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XTRPC1-dependent chemotropic guidance of neuronal growth cones

Nature Neuroscience volume 8, pages 730–735 (2005)Cite this article

Abstract

Calcium arising through release from intracellular stores and from influx across the plasma membrane is essential for signalling by specific guidance cues and by factors that inhibit axon regeneration. The mediators of calcium influx in these cases are largely unknown. Transient receptor potential channels (TRPCs) belong to a superfamily of Ca2+-permeable, receptor-operated channels that have important roles in sensing and responding to changes in the local environment. Here we report that XTRPC1, a Xenopus homolog of mammalian TRPC1, is required for proper growth cone turning responses of Xenopus spinal neurons to microscopic gradients of netrin-1, brain-derived neurotrophic factor and myelin-associated glycoprotein, but not to semaphorin 3A. Furthermore, XTRPC1 is required for midline guidance of axons of commissural interneurons in the developing Xenopus spinal cord. Thus, members of the TRPC family may serve as a key mediator for the Ca2+ influx that regulates axon guidance during development and inhibits axon regeneration in adulthood.

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Figure 1: Requirement of XTRPC1 for growth cone turning responses to a gradient of netrin-1.
Figure 2: Summary of growth cone turning responses to a gradient of netrin-1.
Figure 3: XTRPC1 is required for formation of longitudinal axonal tracts by commissural interneurons in developing Xenopus spinal cord.
Figure 4: XTRPC1 is required for attraction of commissural interneuron axons to the CNS midline.
Figure 5: XTRPC1 as a general mediator for Ca2+-dependent neuronal growth cone guidance.

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Acknowledgements

We would like to thank A. Kolodkin, C. Montell and T. Dawson for critical comments, N. Marsh-Armstrong, L.N. Borodinsky and N.C. Spitzer for their help during this study, and L. Liu for her technical support. This work was supported by National Institute of Neurological Disorders and Stroke, Charles E. Culpeper Scholarships in Medical Science, Whitehall Foundation, and Basal O'Connor Starter Scholar Research Award Program to G.L.M. S.S. is partially supported by a postdoctoral fellowship from the Korea Science and Engineering Foundation. H.L.R and M.D.B. would like to gratefully acknowledge support from the Biotechnology and Biological Sciences Research Council and Royal Society.

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Authors and Affiliations

  1. Institute for Cell Engineering, Johns Hopkins University School of Medicine, 733 N. Broadway, Baltimore, 21205, Maryland, USA

    Sangwoo Shim, Eyleen L Goh, Shaoyu Ge, Kurt Sailor, Hongjun Song & Guo-li Ming

  2. Department of Neurology, Johns Hopkins University School of Medicine, 733 N. Broadway, Baltimore, 21205, Maryland, USA

    Sangwoo Shim, Eyleen L Goh, Shaoyu Ge, Kurt Sailor, Hongjun Song & Guo-li Ming

  3. Department of Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, 21205, Maryland, USA

    Joseph P Yuan, Paul F Worley, Hongjun Song & Guo-li Ming

  4. Laboratory of Molecular Signalling, The Babraham Institute, Babraham, CB2 4AT, Cambridge, UK

    H Llewelyn Roderick & Martin D Bootman

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Supplementary information

Supplementary Fig. 1

Neurite extension in the presence or absence of gradients of guidance cues. (a) Summary of neurite extension rates during the turning assay in a gradient of netrin-1 (5 g/ml in the pipette) for different neurons. Values represent mean ± s.e.m. (n = 20-29). (b) Summary of neurite length of neurons derived from embryos injected with either the XTRPC1 morpholino or a control morpholino cultured without the addition of any guidance cues. Values represent mean ± s.e.m. (n = 25-27). No statistical significance was found (P > 0.05, Bootstrap test). (JPG 150 kb)

Supplementary Fig. 2

Density of commissural interneurons in the developing spinal cord of Xenopus embryos. (a-d) Sample projections of Z-stack confocal images of 3A10 staining (red) of Xenopus spinal cord (stage 30) from the sagittal views. The same segment of spinal cord was imaged from the side injected with the XTRPC1 morpholino and fixable FITC-dextran as a lineage tracer (green) as shown in (a) and from the uninjected side as shown in (c). Enlarged images of 3A10 staining were shown in (b, d). The cell bodies of commissural interneurons exhibit specific 3A10 staining, which is not as strong as those for axons. Note a reduction of 3A10+ fiber tracks in the un-injected side when compared to that in the injected side from these sagittal views. Scale bar: 100 m for (a,c) and 40 m for (b,d). (e) Quantification of the density of commissural interneurons in stage 30 embryos. The number of 3A10+ labelled cell bodies was counted from the uninjected and injected side within the same segment of the spinal cord of embryos unilaterally injected with the XTRPC1 morpholino at the two-cell stage. Values represent mean ± s.e.m. (n = 6). No statistical significance was found (P > 0.05, Bootstrap test) (JPG 209 kb)

Supplementary Fig. 3

A model of the involvement of TRPCs in cytoplasmic signalling of guidance cues. (JPG 145 kb)

Supplementary Video 1

3D reconstruction of Z-series confocal images of the longitudinal axonal tracks of commissural interneurons in the ventral portion of the developing Xenopus spinal cord of stage 30 embryos injected with XTRPC1 morpholino. Shown are axons of commissural interneurons stained with monoclonal antibody 3A10 (red). The XTRPC1 morpholino was delivered to half of the embryo together with fixable FITC-dextran as a lineage tracer (green; see Methods). (MOV 2494 kb)

Supplementary Video 2

3D reconstruction of Z-series confocal images of the longitudinal axonal tracks of commissural interneurons in the ventral portion of the developing Xenopus spinal cord of stage 30 embryos injected with control morpholino. Shown are axons of commissural interneurons stained with monoclonal antibody 3A10 (red). Control morpholino was delivered to half of the embryo together with fixable FITC-dextran as a lineage tracer (green; see Methods). (MOV 2494 kb)

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Shim, S., Goh, E., Ge, S. et al. XTRPC1-dependent chemotropic guidance of neuronal growth cones. Nat Neurosci 8, 730–735 (2005). https://doi.org/10.1038/nn1459

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