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.

Department of Biology Metropolitan State University Denver CO USA

Department of Ecology and Evolutionary Biology University of Colorado Boulder CO USA

Department of Molecular and Cellular Biology University of California Berkeley CA USA

Department of Zoology Charles University Prague Prague Czech Republic

Department of Zoology Comenius University in Bratislava Bratislava Slovakia

Gymnázium Jiřího Wolkera Prostějov Czech Republic

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Meulemans, D. & Bronner-Fraser, M. Gene-regulatory interactions in neural crest evolution and development. Dev. Cell 7, 291–299 (2004). PubMed

Martik, M. L. et al. Evolution of the new head by gradual acquisition of neural crest regulatory circuits. Nature 574, 675–678 (2019). PubMed PMC

Martinez-Morales, J. R., Henrich, T., Ramialison, M. & Wittbrodt, J. New genes in the evolution of the neural crest differentiation program. Genome Biol. 8, R36 (2007). PubMed PMC

Ohno, S. Evolution by Gene Duplication (Springer–Verlag, 1970).

Braasch, I. & Schartl, M. Evolution of endothelin receptors in vertebrates. Gen. Comp. Endocrinol. 209, 21–34 (2014). PubMed

Braasch, I., Volff, J. N. & Schartl, M. The endothelin system: evolution of vertebrate-specific ligand-receptor interactions by three rounds of genome duplication. Mol. Biol. Evol. 26, 783–799 (2009). PubMed

Miller, C. T., Schilling, T. F., Lee, K., Parker, J. & Kimmel, C. B. sucker encodes a zebrafish Endothelin-1 required for ventral pharyngeal arch development. Development 127, 3815–3828 (2000). PubMed

Miller, C. T., Yelon, D., Stainier, D. Y. & Kimmel, C. B. Two endothelin 1 effectors, hand2 and bapx1, pattern ventral pharyngeal cartilage and the jaw joint. Development 130, 1353–1365 (2003). PubMed

Krauss, J. et al. Endothelin signalling in iridophore development and stripe pattern formation of zebrafish. Biol. Open 3, 503–509 (2014). PubMed PMC

Baynash, A. G. et al. Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 79, 1277–1285 (1994). PubMed

Square, T. et al. CRISPR/Cas9-mediated mutagenesis in the sea lamprey Petromyzon marinus: a powerful tool for understanding ancestral gene functions in vertebrates. Development 142, 4180–4187 (2015). PubMed PMC

Stock, D. W. & Whitt, G. S. Evidence from 18S ribosomal RNA sequences that lampreys and hagfishes form a natural group. Science 257, 787–789 (1992). PubMed

Clouthier, D. E. et al. Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice. Development 125, 813–824 (1998). PubMed

Ruest, L. B., Xiang, X., Lim, K. C., Levi, G. & Clouthier, D. E. Endothelin-A receptor-dependent and -independent signaling pathways in establishing mandibular identity. Development 131, 4413–4422 (2004). PubMed PMC

Tavares, A. L. P. et al. Ectodermal-derived Endothelin1 is required for patterning the distal and intermediate domains of the mouse mandibular arch. Dev. Biol. 371, 47–56 (2012). PubMed PMC

Charité, J. et al. Role of Dlx6 in regulation of an endothelin-1-dependent, dHAND branchial arch enhancer. Genes Dev. 15, 3039–3049 (2001). PubMed PMC

Parichy, D. M. et al. Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio. Dev. Biol. 227, 294–306 (2000). PubMed

Kawasaki-Nishihara, A., Nishihara, D., Nakamura, H. & Yamamoto, H. ET3/Ednrb2 signaling is critically involved in regulating melanophore migration in Xenopus. Dev. Dyn. 240, 1454–1466 (2011). PubMed

Metallinos, D. L., Bowling, A. T. & Rine, J. A missense mutation in the endothelin-B receptor gene is associated with lethal white foal syndrome: an equine version of Hirschsprung disease. Mamm. Genome 9, 426–431 (1998). PubMed

Sánchez-Mejías, A., Fernández, R. M., López-Alonso, M., Antiñolo, G. & Borrego, S. New roles of EDNRB and EDN3 in the pathogenesis of Hirschsprung disease. Genet. Med. 12, 39–43 (2010). PubMed

Square, T., Jandzik, D., Cattell, M., Hansen, A. & Medeiros, D. M. Embryonic expression of endothelins and their receptors in lamprey and frog reveals stem vertebrate origins of complex Endothelin signaling. Sci. Rep. 6, 34282 (2016). PubMed PMC

Cerny, R. et al. Evidence for the prepattern/cooption model of vertebrate jaw evolution. Proc. Natl Acad. Sci. USA 107, 17262–17267 (2010). PubMed

Kuraku, S., Takio, Y., Sugahara, F., Takechi, M. & Kuratani, S. Evolution of oropharyngeal patterning mechanisms involving Dlx and endothelins in vertebrates. Dev. Biol. 341, 315–323 (2010). PubMed

Johnels, A. G. On the development and morphology of the skeleton of the head of Petromyzon. Acta Zool. 29, 139–277 (1948).

