digoxigenin coupled to biotin (1/500) from Jackson. Secondary antibodies coupled to Alexa 488, Alexa 555 (Molecular Probes) or to biotin (Jackson) were used at a 1/500 dilution. Constructs, transgenic lines, biological readouts, and quantification procedures AbdA variant were generated by PCR. Domain mutations were YPWMRAAAA; TDWMRAVAI; KEINERKAAAA. The ho- meodomain point mutation alleviating DNA binding is a mutation of position 50 (QRK; [47]). Constructs were cloned in pUAST or pUASTattB vectors for transgenic line establishment. Lines were crossed with the appropriate driver, and collected embryos were stained with anti-AbdA to select the conditions (line and temperature) that result in expression levels similar (+/215%) to AbdA wild type levels in A2 (see [22] for a detailed description of the procedure). Procedures used for quantification of biological readouts using at least 10 embryos of each genotype are provided in Text S1. Hierarchical clustering of domain requirements A matrix containing the values corresponding to the readout was built. The extreme values were given to the total loss of activity (value 0), and to the wild type activity (value 1 for 100% of activity). A hierarchical clustering algorithm (with Euclidian distance and average linking) was applied to the matrix using the MeV software suite [63]. The jacknife method was used for re- sampling the data and provides a statistical support for each tree node. Boxplot data representation Boxplots drawn using the R-Software. Boxplot depicts the value distribution obtained for each tested genotype. Black points correspond to individual counts. Supporting Information Figure S1 (Full data for Figure 2.) AbdA protein domain requirements for somatic muscle specification. Somatic muscle cells are visualized by Nau immunostaining (green). A represen- tative embryo is shown for each AbdA variant, as indicated. AbdA variants were ubiquitously expressed (red) in the mesoderm with the 24B-Gal4 driver. Dotted white rectangles highlights segments (thoracic or abdominal segment A8) where the effect of AbdA variants was determined. (PDF) Figure S2 (Full data for Figure 4A.) AbdA protein domain requirements for the regulation of the dpp direct target gene. The regulatory effect of AbdA variants on dpp expression was determined by in situ hybridisation to dpp transcripts (green). Arrow indicates the expression of dpp in PS7 of the visceral mesoderm. Gain of dpp expression in the visceral mesoderm is indicated by white dots, while loss of PS7 expression is denoted by the absence of arrow. AbdA variants were ubiquitously expressed (red) in the mesoderm with the 24B-Gal4 driver. A representative embryo is shown for each AbdA variant. (PDF) Figure S3 (Full data for Figure 4B.) AbdA protein domain requirements for the regulation of the wg direct target gene. The regulatory effect of AbdA variants on wg expression was determined by in situ hybridisation to wg transcripts (green). Arrow indicates the expression of wg in PS8 of the visceral mesoderm. Gain of wg expression in the visceral mesoderm is indicated by white dots, while loss of PS8 expression is denoted by Protein Domains Driving Hox Protein Function the absence of arrow. 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 showed that the HX mutation results in a protein that activates dpp instead of repressing it, and consequently more efficiently activates wg [41]. (PDF) Figure S4 (Full data for Figure 5.) AbdA protein domain requirements for oenocytes specification. Oenocytes, restricted to A1–A7 abdominal segments, were marked using a seven-up svp- lacZ construct (b-galactosidase staining in green). Ubiquitous expression of AbdA variants (red) with arm-Gal4 induces the formation of ectopic oenocytes in thoracic segments. A represen- tative embryo is shown for each AbdA variant. Boxed areas highlight thoracic segments. (PDF) Figure S5 (Full data for Figure 6A.) AbdA protein domain requirements for cerebral branch specification. The breathless btl- Gal4 driver, specific to tracheal branches, was used to simulta- neously express the AbdA variants (red) and the GFP reporter protein (green), allowing visualisation of tracheal defects. Presence (white arrow) or absence (red star) of the cerebral branch following ectopic expression of the AbdA variants is shown. (PDF) Figure S6 (Full data for Figure 6B.) AbdA protein domain requirements for the specification of heart cells. Thoracic segments are formed of four pairs of cardiac cells, while abdominal ones are composed of six pairs of cardiac cells. The two supplementary pairs of abdominal cardiac cells express Doc1 (green). Ectopic expression of AbdA (red) in the mesoderm driven with the 24B- Gal4 driver induces additional Doc1-expressing cells in thoracic segments that are now composed of six pairs of cells. A representative embryo is shown for each AbdA variant. (PDF) Figure S7 (Full data for Figure 7A.) AbdA protein domain requirements for the regulation of the Dll direct target gene. The regulatory effect of AbdA variants (red) on Dll expression was determined by the activity of the Dll DME enhancer (DME-lacZ, b-Galactosidase immunostaining (green). AbdA variants were ubiquitously expressed with the arm-Gal4 driver. A representative embryo for each AbdA variant is shown. Boxed areas highlight thoracic segments where the effect of AbdA variants was determined. (PDF) Figure S8 (Full data for Figure 7B.) AbdA protein domain requirements for the regulation of the Antp target gene. The regulatory effect of AbdA variants on Antp expression was determined by Antp immunostainings (green). AbdA variants were ubiquitously expressed with the arm-Gal4 driver. A representative embryo for each AbdA variant (red) is shown. Boxed areas highlight thoracic segments where the effect of AbdA variants was determined. (PDF) Figure S9 (Full data for Figure 7C.) AbdA protein domain requirements for A2 epidermal morphology. Abdominal segments harbour large and refringent denticles, organised in a trapezoid in A2 but not A1, while thoracic segments T2–T3 harbour smaller and less refringent denticles organised in a linear manner. The first thoracic segment T1 in addition harbours a specific feature termed PLoS Genetics | www.plosgenetics.org 12 October 2011 | Volume 7 | Issue 10 | e1002302