Generating Transgenic Reporter Lines for Studying Nervous System Development in the Cnidarian Nematostella vectensis

  • Fabian RentzschEmail author
  • Eduard Renfer
  • Ulrich Technau
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)


Neurons often display complex morphologies with long and fine processes that can be difficult to visualize, in particular in living animals. Transgenic reporter lines in which fluorescent proteins are expressed in defined populations of neurons are important tools that can overcome these difficulties. By using membrane-attached fluorescent proteins, such reporter transgenes can identify the complete outline of subsets of neurons or they can highlight the subcellular localization of fusion proteins, for example at pre- or postsynaptic sites. The relative stability of fluorescent proteins furthermore allows the tracing of the progeny of cells over time and can therefore provide information about potential roles of the gene whose regulatory elements are controlling the expression of the fluorescent protein. Here we describe the generation of transgenic reporter lines in the sea anemone Nematostella vectensis, a cnidarian model organism for studying the evolution of developmental processes. We also provide an overview of existing transgenic Nematostella lines that have been used to study conserved and derived aspects of nervous system development.


Transgenic reporter Nematostella Cnidaria Fluorescent protein Neurogenesis Microinjection 



This work is supported by funding from the University of Bergen and the Research Council of Norway (to F.R.) and by the Austrian Science Fund (FWF) (P27353) to U.T.