Green, S. A., Uy, B. R. & Bronner, M. E. Ancient evolutionary origin of vertebrate enteric neurons from trunk-derived neural crest. Nature 544, 88–91 (2017). PubMed PMC

Kuratani, S. Evolution of the vertebrate jaw from developmental perspectives. Evol. Dev. 14, 76–92 (2012). PubMed

Smith, J. J. et al. The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution. Nat. Genet. 50, 270–277 (2018). PubMed PMC

Camargo Sosa, K. et al. Endothelin receptor Aa regulates proliferation and differentiation of Erb-dependant pigment progenitors in zebrafish. PLOS Genet. 15, e1007941 (2019). PubMed DOI PMC

Yao, T., Ohtani, K., Kuratani, S. & Wada, H. Development of lamprey mucocartilage and its dorsal–ventral patterning by endothelin signaling, with insight into vertebrate jaw evolution. J. Exp. Zoolog. B 316, 339–346 (2011).

Clouthier, D. E., Garcia, E. & Schilling, T. F. Regulation of facial morphogenesis by endothelin signaling: insights from mice and fish. Am. J. Med. Genet. A. 152A, 2962–2973 (2010). PubMed PMC

Fujimoto, S., Oisi, Y., Kuraku, S., Ota, K. G. & Kuratani, S. Non-parsimonious evolution of hagfish Dlx genes. BMC Evol. Biol. 13, 15 (2013). PubMed PMC

Tahara, Y. Normal stages of development in the lamprey Lampetra reissneri (Dybowski). Zool. Sci. 5, 109–118 (1988).

Nair, S., Li, W., Cornell, R. & Schilling, T. F. Requirements for Endothelin type-A receptors and Endothelin-1 signaling in the facial ectoderm for the patterning of skeletogenic neural crest cells in zebrafish. Development 134, 335–245 (2007). PubMed

Bonano, M. et al. A new role for the Endothelin-1/Endothelin-A receptor signaling during early neural crest specification. Dev. Biol. 323, 114–129 (2008). PubMed

Asai, R. et al. Endothelin receptor type A expression defines a distinct cardiac subdomain within the heart field and is later implicated in chamber myocardium formation. Development 137, 3823–3833 (2010). PubMed

Jandzik, D. et al. Roles for FGF in lamprey pharyngeal pouch formation and skeletogenesis highlight ancestral functions in the vertebrate head. Development 141, 629–638 (2014). PubMed

Bondurand, N., Dufour, S. & Pingault, V. News from the endothelin-3/EDNRB signaling pathway: role during enteric nervous system development and involvement in neural crest-associated disorders. Dev. Biol. 444 (Suppl 1), S156–S169 (2018). PubMed

Higashiyama, H. et al. On the vagal cardiac nerves, with special reference to the early evolution of the head-trunk interface. J. Morphol. 277, 1146–1158 (2016). PubMed

Thiagarajah, J. R. et al. Altered goblet cell differentiation and surface mucus properties in Hirschsprung disease. PLoS ONE 9, e99944 (2014). PubMed PMC

von Boyen, G. B. et al. Abnormalities of the enteric nervous system in heterozygous endothelin B receptor deficient (spotting lethal) rats resembling intestinal neuronal dysplasia. Gut 51, 414–419 (2002).

Karne, S., Jayawickreme, C. K. & Lerner, M. R. Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores. J. Biol. Chem. 268, 19126–19133 (1993). PubMed

Spiewak, J. E. et al. Evolution of Endothelin signaling and diversification of adult pigment pattern in Danio fishes. PLoS Genet. 14, e1007538 (2018). PubMed PMC

Woodcock, M. R. et al. Identification of mutant genes and introgressed tiger salamander DNA in the laboratory axolotl, Ambystoma mexicanum. Sci. Rep. 7, 6 (2017). PubMed PMC

Simakov, O. et al. Deeply conserved synteny resolves early events in vertebrate evolution. Nat. Ecol. Evol. 4, 820–830 (2020). PubMed PMC

Yanagisawa, H. et al. Dual genetic pathways of endothelin-mediated intercellular signaling revealed by targeted disruption of endothelin converting enzyme-1 gene. Development 125, 825–836 (1998). PubMed

Sive, H. L., Grainger, R. M. & Harland, R. M. Early development of Xenopus laevis: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2000).

