Analyses of Gene Function in Amphioxus Embryos by Microinjection of mRNAs and Morpholino Oligonucleotides

Part of the Methods in Molecular Biology book series (MIMB, volume 770)


The invertebrate chordate amphioxus (Branchiostoma), which is the most basal living chordate, has become an accepted model for the vertebrate ancestor in studies of development and evolution. Amphioxus resembles vertebrates in regard to morphology, developmental gene expression, and gene function. In addition, the amphioxus genome has representatives of most vertebrate gene families. Although it has not undergone the two rounds of whole genome duplications that occurred early in the vertebrate lineage, the amphioxus genome has retained considerable synteny with vertebrate genomes. Thus, studies of genes and development in amphioxus embryos can reveal the fundamental genetic basis of the vertebrate body plan, giving insights into the developmental mechanisms of such organs as the somites, pharynx, kidney, and the central nervous system. Moreover, amphioxus is very useful for understanding how these characters evolved. This chapter details methods for microinjection of amphioxus eggs with mRNAs or morpholino antisense oligonucleotides to analyze gene networks operating in early development.

Key words

Amphioxus gene network microinjection Branchiostoma morpholino oligonucleotides gene function 


  1. 1.
    Putnam, N., Butts, T., Ferrier, D.E.K., Furlong, R.F., Hellsten, U., Kawashima, T., et al. (2008) The amphioxus genome and the evolution of the chordate karyotype. Nature 453, 1064–1071.PubMedCrossRefGoogle Scholar
  2. 2.
    Kuraku, S., Meyer, A., and Kuratani, S. (2009) Timing of genome duplications relative to the origin of the vertebrates: did cyclostomes diverge before or after? Mol. Biol. Evol. 26, 47–59.PubMedCrossRefGoogle Scholar
  3. 3.
    Yu, J.-K., Satou, Y., Holland, N.D., Shin-I, T., Kohara, Y., Satoh, N., et al. (2007) Axial patterning in cephalochordates and the evolution of the organizer. Nature 445, 613–617.PubMedCrossRefGoogle Scholar
  4. 4.
    Schubert, M., Holland, N.D., Laudet, V., and Holland, L.Z. (2006) A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and neuronal specification in the developing central nervous system of the cephalochordate amphioxus. Dev. Biol. 296, 190–202.PubMedCrossRefGoogle Scholar
  5. 5.
    Holland, L.Z. (2002) Heads or tails? Amphioxus and the evolution of anterior-posterior patterning in deuterostomes. Dev. Biol. 241, 209–228.PubMedCrossRefGoogle Scholar
  6. 6.
    Yu, J.K., Meulemans, D., Mckeown, S.J., and Bronner-Fraser, M. (2008) Insights from the amphioxus genome on the origin of vertebrate neural crest. Genome Res. 18, 1127–1132.PubMedCrossRefGoogle Scholar
  7. 7.
    Knecht, A.K. and Bronner-Fraser, M. (2002) Induction of the neural crest: a multigene process. Nat. Rev. Genet. 3, 453–461.PubMedCrossRefGoogle Scholar
  8. 8.
    Wilson, E.B. (1893) Amphioxus, and the mosaic theory of development. J. Morphol. 8, 579–639+ pl. XXIX–XXXVIII.CrossRefGoogle Scholar
  9. 9.
    Morgan, T.H. (1896) The number of cells in larvae from isolated blastomeres of amphioxus. Arch. Entwicklungsmech. 3, 269–294+ pl. XVII.CrossRefGoogle Scholar
  10. 10.
    Tung, T.C., Wu, S.C., and Tung, Y.Y.F. (1958) The development of isolated blastomeres of amphioxus. Sci. Sinica 7, 1280–1320.PubMedGoogle Scholar
  11. 11.
    Tung, T.C., Wu, S.C., and Tung, Y.Y.F. (1960) The developmental potencies of the blastomere layers in Amphioxus egg at the 32-cell stage. Sci. Sinica 9, 119–141.PubMedGoogle Scholar
  12. 12.
    Tung, T.C., Wu, S.C., and Tung, Y.Y.F. (1962) The presumptive areas of the egg of amphioxus. Sci. Sinica 11, 629–644.Google Scholar
  13. 13.
    Tung, T.C., Wu, S.C., and Tung, Y.Y.F. (1962) Experimental studies on the neural induction in amphioxus. Sci. Sinica 11, 805–820.Google Scholar
  14. 14.
    Holland, N.D. and Holland, L.Z. (1989) Fine structural study of the cortical reaction and formation of the egg coats in a lancelet (= amphioxus), Branchiostoma floridae (phylum Chordata: supphylum Cephalochordata = Acrania). Biol. Bull. 176, 111–122.CrossRefGoogle Scholar
  15. 15.
    Fuentes, M., Benito, E., Bertrand, S., Paris, M., Mignardot, A., Godoy, L., et al. (2004) Insights into the spawning behavior of the European amphioxus (Branchiostoma lanceolatum) J. Exp. Zool. 302B, 384–391.Google Scholar
  16. 16.
    Zhang, Q.J., Sun, Y., Zhong, J., Li, G., Lü, X.M., and Wang, Y.Q. (2007) Continuous culture of two lancelets and production of the second filial generations in the laboratory. J. Exp. Zool. 308B, 464–472.CrossRefGoogle Scholar
  17. 17.
    Holland, L.Z. and Yu, J.K. (2004) Cephalochordate (Amphioxus) embryos: procurement, culture, basic methods. Methods Cell. Biol. 74, 195–215.PubMedCrossRefGoogle Scholar
  18. 18.
    Stokes, M.D. (1996) Larval settlement, post-settlement growth and secondary production of the Florida lancelet (=amphioxus). Branchiostoma floridae Mar. Ecol. Prog. Ser. 130, 71–84.CrossRefGoogle Scholar
  19. 19.
    Holland, N.D., Paris, M., and Koop, D. (2009) The club-shaped gland of amphioxus: export of secretion to the pharynx in pre-metamorphic larvae and apoptosis during metamorphosis. Acta Zool. (Stockh) 90, 372–379.CrossRefGoogle Scholar
  20. 20.
    Paris, M., Escriva, H., Schubert, M., Brunet, F., Brtko, J., Ciesielski, F., et al. (2008) Amphioxus postembryonic development reveals the homology of chordate metamorphosis. Curr. Biol. 18, 825–830.PubMedCrossRefGoogle Scholar
  21. 21.
    Holland, N.D. and Holland, L.Z. (2006) Stage- and tissue-specific patterns of cell division in embryonic and larval tissues of amphioxus during normal development. Evol. Dev. 8, 142–149.PubMedCrossRefGoogle Scholar
  22. 22.
    Bayascas, J.R., Yuste, V.J., Benito, E., Garcia-Fernandez, J., and Comella, J.X. (2002) Isolation of AmphiCASP-2/7, an ancestral caspase from amphioxus (Branchiostoma floridae). Evolutionary considerations for vertebrate caspases. Cell Death Diffferen. 9, 1078–1089.CrossRefGoogle Scholar
  23. 23.
    Holland, L.Z., Holland, P.W.H., and Holland, N.D. (1996) Revealing homologies between body parts of distantly related animals by in situ hybridization to developmental genes: amphioxus versus vertebrates. In Molecular Approaches to Zoology and Evolution, ed. Ferraris, J.D. Wiley, New York, NY, pp. 267–282; pp. 473–483.Google Scholar
  24. 24.
    Yu, J.K.S. and Holland, L.Z. (2009) Cephalochordates (Amphioxus or Lancelets): a model for understanding the evolution of chordate characters. Cold Spring Harb Protoc 130, pdb.emo130.CrossRefGoogle Scholar
  25. 25.
    Dos Santos, S., Bardet, C., Bertrand, S., Escriva, H., Habert, D., and Querat, B. (2009) Distinct expression patterns of glycoprotein hormone-α2 and -β5 in a basal chordate suggest independent developmental functions. Endocrinology 150, 3815–3822.PubMedCrossRefGoogle Scholar
  26. 26.
    Li, X.Y., Zhang, W., Chen, D.Y., Lin, Y.S., Huang, X.W., Shi, D.L., et al. (2006) Expression of a novel somite-formation-related gene, AmphiSom, during amphioxus development. Dev. Genes Evol. 216, 52–55.PubMedCrossRefGoogle Scholar
  27. 27.
    Holland, L.Z. and Holland, N.D. (1992) Early development in the lancelet (= amphioxus) Branchiostoma floridae from sperm entry through pronuclear fusion: presence of vegetal pole plasm and lack of conspicuous ooplasmic segregation. Biol. Bull. 182, 77–96.CrossRefGoogle Scholar
  28. 28.
    Deheyn, D.D., Kubokawa, K., McCarthy, J.K., Murakami, A., Porrachia, M., Rouse, G.W., et al. (2007) Endogenous green fluorescent protein (GFP) in amphioxus. Biol. Bull. 213, 95–100.PubMedCrossRefGoogle Scholar
  29. 29.
    Eisen, J.S. and Smith, J.C. (2008) Controlling morpholino experiments: don’t stop making antisense. Development 135, 1735–1743.PubMedCrossRefGoogle Scholar
  30. 30.
    Holland, L.Z., Panfilio, K.A., Chastain, R., Schubert, M., and Holland, N.D. (2005) Nuclear –β catenin promotes non-neural ectoderm and posterior cell fates in amphioxus embryos. Dev. Dyn. 233, 1430–1443.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Marine Biology Research Division, Scripps Institution of OceanographyUniversity of California San DiegoLa JollaUSA

Personalised recommendations