Protein Domains Driving Hox Protein Function Figure 3. Single protein domain requirement for NB5–6 lineage truncation. Neuroblast 5–6 are visualized using the ladybird early lbe(K)- Gal4 driver expressing nuclear GFP (green; upper panels). Dotted rectangles highlight differences in cell number of the T2/3 thoracic NB5–6 lineage. Expression of AbdA (red) in NB5–6, through the lbe(K)-Gal4 driver, leads to thoracic lineage truncation (middle panels). The effect of AbdAUA is illustrated in the lower panel. Graphs (% of remaining activities compared to the wild type AbdA protein (WT) following domain mutations) using the boxplot representation on the right summarize quantitative analysis (see Text S1). A graded color-coded bar above the graphs illustrates the level of protein activity, ranging from light green (full activity) to black (no activity). doi:10.1371/journal.pgen.1002302.g003 Results obtained allow two conclusions. First, single domain mutations result in strong modification of AbdA activity. Second, domain mutations often result not only in a quantitative, but also in a qualitative (neomorphic) modification of activity, changing AbdA from an activator to a repressor, or reversely from a repressor to an activator. Additive contribution of protein domains for oenocyte specification Oenocytes form under AbdA control in segments A1–A7. This occurs through AbdA-dependent activation of Rhomboid (Rho) in a chordotonal organ precursor cell called C1. Expression of Rho then enables the secretion of the EGF ligand Spitz that will instruct neighboring epidermal cells to differentiate into oenocytes [30]. In absence of AbdA, the EGF pathway is not locally activated and oenocytes are not specified [30]. Reversely, ectopic expression of AbdA induces oenocytes in thoracic segments. AbdA variants were ubiquitously expressed with the armadillo (arm)-Gal4 driver. Oenocyte inducing potential of AbdA variants, visualised with the seven-up (svp)-lacZ enhancer trap reporter construct, was deduced from the number of thoracic segments that contain ectopic oenocytes (see Text S1). This inductive potential is reduced following single mutations of the UA domain and combined mutation of the HX/TD or TD/UA domains, and is abolished following HX/UA and HX/TD/UA mutations (Figure 5 and Figure S4). These observations suggest an additive contribution of the HX, TD and UA protein domains for oenocyte induction by AbdA, consistent with protein domains acting independently of each other, and contributing uniquely through additive contribution to protein activity. Functional redundancy in protein domain usage for trachea and heart lineage specification The tracheal cerebral branch forms dorsally exclusively in the second thoracic segment T2, in response to repressive activities of Bithorax Hox proteins in T3-A8 segments [42]. This phenotypic trait can be followed by a breathless (btl) driven GFP reporter that extends posteriorly in the absence of Bithorax complex genes, and that is suppressed in T2 following Btl-driven expression of AbdA in the tracheal system (Figure 6A). Only full repression of cerebral branches was considered and repressive activities of AbdA variants thus correspond to either 0% (no repression) or 100% (full repression) (see Text S1). We found that the repression of the cerebral branch by AbdA is impaired following TD/UA and HX/ TD/UA but not HX/UA or HX/TD mutations, revealing a functional redundancy between the TD and UA domains (Figure 6A, and Figure S5). In the embryonic heart, abdominal segments are made of six pairs of cells, instead of four in thoracic segments [37]. This difference was shown to result from AbdA (and Ubx) promoting the six cell lineage in the abdomen [37], and in the thorax following AbdA ubiquitous expression in the mesoderm driven by the 24B-Gal4 driver ([37], Figure 6B). The visualization of the lineage is facilitated by a Dorsocross (Doc) staining, that labels two cells in each hemisegment, allowing to unambiguously identify each hemisegment. Effects of AbdA variants in cardiac cells specification were visualized by double fluorescent immunostain- ing against AbdA and Dorsocross (Doc). The six cell lineage inductive capacity of AbdA was scored by counting the number of cardiac cells in the T2 and T3 segments (see Text S1). Results showed that the six cell lineage inductive ability of AbdA is lost following HX/UA and HX/TD/UA mutations (Figure 6B and Figure S6). These observations again highlight functional redun- dancy, but between the UA and HX domains, instead of TD and UA domain as observed in cerebral branch specification. Additional examples of functional redundancy, yet in more complex pattern of interactions between protein domains were found in the biological contexts described below. Mutually suppressive interaction of protein domains in the regulation of the Dll and Antp direct target genes, the specification of epidermal morphology, and larval locomotion The limb-promoting gene Distalles (Dll) and Hox gene Antennapedia (Antp) are direct targets of AbdA [26,43]. The ability of AbdA variants, following ubiquitous expression through the PLoS Genetics | www.plosgenetics.org 4 October 2011 | Volume 7 | Issue 10 | e1002302