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.

FGF-induced vesicular release of Sonic hedgehog and retinoic acid in leftward nodal flow is critical for left–right determination

Abstract

The precise specification of left–right asymmetry is an essential process for patterning internal organs in vertebrates. In mouse embryonic development, the symmetry-breaking process in left–right determination is initiated by a leftward extraembryonic fluid flow on the surface of the ventral node. However, it is not known whether the signal transduction mechanism of this flow is chemical or mechanical. Here we show that fibroblast growth factor (FGF) signalling triggers secretion of membrane-sheathed objects 0.3–5 µm in diameter termed ‘nodal vesicular parcels’ (NVPs) that carry Sonic hedgehog and retinoic acid. These NVPs are transported leftward by the fluid flow and eventually fragment close to the left wall of the ventral node. The silencing effects of the FGF-receptor inhibitor SU5402 on NVP secretion and on a downstream rise in Ca2+ were sufficiently reversed by exogenous Sonic hedgehog peptide or retinoic acid, suggesting that FGF-triggered surface accumulation of cargo morphogens may be essential for launching NVPs. Thus, we propose that NVP flow is a new mode of extracellular transport that forms a left–right gradient of morphogens.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Expression of FGF receptors on nodal cilia.
Figure 2: FGF regulates left-specific Ca2+ elevation.
Figure 3: Leftward fluid flow is not affected by SU5402 treatment.
Figure 4: Nodal flow of NVPs is dependent on FGF.
Figure 5: SHH and RA are associated with NVPs.

References

  1. Hirokawa, N. Stirring up development with the heterotrimeric kinesin KIF3. Traffic 1, 29–34 (2000)

    CAS  Article  PubMed  Google Scholar 

  2. Nonaka, S. et al. Randomization of left–right asymmetry due to loss of nodal cilia generating leftward flow of extra embryonic fluid in mice lacking KIF3B motor protein. Cell 95, 829–837 (1998)

    CAS  Article  PubMed  Google Scholar 

  3. Takeda, S. et al. Left-right asymmetry and kinesin superfamily protein KIF3A: new insights in determination of laterality and mesoderm induction by kif3A-/- mice analysis. J. Cell Biol. 145, 825–836 (1999)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Okada, Y. et al. Abnormal nodal flow precedes situs inversus in iv and inv mice. Mol. Cell 4, 459–468 (1999)

    CAS  Article  PubMed  Google Scholar 

  5. Nonaka, S., Shiratori, H., Saijoh, Y. & Hamada, H. Determination of left-right patterning of the mouse embryo by artificial nodal flow. Nature 418, 96–99 (2002)

    ADS  CAS  Article  PubMed  Google Scholar 

  6. Afzelius, B. A. Cilia-related diseases. J. Pathol. 204, 470–477 (2004)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Tabin, C. J. & Vogan, K. J. A two-cilia model for vertebrate left-right axis specification. Genes Dev. 17, 1–6 (2003)

    CAS  Article  PubMed  Google Scholar 

  8. Yokoyama, T. Motor or sensor: A new aspect of primary cilia function. Anat. Sci. Int. 79, 47–54 (2004)

    Article  PubMed  Google Scholar 

  9. McGrath, J., Somlo, S., Makova, S., Tian, X. & Brueckner, M. Two populations of node monocilia initiate left-right asymmetry in the mouse. Cell 114, 61–73 (2003)

    CAS  Article  PubMed  Google Scholar 

  10. Dubrulle, J., McGrew, M. J. & Pourquie, O. FGF signalling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106, 219–232 (2001)

    CAS  Article  PubMed  Google Scholar 

  11. Dubrulle, J. & Pourquie, O. fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo. Nature 427, 419–422 (2004)

    ADS  CAS  Article  PubMed  Google Scholar 

  12. Meyers, E. N. & Martin, G. R. Differences in left-right axis pathways in mouse and chick: functions of FGF8 and SHH. Science 285, 403–406 (1999)

