescued in gfr, H99 double mutant clones. However, blocking cell death did not rescue the reduced number of cone cells or the disrupted ommatidial patterning in gfr clones (Fig. 3D–D′). The H99 deficiency also does not rescue bristle complex number or organization as visualized by anti-BarH1 staining (Fig. 3D″). Similar data were obtained using the UAS-p35 transgene to block cell death (data not shown). Thus, mutations in gfr alter cell number and patterning in the developing pupal eye by a cell death-independent mechanism.

gfr mutations confer a growth advantage and synergize with a block in cell death

Based on the forward scatter data for gfr cells in larval eye discs (data not shown) and the relative size of Dlg-positive apical profiles in gfr and control areas of pupal eye discs (e.g., Fig. 3B′ and C′), gfr mutant cells are not significantly different in size from normal cells. The apparent growth advantage conferred by the gfr alleles might therefore result from increased cell proliferation, decreased cell death, or a combination of these processes. To test in more detail if gfr alleles affect these processes, we first determined whether loss of gfr could alter organ size. This was accomplished by using the eyFLP;FRT system in combination with the cell-lethal Minute (M) mutation RpL141 to generate eyes and head cuticle composed almost entirely of gfr mutant tissue (reviewed in ref. 21).22 The heads of gfr/M(3) adult female flies are consistently larger than control FRT80B/M(3) heads generated from the FRT80B parental chromosome (Fig. 4A and B); simultaneously making the head mutant for gfr and blocking death with Df(3L)H99 promotes an even greater organ overgrowth (Fig. 4C). Quantification of the en face two-dimensional eye area normalized to an invariant body metric (size of wing compartment bounded by L3 and L4 veins and posterior cross-vein) showed that homozygosity for the gfr1, gfr2 or gfrx alleles led to a statistically significant increase in eye size of approximately 10% relative to FRT80B (gfr1 = 0.45 ± 0.01, gfr2 = 0.43 ± 0.01, gfrx = 0.43 ± 0.01 vs. FRT80B = 0.39 ± 0.01; p < 0.001; Fig. 5F). Combining a gfr allele with the H99 deletion indeed produced an increase in adult eye size: gfrX, H99/M(3) eyes are an additional 9% larger than gfrx/M(3) eyes (0.47 ± 0.02 vs. 0.43 ± 0.01, respectively; p < 0.001; Fig. 5B, C and F) and 15% larger than H99/M(3) control eyes (p < 0.001; Fig. 5F). This synergistic effect between gfr and a block in cell death suggests that a proliferative mechanism underlies the growth advantage of gfr mutant cells. Likewise, gfr alleles do not obviously alter rates of developmental cell death in larval or pupal eye discs (data not shown). Thus, the data collectively support the hypothesis that gfr alleles promote growth and differentiation phenotypes via a primarily proliferative mechanism.

Figure 4
Mutations in gfr increase organ size and synergize with a block in cell death. Composite image of (A) FRT80B/M(3), (B) gfrx/M(3) and (C) gfrxDf(3L)H99/M(3) adult female heads. Black brackets in (A–C) are standardized to the width of the gfrxDf(3L)H99/M(3) ...
Figure 5
Modification of gfr eye size by growth regulators and signaling components. Light microscopic images of (A) FRT80B/M(3), (B) gfr1/M(3), (C) gfrxDf(3L)H99/M(3), (D) N54/9/+;gfr1/M(3) and (E) gfr1, FRT80B: Ras85De2f/M(3) adult female eyes. Note the eyes ...

gfr alleles have Notch gain-of-function phenotypes

To assess the tissue specificity of the gfr overgrowth phenotype, we utilized the cell lethal technique in conjunction with the pan wing FLPase transgene Ubx>Flp to generate wings composed almost entirely of gfr mutant cells. As previously described, this technique uses the RpL141 Minute (M) mutation to eliminate M/M cells and allow gfr/gfr cells to populate the wing disc and adult structures derived from it.22 gfr1/M(3) wings display patterning defects—most notably, the L5 vein does not reach the margin (~70% penetrance, n = 10) (compare Fig. 6A and B)—but unlike gfr/M(3) eyes, they are consistently smaller along the proximal-distal axis relative to FRT80B/M(3) wings (Fig. 6C, quantification in Fig. S2). The effect of gfr alleles on organ size is thus tissue-specific.

