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Characterizing the spatiotemporal expression of RNAs and proteins in the starlet sea anemone, Nematostella vectensis

Nature Protocols volume 8, pages 900–915 (2013)Cite this article

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Abstract

In an effort to reconstruct the early evolution of animal genes and proteins, there is an increasing focus on basal animal lineages such as sponges, cnidarians, ctenophores and placozoans. Among the basal animals, the starlet sea anemone Nematostella vectensis (phylum Cnidaria) has emerged as a leading laboratory model organism partly because it is well suited to experimental techniques for monitoring and manipulating gene expression. Here we describe protocols adapted for use in Nematostella to characterize the expression of RNAs by in situ hybridization using either chromogenic or fluorescence immunohistochemistry (∼1 week), as well as to characterize protein expression by whole-mount immunofluorescence (∼3 d). We also provide a protocol for labeling cnidocytes (∼3 h), the phylum-specific sensory-effector cell type that performs a variety of functions in cnidarians, including the delivery of their venomous sting.

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Figure 1: Principal steps in chromogenic ISH and FISH.
Figure 2: Example of ISH in Nematostella.
Figure 3: Example of indirect immunofluorescence.
Figure 4: Example of cnidocyte staining.
Figure 5: Overview of the four protocol options described here.

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Acknowledgements

This research was supported by National Science Foundation grant no. MCB-0924749 to T.D.G. and J.R.F. and by the US National Institutes of Health (NIH) grant no. 1R21RR032121 to M.Q.M. F.S.W. was supported by a predoctoral grant from the Superfund Basic Research Program at Boston University (no. 5 P42 E507381) and by 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.

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Authors and Affiliations

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

    Francis S Wolenski, Thomas D Gilmore & John R Finnerty

  2. Pacific Biosciences Research Center, Kewalo Marine Laboratory, University of Hawaii, Honolulu, Hawaii, USA

    Michael J Layden & Mark Q Martindale

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  1. Francis S Wolenski
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Contributions

F.S.W. optimized immunofluorescence and cnidocyte-labeling protocols. M.J.L. optimized the in situ hybridization protocols. J.R.F., T.D.G. and M.Q.M. provided technical advice on protocol development. All authors participated in writing the manuscript.

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Correspondence to John R Finnerty.

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Wolenski, F., Layden, M., Martindale, M. et al. Characterizing the spatiotemporal expression of RNAs and proteins in the starlet sea anemone, Nematostella vectensis. Nat Protoc 8, 900–915 (2013). https://doi.org/10.1038/nprot.2013.014

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