Article

Gut-like ectodermal tissue in a sea anemone challenges germ layer homology

  • Nature Ecology & Evolution 115351542 (2017)
  • doi:10.1038/s41559-017-0285-5
  • Download Citation
Received:
Accepted:
Published online:

Abstract

Cnidarians (for example, sea anemones and jellyfish) develop from an outer ectodermal and inner endodermal germ layer, whereas bilaterians (for example, vertebrates and flies) additionally have a mesodermal layer as intermediate germ layer. Currently, cnidarian endoderm (that is, ‘mesendoderm’) is considered homologous to both bilaterian endoderm and mesoderm. Here we test this hypothesis by studying the fate of germ layers, the localization of gut cell types, and the expression of numerous ‘endodermal’ and ‘mesodermal’ transcription factor orthologues in the anthozoan sea anemone Nematostella vectensis. Surprisingly, we find that the developing pharyngeal ectoderm and its derivatives display a transcription-factor expression profile (foxA, hhex, islet, soxB1, hlxB9, tbx2/3, nkx6 and nkx2.2) and cell-type combination (exocrine and insulinergic) reminiscent of the developing bilaterian midgut, and, in particular, vertebrate pancreatic tissue. Endodermal derivatives, instead, display cell functions and transcription-factor profiles similar to bilaterian mesoderm derivatives (for example, somatic gonad and heart). Thus, our data supports an alternative model of germ layer homologies, where cnidarian pharyngeal ectoderm corresponds to bilaterian endoderm, and the cnidarian endoderm is homologous to bilaterian mesoderm.

  • Subscribe to Nature Ecology & Evolution for full access:

    $99

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    Ruppert, E. E., Fox, S. R. & Barnes, R. D. Invertebrate Zoology: A Functional Evolutionary Approach 7th edn (Belmont, CA, Brooks/Cole, 2004).

  2. 2.

    Hejnol, A. & Martin-Duran, J. M. Getting to the bottom of anal evolution. Zool. Anz. 256, 61–74 (2015).

  3. 3.

    Byrum, C. A. & Martindale, M. Q. in Gastrulation: from Cells to Embryo (ed Stern, C. D.) Ch. 3, 33–50 (Cold Spring Harbor Laboratory Press, New York, 2004).

  4. 4.

    Hashimshony, T., Feder, M., Levin, M., Hall, B. K. & Yanai, I. Spatiotemporal transcriptomics reveals the evolutionary history of the endoderm germ layer. Nature 519, 219–222 (2015).

  5. 5.

    Hall, B. K. in Evolutionary Biology 121–186 (Springer, New York, 1998).

  6. 6.

    Huxley, T. H. On the anatomy and the affinities of the family of the Medusae. Phil. Trans. R. Soc. Lond. B 139, 413–434 (1849).

  7. 7.

    Grapin-Botton, A. & Constam, D. Evolution of the mechanisms and molecular control of endoderm formation. Mech. Dev. 124, 253–278 (2007).

  8. 8.

    Haeckel, E. Die Gastraea-Theorie, die phylogenetische Classification des Thierreiches und die Homologie der Keimblätter. Jena Z. Naturwiss. 8, 1–55 (1873).

  9. 9.

    Seipel, K. & Schmid, V. Mesodermal anatomies in cnidarian polyps and medusae. Int. J. Dev. Biol. 50, 589–599 (2006).

  10. 10.

    Seipel, K. & Schmid, V. Evolution of striated muscle: jellyfish and the origin of triploblasty. Dev. Biol. 282, 14–26 (2005).

  11. 11.

    Martindale, M. Q., Pang, K. & Finnerty, J. R. Investigating the origins of triploblasty: ‘mesodermal’ gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum Cnidaria; class Anthozoa). Development 131, 2463–2474 (2004).

  12. 12.

    Rottinger, E., Dahlin, P. & Martindale, M. Q. A framework for the establishment of a cnidarian gene regulatory network for “endomesoderm” specification: the inputs of β-catenin/TCF signaling. PLoS Genet. 8, e1003164 (2012).

  13. 13.

    Technau, U. & Scholz, C. B. Origin and evolution of endoderm and mesoderm. Int. J. Dev. Biol. 47, 531–539 (2003).

  14. 14.

    Wijesena, N., Simmons, D. K. & Martindale, M. Q. Antagonistic BMP–cWNT signaling in the cnidarian Nematostella vectensis reveals insight into the evolution of mesoderm. Proc. Natl Acad. Sci. USA 114, E5608–E5615 (2017).

