Protein Domains Driving Hox Protein Function Figure 5. Additive contribution of protein domains. Oenocytes ventrally located in the abdomen are visualized by b-gal (green) driven by the seven-up (svp) promoter (white arrows, upper panel). Expression of AbdA (red) in the thorax through the arm-Gal4 driver induces ectopic oenocytes in the thorax (red arrows, middle panels). The effect of the AbdAHX/UA variants is illustrated (lower panels). Boxed areas highlight thoracic segments. Right panels are magnifications of the 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 S4 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.g005 activities, while in the case of Antp simultaneous mutation of the HX and UA domains or TD and UA domains results in a loss of 70% of AbdA repressive activity. More surprisingly, simultaneous mutation of the HX, TD and UA domains does not compromise further AbdA activity but instead restores a significant level of repressive activity, comparable to that of single domain mutated AbdA variants. This indicates that the three protein domains do not provide independent regulatory input, but likely act in interactive and mutually inhibitory ways. A similar yet more complex pattern of domain interactions was observed in the specification of A2 epidermal morphology. In this tissue, AbdA promotes the formation of a stereotyped trapezoidal arrangement of denticle belts (Figure 7C). The potential of AbdA variants to specify A2 epidermal morphology was assessed following arm-Gal4 driven expression by scoring the denticle belts morphology and organisation in transformed A1 and thoracic segments (Figure 7C and Figure S9). Epidermal specification was not impaired by HX and slightly reduced by UA or TD mutations. Simultaneous mutation in two domains suggests functional redundancy between HX and TD, UA and HX but not between UA and TD domains. As noticed previously for the regulation of Dll and Antp in the epidermis, mutating the three domains simultaneously restores the activity, generating a protein that displays an activity close to the wild type protein. In many animals including vertebrates, locomotion results from the coordinated action of regionally distinct sets of movements. Drosophila larvae crawl by means of three region specific move- ments [38]. The locomotion cycle starts by a contraction of the most posterior abdominal segments (A8/A9), followed by a wave of peristaltic movement in A1–A7, where each segment is transiently lifted up (D/V movement), pulled forward and lowered, starting from A7. When the wave reaches A1, the thoracic and head segments start moving by a telescopic type of movement (A/P movement), occurring through contraction of anterior segments [38]. It was established that AbdA is necessary and sufficient to specify the abdominal type of movement, namely abdominal peristalsis [38]. The potential of wild type and AbdA variants to promote abdominal peristalsis was evaluated following arm-Gal4 driven expression (Figure 7B), by scoring in the T3 thoracic segment D/V movements (see Text S1). Single domain mutations do not significantly alter promotion of abdominal peristalsis (Figure 7D and Figure S10). Again, two types of functional redundancy were observed: between the TD and UA domains, and to a lesser extent between the HX and UA domains. As in the case of Dll and Antp regulation and A2 epidermal morphology specification, triple domain mutation corrected the effects of double mutations, with a protein promoting abdominal peristalsis as efficiently as the wild type protein, providing an additional example of mutually suppressive activity of protein domains. Multifunctionality of protein domains revealed by Exd- dependency Previous studies have established that Exd is required for Dll [25] and wg [45] regulation, oenocytes [30] and epidermal morphology specification [46], and neuroblast lineage commit- ment [37], while dispensable for Antp [46] and dpp [47] regulation. In the case of cerebral branch specification, no conclusion could be reached since loss of Exd results in the absence of cerebral branch formation in the T2 segment [48]: this positive input of Exd hinders the assessment of a possible contribution for AbdA mediated cerebral branch repression in abdominal segments. The potential implication of Exd in AbdA-mediated heart lineage commitment and larval locomotion is not known. Staining for Doc1 in embryos deprived for maternal and zygotic Exd showed that the abdominal hemi segments adopt the AbdA- dependent six cell lineage, showing the dispensability of Exd for this AbdA function (Figure 6C). The requirement of Exd for larval locomotion has been examined in homothorax (hth) mutant that impairs Exd nuclear transport and mimics exd maternal and PLoS Genetics | www.plosgenetics.org 6 October 2011 | Volume 7 | Issue 10 | e1002302