Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Cell-surface heparan sulfate is involved in the repulsive guidance activities of Slit2 protein

Nature Neuroscience volume 4, pages 695–701 (2001)Cite this article

Abstract

Slit proteins are a family of secreted guidance proteins that can repel neuronal migration and axon growth via interaction with their cellular roundabout receptors (Robos). Here it was shown that Slit2–Robo-1 interactions were enhanced by cell-surface heparan sulfate. Removal of heparan sulfate decreased the affinity of Slit for Robo by about threefold. In addition, removal of cell-surface heparan sulfate by heparinase III abolished the chemorepulsive response to Slit2 normally shown by both the migrating neurons and growing axons. These results indicate essential roles for cell-surface heparan sulfate in the repulsive activities of Slit2.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Heparan sulfate promotes Slit2 protein binding to Robo-1.
Figure 2: Effect of heparan sulfate removal on the binding of 125I-Slit to the Robo-1 receptor expressed in 293 cells.
Figure 3: Heparinase III treatment and excess exogenous heparan sulfate abolished repulsive activity of Slit2 for olfactory interneuron precursors.
Figure 4: Heparan sulfate is required on the olfactory interneuron precursors for their repulsive response toward Slit2 protein.
Figure 5: Heparinase III treatment or excess heparan sulfate abolished repulsive activity of Slit2 toward olfactory bulb axons.
Figure 6: Heparan sulfate is required on the olfactory bulb axons for their repulsive response to Slit2.

Similar content being viewed by others

References

  1. Kennedy, T. E., Serafini, T., de la Torre, J. R. & Tessier-Lavigne, M. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78, 425–435 (1994).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Luo, Y., Raible, D. & Raper, J. A. Collapsin: a protein in brain that induces the collapse and paralysis of neuronal growth cones. Cell 75, 217–227 (1993).

    Article  CAS  Google Scholar 

  5. Kolodkin, A. L., Matthes, D. J. & Goodman, C. S. The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 75, 1389–1399 (1993).

    Article  CAS  Google Scholar 

  6. Brose, K. et al. Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795–806 (1999).

    Article  CAS  Google Scholar 

  7. Kidd, T., Bland, K. S. & Goodman, C. S. Slit is the midline repellent for the robo receptor in Drosophila. Cell 96, 785–794 (1999).

    Article  CAS  Google Scholar 

  8. Li, H. S. et al. Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons. Cell 96, 807–818 (1999).

    Article  CAS  Google Scholar 

  9. Ba-Charvet, K. T. N. et al. Slit2-mediated chemorepulsion and collapse of developing forebrain axons. Neuron 22, 463–473 (1999).

    Article  CAS  Google Scholar 

  10. Rothberg, J. M., Jacobs, J. R., Goodman, C. S. & Artavanis-Tsakonas, S. Slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev. 4, 2169–2187 (1990).

    Article  CAS  Google Scholar 

  11. Kidd, T. et al. Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell 92, 205–215 (1998).

    Article  CAS  Google Scholar 

  12. Zallen, J. A., Yi, B. A. & Bargmann, C. I. The conserved immunoglobulin superfamily member SAX-3/Robo directs multiple aspects of axon guidance in C. elegans. Cell 92, 217–227 (1998).

    Article  CAS  Google Scholar 

  13. Rajagopalan, S., Nicolas, E., Vivancos, V., Berger, J. & Dickson, B. J. Crossing the midline: roles and regulation of Robo receptors. Neuron 28, 767–777 (2000).

    Article  CAS  Google Scholar 

  14. Rajagopalan, S., Vivancos, V., Nicolas, E. & Dickson, B. J. Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS. Cell 103, 1033–1045 (2000).

    Article  CAS  Google Scholar 

  15. Simpson, J. H., Kidd, T., Bland, K. S. & Goodman, C. S. Short-range and long-range guidance by slit and its Robo receptors. Robo and Robo2 play distinct roles in midline guidance. Neuron 28, 753–766 (2000).

    Article  CAS  Google Scholar 

  16. Simpson, J. H., Bland, K. S., Fetter, R. D. & Goodman, C. S. Short-range and long-range guidance by Slit and its Robo receptors: a combinatorial code of Robo receptors controls lateral position. Cell 103, 1019–1032 (2000).

    Article  CAS  Google Scholar 

  17. Battye, R., Stevens, A. & Jacobs, J. R. Axon repulsion from the midline of the Drosophila CNS requires slit function. Development 126, 2475–2481 (1999).

    CAS  Google Scholar 

  18. Bashaw, G. J. & Goodman, C. S. Chimeric axon guidance receptors: the cytoplasmic domains of slit and netrin receptors specify attraction versus repulsion. Cell 97, 917–926 (1999).

    Article  CAS  Google Scholar 

  19. Nakayama, M. et al. Identification of high-molecular-weight proteins with multiple EGF-like motifs by motif-trap screening. Genomics 51, 27–34 (1998).

    Article  CAS  Google Scholar 

  20. Holmes, G. P. et al. Distinct but overlapping expression patterns of two vertebrate slit homologs implies functional roles in CNS development and organogenesis. Mech. Dev. 79, 57–72 (1998).

    Article  CAS  Google Scholar 

  21. Itoh, A., Miyabayashi, T., Ohno, M. & Sakano, S. Cloning and expressions of three mammalian homologues of Drosophila slit suggest possible roles for Slit in the formation and maintenance of the nervous system. Brain Res. Mol. Brain Res. 62, 175–186 (1998).

    Article  CAS  Google Scholar 

  22. Erskine, L. et al. Retinal ganglion cell axon guidance in the mouse optic chiasm: expression and function of Robos and Slits. J. Neurosci. 20, 4975–4982 (2000).

    Article  CAS  Google Scholar 

  23. Ringstedt, T. et al. Slit inhibition of retinal axon growth and its role in retinal axon pathfinding and innervation patterns in the diencephalon. J. Neurosci. 20, 4983–4991 (2000).

    Article  CAS  Google Scholar 

  24. Niclou, S. P., Jia, L. & Raper, J. A. Slit2 is a repellent for retinal ganglion cell axons. J. Neurosci. 20, 4962–4974 (2000).

    Article  CAS  Google Scholar 

  25. Wang, K. H. et al. Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell 96, 771–784 (1999).

    Article  CAS  Google Scholar 

  26. Wu, W. et al. Directional guidance of neuronal migration in the olfactory system by the protein Slit. Nature 400, 331–336 (1999).

    Article  CAS  Google Scholar 

  27. Zhu, Y., Li, H., Zhou, L., Wu, J. Y. & Rao, Y. Cellular and molecular guidance of GABAergic neuronal migration from an extracortical origin to the neocortex. Neuron 23, 473–485 (1999).

    Article  CAS  Google Scholar 

  28. Hu, H. Chemorepulsion of neuronal migration by Slit2 in the developing mammalian forebrain. Neuron 23, 703–711 (1999).

    Article  CAS  Google Scholar 

  29. Chen, J. H., Wen, L., Dupuis, S., Wu, J. Y. & Rao, Y. The N-terminal leucine-rich regions in Slit are sufficient to repel olfactory bulb axons and subventricular zone neurons. J. Neurosci. 21, 1548–1556 (2001).

    Article  CAS  Google Scholar 

  30. Liang, Y. et al. Mammalian homologues of the Drosophila slit protein are ligands of the heparan sulfate proteoglycan glypican-1 in brain. J. Biol. Chem. 274, 17885–17892 (1999).

    Article  CAS  Google Scholar 

  31. Nugent, M. A. & Edelman, E. R. Kinetics of basic fibroblast growth factor binding to its receptor and heparan sulfate proteoglycan: a mechanism for cooperactivity. Biochemistry 31, 8876–8883 (1992).

    Article  CAS  Google Scholar 

  32. Hu, H. & Rutishauser, U. A septum-derived chemorepulsive factor for migrating olfactory interneuron precursors. Neuron 16, 933–940 (1996).

    Article  CAS  Google Scholar 

  33. Pini, A. Chemorepulsion of axons in the developing mammalian central nervous system. Science 261, 95–98 (1993).

    Article  CAS  Google Scholar 

  34. Bernfield, M. et al. Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68, 729–777 (1999).

    Article  CAS  Google Scholar 

  35. Binari, R. C. et al. Genetic evidence that heparin-like glycosaminoglycans are involved in wingless signaling. Development 124, 2623–2632 (1997).

    CAS  Google Scholar 

  36. Lin, X., Buff, E. M., Perrimon, N. & Michelson, A. M. Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development. Development 126, 3715–3723 (1999).

    CAS  Google Scholar 

  37. Lin, X. & Perrimon, N. Dally cooperates with Drosophila Frizzled 2 to transduce Wingless signalling. Nature 400, 281–284 (1999).

    Article  CAS  Google Scholar 

  38. Hacker, U., Lin, X. & Perrimon, N. The Drosophila sugarless gene modulates Wingless signaling and encodes an enzyme involved in polysaccharide biosynthesis. Development 124, 3565–3573 (1997).

    CAS  Google Scholar 

  39. Haerry, T. E., Heslip, T. R., Marsh, J. L. & O'Connor, M. B. Defects in glucuronate biosynthesis disrupt Wingless signaling in Drosophila. Development 124, 3055–3064 (1997).

    CAS  Google Scholar 

  40. The, I., Bellaiche, Y. & Perrimon, N. Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan. Mol. Cell 4, 633–639 (1999).

    Article  CAS  Google Scholar 

  41. Bellaiche, Y., The, I. & Perrimon, N. Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion. Nature 394, 85–88 (1998).

    Article  CAS  Google Scholar 

  42. Bullock, S. L., Fletcher, J. M., Beddington, R. S. & Wilson, V. A. Renal agenesis in mice homozygous for a gene trap mutation in the gene encoding heparan sulfate 2-sulfotransferase. Genes Dev. 12, 1894–1906 (1998).

    Article  CAS  Google Scholar 

  43. Kinnunen, T. et al. Neurite outgrowth in brain neurons induced by heparin-binding growth-associated molecule (HB-GAM) depends on the specific interaction of HB-GAM with heparan sulfate at the cell surface. J. Biol. Chem. 271, 2243–2248 (1996).

    Article  CAS  Google Scholar 

  44. Kinnunen, A. et al. Heparan sulphate and HB-GAM (heparin-binding growth-associated molecule) in the development of the thalamocortical pathway of rat brain. Eur. J. Neurosci. 11, 491–502 (1999).

    Article  CAS  Google Scholar 

  45. Kinnunen, A. et al. N-syndecan and HB-GAM (heparin-binding growth-associated molecule) associate with early axonal tracts in the rat brain. Eur. J. Neurosci. 10, 635–648 (1998).

    Article  CAS  Google Scholar 

  46. Karthikeyan, L. et al. Immunocytochemical and in situ hybridization studies of the heparan sulfate proteoglycan, glypican, in nervous tissue. J. Cell Sci. 107, 3213–3222 (1994).

    CAS  Google Scholar 

  47. Saunders, S., Paine-Saunders, S. & Lander, A. D. Expression of the cell surface proteoglycan glypican-5 is developmentally regulated in kidney, limb, and brain. Dev. Biol. 190, 78–93 (1997).

    Article  CAS  Google Scholar 

  48. Litwack, E. D. et al. Expression of the heparan sulfate proteoglycan glypican-1 in the developing rodent. Dev. Dyn. 211, 72–87 (1998).

    Article  CAS  Google Scholar 

  49. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P. & Ornitz, D. M. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64, 841–848 (1991).

    Article  CAS  Google Scholar 

  50. Rapraeger, A. C., Krufka, A. & Olwin, B. B. Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science 252, 1705–1708 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Thanks to J. Y. Wu and Y. Rao for rat Robo-1 expression plasmid, M. Tessier-Lavigne for Slit2 expression plasmid, V. Lemmon for anti-L1 antibody, V. Lemmon, B. Wible and B. Wang for discussions during this project, D. Kunze and V. Lemmon for reading the manuscript and M. Pendergast for help with the confocal microscope. Supported by the National Institutes of Health grant NS38877.

Author information

Authors and Affiliations

  1. Rammelkamp Center for Education and Research, MetroHealth Medical Center, 2500 MetroHealth Drive, Cleveland, 44109, Ohio, USA

    Huaiyu Hu

Authors
  1. Huaiyu Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Huaiyu Hu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hu, H. Cell-surface heparan sulfate is involved in the repulsive guidance activities of Slit2 protein. Nat Neurosci 4, 695–701 (2001). https://doi.org/10.1038/89482

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/89482

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing