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Spinal cord regeneration in Xenopus laevis

Nature Protocols volume 12, pages 372–389 (2017)Cite this article

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Abstract

Here we present a protocol for the husbandry of Xenopus laevis tadpoles and froglets, and procedures to study spinal cord regeneration. This includes methods to induce spinal cord injury (SCI); DNA and morpholino electroporation for genetic studies; in vivo imaging for cell analysis; a swimming test to measure functional recovery; and a convenient model for screening for new compounds that promote neural regeneration. These protocols establish X. laevis as a unique model organism for understanding spinal cord regeneration by comparing regenerative and nonregenerative stages. This protocol can be used to understand the molecular and cellular mechanisms involved in nervous system regeneration, including neural stem and progenitor cell (NSPC) proliferation and neurogenesis, extrinsic and intrinsic mechanisms involved in axon regeneration, glial response and scar formation, and trophic factors. For experienced personnel, husbandry takes 1–2 months; SCI can be achieved in 5–15 min; and swimming recovery takes 20–30 d.

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Figure 1: Spinal cord transection surgery in stage 50 larvae.
Figure 2: Spinal cord transection surgery in stage 66 froglets.
Figure 3: Swimming behavior test.
Figure 4: Platinum electrodes for larvae electroporation.
Figure 5: Sox3pXt-GFP+ cells after spinal cord electroporation.

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Acknowledgements

We thank members of our laboratory for their support, and especially our dedicated animal caretaker A. Farias. We thank H.T. Cline (The Scripps Research Institute) for our collaborative work with two-photon microscopy. This work was supported by funds from the CARE Chile UC-Centro de Envejecimiento y Regeneración (PFB 12/2007), MINREB (RC120003), FONDECYT (1141162), and ICGEB (CRP/CHI-13-01), FONDEF Idea (ID15I10349) to J.L. and Gastos Operacionales (21110043) to G.E.-F. G.E.-F., D.L.-L. and E.E.M.-O. are CONICYT PhD fellows.

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Authors and Affiliations

  1. Center for Aging and Regeneration, Millennium Nucleus in Regenerative Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile

    Gabriela Edwards-Faret, Rosana Muñoz, Emilio E Méndez-Olivos, Dasfne Lee-Liu, Victor S Tapia & Juan Larraín

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  1. Gabriela Edwards-Faret
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  4. Dasfne Lee-Liu
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  5. Victor S Tapia
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Contributions

G.E.-F., R.M., E.E.M.-O., D.L.-.L. and V.S.T. contributed to developing and improving the protocols described here. G.E.-F. prepared the figures and videos. G.E.-F. and J.L. conceived and wrote the paper.

Corresponding author

Correspondence to Juan Larraín.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Spinal cord injury in Xenopus laevis larvae.

Video explains surgery to induce spinal cord injury and the control sham surgery in larvae (Steps 10–12). (AVI 26179 kb)

Spinal cord injury in Xenopus laevis froglets.

Video explains surgery to induce spinal cord injury and the control sham surgery in froglets (Steps 16–20). (AVI 20720 kb)

Intracoelomic injections in Xenopus laevis larvae.

Video explains the intracoelomic injection of small molecules into larvae (Steps 25A(viii) and Box 1). (AVI 6844 kb)

Intracoelomic injections in Xenopus laevis froglets.

Video explains the intracoelomic injection of small molecules into froglets (Box 1). (AVI 6214 kb)

Spinal cord dissection for whole-mount immunofluorescence.

Video explains larva spinal cord dissection for whole-mount immunofluorescence (Steps 25A(xi–xvi)). (AVI 22272 kb)

Spinal cord isolation from Xenopus laevis larvae.

Video explains spinal cord isolation for -omics analysis in larvae (Box 3, step 4A). (AVI 17736 kb)

Spinal cord isolation from Xenopus laevis froglets.

Video explains spinal cord isolation for -omics analysis in froglets (Box 3, step 4B). (AVI 23323 kb)

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Edwards-Faret, G., Muñoz, R., Méndez-Olivos, E. et al. Spinal cord regeneration in Xenopus laevis. Nat Protoc 12, 372–389 (2017). https://doi.org/10.1038/nprot.2016.177

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