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Genetics. 2005 Apr; 169(4): 2075–2086.
PMCID: PMC1449616

RacGap50C Negatively Regulates Wingless Pathway Activity During Drosophila Embryonic Development


The Wingless (Wg)/Wnt signal transduction pathway directs a variety of cell fate decisions in developing animal embryos. Despite the identification of many Wg pathway components to date, it is still not clear how these elements work together to generate cellular identities. In the ventral epidermis of Drosophila embryos, Wg specifies cells to secrete a characteristic pattern of denticles and naked cuticle that decorate the larval cuticle at the end of embryonic development. We have used the Drosophila ventral epidermis as our assay system in a series of genetic screens to identify new components involved in Wg signaling. Two mutant lines that modify wg-mediated epidermal patterning represent the first loss-of-function mutations in the RacGap50C gene. These mutations on their own cause increased stabilization of Armadillo and cuticle pattern disruptions that include replacement of ventral denticles with naked cuticle, which suggests that the mutant embryos suffer from ectopic Wg pathway activation. In addition, RacGap50C mutations interact genetically with naked cuticle and Axin, known negative regulators of the Wg pathway. These phenotypes suggest that the RacGap50C gene product participates in the negative regulation of Wg pathway activity.

THE Wnt signaling pathway plays a critical role in many developmental processes. Of the 19 mouse Wnt genes, 10 have been characterized mutationally and each directs patterning and cell fate decisions in a different tissue, such as brain, somites, kidneys, and limbs (reviewed in Wodarz and Nusse 1998; Logan and Nusse 2004). In Drosophila, a single Wnt molecule, encoded by the wingless (wg) gene, alone directs a similar range of cell fate and patterning decisions, specifying cell fate in the central nervous system, larval muscle, Malpighian tubules, and appendages (reviewed in Dierick and Bejsovec 1999). Although other Wnt genes are present in the fly genome, they do not appear to have major roles in development (reviewed in Logan and Nusse 2004).

Wg signaling is most easily assayed in the embryonic epidermis. Epidermal cells are exquisitely sensitive to Wg signaling levels and so this tissue provides a useful model to investigate the proteins involved in the pathway and to explore how they work together to generate pattern. Ventral epidermal cells secrete a segmentally repeating pattern of small hooks, called denticles, separated by expanses of naked cuticle (Lohs-Schardin et al. 1979; Wieschaus and Nüsslein-Volhard 1986). These belts of denticles provide traction for the crawling larva after it hatches from the egg. In abdominal segments 2–7, each belt contains six rows of denticles. Each row of denticles shows a characteristic morphology that requires input from Wg signaling in the epidermal cells prior to stage 10 or ~6 hr after egg laying (Bejsovec and Martinez-Arias 1991; Moline et al. 1999). Wg signaling at later stages specifies the expanses of naked cuticle that separate the denticle belts (Bejsovec and Martinez-Arias 1991). Loss-of-function wg mutations alter the cuticle pattern profoundly: mutant embryos secrete no naked cuticle and instead produce a continuous lawn of denticles, all with a similar morphology (sslein-Volhard et al. 1984; Bejsovec and Wieschaus 1993). Conversely, ectopic overexpression of wg redirects all of the epidermal cells into the naked cuticle cell fate, resulting in a uniform expanse of smooth cuticle (Noordermeer et al. 1992).

Wg protein specifies cell fates by activating an intracellular signaling cascade, the components of which are highly conserved among animal species (reviewed in Cadigan and Nusse 1997; Wodarz and Nusse 1998; Dierick and Bejsovec 1999). The activity of the pathway hinges on regulation of Armadillo (Arm), the fly homolog of β-catenin. In the absence of Wg signal, Arm is present at adherens junctions, but is kept at low levels in the cytoplasm. This downregulation is accomplished by a set of proteins collectively known as the destruction complex, which includes Axin, Apc2, and the kinase Zeste-white 3 (Zw3). The complex phosphorylates Arm, targeting it for destruction by the proteasome (reviewed in Peifer and Polakis 2000; Jones and Bejsovec 2003). When Wg binds to its receptor, Dfz2, and coreceptor, Arrow, the clustering of the cell surface molecules somehow activates Dishevelled (Dsh) and leads to the degradation of Axin (Tolwinski et al. 2003). Degradation of Axin and disruption of the destruction complex allows Arm to accumulate in the cytoplasm and in the nucleus. In the nucleus, Arm binds to the DNA-binding protein Tcf (also known as Pangolin or LEF) and activates downstream gene transcription (Brunner et al. 1997; van de Wetering et al. 1997). Depending on the tissue and the time of development, different downstream genes are activated.