Nieuwkoop, P. D. & Faber, J. Normal Table of Xenopus laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg Till the End of Metamorphosis (Garland, 1994).

Zu, Y. et al. Biallelic editing of a lamprey genome using the CRISPR/Cas9 system. Sci. Rep. 6, 23496 (2016). PubMed PMC

York, J. R., Yuan, T., Lakiza, O. & McCauley, D. W. An ancestral role for Semaphorin3F-Neuropilin signaling in patterning neural crest within the new vertebrate head. Development 145, dev164780 (2018). PubMed

York, J. R., Yuan, T., Zehnder, K. & McCauley, D. W. Lamprey neural crest migration is Snail-dependent and occurs without a differential shift in cadherin expression. Dev. Biol. 428, 176–187 (2017). PubMed

Yuan, T., York, J. R. & McCauley, D. W. Gliogenesis in lampreys shares gene regulatory interactions with oligodendrocyte development in jawed vertebrates. Dev. Biol. 441, 176–190 (2018). PubMed

Wang, F. et al. Targeted gene disruption in Xenopus laevis using CRISPR/Cas9. Cell Biosci. 5, 15 (2015). PubMed PMC

Rosen, J. N., Sweeney, M. F. & Mably, J. D. Microinjection of zebrafish embryos to analyze gene function. J. Vis. Exp. 25,1115 (2009).

Session, A. M. et al. Genome evolution in the allotetraploid frog Xenopus laevis. Nature 538, 336–343 (2016). PubMed PMC

Flowers, G. P., Timberlake, A. T., McLean, K. C., Monaghan, J. R. & Crews, C. M. Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease. Development 141, 2165–2171 (2014). PubMed PMC

Square, T. et al. A gene expression map of the larval Xenopus laevis head reveals developmental changes underlying the evolution of new skeletal elements. Dev. Biol. 397, 293–304 (2015). PubMed

Aigler, S. R., Jandzik, D., Hatta, K., Uesugi, K. & Stock, D. W. Selection and constraint underlie irreversibility of tooth loss in cypriniform fishes. Proc. Natl Acad. Sci. USA 111, 7707–7712 (2014). PubMed

Sauka-Spengler, T., Meulemans, D., Jones, M. & Bronner-Fraser, M. Ancient evolutionary origin of the neural crest gene regulatory network. Dev. Cell 13, 405–420 (2007). PubMed

Meulemans, D., McCauley, D. & Bronner-Fraser, M. Id expression in amphioxus and lamprey highlights the role of gene cooption during neural crest evolution. Dev. Biol. 264, 430–442 (2003). PubMed

Haming, D. et al. Expression of sympathetic nervous system genes in lamprey suggests their recruitment for specification of a new vertebrate feature. PLoS ONE 6, 0026543 (2011).

McCauley, D. W. & Bronner-Fraser, M. Importance of SoxE in neural crest development and the evolution of the pharynx. Nature 441, 750–752 (2006). PubMed

Yuan, T., York, J. R. & McCauley, D. W. Neural crest and placode roles in formation and patterning of cranial sensory ganglia in lamprey. Genesis 58, e23356 (2020). PubMed

Cattell, M. V., Garnett, A. T., Klymkowsky, M. W. & Medeiros, D. M. A maternally established SoxB1/SoxF axis is a conserved feature of chordate germ layer patterning. Evol. Dev. 14, 104–115 (2012). PubMed

Talikka, M., Stefani, G., Brivanlou, A. H. & Zimmerman, K. Characterization of Xenopus Phox2a and Phox2b defines expression domains within the embryonic nervous system and early heart field. Gene Expr. Patterns 4, 601–607 (2004). PubMed

McCauley, D. W. & Bronner-Fraser, M. Conservation of Pax gene expression in ectodermal placodes of the lamprey. Gene 287, 129–139 (2002). PubMed

Ware, M., Dupé, V. & Schubert, F. R. Evolutionary conservation of the early axon scaffold in the vertebrate brain. Dev. Dyn. 244, 1202–1214 (2015). PubMed

Modrell, M. S. et al. A fate-map for cranial sensory ganglia in the sea lamprey. Dev. Biol. 385, 405–416 (2014). PubMed PMC

Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012). PubMed PMC

Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729 (2013). PubMed PMC

Efficient CRISPR Mutagenesis in Sturgeon Demonstrates Its Utility in Large, Slow-Maturing Vertebrates