Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis

Nature Protocols volume 8, pages 924–934 (2013)Cite this article

Subjects

Abstract

We describe a protocol for microinjection of embryos for an emerging model system, the cnidarian sea anemone, Nematostella vectensis. In addition, we provide protocols for carrying out overexpression and knockdown of gene function through microinjection of in vitro–translated mRNAs or gene-specific oligonucleotide morpholinos (MOs), respectively. Our approach is simple, and it takes advantage of the natural adherence properties of the early embryo to position them in a single layer on a polystyrene dish. Embryos are visualized on a dissecting microscope equipped with epifluorescence and injected with microinjection needles using a picospritzer forced-air injection system. A micromanipulator is used to guide the needle to impale individual embryos. Injection takes ∼1.5 h, and an experienced researcher can inject ∼2,000 embryos in a single session. With the availability of the published Nematostella genome, the entire protocol, including cloning and transcription of mRNAs, can be carried out in ∼1 week.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Microinjection.
Figure 2: Expression of Venus protein in Nematostella injected with NvashA:venus mRNA.
Figure 3: Assaying MO efficiency.
Figure 4: Development after injection.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Kusserow, A. et al. Unexpected complexity of the Wnt gene family in a sea anemone. Nature 433, 156–160 (2005).

    Article  CAS  Google Scholar 

  3. Burton, P.M. & Finnerty, J.R. Conserved and novel gene expression between regeneration and asexual fission in Nematostella vectensis. Dev. Genes Evol. 219, 79–87 (2009).

    Article  Google Scholar 

  4. Reitzel, A.M., Burton, P.M., Krone, C. & Finnerty, J.R. Comparison of developmental trajectories in the starlet sea anemone Nematostella vectensis: embryogenesis, regeneration, and two forms of asexual fission. Invertebr. Biol. 126, 99–112 (2007).

    Article  Google Scholar 

  5. Fritzenwanker, J., Genikhovich, G., Kraus, Y. & Technau, U. Early development and axis specification in the sea anemone Nematostella vectensis. Dev. Biol. 310, 264–279 (2007).

    Article  CAS  Google Scholar 

  6. Darling, J.A. et al. Rising starlet: the starlet sea anemone, Nematostella vectensis. Bioessays 27, 211–221 (2005).

    Article  CAS  Google Scholar 

  7. Gordon, J.W. et al. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl Acad. Sci. USA 77, 7380–73841 (2012).

    Article  Google Scholar 

  8. Cheers, M.S. & Ettensohn, C.A. Rapid microinjection of fertilized eggs. Methods Cell Biol. 74, 287–310 (2004).

    Article  Google Scholar 

  9. Layden, M.J., Boekhout, M. & Martindale, M.Q. Nematostella vectensis achaete-scute homolog NvashA regulates embryonic ectodermal neurogenesis and represents an ancient component of the metazoan neural specification pathway. Development 139, 1013–1022 (2012).

    Article  CAS  Google Scholar 

  10. Lee, P.N., Kumburegama, S., Marlow, H.Q., Martindale, M.Q. & Wikramanayake, A.H. Asymmetric developmental potential along the animal–vegetal axis in the anthozoan cnidarian, Nematostella vectensis, is mediated by Dishevelled. Dev. Biol. 310, 169–186 (2007).

    Article  CAS  Google Scholar 

  11. Wikramanayake, A.H. et al. An ancient role for nuclear β-catenin in the evolution of axial polarity and germ layer segregation. Nature 426, 442–446 (2003).

    Article  Google Scholar 

  12. Holley, S.A. et al. The Xenopus dorsalizing factor noggin ventralizes Drosophila embryos by preventing DPP from activating its receptor. Cell 86, 607–617 (1996).

    Article  CAS  Google Scholar 

  13. Smith, W.C. & Harland, R.M. Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center. Cell 67, 741–752 (1991).

    Article  Google Scholar 

  14. Marlow, H., Roettinger, E., Boekhout, M. & Martindale, M.Q. Functional roles of Notch signaling in the cnidarian Nematostella vectensis. Dev. Biol. 362, 295–308 (2012).

    Article  CAS  Google Scholar 

  15. Genikhovich, G. & Technau, U. Complex functions of Mef2 splice variants in the differentiation of endoderm and of a neuronal cell type in a sea anemone. Development 138, 4911–4919 (2011).

    Article  CAS  Google Scholar 

  16. Draper, B.W., Morcos, P.A. & Kimmel, C.B. Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown. Genesis 30, 154–156 (2001).

