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Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.

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 potencies1. The evolution of these developmental modules is poorly understood. Endothelin (Edn) signaling is unique to vertebrates, and performs various functions in different NC subpopulations2–5. 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 ago6. Thus, comparisons between the two groups can identify deeply conserved and divergent features of vertebrate development. UsingXenopus laevisto 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 regulateshandtranscription factors, which are required for mandible development in gnathostomes. We also identify conserved roles for Edn signaling indlxgene 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.

Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks

The evolutionary origin of the autopod involved a loss of the fin-fold and associated dermal skeleton with a concomitant elaboration of the distal endoskeleton to form a wrist and digits. Developmental studies, primarily from teleosts and amniotes, suggest a model for appendage evolution in which a delay in the AER-to-fin-fold conversion fuelled endoskeletal expansion by prolonging the function of AER-mediated regulatory networks. Here, we characterize aspects of paired fin development in the paddlefishPolyodon spathula(a non-teleost actinopterygian) and catsharkScyliorhinus canicula(chondrichthyan) to explore aspects of this model in a broader phylogenetic context. Our data demonstrate that in basal gnathostomes, the autopod markerHoxA13co-localizes with the dermoskeleton componentAnd1to mark the position of the fin-fold, supporting recent work demonstrating a role forHoxA13in zebrafish fin ray development. Additionally, we show that in paddlefish, the proximal fin and fin-fold mesenchyme share a common mesodermal origin, and that components of the Shh/LIM/Gremlin/Fgf transcriptional network critical to limb bud outgrowth and patterning are expressed in the fin-fold with a profile similar to that of tetrapods. Together these data draw contrast with hypotheses of AER heterochrony and suggest that limb-specific morphologies arose through evolutionary changes in the differentiation outcome of conserved early distal patterning compartments.

The role of Homeobox transcription factors during fin and limb development have been the focus of recent work investigating the evolutionary origin of limb-specific morphologies. Here we characterize the expression of HoxD genes, as well as the cluster-associated genes Evx2 and LNP, in the paddlefish Polyodon spathula, a basal ray-finned fish. Our results demonstrate a collinear pattern of nesting in early fin buds that includes HoxD14, a gene previously thought to be isolated from global Hox regulation. We also show that in both Polyodon and the catshark Scyliorhinus canicula (a representative chondrichthyan) late phase HoxD transcripts are present in cells of the fin-fold and co-localize with And1, a component of the dermal skeleton. These new data support an ancestral role for HoxD genes in patterning the fin-folds of jawed vertebrates, and fuel new hypotheses about the evolution of cluster regulation and the potential downstream differentiation outcomes of distinct HoxD-regulated compartments.