Stefanie D. Hueber, Georg F. Weiller, Michael A. Djordjevic, Tancred Frickey

The family of Hox-proteins has been a major focus of research for over 30 years. Hox-proteins are crucial to the correct development of bilateral organisms, however, some uncertainty remains as to which Hox-proteins are functionally equivalent across different species. Initial classification of Hox-proteins was based on phylogenetic analysis of the 60 amino acid homeodomain. This approach was successful in classifying Hox-proteins with differing homeodomains, but the relationships of Hox-proteins with nearly identical homeodomains, yet distinct biological functions, could not be resolved. Correspondingly, these 'problematic' proteins were classified into one large unresolved group. Other classifications used the relative location of the Hox-protein coding genes on the chromosome (synteny) to further resolve this group. Although widely used, this synteny-based classification is inconsistent with experimental evidence from functional equivalence studies. These inconsistencies led us to re-examine and derive a new classification for the Hox-protein family using all Hox-protein sequences available in the GenBank non-redundant protein database (NCBI-nr). We compare the use of the homeodomain, the homeodomain with conserved flanking regions (the YPWM and linker region), and full length Hox-protein sequences as a basis for classification of Hox-proteins. In contrast to previous attempts, our approach is able to resolve the relationships for the 'problematic' as well as ABD-B-like Hox-proteins. We highlight differences to previous classifications and clarify the relationships of Hox-proteins across the five major model organisms, Caenorhabditis elegans, Drosophila melanogaster, Branchiostoma floridae, Mus musculus and Danio rerio. Comparative and functional analysis of Hox-proteins, two fields crucial to understanding the development of bilateral organisms, have been hampered by difficulties in predicting functionally equivalent Hox-proteins across species. Our classification scheme offers a higher-resolution classification that is in accordance with phylogenetic as well as experimental data and, thereby, provides a novel basis for experiments, such as comparative and functional analyses of Hox-proteins.