The Wg pathway components characterized to date were identified primarily through their mutant phenotypes in Drosophila. Loss of a pathway activator mimics the phenotype of wg null mutants, resulting in embryos that secrete a uniform lawn of denticles. Loss of a negative regulatory component mimics the effect of ectopic wg overexpression, producing a uniform naked cuticle phenotype. Despite the many components implicated in pathway modulation, their interactions and precise mechanisms of action are still somewhat mysterious. For example, it is not clear how Dsh, the most upstream cytosolic component in the Wg pathway, interacts with the receptor complex to mediate inactivation of the destruction complex. Our laboratory has sought to fill the gaps by isolating mutations that modify Wg pathway activity. Here we describe loss-of-function mutations in one such component, encoded by the RacGap50C locus, which were isolated through their suppression of a hypomorphic wg mutant allele.


Drosophila stocks and culture conditions:

All deficiencies and P-element insertions were obtained from the Bloomington Stock Center. P{UAS-DRacGap+} and P{UAS-DRacGapΔEIE}, a dominant-negative form of RacGap50C with GAP domain amino acids 405–407 deleted, were kindly provided by S. Campuzano (Sotillos and Campuzano 2000). The P{UAS-nkd} transgenes (Zeng et al. 2000) were a gift from K. Wharton and M. Scott. P{UAS-Rac1.V12} and P{UAS-Rac1.N17}, the constitutively active and dominant-negative forms of Rac1, respectively (Luo et al. 1994), and P{UAS-AxinGFP} (Cliffe et al. 2003) were obtained from the Bloomington Stock Center. Ubiquitous embryonic expression of UAS transgenes was achieved with either the E22C-Gal4 or the arm-Gal4 driver lines. Both were tested in each assay and produced similar results unless otherwise noted. More restricted embryonic expression was achieved using the prd-Gal4 and wg-Gal4 driver lines. All driver lines are available from the Bloomington Stock Center.

The naked cuticle (nkd) allele used in this study, nkd7E89, is a strong hypomorph (rgens et al. 1984; Zeng et al. 2000). The AR2 and DH15 mutations were induced on a wgIL114 chromosome, as described below, and were subsequently recombined away from wg for further analysis. The mutant chromosomes were extensively recombined with wild type (Oregon-R) to remove unrelated lethal mutations and were rebalanced over a CyO chromosome carrying Kruppel-Gal4 UAS-GFP inserts (obtained from Bloomington) to allow identification of homozygous-mutant embryos by their failure to fluoresce.

Flies were reared on cornmeal-agar-molasses unless otherwise noted. For analysis of embryonic stages, eggs were collected on apple juice-agar plates and aged at 25°. To examine embryonic cuticles, eggs were allowed to develop fully (24 hr at 25°), dechorionated in bleach, and transferred to a drop of Hoyer's medium, mixed 1:1 with lactic acid (Wieschaus and Nüsslein-Volhard 1986), on a microscope slide. Mechanical devitellinization was performed by exerting gentle pressure on the coverslip to burst the vitelline membrane. Cuticle preparations were baked at 65° overnight before viewing on a Zeiss Axioplan microscope with phase optics.

Isolation and characterization of mutant alleles:

The AR2 and DH15 mutant lines were isolated independently in a genetic screen for modifiers of the wg mutant phenotype. The temperature-sensitive wg allele, wgIL114, used in this screen is also known as wgts or wgl-12. Male wgIL114/CyO flies were fed overnight on ethyl methanesulfonate (EMS) at a concentration of 25 mm in 1% sucrose. They were allowed to recover and mated with virgin Dominant temperature sensitive (DTS)/CyO females (sslein-Volhard et al. 1984). At 29°, only flies bearing the mutagenized wgIL114 chromosome over CyO survive, and these were mated singly with DTS/CyO flies and raised at 29° to establish isogenic lines. A total of 1768 lines were established, and their F3 progeny were examined for alteration of the wgIL114 cuticle pattern at 25°.

The AR2 and DH15 mutations were tested for complementation because they yielded similar modifications of the wgIL114 pattern. They failed to complement each other in this modification, and when recombined away from the linked wgIL114 mutation, their homozygous-recessive embryonic-lethal phenotypes also failed to complement. The mutations were mapped using meiotic recombination against a second chromosome marked with the adult visible mutations black, purple, cinnabar, curved, plexus, and speck. The mutations mapped between cinnabar and curved at ~70 MU, and deficiencies in this region were tested for their failure to complement. Both alleles failed to complement Df(2R)CX1, which removes cytological position 49C1-4;50C23-D2, and to complement Df(2R)vgB, which removes 49D3-4;49F15-50A3. We mapped AR2 and DH15 more precisely using site-specific P-element-mediated recombination in males (Chen et al. 1998). The P elements used for mapping the two alleles include P{lacW}drkk02401, P{PZ}drk10626, P{EP}EP2007, P{PZ}fas05488, P{lacW}v(2)k16105k16105, P{EP}EP2423, P{lacW}shotk03010, P{lacW}AGO1k08121, P{lacW}AGO1k00208, P{PZ}AGO104845, and P{lacW}Cp1k15606.

