Reverse Genetic Approaches to Investigate the Neurobiology of the Cnidarian Sea Anemone Nematostella vectensis

  • Jamie A. Havrilak
  • Michael J. LaydenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)


The cnidarian sea anemone Nematostella vectensis has grown in popularity as a model system to complement the ongoing work in traditional bilaterian model species (e.g. Drosophila, C. elegans, vertebrate). The driving force behind developing cnidarian model systems is the potential of this group of animals to impact EvoDevo studies aimed at better determining the origin and evolution of bilaterian traits, such as centralized nervous systems. However, it is becoming apparent that cnidarians have the potential to impact our understanding of regenerative neurogenesis and systems neuroscience. Next-generation sequencing and the development of reverse genetic approaches led to functional genetics becoming routine in the Nematostella system. As a result, researchers are beginning to understand how cnidarian nerve nets are related to the bilaterian nervous systems. This chapter describes the methods for morpholino and mRNA injections to knockdown or overexpress genes of interest, respectively. Carrying out these techniques in Nematostella requires obtaining and preparing embryos for microinjection, designing and generating effective morpholino and mRNA molecules with controls for injection, and optimizing injection conditions.


Nematostella Morpholino mRNA Embryo microinjection Gene knockdown Overexpression 


  1. 1.
    Layden MJ, Boekhout M, Martindale MQ (2012) Nematostella vectensis achaete-scute homolog NvashA regulates embryonic ectodermal neurogenesis and represents an ancient component of the metazoan neural specification pathway. Development 139:1013–1022CrossRefGoogle Scholar
  2. 2.
    Richards GS, Rentzsch F (2014) Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis. Development 141:4681–4689CrossRefGoogle Scholar
  3. 3.
    Richards GS, Rentzsch F (2015) Regulation of Nematostella neural progenitors by SoxB, Notch and bHLH genes. Development 142:3332–3342CrossRefGoogle Scholar
  4. 4.
    Layden MJ, Rentzsch F, Röttinger E (2016) The rise of the starlet sea anemone Nematostella vectensis as a model system to investigate development and regeneration. Wiley Interdiscip Rev Dev Biol:1–21Google Scholar
  5. 5.
    Rentzsch F, Layden M, Manuel M (2016) The cellular and molecular basis of cnidarian neurogenesis. Wiley Interdiscip Rev Dev Biol:1–19Google Scholar
  6. 6.
    Hayakawa E, Fujisawa C, Fujisawa T (2004) Involvement of Hydra achaete?scute gene CnASH in the differentiation pathway of sensory neurons in the tentacles. Dev Genes EvolGoogle Scholar
  7. 7.
    Käsbauer T, Towb P, Alexandrova O et al (2007) The Notch signaling pathway in the cnidarian Hydra. Dev Biol 303:376–390CrossRefGoogle Scholar
  8. 8.
    Münder S, Tischer S, Grundhuber M et al (2013) Dev Biol 383:146–157CrossRefGoogle Scholar
  9. 9.
    Jager M, Quéinnec E, Le Guyader H et al (2011) Multiple Sox genes are expressed in stem cells or in differentiating neuro-sensory cells in the hydrozoan Clytia hemisphaerica. EvoDevo 2:12CrossRefGoogle Scholar
  10. 10.
    Gahan JM, Schnitzler CE, DuBuc TQ et al (2017) Functional studies on the role of Notch signaling in Hydractinia development. Dev Biol 428:224–231CrossRefGoogle Scholar
  11. 11.
    Dunn CW, Hejnol A, Matus DQ et al (2008) Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452:745–749CrossRefGoogle Scholar
  12. 12.
    Hejnol A, Obst M, Stamatakis A et al (2009) Assessing the root of bilaterian animals with scalable phylogenomic methods. Proc R Soc B Biol Sci 276:4261–4270CrossRefGoogle Scholar
  13. 13.
    Dupre C, Yuste R (2017) Non-overlapping neural networks in Hydra vulgaris. Curbio 27:1085–1097Google Scholar
  14. 14.
    Amiel AR, Johnston HT, Nedoncelle K et al (2015) Characterization of morphological and cellular events underlying oral regeneration in the sea anemone, Nematostella vectensis. Int J Mol Sci 16:28449–28471CrossRefGoogle Scholar
  15. 15.
    DuBuc TQ, Traylor-Knowles N, Martindale MQ (2014) Initiating a regenerative response; cellular and molecular features of wound healing in the cnidarian Nematostella vectensis. BMC Biol 12:24–138CrossRefGoogle Scholar
  16. 16.
    Schaffer AA, Bazarsky M, Levy K et al (2016) A transcriptional time-course analysis of oral vs. aboral whole-body regeneration in the sea anemone Nematostella vectensis. BMC Genomics:1–22Google Scholar
  17. 17.
    Sebe-Pedros A, Chomsky E, Saudemont B et al (2017) Cnidarian cell type diversity revealed by whole-organism single-cell RNA-seq analysis, pp 1–29Google Scholar
  18. 18.
    Layden MJ, Röttinger E, Wolenski FS et al (2013) Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis. Nat Protoc 8:924–934CrossRefGoogle Scholar
  19. 19.
    Ikmi A, McKinney SA, Delventhal KM et al (2014) TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis. Nat Commun 5:5486–5488CrossRefGoogle Scholar
