Groucho Central Domains Instruct Development Figure 6. Cuticle phenotypes resulting from overexpression of Gro variants. The Mat-Gal4 driver was used to drive maternal overexpression of the indicated Gro variants. (A–F) Representative cuticles resulting from overexpression of Gro variants. Cuticles images were obtained using a 106 objective. Scale bars = 100 mm (A) Embryos containing the driver but no UAS-Gro construct show no phenotype. Overexpression of GroWT (B) and GroDSP (E) resulted in moderate to severe cuticle defects. Overexpression of GroDGP (C) resulted in no defects, while overexpression of GroDCcN (D) resulted in a truncated or missing 4th or 6th abdominal denticle belt (indicted by arrow), a phenotype sometimes observed in weak gro hypomorphic embryos, and thus consistent with a weak dominant negative function for this deletion variant. Overexpression of GroDCR (F) often resulted in a pair-rule defect reminiscent of that seen in eve mutant embryos. This is consistent with the notion that GroDCR is a dominant negative, since Gro is required for Eve function [35,61]. (G) Cuticles of 100 embryos laid by females overexpressing each of the five Gro variants were assigned to the following phenotypic categories: Wild-type - no observable defects; Minor defects - 1–2 missing or fused denticle belts; Moderate defects - 3–4 missing and/or fused denticle belts; Severe defects - 5 or more missing denticle belts. (H) 100 embryos overexpressing each variant were scored to determine percent of embryos that deposited cuticle and that hatched. doi:10.1371/journal.pone.0030610.g006 In accord with the results of maternal expression in embryos, clonal misexpression of GroWT or GroDSP in wing discs results in vgQ-lacZ repression (Figure 7B, E, compare to Figure 5A), while misexpression of GroDGP or GroDCcN does not (Figure 7C, D). Misexpression of GroDCR does not lead to repression, but instead leads to a very slight expansion of the vgQ-lacZ expression domain into regions at the anterior and posterior edges of the disc where the reporter is not normally expressed (Figure 7F). This is reminiscent of what is observed in gro loss-of-function clones [1,16] suggesting, once again, that GroDCR behaves as a dominant negative form of Gro. This is proven by an experiment in which co-expression of GroWT and GroDCR significantly attenuates repression by GroWT (Figure 7G). This dominant negative effect presumably reflects Q domain mediated association of GroDCR with endogenous GroWT. The adult wing phenotypes resulting from overexpression of the Gro deletion variants in the wing disc are consistent with the conclusions drawn from the vgQ-lacZ reporter assays. Figures 8A–F show representative wings from flies of each genotype, while Figure 8G displays the quantitative results of examining multiple discs from flies of each genotype and assigning them to phenotypic classes. All variants were expressed at very similar levels (Figure 8H). In accord with previous studies, we find that expression of wild-type Gro using the Ser-Gal4 driver (which directs expression in the dorsal compartment of the wing pouch) leads to wing blistering due to inappropriate repression of Gro targets (Figure 8B, G). The GP and CcN domains are required for this overexpression phenotype (Figure 8C, D, G), while the SP domain partially ameliorates it (Figure 8E, G). Finally, overex- pression of GroDCR (and to a lesser extent GroDCcN) results in wing scalloping and supernumerary wing bristles, which are wing phenotypes reminiscent of those that result from reductions in Notch signaling (Figure 8I) [49]. This is consistent with dominant negative functions for these variants since Gro is required for the function of E(spl) bHLH proteins, which are downstream effectors of Notch signaling [2,50]. In summary, the wing disc misexpression studies suggest that the positive roles of the GP and CcN domains and the negative role of the SP domain are maintained throughout development. A requirement for the GP domain in nuclear localization The CcN domain contains a conserved sequence resembling an NLS, and was thus assumed to be responsible for Gro nuclear import [14]. To determine the requirements of the CcN domain and other domains in nuclear localization, we examined the subcellular localization of central region deletion variants through immunofluorescence imaging of third instar larval wing discs and Drosophila S2 cells expressing tagged forms of these variants. PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e30610