The mutations mapped between P{EP}EP2423 and P{lacW}shotk03010, narrowing down the interval to 13 annotated open reading frames, 6 of which were predicted to encode “housekeeping” enzymes. The complete sequences of all exons from the 7 other candidate genes in the AR2 and DH15 mutant lines were compared to the consensus sequence to locate the EMS-induced mutations. PCR primers were designed using MacVector software and tested using Amplify (W. R. Engels). PCR was performed with both homozygous and heterozygous genomic DNA. The products were purified using the QIAquick PCR purification kit (QIAGEN, Chatsworth, CA) and sequenced using ABI PRISM dye terminator cycle sequencing ready reaction kit with Amplitaq DNA polymerase according to manufacturer's protocols. Both alleles were found to encode nonsense mutations in the RacGAP50C gene, CG13345. The PCR primers used to identify the single nucleotide changes in the RacGAP50C gene include 5′-CGAGTGAAAACCGATTCGTAG-3′, 5′-AGGTGTGGTTCCTCATCAG-3′, 5′-GGTAAATGCTATGGCACCTC-3′, and 5′-CGTCAAATGCTTTCCAACAAGG-3′. Additional primers used for sequencing DRacGAP50C were 5′-ATCTGAACAACGAGACCC-3′, 5′-GGTATCTGCTCACCGTTAGC-3′, and 5′-TCCTGGGAGTTTTGGTGTC-3′.

Antibody staining and in situ hybridization:

RNA in situ hybridization was performed with antisense digoxigenin-labeled probes following standard protocols (Ashburner 1989; Tautz and Pfeifle 1989). Antibody staining was as described in Dierick and Bejsovec (1998). Anti-Wg antibody was used at 1:1000 and anti-Arm was used at 1:50 (both from the Developmental Studies Hybridoma Bank). Anti-Coracle antibody (kindly provided by R. Fehon) was used at 1:10,000. DAPI stain was used at 1:20,000. Cy3 and Cy5 IgG secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used to detect fluorescence staining. Embryos were mounted in Aquapolymount (Polysciences, Niles, IL) and viewed with a Zeiss 410 confocal microscope.

Hatching efficiencies and cuticle pattern analysis:

Fly crosses were performed at 25°, and eggs were collected in 2- to 4-hr time windows. Eggs were arrayed on apple juice agar plates and counted before being allowed to age for 24 hr at 25°. Unhatched eggs were then dechorionated with bleach and mounted in Hoyer's medium for scoring; in some cases hatched larvae were mounted as well. The number of unfertilized eggs was subtracted from the total egg count, prior to calculating proportions for different phenotypic classes. Denticle rows in abdominal segments 3–6 were examined in at least 20 embryos for each genotype, concentrating on the middle one-third of the belt centered over the ventral midline.

Immunoblot analysis:

Five- to 7-hr-old embryos were collected, dechorionated, and examined for the presence or absence of GFP from the Kr-GFP CyO balancer. Fifteen homozygous-mutant embryos (identified by lack of fluorescence) as well as 15 wild-type siblings (identified by twofold brightness of fluorescence) were collected and immunoblots were performed as described in Chao et al. (2003). Filters were stained with anti-Arm protein (Developmental Studies Hybridoma Bank) at 1:100 and with antitubulin at 1:5000 (Labvision). Cross-reacting proteins were detected and quantified using the Odyssey infrared imaging system and reagents (Li-Cor Technologies).


AR2 and DH15 modify wg phenotypes:

In a genetic screen to identify mutations that suppress or enhance the mutant phenotype of the wg temperature-sensitive allele wgIL114, we isolated two EMS-induced mutations that subtly modify the wgIL114 mutant cuticle pattern. These modifier mutations, AR2 and DH15, fail to complement each other and thus identify a single complementation group, linked to wg on the second chromosome. These two alleles produce identical phenotypes in our assays: all figures shown use the AR2 line; however, similar results were obtained using DH15. Both mutations show no increase in severity when placed in trans to a deficiency for the region and so are likely to represent loss-of-function alleles.

wgIL114 embryos at the restrictive temperature are much smaller than wild-type embryos and secrete the “lawn of denticles” cuticle pattern typical of low pathway activity (Figure 1A). The modifier lines AR2 and DH15 were found to alter the wgIL114 phenotype in a similar way, increasing the spacing between denticles and the overall body size of the doubly mutant embryos (Figure 1B). This phenotype indicates that the modifier mutations considerably reduce the severity of the wg loss-of-function phenotype, resulting in a larger, healthier embryo even though the cuticle pattern is only subtly altered. Both AR2 and DH15 also modify other wg alleles, including wgCX4, an RNA null allele of wg (not shown). The cuticle pattern defects of weak wg alleles are more dramatically suppressed by the AR2 and DH15 mutations. The wgPE2 hypomorphic allele produces a protein with a lower affinity for the receptor complex (Bejsovec and Wieschaus 1995; Moline et al. 2000). wgPE2 homozygous mutants show segmental denticle diversity, but little or no naked cuticle separating the belts (Figure 1C). The wgPE2 AR2 doubly homozygous mutants show an increase in the amount of naked cuticle, indicating that some Wg signaling is restored (Figure 1D).

