Protein Domains Driving Hox Protein Function Figure 9. Phylogeny of the HX, TD, and UbdA protein domains. Abbreviations are as follows: B: bilaterians; P: protostomes; D: deuterostomes; L: lophotrochozoa; E:ecdysozoa; Cy, cycloneuralia. PA: pan-arthopods; O: onycophores; T: tardigrades; Ch: chelicerates; M; myriapods; Cr: crustaceans; H:hexapods. Sequence alignment spanning the HX, TD and UA domains are shown for representatives of the four main arthropod branches (Tc: Tribolium castaneum; Mr: Myrmica rubra; Ag: Anophela gambiae; Dm: Drosophila melanogaster) and for a representative of deuterostomes (Mus musculus, Mm) and lophotrochozoa (Hirudo medicinalis, Hme). doi:10.1371/journal.pgen.1002302.g009 ones. This is for example the case for the acquisition of the QA domain that confers repressive function to Ubx [6], and the acquisition/loss of HX or LRALLT domains by Futzitarazu (Ftz) from distinct insect species, which provides Ftz with the capacity to recruit either Exd or FtzF1 cofactors and switches its activity from a Hox to a segmentation protein [53]. In the second model, the acquisition of a novel protein domain interferes with the activity of pre existing domains, reorganizing the intrinsic functional organization of the protein. This view is supported by the predominant role of the UA domain and the widespread domain interactivity seen in this study. More room for changes in protein activity during morphological evolution Evolutionary changes in animal morphology is thought to mostly rely on changes in cis-regulatory sequences [1]. This is conceptually supported by the modular organization of cis- regulatory sequences, allowing subtle and cell specific changes in gene expression not deleterious for the animal. Experimentally, it is largely supported by the correlation between expression of key developmental regulatory genes and morphological changes (for example see [54]), and by changes in cis-regulatory sequences that impact on morphological traits [55–59]. Changes in animal morphology could also result from changes in protein sequence and function, as shown for Hox proteins in the morphological diversification in arthropods [4,6]. However, changes in protein function are not believed to broadly contribute to morphological diversification during animal evolution, based on the assumption that changes in protein sequences are expected to have pleiotropic effects, which as such, do not provide a mean to convey subtle and viable evolutionary changes. Our work grasps redundancy and selectivity in protein domain usage and as salient features of AbdA transcription factor intrinsic regulatory logic: even the HX domain, evolutionarily conserved in all Hox proteins, is essential for only one AbdA function, and often acts in a redundant way with the TD or the UA protein domains. Selective use of protein domains is also supported by findings of a few smaller scale studies of three other Drosophila Hox proteins: viable missense or small deletion mutations within the Scr protein coding sequences falls in different allelic series when examined for three distinct biological readouts [60]; deletion of C-terminal sequences of the Ubx protein, starting from an insect specific QA protein domain preferentially affects a subset of Ubx function [61]; dispensability of the HX was reported for the leg inducing capabilities of the Antp Hox protein, while required for other Antp functions [62]. This context dependent selective mode of protein domain usage, or differential pleiotropy, may be essential for the evolution of Hox protein functions, as it ensures developmental robustness of a Hox-controlled program while being permissive to evolutionary changes endowing novel functions to preexisting protein domains. In addition, our work also establishes that interactivity between protein domains is highly context dependent, suggesting that Hox protein function not only relies on selective mode of protein domain usage but also on selective mode of protein domain interactivity. Altogether, these observations challenge the view that changes in protein sequences necessarily have pleiotropic effects, giving more room for protein changes in the evolution of animal body plans. Materials and Methods Flies, egg collections, cuticle preparations, in situ hybridization, and immunostaining 24B-Gal4 and arm-Gal4 were used as embryonic mesodermal and ubiquitous drivers, respectively. Btl-Gal4 and lbe(K)-Gal4 for specific expression in the tracheal system and NB5–6 neuroblasts, respectively. The DME-lacZ and svp-lacZ lines are respectively from R. Mann (Columbia Univ., NY, USA) and S. Zaffran (IBDML, Marseille, France). exdXP11 and hthP2 alleles were used. Embryo collections, cuticle preparations, in situ hybridizations, and immunodetections were performed according to standard proce- dures. Digoxigenin RNA-labelled probes were generated accord- ing to the manufacturer’s protocol (Boehringer Mannheim, Gaithersburg, MD) from wg and dpp cDNAs cloned in Bluescript. Primary antibodies used are: anti-Antp (4C3, dilution 1/100, Developmental Studies Hybridoma Bank (DSHB)); rabbit anti- AbdA (1/1000); guinea-pig anti-Doc2+3 (1/400) and rabbit anti- Dmef2 (1/2000) from L. Perrin (IBDML, Marseille, France); rabbit anti-Exd (1/1000) from R. Mann; rabbit anti-Nau (1/100) from BM Paterson (University of Texas Southwestern Medical Center, Dallas, TX); rabbit (1/500) or mouse (1/200) anti-GFP (1/500) from Molecular Probes; chicken anti-GFP (1/1000) from Aves labs; mouse anti-b-galactosidase (1/1000) from Promega; rabbit anti-b-galactosidase (1/1000) from MP Biomedical; anti- PLoS Genetics | www.plosgenetics.org 11 October 2011 | Volume 7 | Issue 10 | e1002302