  15. 15.

    Rodaway, A. & Patient, R. Mesendoderm: an ancient germ layer? Cell 105, 169–172 (2001).

  16. 16.

    Tardent, P. in Morphogenese der Tiere (ed. Seidel, F.) (VEB Gustav Fischer Verlag, Jena, 1978).

  17. 17.

    Kraus, Y. A. & Markov, A. V. The gastrulation in Cnidaria: a key to understanding phylogeny or the chaos of secondary modifications? Zh. Obshch. Biol. 77, 83–105 (2016).

  18. 18.

    Kraus, Y. & Technau, U. Gastrulation in the sea anemone Nematostella vectensis occurs by invagination and immigration: an ultrastructural study. Dev. Genes Evol. 216, 119–132 (2006).

  19. 19.

    Magie, C. R., Daly, M. & Martindale, M. Q. Gastrulation in the cnidarian Nematostella vectensis occurs via invagination not ingression. Dev. Biol. 305, 483–497 (2007).

  20. 20.

    Wilson, E. B. The mesenterial filaments of the Alcyonaria. Mitt. Zool. Stat. Neapel 5, 1–27 (1884).

  21. 21.

    Yuan, D., Nakanishi, N., Jacobs, D. K. & Hartenstein, V. Embryonic development and metamorphosis of the scyphozoan. Aurelia. Dev. Genes Evol. 218, 525–539 (2008).

  22. 22.

    Mayorova, T., Kosevich, I. & Melekhova, O. On some features of embryonic development and metamorphosis of Aurelia aurita (Cnidaria, Scyphozoa). Russ. J. Dev. Biol. 43, 271–285 (2012).

  23. 23.

    Gold, D. A., Nakanishi, N., Hensley, N. M., Hartenstein, V. & Jacobs, D. K. Cell tracking supports secondary gastrulation in the moon jellyfish Aurelia. Dev. Genes Evol. 226, 383–387 (2016).

  24. 24.

    Shick, J. M. A Functional Biology of Sea Anemones (Springer Science & Business Media, Dordrecht, 2012).

  25. 25.

    Raz-Bahat, M., Douek, J., Moiseeva, E., Peters, E. C. & Rinkevich, B. The digestive system of the stony coral Stylophora pistillata. Cell Tissue Res. 368, 311–323 (2017).

  26. 26.

    Rhodes, C. J. & White, M. F. Molecular insights into insulin action and secretion. Eur. J. Clin. Invest. 32, 3–13 (2002).

  27. 27.

    Lecroisey, C., Le Pétillon, Y., Escriva, H., Lammert, E. & Laudet, V. Identification, evolution and expression of an insulin-like peptide in the cephalochordate Branchiostoma lanceolatum. PLoS ONE 10, e0119461 (2015).

  28. 28.

    Guo, B., Zhang, S., Wang, S. & Liang, Y. Expression, mitogenic activity and regulation by growth hormone of growth hormone/insulin-like growth factor in Branchiostoma belcheri. Cell Tissue Res. 338, 67–77 (2009).

  29. 29.

    Brogiolo, W., Stocker, H., Rintelen, F., Fernandez, R. & Hafen, E. An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control. Curr. Biol. 11, 213–221 (2001).

  30. 30.

    Perillo, M. & Arnone, M. I. Characterization of insulin-like peptides (ILPs) in the sea urchin Strongylocentrotus purpuratus: insights on the evolution of the insulin family. Gen. Comp. Endocrinol. 205, 68–79 (2014).

  31. 31.

    Fritzenwanker, J. H., Saina, M. & Technau, U. Analysis of forkhead and snail expression reveals epithelial–mesenchymal transitions during embryonic and larval development of Nematostella vectensis. Dev. Biol. 275, 389–402 (2004).

  32. 32.

    Sun, Z. & Hopkins, N. vhnf1, the MODY5 and familial GCKD-associated gene, regulates regional specification of the zebrafish gut, pronephros, and hindbrain. Genes Dev. 15, 3217–3229 (2001).

  33. 33.

    Yu, J.-K. et al. Axial patterning in cephalochordates and the evolution of the organizer. Nature 445, 613–617 (2007).

  34. 34.

    Howard-Ashby, M. et al. Identification and characterization of homeobox transcription factor genes in Strongylocentrotus purpuratus, and their expression in embryonic development. Dev. Biol. 300, 74–89 (2006).

  35. 35.

    Lowe, C. J. et al. Dorsoventral patterning in hemichordates: insights into early chordate evolution. PLoS Biol. 4, e291 (2006).

  36. 36.

    Tomancak, P. et al. Global analysis of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 8, R145 (2007).

  37. 37.

    Zorn, A. M. & Wells, J. M. Vertebrate endoderm development and organ formation. Annu. Rev. Cell Dev. Biol. 25, 221–251 (2009).

  38. 38.

    Gittes, G. K. Developmental biology of the pancreas: a comprehensive review. Dev. Biol. 326, 4–35 (2009).

  39. 39.

    Arntfield, M. E. & van der Kooy, D. β-Cell evolution: how the pancreas borrowed from the brain: the shared toolbox of genes expressed by neural and pancreatic endocrine cells may reflect their evolutionary relationship. BioEssays 33, 582–587 (2011).

  40. 40.

    Wisely, G. B. et al. Hepatocyte nuclear factor 4 is a transcription factor that constitutively binds fatty acids. Structure 10, 1225–1234 (2002).

  41. 41.

    Kozmik, Z. et al. PaxSixEyaDach network during amphioxus development: conservation in vitro but context specificity in vivo. Dev. Biol. 306, 143–159 (2007).

  42. 42.

    Ciglar, L. & Furlong, E. E. Conservation and divergence in developmental networks: a view from Drosophila myogenesis. Curr. Opin. Cell Biol. 21, 754–760 (2009).

  43. 43.

    Steinmetz, P. R. et al. Independent evolution of striated muscles in cnidarians and bilaterians. Nature 487, 231–234 (2012).

  44. 44.

    Brunet, T. et al. The evolutionary origin of bilaterian smooth and striated myocytes. eL ife 5, e19607 (2016).

  45. 45.

    Martindale, M. Q. & Henry, J. Q. Intracellular fate mapping in a basal metazoan, the ctenophore Mnemiopsis leidyi, reveals the origins of mesoderm and the existence of indeterminate cell lineages. Dev. Biol. 214, 243–257 (1999).

  46. 46.

    Bumann, D. & Puls, G. The ctenophore Mnemiopsis leidyi has a flow-through system for digestion with three consecutive phases of extracellular digestion. Physiol. Zool. 70, 1–6 (1997).

  47. 47.

    Arendt, D. & Nübler-Jung, K. Dorsal or ventral: similarities in fate maps and gastrulation patterns in annelids, arthropods and chordates. Mech. Dev. 61, 7–21 (1997).

  48. 48.

    Fritzenwanker, J. H. & Technau, U. Induction of gametogenesis in the basal cnidarian Nematostella vectensis (Anthozoa). Dev. Genes Evol. 212, 99–103 (2002).

  49. 49.

    Renfer, E., Amon-Hassenzahl, A., Steinmetz, P. R. & Technau, U. A muscle-specific transgenic reporter line of the sea anemone, Nematostella vectensis. Proc. Natl Acad. Sci. USA 107, 104–108 (2010).

  50. 50.

    Papapetrou, E. P. & Sadelain, M. Derivation of genetically modified human pluripotent stem cells with integrated transgenes at unique mapped genomic sites. Nat. Protoc. 6, 1274–1289 (2011).

  51. 51.

    Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

  52. 52.

    Castresana, J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17, 540–552 (2000).

  53. 53.

    Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27, 1164–1165 (2011).

  54. 54.

    Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. The Clustal_X windows interface: flexible strategiesfor multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 24, 4876–4882 (1997).

  55. 55.

    Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010).

  56. 56.

    Putnam, N. H. et al. Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317, 86–94 (2007).

  57. 57.

    Technau, U. et al. Maintenance of ancestral complexity and non-metazoan genes in two basal cnidarians. Trends Genet. 21, 633–639 (2005).

  58. 58.

    Fredman, D., Schwaiger, M., Rentzsch, F. & Technau, U. Nematostella vectensis transcriptome and gene models v2.0 https://figshare.com/articles/Nematostella_vectensis_transcriptome_and_gene_models_v2_0/807696 (2013).

  59. 59.

    Kraus, J. E., Fredman, D., Wang, W., Khalturin, K. & Technau, U. Adoption of conserved developmental genes in development and origin of the medusa body plan. Evodevo 6, 23 (2015).

