New Insights From Hemichordate Genomes: Prebilaterian Origin and Parallel Modifications in the Paired Domain of the Pax gene eyegone MARKUS FRIEDRICH 1,2Ã AND JASON CARAVAS 1 1 Department of Biological Sciences, Wayne State University, Detroit, Michigan 2 Department of Anatomy and Cell Biology, Wayne State University, School of Medicine, Detroit, Michigan The evolutionary origin of the Drosophila Pax transcription factor gene eyegone (eyg) has long been enigmatic owing to the failure in detecting orthologs in other species and the unusual N-terminal truncation of the DNA-binding paired domain (PD). Based on the discovery of eyg orthologs in representatives of hemichordate phyla, we show that the origin of eyg predated metazoan diversification and that the PD experienced similar but independent N-terminal modifications in the lineages to sea urchins and insects. Sequence conservation patterns further raise the possibility of persisting functionality in the N-terminal PD of strongly modified eyg orthologs. Finally, we note that the evolutionary histories of eyg and the vertebrate Pax6 isoform 5a, which have been considered functional homologs, are not correlated. Taken together, these findings identify Drosophila eyg as the baptizing member of an ancient Pax gene subfamily and recommend abandoning its classification as Pax6(5a)-related gene. J. Exp. Zool. (Mol. Dev. Evol.) 316:387–392, 2011. & 2011 Wiley-Liss, Inc. How to cite this article: Friedrich M, Caravas J. 2011. New insights from hemichordate genomes: prebilaterian origin and parallel modifications in the paired domain of the Pax gene eyegone. J. Exp. Zool. (Mol. Dev. Evol.) 316:387–392. RESULTS Pax genes constitute an ancient transcription factor gene family with important functions in development and body plan evolution (Chi and Epstein, 2002). Previous studies recognized up to six ancient subfamilies, most of which include insect and vertebrate homologs (Table 1) (Groger et al., 2000; Sun et al., 2002; Matus et al., 2007). Two DNA-binding domains are found in Pax genes: the paired domain (PD) and the homeodomain (HD). The PD is present in all Pax genes, whereas the HD is absent from the members of some Pax gene superfamilies. The most atypical Pax gene reported so far is encoded by the Drosophila gene eyegone (eyg), which possesses a canonical HD but unusually organized PD (Jun et al., ’98). Canonical PDs are 128 amino acids long, encompassing three DNA-contacting modules: the N-terminal PAI subdomain, the linker region, and the C-terminal RED subdomain (Czerny et al., ’93; Xu et al., ’95, ’99). The PAI subdomain is the most complex, containing two b-sheets in its N-terminal flank followed by the PD a-helices 1–3 at the C-terminal flank. Drosophila eyg contains only sequence regions with strong similarity to the linker region and RED subdomain of other Pax genes. It has, therefore, been concluded that Drosophila eyg lost the PAI subdomain during evolution (Jun et al., ’98). Adding to the ‘‘mystery’’ of the eyg gene (Pichaud and Desplan, 2002), no orthologs could be identified in species other than Drosophila, suggesting a more recent origin. The taxonomic uniqueness of eyg was further enhanced by the discovery of a closely related paralog of eyg in Drosophila, named twin of eyegone (toe) (Aldaz et al., 2003; Yao and Sun, 2005). Protein sequence comparisons revealed domain-specific similarities of Published online 19 April 2011 in Wiley Online Library (wileyonline library.com). DOI: 10.1002/jez.b.21412 Received 20 January 2011; Revised 26 February 2011; Accepted 2 March 2011 Grant Sponsor: National Science Foundation; Grant number: 0951886. Additional Supporting Information may be found in the online version of this article. Ã Correspondence to: Markus Friedrich, Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Detroit, MI 48202. E-mail: mf@biology.biosci.wayne.edu ABSTRACT J. Exp. Zool. (Mol. Dev. Evol.) 316:387–392, 2011 & 2011 WILEY-LISS, INC. PERSPECTIVE AND HYPOTHESIS