Groucho Central Domains Instruct Development Figure 4. Central domains have both positive and negative roles during Gro-mediated repression in the embryo. (A) qRT-PCR analysis of the gro transcript in embryos shows that Mat-Gal4 driven expression results in very similar levels of overexpression of each variant. (B) An anti-Gro immunoblot verifies equal expression of the variants. An anti-tubulin immunoblot serves as a control for relative total protein levels. (C) qRT-PCR was performed on embryos lacking maternally overexpressed Gro; or containing maternally overexpressed GroWT (green), GroDGP (blue), GroDCcN (pink), GroDSP (orange), or GroDCR (yellow). Expression levels of zen, dpp, twi, ftz, hkb, tll, sna, kni, Rpt3, and cin were normalized for expression levels of RpL32. Each fold repression value was obtained by dividing the normalized expression level of a gene in the absence of overexpressed Gro by the normalized expression level of the same gene in the presence of one of the overexpressed Gro variants. Values were graphed on a log2 scale. Statistical significance of each value relative to the value in embryos lacking overexpressed Gro was determined from the two-tailed unpaired Student’s T-test. * signifies p,0.05, ** signifies p,0.01, *** signifies p,0.005. No asterisk signifies p.0.05. doi:10.1371/journal.pone.0030610.g004 Y). sna expression retracts significantly from the termini, and its expression domain is narrowed along the dorsoventral axis (Figure 5G, Z). In contrast, overexpression of GroDGP or GroDCcN leads to no observable changes in spatial patterning of target genes (Figure 5I–P, Y, Z) in support of the conclusion that the GP and CcN domains are essential for Gro activity. As with overexpression of GroWT, overexpression of GroDSP disrupts the expression patterns of hkb, tll and sna (Figure 5Q–T, Y, Z). However, the effects are more severe than those resulting from overexpression of GroWT. No hkb or tll expression is observed at the embryonic termini (Figure 5Q, R, Y). sna expression retracts from both termini in these embryos to a greater degree than is observed in embryos overexpressing GroWT (Figure 5S, Z). Consistent with the notion that GroDCR has a dominant negative function, overexpression of this variant results in expansion of the expression domains. Slight expansion of hkb at the posterior pole of the embryo (Figure 5U, Y) and ventral expansion of tll at the anterior pole (Figure 5V, Y) are observed. In the case of sna, we observe loss of repression at the poles as well as an increase in the width of the ventral stripe (Figure 5W, Z). The cuticle phenotypes resulting from maternal overexpression of the Gro variants are consistent with the effects on target gene expression (Figure 6A–G). While many of the embryos overex- pressing GroWT and GroDSP fail to form cuticle (Figure 6H), those that do show pleiotropic patterning defects, reflecting the repression of multiple genes directing pattern formation (Figure 6B, E). GroDCR overexpression also results in severely defective cuticles, presumably due to a dominant negative function for this variant (Figure 6F). In contrast, overexpression of GroDGP or GroDCcN results in no defects or only mild defects validating the critical roles played by the GP and CcN domains in Gro function (Figure 6C, D). We conclude that the GP and CcN domains are crucial for Gro- mediated repression in the embryo, while the SP domain serves to temper Gro activity and restrict target gene selection. Further- more, Gro overexpression modulates target gene expression patterns in addition to target gene expression levels. The Gro central region is also required for transcriptional repression in the wing disc To determine if the above conclusions are applicable in another developmental system, we examined the effect of misexpressing the Gro variants in the developing wing. Anteroposterior (a/p) patterning of the wing imaginal disc requires the Gro-dependent repressor Brk, which silences the expression of genes such as optomotor blind (omb) and vestigial (vg) at the anterior and posterior PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e30610