Figure 1.
Epidermal patterning is altered in AR2 and DH15 homozygous-mutant embryos. (A) The wgIL114 allele at the restrictive temperature produces the “lawn of denticles” phenotype characteristic of wg loss-of-function mutations. (B) This severe ...

In an otherwise wild-type background, the AR2 and DH15 mutations are recessive lethal, and homozygous embryos show an excess specification of naked cuticle at the expense of denticles. Wild-type embryos (Figure 1E) secrete an average of 5.95 ± 0.15 rows of denticles/belt (n = 40) in abdominal segments 3–6. In contrast, AR2 mutant embryos secrete an average of only 3.95 ± 0.64 rows of denticles/belt (n = 80) (Figure 1F). The mutant denticles also differ morphologically from the wild type. Wild-type denticles show a wide range in size, with those in row 5 being largest (Bejsovec and Wieschaus 1993). The AR2 and DH15 mutant denticle belts contain fewer of these large denticles: most denticles observed are similar in size to the smaller denticles normally found in rows 1–4 of the wild-type belt pattern. The slight suppression of wg mutant phenotypes and the ectopic specification of naked cuticle are consistent with an increase in Wg pathway activity, suggesting that the AR2 and DH15 mutations might disrupt a negative regulator of the pathway.

AR2 and DH15 carry nonsense mutations disrupting the RacGap50C locus:

The AR2 and DH15 mutations were mapped on the second chromosome, using standard meiotic recombination and deficiency analysis. Male site-specific recombination further refined the interval to cytological region 50C06-09. Seven candidate genes within this interval were sequenced and lesions in only the RacGap50C locus were detected for both the AR2 and DH15 lines. RacGap50C encodes one of two RacGaps annotated in the Drosophila genome (Adams et al. 2000; FlyBase at http://flybase.bio.indiana.edu/). While the genome contains 21 predicted Rho family GTPase-activating proteins (Bernards 2003), only RacGap50C and RacGap84C are strongly conserved with the human RacGap1. The predicted RacGap50C polypeptide contains a protein kinase C (PKC) conserved region, which is predicted to bind diacylglycerol, as well as a RhoGap motif, a 140-amino-acid GTPase-activating domain that stimulates the Rho-like GTPases, Rac, Rho, or Cdc42 (Figure 2A). AR2 carries a point mutation in the RacGap50C coding region that changes amino acid 470, a tryptophan, to a premature stop. This is predicted to truncate the protein within the RhoGap domain. DH15 encodes a mutation at amino acid 195, changing an arginine to a stop and truncating the protein prior to both the PKC domain and the RhoGap domain.

Figure 2.
RacGap50C is mutated in AR2 and DH15. (A) Structure of RacGap50C predicted protein, with position of premature termination codons noted and positions of the PKC and GTPase-activating domain homologies highlighted. Overall, the predicted RacGap50C protein ...

Previous work using misexpression constructs indicated that RacGap50C modulates EGF pathway activity in the developing wing (Sotillos and Campuzano 2000) and plays a critical role in cytokinesis (Somers and Saint 2003). Sotillos and Campuzano constructed a dominant-negative form of RacGap50C, which abolishes the GAP activity while retaining the ability to bind Rac. When expressed in the wing, this construct caused a range of defects, including increased width and fusion of veins, enlarged cells and an increased number of sensory organs, as well as an expansion of the EGF signaling marker, diphosphoERK, in the presumptive veins (Sotillos and Campuzano 2000). These wing phenotypes are enhanced by coexpression of Rac1, leading these authors to conclude that Rac1 is a target of RacGap50C activity in the wing. In contrast, Rac1 has been shown to be irrelevant for the cytokinesis role of RacGap50C, where the primary function of RacGap50C is to serve as an adaptor molecule linking the microtubule and actin cytoskeletons. This linkage is essential for positioning the contractile ring of dividing cells; expression of a dominant-negative form of RacGap50C results in accumulation of multinucleate cells (Somers and Saint 2003). The other Drosophila RacGap, RacGap84C, encodes a protein 47% similar to RacGap50C. No point mutations in RacGap84C have been identified, but deficiencies in the area are known to cause defects in spermatid and retinal differentiation (Bergeret et al. 2001); thus both RacGaps are likely to be functional.

