Abstract; ; The neural crest (NC) is a vertebrate-specific embryonic tissue that forms an array of clade-defining adult features. A key step in the formation of these diverse derivatives is the partitioning of NC cells into subpopulations with distinct migration routes and potencies; 1; . The evolution of these developmental modules is poorly understood. Endothelin (Edn) signaling is unique to vertebrates, and performs various functions in different NC subpopulations; 2–5; . To better understand the evolution of NC patterning, we used CRISPR/Cas9-driven mutagenesis to disrupt Edn receptors, ligands, and Dlx transcription factors in the sea lamprey,; Petromyzon marinus; . Lampreys and modern gnathostomes last shared a common ancestor 500 million years ago; 6; . Thus, comparisons between the two groups can identify deeply conserved and divergent features of vertebrate development. Using; Xenopus laevis; to facilitate side-by-side analyses, we show here that lamprey and gnathostomes display fundamental differences in Edn signaling function. Unlike gnathostomes, both lamprey Ednrs cooperate during oropharyngeal skeleton development. Furthermore, neither paralog regulates; hand; transcription factors, which are required for mandible development in gnathostomes. We also identify conserved roles for Edn signaling in; dlx; gene regulation, pigment cell, and heart development. Together our results illustrate the stepwise neofunctionalization and specialization of this vertebrate-specific signaling pathway, and suggest key intermediate stages in the early evolution of the NC.;
