Eric Van Otterloo, Robert A. Cornell, Daniel Meulemans Medeiros, Aaron T. Garnett

The appearance of novel anatomic structures during evolution is driven by changes to the networks of transcription factors, signaling pathways, and downstream effector genes controlling development. The nature of the changes to these developmental gene regulatory networks (GRNs) is poorly understood. A striking test case is the evolution of the GRN controlling development of the neural crest (NC). NC cells emerge from the neural plate border (NPB) and contribute to multiple adult structures. While all chordates have a NPB, only in vertebrates do NPB cells express all the genes constituting the neural crest GRN (NC-GRN). Interestingly, invertebrate chordates express orthologs of NC-GRN components in other tissues, revealing that during vertebrate evolution new regulatory connections emerged between transcription factors primitively expressed in the NPB and genes primitively expressed in other tissues. Such interactions could have evolved by two mechanisms. First, transcription factors primitively expressed in the NPB may have evolved new DNA and/or cofactor binding properties (protein neofunctionalization). Alternately, cis-regulatory elements driving NPB expression may have evolved near genes primitively expressed in other tissues (cis-regulatory neofunctionalization). Here we discuss how gene duplication can, in principle, promote either form of neofunctionalization. We review recent published examples of interspecies gene-swap, or regulatory-element-swap, experiments that test both models. Such experiments have yielded little evidence to support the importance of protein neofunctionalization in the emergence of the NC-GRN, but do support the importance of novel cis-regulatory elements in this process. The NC-GRN is an excellent model for the study of gene regulatory and macroevolutionary innovation.