Groucho Central Domains Instruct Development Figure 3. Rescue of gro mutant alleles with genomic rescue constructs encoding Gro deletion variants. (A) Intron/exon organization of gro and flanking regions from the left arm of chromosome 3. The 10 kb region used in the rescue constructs is indicated by the bracket. Exons encoding to the Q domain are in green, GP domain in blue, CcN domain in magenta, SP domain in orange, WD repeat domain in red, and non- encoding exons in gray. (B) The hypomorphic (MB12) and (MB36) gro alleles [16]. The black lines indicate the sequences included in each deletion variant or mutant allele. The green lines represent the coiled-coil motifs in the Q domain that are known to be required for self-association [18]. (C) The indicated Gro rescue constructs were tested, as described in Materials and Methods, for their ability to rescue the lethality associated with the MB12 or MB36 gro alleles. Each data point is the average (6 S.D.) of three independent trials (for each of trials 1 and 2, 120 flies were analyzed; for trial 3, 100 flies were analyzed). Asterisks (*) signify p,1025 as determined from the two-tailed unpaired student’s T test. doi:10.1371/journal.pone.0030610.g003 immunoprecipitation analysis in embryos (manuscript in prepara- tion) indicates that Gro is absent from the Rpt3 and cinnamon (cin) loci suggesting that these genes are not likely to be Gro repression targets. As predicted, overexpression of GroWT, GroDGP, GroDCcN, and GroDCR results in no change in Rpt3 and cin transcript levels. In contrast, overexpression of GroDSP results in ,3-fold repression of both these genes (Figure 4C). In conclusion, the SP domain seems to have a broad role in Gro specificity. In its absence, we observe increased levels of repression and promiscuous repression of genes that are not normally targeted by Gro. To determine if the central region modulates the spatial patterning of embryonic target gene expression or just the overall level of expression, we performed fluorescence in situ hybridiza- tion looking at the expression of Gro targets hkb, tll, and sna in the cellular blastoderm embryo. Gro participates in terminal pattern- ing by restricting the expression of hkb and tll to the embryonic termini through interaction with the repressor Cic [43,44] (Figure 5A–B), and mediates dorsoventral (d/v) pattern formation through interaction with Dorsal, a transcription factor that can function as both an activator of genes such as twi and sna and a repressor of genes such as dpp and zen [3,45]. Activation targets such as sna are expressed in a stripe around the ventral midline (Figure 5C), while repression targets such as dpp are expressed in the dorsal region of the embryo. The dual functionality of Dorsal relies on the fact that Dorsal has an intrinsically low affinity for Gro [46], and previous studies have shown that addition of a high affinity Gro interaction motif to Dorsal converts it into a dedicated repressor leading to sna repression rather than activation [47]. Targets of the d/v system, such as sna, are also under the regulation of the terminal pattern forming system, which works through Cic to repress these genes at the embryonic termini [43,48]. Thus, the effects of Gro variant overexpression on sna expression that we describe below reflect the Gro dependence of both the terminal and dorsoventral systems. In the following description of the effects of Gro variant overexpression on target gene expression pattern, we describe the most common phenotypes and present representative data (Figure 5A–X). We have quantified the data by looking at multiple embryos and assigning them to classes based on expression pattern (Figure 5Y–Z). The results of this quanitification are consistent with the conclusions presented below. In the case of each target gene, overexpression of GroWT not only results in decreased levels of expression, but also leads to a disruption of transcriptional patterning consistent with the idea that excess Gro activity leads to a resetting of the threshold concentrations at which graded repressors such as Dorsal and Cic repress transcription (Figure 5E–H, compare to Figure 5A–D). hkb is completely repressed at the posterior terminus of embryos containing excess GroWT, but only partially repressed at the anterior terminus of such embryos (Figure 5E, Y). tll is significantly reduced and restricted to the ventral side of such embryos at the posterior terminus and reduced at the anterior terminus (Figure 5F, PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e30610