Groucho Central Domains Instruct Development Figure 8. Phenotypes resulting from overexpression of Gro variants in the wing disc. The Ser-Gal4 driver was used to drive expression of GroWT or Gro internal deletion variants in the wing. (A) A wing containing the driver but no UAS-Gro construct shows no phenotype. Overexpression of GroWT (B) and GroDSP (E) resulted in moderate to severe blistering and vein deformation. Overexpression of GroDGP (C) resulted in no defects, while overexpression of GroDCcN (D) resulted in bifurcation of the 4th and/or 5th longitudinal vein (LV, arrows). Overexpression of GroDCR (F) resulted in severe blistering and vein deformation as well as ectopic bristles along the wing vein (inset, yellow arrows). (G) 100 wings of each genotype were scored according to phenotype as indicated showing the differences in the relative severity of the phenotypes generated by overexpression of the various deletion variants. (H) Gro immunoblot of third instar wing discs verifies equal expression levels. Tubulin serves as a control for equal protein levels. (I) In addition to the blistering and wing veination defects, GroDCcN and GroDCR Gro overexpression also resulted in 13% and 29% wing scalloping phenotypes (arrows). The severe defects in the GroDCR overexpressing wings and the milder defects in the GroDCcN overexpressing wings probably results from a dominant negative function for these variant as discussed in the text. The wing scalloping (J) and the ectopic wing bristles (F and I, indicated by arrows in the high magnification insets), both of which are reminiscent of a Notch pathway hypomorphic phenotypes [49]. Except for the high magnification insets in panels F and I, the wing images were obtained using a 46 objective. Scale bars = 400 mm. For the high magnification insets in panels F and I, the images were obtained using a 406 objective. doi:10.1371/journal.pone.0030610.g008 repeat domain. Furthermore, if the deletions were leading to the misfolding of the conserved domains, we might expect to detect changes in protein stability in vivo, which is something that we do not observe. In addition, the observation that deletion of the CcN domain or of the entire central region leads to dominant negative phenotypes consistent with the known roles of Gro in development (e.g., its role in Notch signaling) suggests that these variants are able to associate normally with endogenous Gro in vivo via a properly folded Q domain. Finally, our finding that deletion of the GP, CcN, and SP domain deletions each result in different phenotypes (see below) is inconsistent with the idea that the only role of these domains is to maintain the spacing between the Q and WD-repeat domains. The sensitivity of development to Gro levels Many of the repressors that interact with Gro (e.g., Dorsal, Cic, and Brk) are distributed in concentration gradients and repress target genes in a concentration dependent manner to direct the formation of multiple distinct domains of gene expression [1,3,5,44]. It is often assumed that Gro merely needs to be present in an active form to allow these repressors to function. Contrary to this idea, however, we find that development is exquisitely sensitive to Gro activity levels. Our overexpression experiments show that a modest increase in Gro levels leads to changes in the spatial domains of target gene expression demonstrating that the threshold concentrations at which targets genes respond to graded repressors are sensitive to Gro activity levels. This is further shown by the sensitivity of development to the gro gene dosage as we have shown that flies bearing, one, two, or three copies of the gro gene are viable, while a fourth copy results in a loss of viability. How can we account for the sensitivity of transcription factor gradient threshold responses to Gro activity levels? One possibility is that the assembly of a transcriptionally silent domain is a highly cooperative process that involves the spreading of Gro across a large chromosomal region. A cooperative process of this type might well be highly sensitive to the concentration of active Gro. An unanticipated role for the SP domain in target gene specificity The SP domain apparently functions to keep Gro activity in check as deletion of this domain results in increased repression of Gro targets, decreased target gene specificity (and therefore promiscuous repression), and severely weakened rescue activity. The role of the SP domain in specificity is completely unanticipated since Gro is not a DNA binding protein and therefore would not be expected to have a role in the selection of target genes. However, Gro is capable of binding to chromatin PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e30610