Figure 6Figure 6
gfr alleles confer tissue-specific growth advantage but have general Notch gain-of-function phenotypes. (A–F) Light microscopic images of female adult wings of indicated genotypes. (A–C) gfr1/M(3) wings (B) have a growth disadvantage relative ...

The interrupted vein phenotype in gfr/M(3) wings resembles wing-vein phenotypes associated with Abruptex (Ax) alleles, which contain gain-of-function mutations in the extracellular domain of Notch that alter its substrate specificity.23 To determine if gfr and Notch interact genetically in wing development, gfr alleles were tested for their ability to dominantly modify the characteristic Notch loss-of-function wing notching phenotype (Fig. 6E; and reviewed in ref. 24). Compared to the FRT80B control, gfr alleles dominantly suppress wing notching in N54l9/+ adult female flies (p < 0.001; Fig. 6D–G). Although gfr alleles behave as genetic antagonists of Notch in the wing, the alleles do not have a clear molecular effect on Notch activity: molecular markers of increased Notch activity in the wing, including Cut and Wingless protein expression, are not significantly altered in gfr mutant tissue as detected by immunofluorescence (data not shown). Considered together with the differential effects of gfr alleles on eye and wing organ size (see Figs. 5A, B, F and 6A–C), these data further indicated that gfr activity is tissue-specific. We, therefore, focused on the ability of gfr to affect eye size and sought to determine whether elevated Notch activity might be a consistent feature of gfr mutant cells in the eye. Indeed, activity of the Notch reporter E(spl)mβ-CD2 is increased in gfr clones in both posterior (Fig. 6H–H″) and anterior (Fig. 6I–I″) regions of the larval eye imaginal disc.25 This effect on pathway activity occurs independently of an effect on overall Notch levels as detected with an antibody that recognizes the Notch intracellular domain (Fig. 6J–J″). While Notch signaling affects both proliferation and patterning in the Drosophila eye (reviewed in ref. 3), the N54l9 allele was unable to dominantly modify either the gfr enlarged eye phenotype (see Fig. 5F) or the bristle defects apparent in gfr clones on the surface of the adult eye (data not shown), suggesting either that the effect of gfr on Notch activity is too strong to be sensitive to halving the genetic dosage of Notch or that a Notch-independent pathway contributes to these gfr phenotypes.

gfr interactions with the puc phosphatase

In testing the effect of gfr alleles on other pathways that are active in developing discs, we found evidence of a strong genetic interaction between gfr and the dual specificity phosphatase puckered (puc). puc acts in an inhibitory feedback loop to antagonize the Jun N-terminal kinase (JNK) pathway, components of which also interact genetically with the Notch pathway (reviewed in ref. 26). While puc or gfr heterozygotes are completely viable with no obvious phenotypes, each gfr allele is fully lethal in trans to either of two different puc loss-of-function alleles, pucE69 (reviewed in ref. 27) and pucA251 (BDSC), at 25° (0% viability; Table 1); this lethality was not dependent on the parent-of-origin of puc and gfr alleles. Because loss of puc can increase JNK-dependent apoptosis,28 we tested whether reducing the genetic dosage of Df(3L)H99 pro-apoptotic genes in the background of gfr/puc trans-heterozygotes might affect gfr/puc lethality. However, the gfrx, H99 chromosome is still completely lethal in trans to either puc allele (Table 1). Thus, gfr/puc synthetic lethality is not likely due to a non-specific increase in apoptosis, but instead to a specific role for gfr in a puc-dependent developmental mechanism.