  20. 20.
    Putnam NH, Srivastava M, Hellsten U et al (2007) Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317:86–94CrossRefGoogle Scholar
  21. 21.
    Genikhovich G, Technau U (2009) The starlet sea anemone Nematostella vectensis: an anthozoan model organism for studies in comparative genomics and functional evolutionary developmental biology. Cold Spring Harb Protoc 2009:pdb.emo129–pdb.emo129PubMedGoogle Scholar
  22. 22.
    Tulin S, Aguiar D, Istrail S et al (2013) A quantitative reference transcriptome for Nematostella vectensis early embryonic development: a pipeline for de novo assembly in emerging model systems. EvoDevo 4:16–11CrossRefGoogle Scholar
  23. 23.
    Helm RR, Siebert S, Tulin S et al (2013) Characterization of differential transcript abundance through time during Nematostellavectensis development. BMC Genomics 14:1–1CrossRefGoogle Scholar
  24. 24.
    Wittlieb J, Khalturin K, Lohmann JU et al (2006) Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis. PNAS 103:6208–6211CrossRefGoogle Scholar
  25. 25.
    Galliot B, Miljkovic-Licina M, Ghila L et al (2007) RNAi gene silencing affects cell and developmental plasticity in hydra. C R Biol 330:491–497CrossRefGoogle Scholar
  26. 26.
    Dunn SR, Phillips WS, Green DR et al (2007) Knockdown of actin and caspase gene expression by RNA interference in the symbiotic anemone. Biol Bull 212:250–258CrossRefGoogle Scholar
  27. 27.
    Warner J, Guerlais V, Amiel A et al (2017) NvERTx: a gene expression database to compare embryogenesis and regeneration in the sea anemone Nematostella vectensis, pp 1–18Google Scholar
  28. 28.
    Sebe-Pedros A, Chomsky E, Saudemont B et al (2017) Cnidarian cell type diversity revealed by whole-organism single-cell RNA-seq analysis, bioRxiv, pp 1–29Google Scholar
  29. 29.
    Nakanishi N, Renfer E, Technau U et al (2012) Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms. Development 139:347–357CrossRefGoogle Scholar
  30. 30.
    Layden MJ, Martindale MQ (2014) Non-canonical Notch signaling represents an ancestral mechanism to regulate neural differentiation. EvoDevo 5:30–14CrossRefGoogle Scholar
  31. 31.
    Layden MJ, Johnston H, Amiel AR et al (2016) MAPK signaling is necessary for neurogenesis in Nematostella vectensis. BMC Biol:1–19Google Scholar
  32. 32.
    Havrilak JA, Faltine-Gonzalez D, Wen Y et al (2017) Characterization of NvLWamide-like neurons reveals stereotypy in Nematostella nerve net development. Dev Biol. 431(2):336–346CrossRefGoogle Scholar
  33. 33.
    Watanabe H, Kuhn A, Fushiki M et al (2014) Sequential actions of β-catenin and Bmp pattern the oral nerve net in Nematostella vectensis. Nat Commun 5:5536–5514CrossRefGoogle Scholar
  34. 34.
    Leclère L, Bause M, Sinigaglia C et al (2016) Development of the aboral domain in Nematostella requires β-catenin and the opposing activities of Six3/6 and Frizzled5/8. Development 143:1766–1777CrossRefGoogle Scholar
  35. 35.
    Sinigaglia C, Busengdal H, Leclère L et al (2013) The bilaterian head patterning gene six3/6 controls aboral domain development in a cnidarian. PLoS Biol 11:e1001488CrossRefGoogle Scholar
  36. 36.
    Marlow H, Matus DQ, Martindale MQ (2013) Ectopic activation of the canonical wnt signaling pathway affects ectodermal patterning along the primary axis during larval development in the anthozoan Nematostella vectensis. Dev Biol 380:324–334CrossRefGoogle Scholar
  37. 37.
    Wikramanayake AH, Hong M, Lee PN et al (2003) An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layder segregation. Nature 426:446–450CrossRefGoogle Scholar
  38. 38.
    Magie CR, Daly M, Martindale MQ (2007) Gastrulation in the cnidarian Nematostella vectensis occurs via invagination not ingression. Dev Biol 305:483–497CrossRefGoogle Scholar
  39. 39.
    Rentzsch F, Fritzenwanker JH, Scholz CB et al (2008) FGF signalling controls formation of the apical sensory organ in the cnidarian Nematostella vectensis. Development 135:1761–1769CrossRefGoogle Scholar
  40. 40.
    Fritzenwanker JH, Technau U (2002) Induction of gametogenesis in the basal cnidarian Nematostella vectensis (Anthozoa). Dev Genes Evol 212:99–103CrossRefGoogle Scholar
  41. 41.
    Hand C, Uhlinger KR (1992) The culture, sexual and asexual reproduction, and growth of the sea anemone Nematostella vectensis. Biol Bull 182:169–176CrossRefGoogle Scholar
  42. 42.
    Renfer E, Technau U Meganuclease-assisted generation of stable transgenics in the sea anemone Nematostella vectensis. Nat Protoc 12:1844 EPCrossRefGoogle Scholar
  43. 43.
    Wolenski FS, Bradham CA, Finnerty JR et al (2012) B is required for cnidocyte development in the sea anemoneNematostella vectensis. Dev Biol:1–11Google Scholar
  44. 44.
    Marlow H,Roettinger E,Boekhout M et al. (2012) Developmental biology. Dev Biol:1–14Google Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Biological SciencesLehigh UniversityBethlehemUSA

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