absent in thoracic segments, a feature that can be visualized by the expression of nautilus (nau) [32]. This distinction was previously shown to result, at least in part, from the activity of AbdA [32]. Accordingly, anterior ectopic expression of AbdA using the mesodermal driver (24B-Gal4) results in ectopic ventral expression of Nau in anterior segments (Figure 2). We found however that none of the AbdA protein domains under study, alone or in combination, was required to specify the abdominal specific features of the somatic musculature (Figure 2 and Figure S1). In the same conditions, a point mutation at position 50 of the homeodomain that impairs AbdA binding to DNA resulted in the loss of Nau inducing capacity (Figure 2 and Figure S1). The dispensability of the HX, TD and UA domains for specifying abdominal features of somatic muscle pattern is consistent with the fact that nau activation by AbdA is not dependent upon Exd activity [32], although results below argue that these domains assume other functions than Exd recruitment. Single protein domain requirement for NB5–6 CNS lineage specification In the embryonic central nervous system, a subset of 30 neuroblasts (NB’s) found in each hemisegment, including the NB5–6, generate a larger lineage in the thorax than in the abdomen. Recent studies demonstrated that posterior Hox genes, such as abdA, impose in the abdomen a smaller NB5–6 lineage by triggering an early cell cycle exit [39]. Misexpression of AbdA within NB5–6 in the thorax using ladybird(K)-Gal4 result in an early lineage truncation, mimicking the situation that normally occurs in the abdomen, ultimately leading to a smaller thoracic NB5–6 lineage size (Figure 3). Average number of NB5–6 cells in wild type thoracic and abdominal segments was previously estimated at 16 and 6 cells respectively: these values were considered as references for full (100%) or complete loss (0%) of repressive activities of AbdA variants on NB5–6 lineage. Intermediate repressive levels upon ectopic expression with ladybird(K)-Gal4 were deduced from Protein Domains Driving Hox Protein Function the quantification of NB5–6 lineage cell numbers in thoracic segments T2/3 (see methods). Results obtained indicate that lineage truncation triggered by AbdA is similarly affected following UA, HX/UA, TD/UA and HX/TD/UA mutations (Figure 3), which can be best explained by a unique requirement of the UA domain for AbdA function. Protein domain mutations induces neomorphic activity in the regulation of the dpp and wg target genes In the visceral mesoderm, AbdA is expressed in parasegment (PS)8–12. The target genes wg and dpp are respectively activated (in PS8) and repressed (in PS8–12) by AbdA in the visceral mesoderm. Restricted (PS8) activation of wg by AbdA results from the action of the Dpp signal, locally produced by PS7 cells under the control of the Ubx protein [40]. Accordingly, anterior ectopic expression of AbdA only results in a mild activation of wg, as activation only occurs in cells experiencing partial repression of dpp [27]. Previous work has shown that the HX mutation results in a protein that activates dpp instead of repressing it, and consequently more efficiently activates wg [41]. AbdA variants were expressed with the 24B-Gal4 driver. Levels of regulatory activities were deduced following fluorescent in situ hybridization against dpp or wg in the visceral mesoderm of stage 14 embryos in PS1–PS7, ie anterior to endogenous AbdA expressing cells (PS8–12; Figure 4 and Figures S2 and S3). Arbitrary values have been assigned to regulatory activities of AbdA variants. For dpp (Figure 4A and Figure S2), no effect on dpp expression was scored by 0, normal repression of dpp expression in PS7 by 100 (partial repression was never observed) and ectopic activation (instead of repression) of dpp was scored by negative values (depending of the number of ectopic sites (see Text S1). For wg, in a manner similar to dpp, no effect was scored by 0, and positive and negative values were respectively assigned to normal (activation) or abnormal (repression) activities on wg expression (Figure 4B and Figure S3; see Text S1). Figure 2. Dispensability of HX, TD, and UA protein domains for somatic muscle specification. Somatic muscles are visualized by Nautilus (Nau, green) expression (upper panels). Expression of AbdA (red) in the thorax using the 24B-Gal4 driver induces abdominal specific muscle pattern (white arrows) in thoracic segments (red arrows) (middle panels). The effect of the AbdAHX,UA variant is illustrated (lower panels). Right panels are magnifications of boxed areas. Graphs (% of remaining activities compared to the wild type AbdA protein (WT) following domain mutations) using the boxplot representation on the right summarize quantitative analyses (see Text S1 and Figure S1 for full illustration). 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.g002 PLoS Genetics | www.plosgenetics.org 3 October 2011 | Volume 7 | Issue 10 | e1002302