Neofunctionalization in Vertebrates: The Example of Retinoic Acid Receptors

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Abstract
Understanding the role of gene duplications in establishing vertebrate innovations is one of the main challenges of Evo-Devo (evolution of development) studies. Data on evolutionary changes in gene expression (i.e., evolution of transcription factor-cis-regulatory elements relationships) tell only part of the story; protein function, best studied by biochemical and functional assays, can also change. In this study, we have investigated how gene duplication has affected both the expression and the ligand-binding specificity of retinoic acid receptors (RARs), which play a major role in chordate embryonic development. Mammals have three paralogous RAR genes—RARα, β, and γ—which resulted from genome duplications at the origin of vertebrates. By using pharmacological ligands selective for specific paralogues, we have studied the ligand-binding capacities of RARs from diverse chordates species. We have found that RARβ-like binding selectivity is a synapomorphy of all chordate RARs, including a reconstructed synthetic RAR representing the receptor present in the ancestor of chordates. Moreover, comparison of expression patterns of the cephalochordate amphioxus and the vertebrates suggests that, of all the RARs, RARβ expression has remained most similar to that of the ancestral RAR. On the basis of these results together, we suggest that while RARβ kept the ancestral RAR role, RARα and RARγ diverged both in ligand-binding capacity and in expression patterns. We thus suggest that neofunctionalization occurred at both the expression and the functional levels to shape RAR roles during development in vertebrates. In eukaryotic organisms, each gene is a stretch of DNA composed of control regions that bind transcription factors and coding regions that transcribe the mRNA that is later translated into proteins. At the molecular level, changes in control regions can affect the time and place at which a protein is synthesized, whereas changes in the coding region can alter the protein's function. Retinoic acid receptors (RARs) are chordate-specific transcription factors which, upon binding the natural morphogen retinoic acid, bind to and activate transcription from target genes. Here, the authors show how the ligand specificity of RARs has changed during vertebrate evolution in parallel with changes in expression. Through functional characterization of the RARs from several vertebrates, the chordate amphioxus, and the reconstructed ancestral RAR sequence, the authors show that of the three vertebrate RARs, RARβ has retained the ancestral characteristics in terms of both function and expression, while RARα and γ have evolved by acquiring new functions, both new binding specificity and new expression patterns. Thus both types of evolution have been important in the diversification of vertebrate RARs.