Since mutations in RacGap50C were detected in both of our mutant lines, it seemed likely that RacGap50C was the gene responsible for the cuticle phenotypes observed. We find that RacGap50C mRNA transcripts appear to be maternally contributed to the egg, because early stage embryos show strong staining in RNA in situ hybridization experiments (Figure 2B). Low uniform levels of transcript are detected throughout embryogenesis (Figure 2C), showing that it is zygotically expressed during the stages when Wg signaling determines epidermal pattern. We do not detect any segmental modulation of RacGap50C expression. To verify that the RacGap50C mutations account for the AR2 and DH15 mutant phenotypes, we expressed a wild-type RacGap50C transgene (Sotillos and Campuzano 2000) in mutant embryos and looked for rescue of the mutant phenotype. Driving ubiquitous expression of the UAS-RacGap50C transgene during embryogenesis partially rescues the embryonic lethality and completely rescues the pattern defect of AR2 homozygous-mutant embryos (Figure 3, A and B). Whereas 25% of the progeny from AR2 heterozygous parents normally fail to hatch (n = 593), <10% fail to hatch (n = 796) when UAS-RacGap50C is uniformly expressed in embryos. However, the homozygous larvae die during larval stages probably because E22C-Gal4 is expressed only embryonically. Denticle belts of rescued homozygous mutants contain 5.23 ± 0.60 rows of denticles/belt (n = 64), compared to 3.95 ± 0.64 (n = 80) observed in unrescued mutant embryos. These results confirm that the cuticle pattern defect observed in our mutant lines is due to loss of RacGap50C function.

Figure 3.
The AR2 and DH15 mutant phenotype is rescued by RacGap50C expression and is not mimicked by Rac1 manipulation. The AR2 homozygous-mutant cuticle pattern defect (A) is substantially rescued by uniformly expressing a UAS-RacGap50C transgene with E22C-Gal4 ...

RacGap50C does not appear to interact with Rac in epidermal patterning:

RacGaps are thought to downregulate activity of Rac by stimulating the intrinsic GTP hydrolysis function of the protein and thereby converting it from the active GTP-bound form to the inactive GDP-bound form (reviewed in Hall 1998). Both the vertebrate and invertebrate RacGTPases are associated with signaling cascades that promote cytoskeletal rearrangements, often during morphological processes. For example, the Drosophila Racs are involved in dorsal closure movements of the embryo, as well as in axon growth and guidance in the developing nervous system (Hakeda-Suzuki et al. 2002; Ng et al. 2002).

To determine whether the effects of RacGap50C in the embryonic epidermis are mediated through a Rho family member, we tested whether the GTPase-activating domain of RacGap50C was required for rescue of the epidermal patterning defect in mutant embryos. The dominant-negative RacGap50CΔEIE construct deletes three conserved amino acids in the GAP domain, producing a protein that still binds Rac but cannot stimulate GTP hydrolysis (Sotillos and Campuzano 2000). As mentioned above, Sotillos and Campuzano observed profound effects of this UAS-RacGap50CΔEIE construct when it is expressed in the wing imaginal disc. In contrast, we find that when we express UAS-RacGap50CΔEIE ubiquitously in a RacGap50C mutant embryo, it is able to rescue the cuticle pattern phenotype (Figure 3C). The rescued homozygous-mutant embryos have 4.81 ± 0.60 rows of denticles/belt (n = 160). This rescue is not as complete as that observed for the wild-type RacGap50C construct (5.23 ± 0.60 rows of denticles/belt, above) and does not include any rescue of the embryonic lethality as was observed for the wild-type construct. However, the cuticle phenotype is significantly different from the unrescued RacGap50C mutant (P = 0.001). Overexpressing the UAS-RacGap50CΔEIE transgene in a wild-type embryo does not produce any detectable patterning defect nor does it appear to affect larval viability (data not shown). These results suggest that RacGap50C has a novel function in epidermal patterning that does not involve interaction with a GTPase.

To further confirm that the GTPase-activating function of RacGap50C is dispensable for epidermal patterning, we tested the effects of Rac activity in patterning. Since GTPase activation would be expected to turn Rac off, a constitutively active Rac would be expected to produce phenotypes similar to loss of RacGap50C in processes where its GAP activity is relevant. We therefore tested whether the patterning defects of our RacGap50C mutants could be mimicked by expression of the constitutively activated Rac1 transgene, UAS-RacV12 (Luo et al. 1994). We used several different embryonic Gal4 drivers to express the UAS-RacV12 transgene, but most caused the embryos to die prior to secretion of the cuticle, making it impossible to score cuticle pattern alterations. Expressing RacV12 in the spatially restricted wg domain in each segment, using the wg-Gal4 driver, does not block cuticle secretion but these embryos produce a cuticle pattern very different from the RacGap50C loss-of-function phenotype (Figure 3D). Rather than the expected hyperactivation, the resulting cuticles show defects more consistent with a reduction in Wg signaling, even though Wg protein levels are unaffected (not shown). Furthermore, overexpression of a dominant-negative form of Rac1, UAS-RacN17 (Luo et al. 1994), with most embryonic drivers tested does not produce any disruption of ventral epidermal patterning, although dorsal closure is clearly disrupted as previously reported (Figure 3E). We conclude that RacGap50C does not act through Rac in promoting its effects on embryonic pattern formation.