  60. 60.

    Petersen, T. N., Brunak, S., von Heijne, G. & Nielsen, H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8, 785–786 (2011).

  61. 61.

    Duckert, P., Brunak, S. & Blom, N. Prediction of proprotein convertase cleavage sites. Protein Eng. Des. Sel. 17, 107–112 (2004).

  62. 62.

    Southey, B. R., Amare, A., Zimmerman, T. A., Rodriguez-Zas, S. L. & Sweedler, J. V. NeuroPred: a tool to predict cleavage sites in neuropeptide precursors and provide the masses of the resulting peptides. Nucleic Acids Res. 34, W267–W272 (2006).

  63. 63.

    Genikhovich, G. & Technau, U. In situ hybridization of starlet sea anemone (Nematostella vectensis) embryos, larvae, and polyps. Cold Spring Harb. Protoc. 2009, pdb.prot5282 (2009).

  64. 64.

    King, R. S. & Newmark, P. A. In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea. BMC Dev. Biol. 13, 8 (2013).

  65. 65.

    Lauter, G., Söll, I. & Hauptmann, G. Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain. Neural Dev. 6, 10 (2011).

  66. 66.

    Hopman, A. H., Ramaekers, F. C. & Speel, E. J. Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for in situ hybridization using CARD amplification. J. Histochem. Cytochem. 46, 771–777 (1998).

  67. 67.

    O’Rourke, E. J., Soukas, A. A., Carr, C. E. & Ruvkun, G. C. elegans major fats are stored in vesicles distinct from lysosome-related organelles. Cell Metab. 10, 430–435 (2009).

  68. 68.

    Schlombs, K., Wagner, T. & Scheel, J. Site-1 protease is required for cartilage development in zebrafish. Proc. Natl. Acad. Sci. USA 100, 14024–14029 (2003).

  69. 69.

    Achilles, J., Müller, S., Bley, T. & Babel, W. Affinity of single S. cerevisiae cells to 2-NBDglucose under changing substrate concentrations. Cytometry A 61A, 88–98 (2004).

  70. 70.

    Zou, C., Wang, Y. & Shen, Z. 2-NBDG as a fluorescent indicator for direct glucose uptake measurement. J. Biochem. Biophys. Methods 64, 207–215 (2005).

Download references

Acknowledgements

We thank M. Owusu, J. Steiner, M. Laplante, W. Wang and K. Khalturin for protocols and help with carrying out gene clonings and in situ hybridization experiments; S. I. Q. Kaul-Strehlow for confocal imaging and the Core Facility Cell Imaging of the Faculty of Life Sciences (University of Vienna) for support with confocal imaging; S. Shimeld (foxC) and F. Rentzsch (nkx2.2A, -B and -E genes) for sharing plasmids of Nematostella gene fragments, and the members of the Technau lab for discussion. This work was supported by grants from the Austrian Science Fund FWF to P.R.H.S. (P26538), U.T. (P27353 and P25993) and HFSP to A.A. (LT000809/2012-L).

Author information

Affiliations

  1. Department for Molecular Evolution and Development, Centre for Organismal Systems Biology, University of Vienna, Althanstraße 14, A-1090, Vienna, Austria

    • Patrick R. H. Steinmetz
    • , Andy Aman
    • , Johanna E. M. Kraus
    •  & Ulrich Technau
  2. Sars International Centre for Marine Molecular Biology, University of Bergen, Thormøhlensgate 55, N-5006, Bergen, Norway

    • Patrick R. H. Steinmetz
    •  & Johanna E. M. Kraus
  3. Department of Biology, University of Virginia, Charlottesville, VA, 22904, USA

    • Andy Aman

Authors

  1. Search for Patrick R. H. Steinmetz in:

  2. Search for Andy Aman in:

  3. Search for Johanna E. M. Kraus in:

  4. Search for Ulrich Technau in:

Contributions

P.R.H.S. designed the study, performed most experiments and wrote the paper. A.A. designed and performed the fate mapping and transgene mapping experiments. J.E.M.K. cloned and analysed the A. aurita foxA gene, and developed an A. aurita in situ hybridization protocol. U.T. also designed the study and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Patrick R. H. Steinmetz or Ulrich Technau.

Electronic supplementary material

  1. Supplementary Information

    Supplementary Figures 1–11; Supplementary Tables 1–3; Supplementary References