  1. 1.
    Telford MJ, Budd GE, Philippe H (2015) Phylogenomic Insights into Animal Evolution. Curr Biol 25(19):R876–R887CrossRefGoogle Scholar
  2. 2.
    Galliot B, Quiquand M (2011) A two-step process in the emergence of neurogenesis. Eur J Neurosci 34(6):847–862CrossRefGoogle Scholar
  3. 3.
    Watanabe H, Fujisawa T, Holstein TW (2009) Cnidarians and the evolutionary origin of the nervous system. Dev Growth Differ 51(3):167–183CrossRefGoogle Scholar
  4. 4.
    Rentzsch F, Layden M, Manuel M (2017) The cellular and molecular basis of cnidarian neurogenesis. Wiley Interdiscip Rev Dev Biol 6(1)CrossRefGoogle Scholar
  5. 5.
    Kelava I, Rentzsch F, Technau U (2015) Evolution of eumetazoan nervous systems: insights from cnidarians. Philos Trans R Soc Lond B Biol Sci 370(1684):20150065CrossRefGoogle Scholar
  6. 6.
    Zapata F, Goetz FE, Smith SA, Howison M, Siebert S, Church SH, Sanders SM, Ames CL, McFadden CS, France SC, Daly M, Collins AG, Haddock SH, Dunn CW, Cartwright P (2015) Phylogenomic Analyses Support Traditional Relationships within Cnidaria. PLoS One 10(10):e0139068CrossRefGoogle Scholar
  7. 7.
    Katsuki T, Greenspan RJ (2013) Jellyfish nervous systems. Curr Biol 23(14):R592–R594CrossRefGoogle Scholar
  8. 8.
    Mackie GO (2004) Central neural circuitry in the jellyfish Aglantha: a model simple nervous system. Neurosignals 13(1-2):5–19CrossRefGoogle Scholar
  9. 9.
    Garm A, Ekstrom P, Boudes M, Nilsson DE (2006) Rhopalia are integrated parts of the central nervous system in box jellyfish. Cell Tissue Res 325(2):333–343CrossRefGoogle Scholar
  10. 10.
    Petie R, Garm A, Nilsson DE (2011) Visual control of steering in the box jellyfish Tripedalia cystophora. J Exp Biol 214(Pt 17):2809–2815CrossRefGoogle Scholar
  11. 11.
    Sebe-Pedros A, Saudemont B, Chomsky E, Plessier F, Mailhe MP, Renno J, Loe-Mie Y, Lifshitz A, Mukamel Z, Schmutz S, Novault S, Steinmetz PRH, Spitz F, Tanay A, Marlow H (2018) Cnidarian cell type diversity and regulation revealed by whole-organism single-cell RNA-seq. Cell 173(6):1520–1534 e20CrossRefGoogle Scholar
  12. 12.
    Bosch TC, Anton-Erxleben F, Hemmrich G, Khalturin K (2010) The Hydra polyp: nothing but an active stem cell community. Dev Growth Differ 52(1):15–25CrossRefGoogle Scholar
  13. 13.
    Watanabe H, Hoang VT, Mattner R, Holstein TW (2009) Immortality and the base of multicellular life: Lessons from cnidarian stem cells. Semin Cell Dev Biol 20(9):1114–1125CrossRefGoogle Scholar
  14. 14.
    Frank U, Plickert G, Muller WA (2009) Cnidarian interstitial cells: The dawn of stem cell research. In: Rinkevich B, Matranga V (eds) Stem cells in marine organisms. Springer, New York, pp 33–59CrossRefGoogle Scholar
  15. 15.
    Martin VJ (1990) Development of Nerve-Cells in Hydrozoan Planulae. 3. Some Interstitial-Cells Traverse the Ganglionic Pathway in the Endoderm. Biol Bull 178(1):10–20CrossRefGoogle Scholar
  16. 16.
    Martin VJ, Archer WE (1986) Migration of interstitial cells and their derivatives in a hydrozoan planula. Dev Biol 116:486–496CrossRefGoogle Scholar
  17. 17.
    Martin VJ, Littlefield CL, Archer WE, Bode HR (1997) Embryogenesis in hydra. Biol Bull 192(3):345–363CrossRefGoogle Scholar
  18. 18.
    Nakanishi N, Renfer E, Technau U, Rentzsch F (2012) Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms. Development 139(2):347–357CrossRefGoogle Scholar
  19. 19.
    Richards GS, Rentzsch F (2014) Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis. Development 141(24):4681–4689CrossRefGoogle Scholar
  20. 20.
    Grens A, Mason E, Marsh JL, Bode HR (1995) Evolutionary conservation of a cell fate specification gene: the Hydra achaete-scute homolog has proneural activity in Drosophila. Development 121(12):4027–4035PubMedGoogle Scholar
  21. 21.
    Richards GS, Rentzsch F (2015) Regulation of Nematostella neural progenitors by SoxB, Notch and bHLH genes. Development 142(19):3332–3342CrossRefGoogle Scholar
  22. 22.
    Watanabe H, Kuhn A, Fushiki M, Agata K, Ozbek S, Fujisawa T, Holstein TW (2014) Sequential actions of beta-catenin and Bmp pattern the oral nerve net in Nematostella vectensis. Nat Commun 5:5536CrossRefGoogle Scholar
  23. 23.
    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(5):1013–1022CrossRefGoogle Scholar
  24. 24.
    Gahan JM, Schnitzler CE, DuBuc TQ, Doonan LB, Kanska J, Gornik SG, Barreira S, Thompson K, Schiffer P, Baxevanis AD, Frank U (2017) Functional studies on the role of Notch signaling in Hydractinia development. Dev Biol 428(1):224–231CrossRefGoogle Scholar
  25. 25.
    Renfer E, Amon-Hassenzahl A, Steinmetz PR, Technau U (2009) A muscle-specific transgenic reporter line of the sea anemone, Nematostella vectensis. Proc Natl Acad Sci U S A 107(1):104–108CrossRefGoogle Scholar
  26. 26.
    Renfer E, Technau U (2017) Meganuclease-assisted generation of stable transgenics in the sea anemone Nematostella vectensis. Nat Protoc 12(9):1844–1854CrossRefGoogle Scholar
  27. 27.
    Wittlieb J, Khalturin K, Lohmann JU, Anton-Erxleben F, Bosch TC (2006) Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis. Proc Natl Acad Sci U S A 103(16):6208–6211CrossRefGoogle Scholar
  28. 28.
    Dupre C, Yuste R (2017) Non-overlapping Neural Networks in Hydra vulgaris. Curr Biol 27(8):1085–1097CrossRefGoogle Scholar
  29. 29.
    Kunzel T, Heiermann R, Frank U, Muller W, Tilmann W, Bause M, Nonn A, Helling M, Schwarz RS, Plickert G (2010) Migration and differentiation potential of stem cells in the cnidarian Hydractinia analysed in eGFP-transgenic animals and chimeras. Dev Biol 348(1):120–129CrossRefGoogle Scholar
  30. 30.
    Havrilak JA, Faltine-Gonzalez D, Wen Y, Fodera D, Simpson AC, Magie CR, Layden MJ (2017) Characterization of NvLWamide-like neurons reveals stereotypy in Nematostella nerve net development. Dev Biol 431(2):336–346CrossRefGoogle Scholar
  31. 31.
    Busengdal H, Rentzsch F (2017) Unipotent progenitors contribute to the generation of sensory cell types in the nervous system of the cnidarian Nematostella vectensis. Dev Biol 431(1):59–68CrossRefGoogle Scholar
  32. 32.
    Columbus-Shenkar YY, Sachkova MY, Macrander J, Fridrich A, Modepalli V, Reitzel AM, Sunagar K, Moran Y (2018) Dynamics of venom composition across a complex life cycle. Elife 7:e35014CrossRefGoogle Scholar
  33. 33.
    Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345CrossRefGoogle Scholar
  34. 34.
    Layden MJ, Rottinger E, Wolenski FS, Gilmore TD, Martindale MQ (2013) Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis. Nat Protoc 8(5):924–934CrossRefGoogle Scholar
  35. 35.
    Schwaiger M, Schonauer A, Rendeiro AF, Pribitzer C, Schauer A, Gilles AF, Schinko JB, Renfer E, Fredman D, Technau U (2014) Evolutionary conservation of the eumetazoan gene regulatory landscape. Genome Res 24(4):639–650CrossRefGoogle Scholar
  36. 36.
    Fritzenwanker JH, Technau U (2002) Induction of gametogenesis in the basal cnidarian Nematostella vectensis(Anthozoa). Dev Genes Evol 212(2):99–103CrossRefGoogle Scholar
  37. 37.
    Genikhovich G, Technau U (2009) Induction of spawning in the starlet sea anemone Nematostella vectensis, in vitro fertilization of gametes, and dejellying of zygotes. Cold Spring Harb Protoc 2009(9):pdb prot5281PubMedGoogle Scholar
  38. 38.
    Stefanik DJ, Friedman LE, Finnerty JR (2013) Collecting, rearing, spawning and inducing regeneration of the starlet sea anemone, Nematostella vectensis. Nat Protoc 8(5):916–923CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Fabian Rentzsch
    • 1
    • 2
    Email author
  • Eduard Renfer
    • 3
  • Ulrich Technau
    • 3
  1. 1.Sars International Centre for Marine Molecular BiologyUniversity of BergenBergenNorway
  2. 2.Department of Biological SciencesUniversity of BergenBergenNorway
  3. 3.Department for Molecular Evolution and Development, Faculty of Life Sciences, Center of Organismal Systems BiologyUniversity of ViennaViennaAustria

Personalised recommendations