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Transgenesis in Hydra to characterize gene function and visualize cell behavior

Nature Protocols volume 14, pages 2069–2090 (2019)Cite this article

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Abstract

The freshwater polyp Hydra is a cnidarian used as a model organism in a number of fields, including the study of the origin and evolution of developmental mechanisms, aging, symbiosis and host–microbe interactions. Here, we describe a procedure for the establishment of stable transgenic Hydra lines by embryo microinjection. The three-stage protocol comprises (i) the design and preparation of a transgenic construct, (ii) the microinjection of the vector into early embryos of Hydra vulgaris, and (iii) the selection and enrichment of mosaic animals in order to develop uniformly transgenic clonal lines. The preparation of a transgenic construct requires ~2 weeks, and transgenic lines can be obtained within 3 months. The method allows constitutive or inducible gain- and loss-of-function approaches, as well as in vivo tracing of individual cells. Hydra polyps carrying transgenic cells reveal functional properties of the ancestral circuitry controlling animal development.

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Fig. 1: Overview of the Hydra transgenesis technology.
Fig. 2: Embryo microinjection allows genetic manipulation of all cell types in the transgenic Hydra lines.
Fig. 3: The diversity of available constructs ensures high versatility of the transgenic Hydra technology.
Fig. 4: High-density culture and regular intensive feeding before sex induction allow maximization of the yield of fertilized eggs from a mass culture of Hydra.
Fig. 5: Two clonal lines should be raised from each mosaic founder polyp.
Fig. 6: Evidence for germline transmission of a transgene.

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Data availability

All data generated or analyzed during the current study are included in this paper. The raw data used in the example results are available from the corresponding authors upon reasonable request.

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Acknowledgements

We are grateful to F. Anton-Erxleben for assistance with confocal microscopy and to J. Lohmann and K. Khalturin for contributions to the development of the genetic constructs. This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG) (grant CRC 190, ‘Mechanisms and factors of gene activation’; grant nos. 846/6-1-4, 848/13-1-4, and 848/15-1; grant CRC 1182, ‘Origin and function of Metaorganisms’; and grants from the DFG Clusters of Excellence ‘Inflammation at Interfaces’ and ‘The Future Ocean’). A.K. was supported by a fellowship from the Alexander von Humboldt Foundation. T.C.G.B. acknowledges support from the Canadian Institute for Advanced Research (CIFAR).

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  1. Zoological Institute, University of Kiel, Kiel, Germany

    Alexander Klimovich, Jörg Wittlieb & Thomas C. G. Bosch

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  1. Alexander Klimovich
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Contributions

A.K. and T.C.G.B. conceived the paper. J.W. and A.K. generated the data and took photos. A.K., J.W. and T.C.G.B. wrote the manuscript.

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Correspondence to Alexander Klimovich or Thomas C. G. Bosch.

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Journal peer review information: Nature Protocols thanks Brigitte Galliot, Rob Steele and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Wittlieb, J., Khalturin, K., Lohmann, J. U., Anton-Erxleben, F. & Bosch, T. C. G. Proc. Natl. Acad. Sci. USA 103, 6208–6211 (2006): https://doi.org/10.1073/pnas.0510163103

Siebert, S., Anton-Erxleben, F. & Bosch, T. C. G. Dev. Biol. 313, 13–24 (2008): https://doi.org/10.1016/j.ydbio.2007.09.007

Klimovich, A. et al. Aging 10, 951–972 (2018): https://doi.org/10.18632/aging.101440

Integrated supplementary information

Supplementary Figure 1 Shortening of tentacles is the first indicator of a polyp’s compromised health.

A healthy Hydra polyp (left) typically has long tentacles armed with nematocytes and displays conspicuous periodically recurring contractions of the body followed by a passive re-extension. Sick polyps (right) can be recognized by shortened tentacles with rounded knobs at their ends (arrowheads). Diseased polyps stay in a permanently elongated or contracted state. Irregular or inappropriate washing of the culture after feeding or exposure to some toxic chemicals from the plastic are main causes for emergence of such diseased polyps.

Supplementary information

Supplementary information

Supplementary Figure 1

Reporting Summary

Supplementary Video 1

Microinjection of a Hydra embryo. (00:00–00:04) A female polyp with a fertilized egg undergoing the first cleavage. (00:05–00:17) Microinjection setup ready for injection. The tips of both the holding capillary (left) and the injection needle (right) are submerged in the injection chamber. (00:18–00:33) The embryo is fixed by the holding capillary (left) and pierced with the injection needle (right) to deliver the transgene into it. (00:34–00:40) The injected embryo continues its development and undergoes a series of cleavages. (00:41–00:43) Three transgenic embryos at the cuticle stage with clearly visible eGFP fluorescence. (00:44–00:51) Transgenic polyp hatches from one of the embryos.

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Klimovich, A., Wittlieb, J. & Bosch, T.C.G. Transgenesis in Hydra to characterize gene function and visualize cell behavior. Nat Protoc 14, 2069–2090 (2019). https://doi.org/10.1038/s41596-019-0173-3

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