Groucho Central Domains Instruct Development Figure 1. The central domains mediate multiple essential functions and are likely disordered. Prediction of disorder in Gro using two disorder prediction algorithms: PONDR-FITTM (Meta, black dashed line) [40] and FoldIndex (green and red shaded plot) [41]. FoldIndex tool also calculated hydrophobicity (blue line) and charge density (pink line). Residues with Meta scores exceeding 0.5 are likely to be disordered, as are residues with FoldIndex scores below 0. The prediction tools strongly suggest that the Gro central region is disordered, while the Q and WD-repeat domains are ordered. The Gro domains (shown along the horizontal axis) are labeled with the many of the functions that have been previously ascribed to them (see text for references). doi:10.1371/journal.pone.0030610.g001 site (attP) [42]. The genomic construct encoding GroWT is approximately 10 kb in length and includes the gro transcription unit as well as flanking sequences reaching to the neighboring genes both upstream and downstream of the gro transcription unit (Figure 3A). In the course of introducing the full-length genomic construct into flies, we noticed that the flies were surprisingly sensitive to gro gene dosage. Specifically, we found that, while flies could tolerate a single copy of the gro transgene (in a background containing two wild-type copies of endogenous gro), a second copy of the transgene resulted in complete lethality. To assess the recessive phenotypes of the deletion variants, the transgenes encoding GroWT, GroDGP, GroDCcN, and GroDSP were crossed into flies homozygous for either of two lethal alleles of gro (the groMB36 allele or the hypomorphic groMB12 allele [16], Figure 3B). The construct encoding GroWT completely rescued the lethality associated with either gro allele (Figure 3C). In contrast, constructs encoding GroDGP and GroDCcN were unable to rescue the lethal gro alleles (Figure 3C), strongly suggesting essential roles for the GP and CcN domains in Gro function. In the presence of endogenous wild-type Gro, the GroDCcN rescue construct rendered the flies weak and led to wing blistering and abnormal abdominal segmentation, while GroDGP had no such dominant phenotype (data not shown). Although the construct encoding GroDSP gave some rescue, this rescue was ,5 to 8-fold less efficient than rescue by the wild-type transgene (Figure 3C). We did not attempt to generate a rescue construct encoding GroDCR because preliminary misexpression experiments (see the following section) strongly suggested that this construct would lead to dominant lethality. In conclusion, the central domains appear to have essential roles in Gro function strongly suggesting that their apparent immuta- bility is a reflection of their disorder. To explore the functions of the central domains further, we have examined the effects of misexpression of the deletion variants in two different develop- mental contexts: the embryo and the wing disc. The GP, SP, and CcN domains are required for embryonic patterning To determine the roles of the central domains in embryonic patterning, we used a maternally active Gal4 driver to direct expression of UASp constructs encoding GroWT, GroDGP, GroDCcN, GroDSP, and GroDCR. qRT-PCR analysis of the transcripts in 0–3 hour embryos encoding the maternally expressed Gro variants indicates that all five variants are overexpressed by ,4-fold relative to endogenous Gro (Figure 4A), a finding that is consistent with the results of an anti-Gro immunoblot (Figure 4B). We examined the effect of maternal overexpression of the deletion variants on the expression of a variety of early embryonic Gro targets by qRT-PCR. If expression levels of Gro target genes were sensitive to Gro levels (as opposed to just its absence or presence), overexpression of active Gro variants would be expected to reduce expression of Gro targets to levels below those seen in wild-type embryos. In accord with this expectation, overexpression of GroWT leads to repression of the Dorsal targets zerknu¨lt (zen), twist (twi), dpp, and snail (sna), the Hairy target fushi tarazu (ftz), and the Cic targets huckebein (hkb) and tailless (tll), and apparent activation of the Tll target knirps (kni) (Figure 4C, repression corresponds to relative repression values greater than 1, while activation corresponds to relative repression values less than 1). The qRT-PCR assays indicate that the GP domain is critical for Gro-mediated repression, since, consistent with the genomic rescue experiments, GroDGP overexpression has essentially no effect on Gro target gene expression. The CcN domain is also critical for repression as GroDCcN overexpression leads to no repression of Gro targets and, in at least one case (dpp), results in significant activation of a Gro target. Similarly, overexpression of GroDCR (in which the entire central region is deleted) leads to no repression of Gro targets and, in several cases (e.g., zen, hkb, and sna), leads to significant activation of these targets. Thus, GroDCcN PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e30610