RacGap50C interacts with negative regulators of the Wg pathway:

The increased specification of naked cuticle in AR2 and DH15 mutant embryos suggests an increase in Wg pathway activity similar to that observed in mutants disrupting known negative pathway regulators. Most of these negative regulator mutations have a strong maternal effect, and the excess naked cuticle phenotype is observed only when maternal contribution is reduced (Siegfried et al. 1992; Hamada et al. 1999; McCartney et al. 1999). The exception is nkd, which acts zygotically (rgens et al. 1984) as do the AR2 and DH15 mutations. nkd encodes a protein that interacts with the Wg pathway at the level of Dsh, to which it binds through the Dsh basic-PDZ region (Zeng et al. 2000; Rousset et al. 2001). nkd mutant embryos show an excess of naked cuticle at the expense of denticle belts, and this phenotype is enhanced by RacGap50C mutations. There is some variability in the nkd mutant phenotype, but on average nkd mutants produce 1.5 abdominal denticle belts (Figure 4A; Table 1), as opposed to the 8 abdominal belts seen in wild type. The denticles formed in the few nkd mutant belts are normally patterned, with typical denticle morphologies (Bejsovec and Wieschaus 1993). In contrast, the average phenotype of RacGap50C; nkd doubly homozygous mutants is uniform naked cuticle, showing no denticle belts at all (Figure 4B; Table 1).

Figure 4.
RacGap50C mutations interact genetically with known negative regulators of Wg pathway activity. (A) Embryos homozygous for a strong nkd allele, nkd7E89, show excessive specification of naked cuticle, but typically retain between one and two denticle belts ...
Genetic interactions betweenAR2 andnaked

The nkd mutant phenotype is sensitive to wg gene dosage: heterozygosity for a wg null allele partially suppresses the ectopic naked cuticle specification of nkd homozygotes (Bejsovec and Wieschaus 1993). RacGap50C mutations remove the ability of lowered Wg dose to suppress the nkd mutant phenotype. Embryos homozygous for nkd and heterozygous for wgCX4 typically show a range of between 4.5 and 7.5 abdominal denticle belts (Figure 4C; Table 1), as opposed to the 1.5 average for nkd single mutants. This rescue is reversed when the AR2 or DH15 mutation is placed in the background. Embryos homozygous for both RacGap50C and nkd, and heterozygous for wgCX4, secrete uniform naked cuticle with no denticle belts (Figure 4D; Table 1). Thus RacGap50C activity is required for the wg dose sensitivity of nkd.

Since RacGap50C mutations are enhancers of the nkd loss-of-function phenotype, we wondered how they would behave in nkd gain-of-function experiments. Ubiquitous overexpression of nkd in a wild-type embryo produces no cuticle pattern defect unless the gene dosage of wg is reduced (Zeng et al. 2000). In wgCX4 heterozygous embryos, overexpressing nkd produces fusions between denticle belts due to loss of the intervening naked cuticle (Figure 4E). Thus, when Wg levels are limiting, ectopic Nkd activity can antagonize the pathway. We find that reducing the dose of RacGap50C reverses this effect. Embryos heterozygous for wgCX4 and RacGap50C show a wild-type cuticle pattern when nkd is overexpressed (Figure 4F). This series of experiments demonstrates that RacGap50C has a synergistic interaction with Nkd, a negative regulator of the Wg pathway, suggesting that RacGap50C also is required for negative pathway regulation.

We find similar interactions with Axin, another negative regulator of the Wg pathway. Overexpression of a GFP-tagged Axin transgene causes cuticle pattern defects consistent with severely compromised Wg signal transduction. The embryos show a “lawn of denticles” pattern similar to wg loss-of-function mutants (Figure 4G). However, when Axin is overexpressed in RacGap50C mutant embryos, the resulting phenotype is not as severe. Some aspects of the segmental pattern are rescued, with more naked cuticle separating the denticle belts (Figure 4H). Thus loss of RacGap50C gene activity can partially suppress Axin overexpression phenotypes, suggesting that the RacGap50C mutations disrupt a pathway component that either is a limiting cofactor for Axin or acts downstream of Axin in the Wg signaling pathway.