Table 1
Summary of genetic interactions among gfr, puc and bru-3

To characterize the interaction between puc and gfr further, we tested whether puc alleles share properties of gfr alleles. Like gfr, pucE69 and pucA251 dominantly suppressed the wing notching observed in N54l9/+ females by approximately 30% (p < 0.001; Fig. 6G), indicating gfr and puc normally operate in the same direction in this wing modification assay and supporting further the specificity of the gfr-puc genetic interaction. Because puc is both a transcriptional target and negative regulator of the JNK pathway, mutations that inactivate puc also result in increased puc transcription.27 To test the hypothesis that gfr alleles phenocopy the effect of puc alleles on JNK targets, we measured puc transcript levels in gfr mutant larval eye imaginal discs by quantitative RT-PCR (qPCR). This analysis found a significant 2- to 6-fold increase in puc transcript in gfr mutant eye discs (Fig. 7A). Thus, gfr regulates puc transcript abundance in the eye disc. Transcript levels of dpp, a second JNK target,29 are only mildly increased (1.2- to 2.3-fold increase) in the same genetic backgrounds (Fig. 7B). Despite these strong genetic data that link gfr to puc, a lethal allele of Drosophila JNK, bsk1,30 does not dominantly modify the gfr/M(3) enlarged eye size (see Fig. 5F). Moreover, gfr mutations do not affect phospho-Bsk levels in western blots of whole eye/antennal discs (Fig. S3A) and do not alter levels or localization of Drosophila Jun (dJun/Jra) in mosaic eye discs (Fig. S3B–B″). Thus, although gfr behaves genetically as a JNK pathway component, it may regulate this pathway either downstream of or in parallel to Bsk and Jra. Together these data can be interpreted to support a model in which gfr alleles elevate JNK pathway activity, and this in turn elevates puc expression as part of the established feedback inhibition mechanism; compound heterozygosity for gfr and puc might then synergistically elevate JNK activity beyond a threshold compatible with viability. However, elevated puc transcript levels observed in gfr eye discs could also indicate that mutations in gfr suppress JNK signaling by increasing puc transcript abundance.

Figure 7
Expression of puc and dpp are elevated in gfr mutant tissue. Quantitative real-time PCR analysis of the expression of (A) puc and (B) dpp mRNAs in FRT80B/M(3) control or gfr/M(3) third instar eye/antennal discs. Error bars represent 95% confidence intervals. ...

gfr alleles decrease Cic levels

JNK signaling controls apoptosis in larval discs and is implicated in the localized outgrowth of groups of cells in the larval wing disc,28,31 but JNK signaling alone is not known to elicit organ-wide hyperplasia. To address the question of how gfr alleles cause eyes to become enlarged, we searched for links between gfr and several known growth-regulatory pathways. Molecular phenotypes in the eye link gfr to the Ras/ERK MAPK module: levels of Capicua (Cic), an HMG-box transcriptional repressor that is destabilized by pro-growth signals from the Ras/ERK MAPK cascade,10,32 are decreased in gfr clones throughout the larval eye imaginal disc (Fig. 8A–A″). The drop in Cic levels in the eye disc is not accompanied by a change in cic transcript levels as detected by qPCR (data not shown). Cic is also decreased in gfr clones in the wing imaginal disc (Fig. S4A–A″), suggesting that while the growth advantage conferred by gfr alleles is specific to the eye, the molecular consequences of gfr mutations may not be. Thus, gfr alleles phenocopy Ras/ERK-induced reduction of Cic levels. Interestingly, cic alleles were also isolated in an eye-specific FLP;FRT screen for overgrowth mutants similar to that which produced the gfr complementation group.10 cic alleles show a relatively subtle growth phenotype similar to gfr but do not affect retinal cell patterning, indicating that while gfr may regulate growth via Cic, it must also act upstream of factors involved in retinal patterning.

Figure 8
Cic is decreased in the eye due to gfr mutation or bru-3 overexpression. (A–A″) Merged confocal sections of gfr third instar larval eye disc clones marked by the absence of GFP (green) and stained with Cic (red). (B) Western blot for Cic ...

In view of the genetic link between gfr and puc and the possibility that the JNK MAPK module might regulate Cic levels in parallel to Ras/ERK, we tested whether clonal loss of puc could also downregulate Cic in eye disc cells. No effect on Cic was observed in pucE69 larval eye clones (Fig. S4B–B″). However, we also tested whether cic alleles could dominantly influence puc viability; indeed, although not nearly as strong as the synthetic lethality observed with gfr alleles, the pucE69/cicQ474X trans-heterozygous combination shows an approximate 35% reduction in viability (Table 1).

In consideration of the effect of gfr on Cic, we tested whether the gfr enlarged eye phenotype was dependent on wild-type dosages of cic and two genes predicted to control Cic through the Ras/ERK pathway, Ras85D and rolled (rl, Drosophila ERK).33,34 While heterozygosity for cicQ474X does not show a strong enhancement gfr eye size, gfr1/M(3) eyes generated in backgrounds heterozygous for the alleles Ras85De2f or rl10a are significantly smaller in size than gfr1/M(3) eyes alone (p < 0.001; Fig. 5B, E–F). These effects are fairly specific: with the exception of an allele of the Warts/Hippo pathway transcriptional effector yki, ykiB5,35 alleles of Notch, bsk, and the mitotic cyclin-dependent kinase cdc2, do not significantly modify gfr/M(3) eye size (Fig. 5F). Additionally, the Ras85De2f/+ genotype was unable to rescue BarHI and Dlg phenotypes in the pupal eye (Fig. 3E–E″). Thus, although activated Ras can result in reduced cone cell numbers,36,37 Ras85D is more strongly required for the effects of gfr alleles on growth than their effects on cellular patterning. In sum, the eye overgrowth produced by gfr alleles correlates with reduced levels of Cic and is sensitive to the dose of two genes, Ras85D and rl, which act within a pathway that promotes clonal overgrowth by repressing Cic levels.10 The significance of the additional effect of the ykiB5 allele on gfr eye size is not clear, but considered with the puc, Notch and cic data, it may indicate that gfr alleles elicit growth and patterning phenotypes via multiple signaling pathways.

gfr alleles are gain-of-function for bru-3

While cellular and molecular phenotypes associated with gfr mutations are quite apparent, the gene representing the gfr complementation group is not identified. As previously described (see above and Materials and Methods), gfr1 lethality is tightly linked to cytological position 70A-B on chromosome 3L. However, gfr alleles do not behave as loss-of-function mutations: they are lethal in trans to each other, yet completely viable and without obvious phenotypes in trans to all available deficiencies and lethal lesions mapping to the 70A-B chromosomal region. Direct sequencing of both coding and non-coding regions of many candidate genes in the area failed to identify any DNA lesions that might cause gain-of-function effects (data not shown). Because the gfr alleles may be regulatory in nature, multiple candidate genes near the P-elements that are most closely linked to gfr lethality (KG01069 and BG00690), including the CG10133, CG17689, CG10089 and bruno-3 genes, were also tested for changes in expression levels by qPCR. Of these, only bruno-3 (bru-3) expression was changed: both annotated transcripts of the bru-3 gene, which encodes an RNA-binding protein that can bind to the EDEN translational repression sequence,14 are overexpressed several fold (2- to 14-fold) relative to levels in eye discs homozygous for the parental FRT80B chromosome. This effect on bru-3 RNA levels is observed in all gfr alleles (Fig. 9). Therefore, to more closely examine putative regulatory regions of bru-3 in gfr alleles, we performed a series of 2.5-kb overlapping genomic PCRs spanning the 25-kb region upstream of bru-3 in genomic DNA isolated from gfr/gfr embryos. We anticipated that any change in PCR product size between the gfr alleles and the FRT parental chromosome could represent an insertion, deletion, or other chromosomal aberration leading to the misexpression of bru-3; however, we observed no changes in size between the gfr alleles and the parental chromosome in any of the PCR products generated (data not shown).

Figure 9
Expression of bru-3 is elevated in gfr mutant eye tissue. Quantitative real-time PCR analysis of the expression of bru-3 transcripts RA (A) and RB (B) in FRT80B/M(3) control or gfr/M(3) third instar eye/antennal discs. Error bars represent 95% confidence ...

The recently reported ability of overexpressed bru-3 to promote hemocyte over-proliferation and enlarged lymph glands15 suggests that excess bru-3 might contribute to the gfr phenotypes. To further characterize the relationship between the gfr complementation group and bru-3, we utilized two bru-3 alleles: l(3)05871, a lethal P-element insertion immediately upstream of bru-3, and Df(3L)Exel6119, a deficiency that removes a portion of the bru-3 gene but does not overlap with the l(3)05871 P-element insertion site (FlyBase). Although not inserted in the gene body, the bru-3l(3)05871 allele fails to complement Df(3L) Exel6119 (Table 1), and acts in a direction opposite to gfr alleles with respect to Notch: gfr alleles are dominant suppressors of N54l9/+ wing notching, but bru-3l(3)05871 dominantly enhances both the penetrance (68% vs. 45%, respectively; p < 0.001; Fig. 6G) and expressivity of N54l9/+ wing notching by increasing the percentage of wings having two or more notches (data not shown). actin-Gal4 driven ubiquitous overexpression of bru-3 from the UAS-containing EY08487 element38 is also lethal (data not shown), suggesting that elevated expression of bru-3 in the gfr background may contribute to the homozygote lethality of gfr alleles. Because bru-3 and gfr are tightly linked to the same chromosomal region, bru-3 alleles cannot be recombined with gfr alleles to test whether reducing bru-3 activity can dominantly rescue gfr homozygote lethality (data not shown). Consequently, we tested whether reducing bru-3 gene dosage with either bru-3l(3)05871 or Df(3L)Exel6119 could rescue the gfr/puc trans-heterozygote lethality. This lead to a complete rescue of gfr/puc lethality by either bru-3 allele and in the background of multiple gfr alleles; this rescue is also observed in multiple bru-3, puc recombinant lines (Table 1). These data demonstrate that loss-of-function alleles of bru-3 restore gfr/puc viability and provide strong genetic evidence that overexpression of bru-3 in gfr alleles is responsible for gfr/puc lethality and, by extension, perhaps gfr/gfr lethality as well.

bru-3 regulates Cic and eye size

To test whether overexpression of bru-3 might phenocopy all or some elements of the gfr mutant eye phenotype, the EY08487 bru-3 UAS-element was initially combined with the eyeless-Gal4 driver. At all temperatures tested, this failed to increase bru-3 transcript in larval eye discs as measured by qPCR (data not shown). Consequently, we utilized the “eyFLP-out” technique to drive EY08487 with the actin-FRT-CD2-FRT-Gal4 transgene specifically in eye disc cells.39 By this method, we achieved a 90-fold increase in bru-3 RB transcript levels in the eye at 25° (see Fig. 8B). We then tested these ey>act>EY08487 eye discs for evidence of an effect on Cic protein levels. As observed in gfr mutant eye tissue (see Fig. 8A–A″), overexpression of bru-3 is also associated with a decrease in the levels of Cic in the larval eye disc as detected both by western blot (Fig. 8B) and by immunofluorescence (compare Figs. 8C and 9C′). Thus, overexpression of bru-3 produces an effect on Cic levels that is similar to that of gfr alleles. Although overexpressing bru-3 in this manner was insufficient to increase adult eye size, adult flies homozygous for the EY08487 bru-3 allele show reduced eye size compared with EY08487/+ flies (p < 0.001; Fig. 10). The EY08487 allele produces reduced viability in trans to either bru-3l(3)05871 or Df(3L)Exel6119 (Table 1), suggesting that EY08487 is a weakly hypomorphic for bru-3. While both increased and decreased bru-3 expression share some phenotypes expected of gfr alleles, gain-of-function of the bru-3 gene product in gfr mutant tissue could be a secondary effect of increased activity from gfr-affected pathways. In light of the links to puc and Cic, we sought to test whether overactivating the JNK or ERK pathways leads to increased bru-3 transcript levels. qPCR analysis of bru-3 RNA in GMR-Gal-4, UAS-hepact and UAS-EGFRElp larval eye discs showed no effect on bru-3 (Fig. S5),40,41 suggesting that overexpression of bru-3 may be a more primary molecular defect in gfr mutant cells.

Figure 10
Hypomorphic allele of bru-3 has decreased eye size. Light microscopic images of (A) EY08487/+ and (B) EY08487/EY08487 adult female eyes. Note the eye in (B) is smaller than the eye in (A). (C) Graphic summary of the effect of the indicated genotypes on ...


Here we show that mutations in the novel complementation group gfr disrupt patterns of cell differentiation in the eye and increase eye size through a proliferative mechanism that can be enhanced by a block in apoptosis. gfr alleles display genetic and molecular phenotypes indicative of a regulatory effect on the JNK/ERK and Notch pathways and map to a fairly small genomic interval at 70A-B. This interval contains a gene bru-3, which is overexpressed in all gfr mutant backgrounds and has been previously identified in a misexpression screen for positive regulators of larval hemocyte proliferation and lymph gland size.15 In addition to bru-3, other loci identified in this screen have known roles in growth control, and several are components of gfr-affected pathways; examples include the bantam microRNA, which controls proliferation and apoptosis,42 the Drosophila CHK1 kinase homolog grapes, the Drosophila Insulin-like receptor, which controls cell size and number,43 the Drosophila Fos homolog kayak, and the EGFR ligand Keren. bru-3 encodes an mRNA binding protein and translational repressor that binds to EDEN sequence elements in mRNAs and is orthologous to Xenopus EDEN-BP and human CUG-BP.14 In mammalian cells, CUG-BP binds the c-Jun mRNA and plays an important role in post-transcriptional control of c-Jun expression.44 Moreover, overexpression of such RNA-binding factors can lead to stabilization of mRNAs containing these target sequences.4547 Thus, bru-3 over-expression in gfr eye disc cells could lead to a stabilization of Jra and subsequent alteration of its downstream transcriptional program. Intriguingly, reducing bru-3 gene dosage can rescue an embryonic lethal combination of gfr and puc alleles, yet ectopic overexpression of bru-3 is unable to phenocopy the effect of gfr alleles on head and eye size. From this, it appears that bru-3 is necessary for gfr mutant phenotypes but may not be sufficient to reproduce them, indicating that other factors are required to drive the full spectrum of gfr growth and patterning phenotypes. However, since our system expressed bru-3 in the eye to levels 90-fold over baseline, it may be that any potential bru-3 growth phenotypes are masked by the cellular consequences of supra-physiologic levels of Bru-3.

The specific molecular mechanisms by which gfr alleles produce excess growth are not known, mainly because the gfr lesions remain unidentified. However, certain molecular aspects of the gfr phenotype are reminiscent of MAPK pathway components—in particular, the Ras/ERK pathway, which has been shown to promote growth by downregulating Cic.10 The pattern of Cyclin E expression anterior to the MF resembles what has been reported in cic mutant clones, and this correlates with a requirement for wild-type gfr to maintain Cic levels in eye cells (see Fig. 8). The genetic dependence of the gfr enlarged-eye mutant phenotype on the MAPK components Ras85D and rl coupled with the synthetic lethality of gfr and puc alleles further argue that gfr alleles elicit a subset of phenotypes via effects on MAPK pathways. Indeed the Cic and puc data support a model in which gfr acts within one or more MAPK cascades, and that interactions between gfr and other pathways (e.g., Notch and yki) are a consequence of this more primary role (see model in Fig. 11). Many gfr phenotypes, from decreased cone cells to increased proliferation, could be explained by an increase in Ras/ERK signaling, which has known roles in both proliferation and differentiation in the eye and interacts with many pathways, including Notch.48 In addition, the Drosophila Jun homolog Jra has well documented roles in the eye downstream of Ras/ERK,49 thereby providing a potential link between decreased Cic levels and the strong genetic interaction between gfr and the putative Jra target, puc. The synthetic lethality of gfr and puc alleles is generally only observed between genes that are tightly linked in the same pathway (e.g., wg and dsh),50 arguing a role for gfr in the JNK pathway or another puc-specific function. Activation of the JNK pathway in the eye is typically pro-apoptotic,31 which is in conflict with the gfr overgrowth phenotype. However, it has been shown that in the presence of activated Ras, JNK switches from pro-apoptotic to pro-growth and cooperates with oncogenic Ras to promote tumor growth and metastasis.51 Thus, gfr could act to regulate JNK/ERK activity in developing tissues. However, genes directly involved in JNK signaling typically have dorsal closure defects,31 and because gfr/gfr animals can survive to an early larval stage, a role in dorsal closure, and by extension, a central role as a core JNK pathway component, seems doubtful. Alternately, gfr may encode a factor that controls MAPK cascades in a more tissue-specific manner such that gfr alleles are insufficient to drive ERK/JNK phenotypes in all cells at all developmental stages.

Figure 11
Pathways affected by gfr alleles in the eye. gfr may be allelic to bru-3 and act to regulate signaling through MAPK modules, perhaps via a role in regulating RNAs encoding signaling components of these pathways. Interactions between gfr and Notch may ...

A major outstanding question remains: what is the gfr gene? Meiotic mapping links gfr closely to the chromosomal region represented by cytological positions 70A-B (see Materials and Methods); however, direct sequencing of many candidate genes in the region has not uncovered any apparent mutations. Though all gfr alleles are lethal in trans to each other, they are viable with no obvious defects in trans to all available deficiencies and lethal alleles in this region. Thus, the underlying assumption that the gfr alleles are caused by recessive, loss-of-function mutations may be incorrect. gfr alleles do not produce dominant morphological phenotypes characteristic of gain-of-function mutations. However, gfr heterozygotes do display a dominant genetic phenotype: they are hypersensitized to loss of a single allele of puc. That this dominant phenotype can then be suppressed by bru-3 alleles (Df(3L)Exel6119 or l(3)05871) tends to argue either that bru-3 is the actual target of the gfr mutations or that bru-3 expression is downstream of the gene affected by gfr lesions. Although this sort of recessive gain-of-function allele is not common, such gfr alleles could have been selected for in the eyFLP screen due to their pro-growth effects. Several gain-of-function alleles of Drosophila genes, such as Tufted and Bearded (Brd), share some phenotypes with gfr mutations, including bristle defects and interactions with the Notch pathway.5254 Additionally, recessive phenotypes other than lethality associated with gain-of-function alleles is not without precedence: dominant Drop mutations are associated with recessive phenotypes such as patterning and bristle defects in the eye,55 and the phenotypes associated with gain-of-function Brd mutations are dosage-sensitive—Brd homozygotes have a more severe phenotype than Brd heterozygotes.54 Yet, the inability of bru-3 to phenocopy all elements of the gfr phenotype either (1) reflects a consequence of insufficient tools to effectively overexpress bru-3 to levels seen in gfr mutant cells or (2) supports a model in which bru-3 is not the sole effector of gfr alleles. However, since gfr maps very close to bru-3, another possibility that must be considered is that gfr alleles affect the expression of multiple genes in the bru-3 region (for example, by disrupting a chromatin insulator element or microRNA) and that the gfr phenotypes are the product of altered expression of multiple genes.

Materials and Methods

Stocks, genetics and statistics

Crosses were performed at 25°C, unless otherwise indicated. The following genotypes were used for gfr analysis: y, w, eyFLP; P[m-w+;ubi>GFP],FRT80B, y, w, eyFLP; P[m-w+]RpL141, FRT80B, y, w, ubxFLP; P[m-w+] RpL141, FRT80B, FRT80B, Df(3L)H99, FRT80B, gfr1FRT80B/TM6B (FBgn0084590), gfr2FRT80B/TM6B, gfrxFRT80B/TM6B, gfrx, Df(3L)H99, FRT80B/TM6B, gfr4FRT80B/TM6B (stock no longer extant), y, w, eyFLP, E(spl)mβ-CD2, P[m-w+;ubi>GFP], FRT80B, cdc2B47/CyO;gfr1, FRT80B/Tm6B, N54l9/Fm7c;gfr1, FRT80B/Tm6B, bsk1/CyO;gfr1, FRT80B/Tm6B, gfr1, FRT80B, Ras85De2F/Tm6B, rl10a/CyO;gfr1, FRT80B/Tm6B, FRT42D, ykiB5/CyO;gfr1, FRT80B/Tm6B, y, wa, N54l9/Fm6, FRT82B, pucE69/Tm6B, pucA251.1F3/Tm3, FRT82B, cicQ474X/Tm6B, gfr1, FRT80B, cicQ474X/Tm6B. The following genotypes were used for bru-3 analysis: Df(3L)Exel6119/Tm6B, P{PZ}l(3)0587105871/Tm3, w1118, P{EPgy2}EY08487, y, w, eyFLP;act>y+>Gal4, l(3)05871, pucE69/Tm6B, Df(3L)Exel6119, pucE69/Tm6B. Mapping stocks: Df(3L)ED4502/Tm6c, Df(3L)Exel6119/Tm6B, P{SUPor-P}tRNA:CR32123:ΨKG01069, P{GT1}BG00690. gfrx, Df(3L)H99, FRT80B/TM6B gift of M.M. Gilbert. pucE69 and rl10a gifts of A. Vrailas Mortimer. FRT42D, ykiB5/CyO gift of K. Irvine. cicQ474X gift of I.K. Hariharan. P{SUPor-P} tRNA:CR32123:ΨKG01069 (FBti0021452), P{GT1}BG00690 (FBti0040536), cdc2B47 (FBal0030731), bsk1 (FBal0001321), Ras85De2F (FBal0030102), pucA251.1F3 (FBal0032523), P{PZ} l(3)0587105871 (FBti0005547), Df(3L)ED4502 (FBab0035750), Df(3L)Exel6119 (FBab0038139), P{EPgy2}EY08487 (FBti0058048) obtained from Bloomington Drosophila Stock Center. All statistical comparisons were performed using Student’s t-test.

Mapping of the gfr locus

The genetic configuration of the original eyFLP;FRT screen indicates that the gfr locus is located on the left arm of chromosome 3. Complementation mapping using every chromosomal deficiency in the 3L Bloomington kit and others that span the remaining gaps in coverage failed to identify a deficiency that uncovers the lethality of gfr alleles. Meiotic mapping with molecularly defined P-element insertions located along the length of chromosome 3L was performed in accordance with the crossing scheme previously described by Zhai et al. 2003.16 Briefly, gfr1 was crossed individually to several w+-marked P-element insertion strains. gfr1/P F1 females were crossed to males heterozygous for another gfr allele and a hs-hid balancer.56 This cross was heat-shocked at 37°C for 1.5 hour 5 days after setting up the cross to kill off progeny carrying the balancer. Surviving F2 progeny were scored for eye color: red-eyed progeny are gfr2/P nonrecombinants; white-eyed progeny are the result of recombination between gfr1 and the P insertion. The progeny represents the percentage of white-eyed flies in the F2 recombination distance in cM between the gfr1 lesion and the P insertion (see Table S1).

Eye/wing pictures and measurements

Adult eyes and wings were photographed with a Leica DFC500 CCD digital camera. For measurements, areas were quantitated with Adobe Photoshop; a minimum of 10 eyes and wings were counted per genotype.

Immunohistochemistry & microscopy

Immunostaining and confocal microscopy was performed using a 4% paraformaldehyde fixative as described previously;57 α-CycE staining was performed according to the same protocol but used a PLP fixative. Primary antibodies and dilutions used in immunostaining: mouse α-CycA 1:50 (A12, DSHB); mouse α-CycB 1:50 (F2F4, DSHB); mouse α-CycE 1:5 (8B10, gift of H. Richardson); guinea pig α-Sens 1:1,000 (gift of H. Bellen);58 mouse α-Dlg 1:50 (4F3, DSHB); rabbit α-BarH1 1:50 (S12; gift of K. Saigo);19 mouse α-Notch 1:200 (9C6, DSHB); rat α-CD2 1:100 (Research Diagnostics, Inc.,); guinea pig α-Cic1501 and guinea pig α-Cic1503 1:300 (gift of I. Hariharan).10 For immunoblotting, imaginal disc extracts were prepared in sample buffer containing DTT and resolved on 7.5% SDS-PAGE prior to western blotting with guinea pig α-Cic1503 (1:1,000), or anti-β-tubulin (1:1,000; Santa Cruz Biotechnology). Secondary antibodies conjugated to Cy3, Cy5 and HRP were used as recommended (Jackson ImmunoResearch).

Real time RT-PCR (qPCR)

Total RNA isolated from 30 eye discs (TRIzol/Invitrogen) was reverse transcribed (SuperScript II RT/Invitrogen) and analyzed by qPCR (SYBR Green 1 Master/Roche) Primers: puc 5′-GCC ACA TCA GAA CAT CAA GC-3′, 5′-CCG TTT TCC GTG CAT CTT-3′; dpp 5′-GTG CGA AGT TTT ACA CAC AAA GA-3′, 5′-CGC CTT CAG CTT CTC GTC-3′; bru-3-RA 5′-TTG CCA TCA TCC ATT AAT ACC A-3′, 5′-TTC AGC TGT AAA GCA CGG TTC-3′; bru-3-RB 5′-CTA CCC TGC AAC ATG CCT TC-3′, 5′-GGT GGT AAA GCT TGT GGA AAC T-3′ β-tub 5′-CGC ACA GAG TCC ATG GTG-3′, 5′-AAA TCG TTC ACA TCC AAG CTG-3′.

Supplementary Material

Supplemantal Figures and Legends


We apologize to those whose work could not be cited due to space constraints. We thank S. Burdick for technical support; A. Locke and M. Zwick for genomic sequencing; M.M. Gilbert, A. Vrailas-Mortimer, D. Marenda, R. Jones, K. Saigo, H. Richardson, K. Irvine and I.K. Hariharan for gifts of stocks and antibodies. We are grateful to members of the Moberg and Yedvobnick laboratories for helpful discussion and comment. K.H.M. was supported by grant GM079242 from the National Institutes of Health.


gang of four
quantitative real-time PCR


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