    Article  CAS  Google Scholar 

  17. Summerton, J. & Weller, D. Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 7, 187–195 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Rentzsch, F., Fritzenwanker, J.H., Scholz, C.B. & Technau, U. FGF signalling controls formation of the apical sensory organ in the cnidarian Nematostella vectensis. Development 135, 1761–1769 (2008).

    Article  CAS  Google Scholar 

  20. Nakanishi, N., Renfer, E., Technau, U. & Rentzsch, F. Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms. Development 139, 347–357 (2011).

    Article  Google Scholar 

  21. Saina, M. et al. BMPs and Chordin regulate patterning of the directive axis in a sea anemone. Proc. Natl Acad. Sci. 106, 18592–18597 (2009).

    Article  CAS  Google Scholar 

  22. Kumburegama, S., Wijesena, N., Xu, R. & Wikramanayake, A.H. Strabismus-mediated primary archenteron invagination is uncoupled from Wnt/β-catenin-dependent endoderm cell fate specification in Nematostella vectensis (Anthozoa, Cnidaria): implications for the evolution of gastrulation. Evodevo 2, 2 (2011).

    Article  CAS  Google Scholar 

  23. Wolenski, F.S., Bradham, C.A., Finnerty, J.R. & Gilmore, T.D. NF-kB is required for the development of subset of cnidocytes in the body column of the sean anemone. Dev. Biol. 373, 205–215 (2012).

    Article  Google Scholar 

  24. Eric, R., Paul, D. & 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).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Hand, C. & Uhlinger, K.R. The culture, sexual and asexual reproduction, and growth of the sea anemone Nematostella vectensis 182, 169–176 (1992).

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

    Article  Google Scholar 

  28. Eisen, J.S. & Smith, J.C. Controlling morpholino experiments: don't stop making antisense. Development 135, 1735–1743 (2008).

    Article  CAS  Google Scholar 

  29. Roure, A. et al. A multicassette gateway vector set for high-throughput and comparative analyses in Ciona and vertebrate embryos. PLoS ONE 2, e916 (2007).

    Article  Google Scholar 

  30. Stefanik, D.J., Friedman, L. & Finnerty, J.R. Collecting, rearing, spawning, and inducing regeneration of the starlet sea anemone, Nematostella vectensis. Nat. Protoc. 8, 916–923 (2013).

    Article  Google Scholar 

Download references

Acknowledgements

We would like to acknowledge T. Lepage (Station Zoologique de Villefranche-sur-Mer, France) and P. Lemaire (CRBM; Montpelier, France) for providing pCS2-gfp and pSPE3–RVenus vectors, respectively. This research was supported by US National Institutes of Health (NIH) grant no. 1R21RR032121 to M.Q.M. and by National Science Foundation grant no. MCB-0924749 to T.D.G. F.S.W. was supported by a predoctoral grant from the Superfund Basic Research Program at Boston University (no. 5 P42 E507381) and Warren-McLeod graduate fellowships in Marine Biology. M.J.L. was supported by a Ruth L. Kirschstein National Research Service Award (no. FHD0550002) from the NIH.

Author information

Authors and Affiliations

  1. The Whitney Marine Laboratory for Marine Science, University of Florida, St. Augustine, Florida, USA

    Michael J Layden, Eric Röttinger & Mark Q Martindale

  2. Department of Biology, Boston University, Boston, Massachusetts, USA

    Francis S Wolenski & Thomas D Gilmore

Authors
  1. Michael J Layden
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eric Röttinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Francis S Wolenski
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas D Gilmore
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mark Q Martindale
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.J.L. and E.R. generated and optimized mRNA injection, and MO-knockdown protocols. M.Q.M. provided technical advice on protocol development. All authors participated in writing the manuscript.

Corresponding author

Correspondence to Mark Q Martindale.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Close-up images of injection rig set up and ready for injection. (a) head on view of rig set up for a right hand injector. Micromanipulator joystick and micromanipulation machinery is mounted to the right of the microscope. (b) View from right side showing injection needle entering dish. Notice the steep angle of the needle in both images. (PDF 1503 kb)

Supplementary Figure 2

GFP fluorescence 6 days after injection of mRNA. (a-b) 200 ng/μl dextran injected alone. (c-d) 200 ng/μl dextran co-injected with 300ng/μl gfp mRNA. No GFP is detected in dextran control injections (b) while ubiquitous GFP expression can be visualized (d) in live animals. GFP expression is observed in ∼90% of the animals (99 GFP expressing animals out of 113 animals scored). (PDF 484 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Layden, M., Röttinger, E., Wolenski, F. et al. Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis. Nat Protoc 8, 924–934 (2013). https://doi.org/10.1038/nprot.2013.009

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.009

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing