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Evolution of the new vertebrate head by co-option of an ancient chordate skeletal tissue

Abstract

A defining feature of vertebrates (craniates) is a pronounced head that is supported and protected by a robust cellular endoskeleton. In the first vertebrates, this skeleton probably consisted of collagenous cellular cartilage, which forms the embryonic skeleton of all vertebrates and the adult skeleton of modern jawless and cartilaginous fish. In the head, most cellular cartilage is derived from a migratory cell population called the neural crest, which arises from the edges of the central nervous system. Because collagenous cellular cartilage and neural crest cells have not been described in invertebrates1, the appearance of cellular cartilage derived from neural crest cells is considered a turning point in vertebrate evolution2. Here we show that a tissue with many of the defining features of vertebrate cellular cartilage transiently forms in the larvae of the invertebrate chordate Branchiostoma floridae (Florida amphioxus). We also present evidence that during evolution, a key regulator of vertebrate cartilage development, SoxE, gained new cis-regulatory sequences that subsequently directed its novel expression in neural crest cells. Together, these results suggest that the origin of the vertebrate head skeleton did not depend on the evolution of a new skeletal tissue, as is commonly thought, but on the spread of this tissue throughout the head. We further propose that the evolution of cis-regulatory elements near an ancient regulator of cartilage differentiation was a major factor in the evolution of the vertebrate head skeleton.

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Figure 1: Development of the amphioxus oral skeleton.
Figure 2: The amphioxus oral skeleton requires FGF-mediated signalling for formation and expresses orthologues of vertebrate cartilage markers.
Figure 3: A reporter construct incorporating the amphioxus SoxE locus recapitulates the amphioxus SoxE expression pattern in zebrafish embryos.
Figure 4: The evolution of the vertebrate head skeleton via co-option of an ancient cellular cartilage gene program.

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Acknowledgements

We thank D. Brunelle, C.-H. Tung and T.-K. Huang for assistance with amphioxus husbandry, and P. Tsai and D. W. Stock for use of their microtomes. D.M.M., D.J., TA.S. and M.V.C. were supported by National Science Foundation grants IOS 1257040 and IOS 1160733 (D.M.M.) and University of Colorado, Boulder start-up funds (D.M.M.). A.T.G. was supported by National Science Foundation grant DBI 0905991. J.-K.Y. was supported by National Science Council Taiwan grants NSC101-2923-B-001-004-MY2 and NSC102-2311-B-001-011-MY3 and Career Development Award AS-98-CDA-L06 from Academia Sinica Taiwan.

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Contributions

D.M.M. designed the project and wrote the manuscript. D.J., A.T.G. and T.A.S. performed the experiments and prepared the images; D.J. assembled the figures; and M.V.C. and J.-K.Y. provided materials and reagents. All authors discussed the results and provided input on the manuscript.

Corresponding author

Correspondence to Daniel M. Medeiros.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Amphioxus oral cartilage differentiation is initiated before cirrus outgrowth and requires FGF-mediated signalling.

a, b, Phase contrast images of a metamorphic amphioxus larva. a, The differentiation of cartilage rods occurs first in the rim of the mouth. b, The cartilage rods later grow outwards into nascent cirri. c, Toluidine blue staining of a JB-4-embedded control larval section showing the oral cartilage rod (red rectangle; arrows in f) embedded in the rim of the mouth. d, e, Toluidine blue staining of two representative SU5402-treated larvae, sectioned at the same level as the larva in c. The differentiation of the oral cartilage bar is completely eliminated in d and is strongly reduced in e. fh, High magnification views of the sections in ce.

Extended Data Figure 2 Expression of SoxE, ColA and FGF signalling components in the oral region of metamorphic amphioxus larvae.

al, In situ hybridization was performed on transverse sections in the planes shown in a. b, A high magnification view of cirri hybridized with a probe against ColA. ColA mRNA was detected in the central cartilage rod, which is a single stack of discoidal chondrocytes. c, A high magnification view of cirri hybridized with a probe against SoxE. SoxE mRNA was detected in the central cartilage rod. d, A high magnification view of cirri hybridized with a probe against FGFR. e, f, High magnification views of cirri hybridized with a probe against Ets. gi, High magnification views of cirri hybridized with a probe against ColA. j, k, High magnification views of cirri hybridized with a probe against DUSP6/7/9. l, A high magnification view of cirri hybridized with a probe against DUSP1/4/5, an orthologue of mouse Dusp4, a gene that is expressed at high levels in mesoderm-derived chondrocytes37. In all panels, the arrows indicate expression in oral chondrocytes, and the arrowheads indicate expression in the associated oral mesothelial cells.

Extended Data Figure 3 Activity of the amphioxus SoxE reporter construct in zebrafish during development.

Injected zebrafish embryos displaying broad GFP fluorescence and normal morphology were processed for in situ hybridization to detect GFP mRNA. Embryos were scored for the expression of GFP in five or more cells in each domain. a, A map of the expression domains scored in 15-somite and 18-somite embryos. b, A representative 4-day larva showing sporadic expression in the heart region and trunk muscles (arrows in insets). The scored domains are outlined. The image is a composite of eight photographs of the same larva taken at different focal planes. c, A representative 18-somite tfap2a and tfap2c dual knockdown morphant (tfap2a/c morphant)28 expressing the amphioxus SoxE reporter. The image is a composite of four photographs of the same embryo taken at different focal planes. The tfap2a/c morphants28 displayed a highly penetrant and almost complete loss of NCC marker expression, including sox10 (a co-orthologue of amphioxus SoxE). Like wildtype embryos, tfap2a/c morphant embryos displayed mosaic expression of the amphioxus SoxE reporter in the neural tube (double arrowheads) and tail bud (arrow). The ability of the amphioxus SoxE reporter to function in tfap2-depleted embryos supports expression data27 suggesting that amphioxus SoxE transcription is Tfap2 independent. d, The numbers and frequencies of embryos and larvae with GFP expression in the indicated domains. The numbers in each column are the pooled results of two to four separate experiments.

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Jandzik, D., Garnett, A., Square, T. et al. Evolution of the new vertebrate head by co-option of an ancient chordate skeletal tissue. Nature 518, 534–537 (2015). https://doi.org/10.1038/nature14000

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