RacGap50C mutations affect Arm accumulation:

To determine where in the Wg pathway RacGap50C acts, we analyzed molecular markers of Wg signaling. The wg gene is expressed in a one-cell-wide stripe in each segment of the developing embryo. wg itself is a target gene activated by Wg signaling: disruption of various pathway components can alter wg gene expression or Wg protein secretion or distribution. However, we find that Wg protein distribution is indistinguishable from wild type in the RacGap50C mutant embryos (Figure 5, A and B), indicating that the mutations disrupt neither expression nor protein accumulation. We also examined expression of engrailed, another target gene for Wg activity (DiNardo et al. 1988; Martinez Arias et al. 1988), and found no significant change in its pattern or level compared with wild type (not shown).

Figure 5.
RacGap50C mutant embryos have normal Wg levels, but show elevated Arm protein accumulation. (A) Anti-Wg antibody staining in wild-type stage 10 embryos shows segmental stripes, with a slightly punctate appearance due to secretion and endocytosis of the ...

In contrast, the levels of Arm protein accumulation are significantly altered in RacGap50C mutants compared with wild type. arm mRNA is expressed at uniform levels throughout the epidermis (Riggleman et al. 1989), but Arm protein accumulates in a striped pattern in wild-type embryos, reflecting the segmental stripes of high Wg signaling activity (Riggleman et al. 1990; Peifer et al. 1994). In RacGap50C mutant embryos, Arm accumulation appears to be elevated compared to wild type (Figure 5, C and D). This increase in Arm protein is confirmed by immunoblot analysis. The RacGap50C mutant chromosomes were balanced over a CyO chromosome bearing a Kruppel-driven GFP transgene insert. This allows identification and selection of homozygous-mutant embryos by their failure to fluoresce. At stage 10, homozygous-mutant embryos show between 20 and 25% higher steady-state levels of Arm protein compared to their fluorescing wild-type siblings (Figure 5E). Although these increases are not as extreme as those observed for maternal and zygotic loss of zw3, the principal agent of Arm destruction (Peifer et al. 1994), they represent the most dramatic zygotic phenotype documented.

The RacGap50C cell cycle defects are unrelated to epidermal patterning defects:

Previously, the Saint lab has shown by RNA interference that RacGap50C is required for cytokinesis in cell culture and in imaginal disc tissue (Somers and Saint 2003). This requirement for RacGap50C in cell division explains why we have been unable to obtain sizable clones of RacGap50C mutant tissue in germline and somatic mosaic experiments (data not shown). This has prevented us from assessing the mutant effects of reducing maternal contribution for the RacGap50C gene product. The abundant RacGap50C mRNA detected in early stage embryos (Figure 2B) suggests that substantial maternal product is contributed to the egg. This maternal product most likely accounts for the relatively normal cell division that we observe in the RacGap50C mutant embryos through most of development. Work on our mutant lines in the Saint laboratory indicates that cell division defects can be detected in late stage embryos, with binucleate cells accumulating in the epidermis during stage 11 (M. Zavortink and R. Saint, personal communication).

The epidermal patterning defects that we observe are not, however, a secondary consequence of late defects in cytokinesis. First, we observe changes in Armadillo stabilization beginning at stage 9, whereas the cell division defects observed by the Saint laboratory do not become apparent until stage 11. Second, we find that the cuticle pattern defect is rescued by ectopic expression of the UAS-RacGap50CΔEIE transgene (Figure 3C), whereas the cytokinesis defect is not (Somers and Saint 2003). Third, we have found another experimental condition that rescues the epidermal patterning defects of RacGap50C homozygotes without rescuing the cytokinesis defects. Antagonizing Wg pathway activity by ectopically overexpressing nkd significantly reverses the cuticle pattern defects of RacGap50C mutant embryos (Figure 6, A and B), although it does not alter their embryonic lethality. RacGap50C homozygous embryos overexpressing nkd produce 5.25 ± 0.47 rows of denticles/belt (n = 80), comparable to the measurements obtained for mutant embryos rescued with the wild-type RacGap50C transgene. Thus ectopic Nkd activity can partially compensate for loss of the RacGap50C activity in Wg-mediated patterning events and allows RacGap50C mutant embryos to secrete a pattern more similar to that of the wild-type state (Figure 6C). However, we observe large, binucleate cells accumulating in nkd-overexpressing RacGap50C mutant embryos at the same frequency as they do in the RacGap50C homozygotes alone (Figure 6, D and E), even though epidermal patterning defects are substantially rescued in the nkd-overexpressing embryos. Wild-type embryos at the same stage (Figure 6F) do not accumulate binucleate cells and show a higher cell density in the epidermal epithelium than do the RacGap50C mutant embryos. Thus the role of RacGap50C in Wg pathway modulation is separable from its role in forming the contractile ring during cytokinesis.

