Letter | Published:

Floor-plate-derived netrin-1 is dispensable for commissural axon guidance

Nature volume 545, pages 350354 (18 May 2017) | Download Citation

Abstract

Netrin-1 is an evolutionarily conserved, secreted extracellular matrix protein involved in axon guidance at the central nervous system midline1,2. Netrin-1 is expressed by cells localized at the central nervous system midline, such as those of the floor plate in vertebrate embryos1,3. Growth cone turning assays and three-dimensional gel diffusion assays have shown that netrin-1 can attract commissural axons2,4,5,6. Loss-of-function experiments further demonstrated that commissural axon extension to the midline is severely impaired in the absence of netrin-1 (refs 3, 7, 8, 9). Together, these data have long supported a model in which commissural axons are attracted by a netrin-1 gradient diffusing from the midline. Here we selectively ablate netrin-1 expression in floor-plate cells using a Ntn1 conditional knockout mouse line. We find that hindbrain and spinal cord commissural axons develop normally in the absence of floor-plate-derived netrin-1. Furthermore, we show that netrin-1 is highly expressed by cells in the ventricular zone, which can release netrin-1 at the pial surface where it binds to commissural axons. Notably, Ntn1 deletion from the ventricular zone phenocopies commissural axon guidance defects previously described in Ntn1-knockout mice. These results show that the classical view that attraction of commissural axons is mediated by a gradient of floor-plate-derived netrin-1 is inaccurate and that netrin-1 primarily acts locally by promoting growth cone adhesion.

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References

  1. 1.

    et al. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 78, 409–424 (1994)

  2. 2.

    , , , & UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans. Neuron 9, 873–881 (1992)

  3. 3.

    et al. Genetic analysis of netrin genes in Drosophila: netrins guide CNS commissural axons and peripheral motor axons. Neuron 17, 203–215 (1996)

  4. 4.

    , , & Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78, 425–435 (1994)

  5. 5.

    et al. cAMP-dependent growth cone guidance by netrin-1. Neuron 19, 1225–1235 (1997)

  6. 6.

    et al. Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC. Neuron 19, 1211–1224 (1997)

  7. 7.

    et al. Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell 87, 1001–1014 (1996)

  8. 8.

    , & Phenotypic analysis of mice completely lacking netrin 1. Development 142, 3686–3691 (2015)

  9. 9.

    et al. Complete loss of netrin-1 results in embryonic lethality and severe axon guidance defects without increased neural cell death. Cell Reports 12, 1099–1106 (2015)

  10. 10.

    Development and plasticity of commissural circuits: from locomotion to brain repair. Trends Neurosci. 37, 551–562 (2014)

  11. 11.

    et al. The divergent Robo family protein Rig-1/Robo3 is a negative regulator of slit responsiveness required for midline crossing by commissural axons. Cell 117, 157–169 (2004)

  12. 12.

    et al. The slit receptor Rig-1/Robo3 controls midline crossing by hindbrain precerebellar neurons and axons. Neuron 43, 69–79 (2004)

  13. 13.

    The development of the cervical spinal cord of the mouse embryo. II. A Golgi analysis of sensory, commissural, and association cell differentiation. J. Comp. Neurol. 222, 96–115 (1984)

  14. 14.

    , , & Axon guidance by diffusible chemoattractants: a gradient of netrin protein in the developing spinal cord. J. Neurosci. 26, 8866–8874 (2006)

  15. 15.

    et al. The Purkinje neuron acts as a central regulator of spatially and functionally distinct cerebellar precursors. Dev. Cell 27, 278–292 (2013)

  16. 16.

    et al. Genetic dissection of the function of hindbrain axonal commissures. PLoS Biol. 8, e1000325 (2010)

  17. 17.

    , , & The Drosophila Netrin receptor Frazzled guides axons by controlling Netrin distribution. Nature 406, 886–889 (2000)

  18. 18.

    et al. Phenotype of mice lacking functional Deleted in colorectal cancer (Dcc) gene. Nature 386, 796–804 (1997)

  19. 19.

    et al. Wnt antagonism of Shh facilitates midbrain floor plate neurogenesis. Nat. Neurosci. 12, 125–131 (2009)

  20. 20.

    , , & Floor plate and netrin-1 are involved in the migration and survival of inferior olivary neurons. J. Neurosci. 19, 4407–4420 (1999)

  21. 21.

    & FoxP2 expression in the cerebellum and inferior olive: development of the transverse stripe-shaped expression pattern in the mouse cerebellar cortex. J. Comp. Neurol. 520, 656–677 (2012)

  22. 22.

    et al. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat. Genet. 23, 99–103 (1999)

  23. 23.

    et al. Structural decoding of the Netrin-1/UNC5 interaction and its therapeutical implications in cancers. Cancer Cell 29, 173–185 (2016)

  24. 24.

    & Netrins guide Drosophila commissural axons at short range. Nat. Neurosci. 9, 188–194 (2006)

  25. 25.

    & Frazzled promotes growth cone attachment at the source of a Netrin gradient in the Drosophila visual system. eLife 5, e20762 (2016)

  26. 26.

    , & Traction on immobilized netrin-1 is sufficient to reorient axons. Science 325, 166 (2009)

  27. 27.

    , , , & Ventral midline cells are required for the local control of commissural axon guidance in the mouse spinal cord. Development 126, 3649–3659 (1999)

  28. 28.

    et al. Nucleolar localization of a netrin-1 isoform enhances tumor cell proliferation. Sci. Signal. 5, ra57 (2012)

  29. 29.

    , & Expression pattern of a Krox-20/Cre knock-in allele in the developing hindbrain, bones, and peripheral nervous system. Genesis 26, 123–126 (2000)

  30. 30.

    et al. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118, 517–528 (2004)

  31. 31.

    & Targeting of Cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures. Dev. Biol. 222, 296–306 (2000)

  32. 32.

    et al. A simple method for 3D analysis of immunolabeled axonal tracts in a transparent nervous system. Cell Reports 9, 1191–1201 (2014)

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Acknowledgements

We thank C. Wright for the Ptf1a:creERT2 line and P. Charnay for the Krox20:cre line. We also thank A. Kolodkin and R. Vigouroux for critical reading of the manuscript and S. Fouquet of the Vision Institute imaging facility for its technical support. This work was supported by grants from the Agence Nationale de la Recherche (ANR-14-CE13-0004-01) (A.C.). It was performed in the frame of the LABEX LIFESENSES (reference ANR-10-LABX-65) supported by French state funds managed by the ANR within the Investissements d’Avenir programme under reference ANR-11-IDEX-0004-02 (A.C.). This work was also supported by grants from INCA, ERC, ANR and Fondation Bettencourt (P.M.). C.D. was recipient of a PhD fellowship from the Fondation pour la recherche médicale.

Author information

Author notes

    • Chloé Dominici
    •  & Juan Antonio Moreno-Bravo

    These authors contributed equally to this work.

    • Patrick Mehlen
    •  & Alain Chédotal

    These authors contributed equally to this work.

Affiliations

  1. Sorbonne Universités, UPMC Paris 06, INSERM, CNRS, Institut de la Vision, 17 Rue Moreau, 75012 Paris, France

    • Chloé Dominici
    • , Juan Antonio Moreno-Bravo
    • , Sergi Roig Puiggros
    • , Quentin Rappeneau
    •  & Alain Chédotal
  2. Apoptosis, Cancer and Development Laboratory, Equipe labellisée ‘La Ligue’, LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon, INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, 69008 Lyon, France

    • Sergi Roig Puiggros
    • , Nicolas Rama
    • , Pauline Vieugue
    • , Agns Bernet
    •  & Patrick Mehlen

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Contributions

A.C., A.B. and P.M. designed the experiments. C.D., J.A.M.B., N.R., P.V., Q.R. and S.R.P. performed the experiments. A.C., C.D. and J.A.M.B. prepared the figures. A.C. and P.M. supervised the project and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Patrick Mehlen or Alain Chédotal.

Reviewer Information Nature thanks T. Gomez and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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    Supplementary Figure

    This file contains the uncropped Western Blot scans with size marker indications (Extended Data Figure 1d and Extended Data Figure 2h).

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https://doi.org/10.1038/nature22331

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