Figure 6.
Naked overexpression rescues the RacGap50C cuticle pattern defect without affecting the cytokinesis defect. (A) AR2 mutants show the characteristic loss of denticles, particularly in the more anterior rows of the denticle belt. (B) Driving the UAS-nkd ...


Involvement of a Rac GTPase-activating protein homolog in Wg signaling is unexpected and unprecedented and may reveal a novel function that does not involve a Rho family GTPase. We find that RacGap50C interacts genetically with nkd and appears to act at the same level or downstream of Axin in the control of Arm stabilization. Our data indicate that RacGap50C probably does not act through Rac1 to negatively regulate Wg activity, nor are other GTPases likely to be involved in this aspect of epidermal patterning since the cuticle defects of mutant embryos can be rescued by a form of RacGap50C that lacks catalytic residues in the GTPase-activating domain. Moreover, previous work shows that other Rho family members are unlikely to be involved in Wg-mediated patterning. Overexpressing either constitutively active or dominant-negative Rho, Rac, or cdc42 transgenes disrupts dorsal closure but does not appear to affect ventral patterning (Luo et al. 1994; Harden et al. 1999). Loss of maternal Rho activity has been found to alter embryonic segmental pattern, but this is due to an early effect on establishing segmentation gene expression patterns (Magie et al. 1999). Ras activation through the EGF signaling cascade has been found to affect epidermal patterning, but in a way that counteracts Wg signaling (O'Keefe et al. 1997; Szuts et al. 1997). Thus a GTPase-activating protein would be expected to positively influence Wg-mediated patterning if it acted through Ras, rather than the negative influence we observe for RacGap50C.

For these reasons, we believe that the role of RacGap50C in Wg signaling may instead parallel its role in cytokinesis, where it seems to function primarily as an adaptor molecule. RacGap50C was identified by the Saint laboratory through a yeast two-hybrid screen for molecules that interact with pebble, a RhoGEF that is essential for cytokinesis (Somers and Saint 2003). Subsequently, they showed that RacGap50C protein also binds to Pavarotti, a kinesin-like molecule, thus forming a bridge between the microtubule and actin cytoskeletons. This adaptor function appears critical for the proper positioning of the acto-myosin contractile ring at the end of mitosis (Somers and Saint 2003).

One could imagine that a structural role for RacGap50C in linking the microtubule and actin cytoskeletons might be relevant to its regulation of Wg pathway activity. Apc2, a scaffolding molecule that is an essential component of the destruction complex, is known to interact with both microtubules and the cortical actin cytoskeleton (McCartney et al. 1999, 2001). Mutations that disrupt the cortical localization of Apc2 compromise function of the destruction complex (McCartney et al. 1999; A. Bejsovec, unpublished results), suggesting that subcellular localization of the complex may be critical. Furthermore, recent work from the Bienz and Wieschaus laboratories demonstrates that Axin, another scaffolding component in the complex, changes its subcellular localization in response to Wg signaling (Cliffe et al. 2003) and is consequently degraded (Tolwinski et al. 2003). Thus the positioning of the destruction complex and/or some of its subunits may play a critical role in regulating its Arm-degrading activity. RacGap50C may be involved directly in linking the destruction complex to the cell cortex to promote its proper activity or may restrict movement of Axin to the cortex for its Wg-mediated destruction. In either case, loss of RacGap50C function would reduce the normal degradation of Arm and thereby cause ectopic Wg pathway activity.

Another possible explanation for RacGap50C's effects on Wg pathway activity would not require direct interaction with any pathway component. Rather, some coordination between the microtubule and actin cytoskeletons may be generally required for many different cellular processes. The loss of this adaptor molecule would “loosen” the connection between these two filamentous networks and compromise many cellular events indirectly. It may be that cytokinesis and Wg signal transduction are particularly sensitive to such perturbations or that they simply produce the earliest or most easily detected phenotypes in response to them. A general requirement for microtubule and actin network coordination in destruction complex function could be of great importance in understanding oncogenic Wnt pathway activity. Current work in the laboratory focuses on distinguishing whether RacGap50C interacts specifically with the destruction complex or acts more generally by controlling cellular architecture.


We thank E. Wieschaus, R. Selinger, R. Leung, R. Hays, and H. Dierick for their contributions to early phases of this work; T. Addy for technical assistance; and A. Chao and other members of the Bejsovec laboratory for insightful discussions and comments on the manuscript. We are grateful to the Fehon laboratory for their cheerful help and advice, as well as their generosity with reagents and equipment. We also thank Robert Saint and Michael Zavortink for helpful suggestions and for communicating information before publication. As always we are deeply indebted to the dedicated staff at the Bloomington Stock Center and FlyBase. This work was supported by National Institutes of Health grant GM59068 to A.B.


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