    CAS  Article  PubMed  Google Scholar 

  13. Kondo, S. et al. KIF3A is a new microtubule-based anterograde motor in the nerve axon. J. Cell Biol. 125, 1095–1107 (1994)

    CAS  Article  PubMed  Google Scholar 

  14. Rosenbaum, J. L. & Witman, G. B. Intraflagellar transport. Nature Rev. Mol. Cell Biol. 3, 813–825 (2002)

    CAS  Article  Google Scholar 

  15. Scholey, J. M. Intraflagellar transport. Annu. Rev. Cell Dev. Biol. 19, 423–443 (2003)

    CAS  Article  PubMed  Google Scholar 

  16. Mohammadi, M. et al. Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 276, 955–960 (1997)

    CAS  Article  PubMed  Google Scholar 

  17. Kos, F. J. & Chin, C. S. Costimulation of T cell receptor-triggered IL-2 production by Jurkat T cells via fibroblast growth factor receptor 1 upon its engagement by CD56. Immunol. Cell Biol. 80, 364–369 (2002)

    CAS  Article  PubMed  Google Scholar 

  18. Tsukui, T. et al. Multiple left-right asymmetry defects in Shh-/- mutant mice unveil a convergence of the Shh and retinoic acid pathways in the control of Lefty-1. Proc. Natl Acad. Sci. USA 96, 11376–11381 (1999)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Zhang, X. M., Ramelho-Santos, M. & McMahon, A. P. Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R asymmetry by the mouse node. Cell 105, 781–792 (2001)

    CAS  Article  PubMed  Google Scholar 

  20. Raya, Á. et al. Notch activity acts as a sensor for extracellular calcium during vertebrate left-right determination. Nature 427, 121–128 (2004)

    ADS  CAS  Article  PubMed  Google Scholar 

  21. Mathieu, J. et al. Nodal and Fgf pathways interact through a positive regulatory loop and synergize to maintain mesodermal cell populations. Development 131, 629–641 (2004)

    CAS  Article  PubMed  Google Scholar 

  22. Kawakami, T. et al. Mouse dispatched mutants fail to distribute hedgehog proteins and are defective in hedgehog signaling. Development 129, 5753–5765 (2002)

    CAS  Article  PubMed  Google Scholar 

  23. Ye, W., Shimamura, K., Rubenstein, J. L. R., Hynes, M. A. & Rosenthal, A. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 93, 755–766 (1998)

    CAS  Article  PubMed  Google Scholar 

  24. Kessaris, N., Jamen, F., Rubin, L. L. & Richardson, W. D. Cooperation between sonic hedgehog and fibroblast growth factor/MAPK signalling pathways in neocortical precursors. Development 131, 1289–1298 (2004)

    CAS  Article  PubMed  Google Scholar 

  25. Scherz, P. J., Harfe, B. D., McMahon, A. P. & Tabin, C. J. The limb bud Shh-Fgf feedback loop is terminated by expansion of former ZPA cells. Science 305, 396–399 (2004)

    ADS  CAS  Article  PubMed  Google Scholar 

  26. Rice, R. et al. Disruption of Fgf10/Fgfr2b-coordinated epithelial-mesenchymal interactions causes cleft palate. J. Clin. Invest. 113, 1692–1700 (2004)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Zeng, X. et al. A freely diffusible form of Sonic hedgehog mediates long-range signalling. Nature 411, 716–720 (2001)

    ADS  CAS  Article  PubMed  Google Scholar 

  28. Huangfu, D. et al. Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426, 83–87 (2003)

    ADS  CAS  Article  PubMed  Google Scholar 

  29. Ramírez-Weber, F.-A. & Kornberg, T. B. Cytonemes: cellular processes that project to the principal signalling center in Drosophila imaginal discs. Cell 97, 599–607 (1999)

    Article  PubMed  Google Scholar 

  30. Sato, M. & Kornberg, T. B. FGF is an essential mitogen and chemoattractant for the air sacs of the Drosophila tracheal system. Dev. Cell 3, 195–207 (2002)

    CAS  Article  PubMed  Google Scholar 

  31. Sturm, K. & Tam, P. P. L. Isolation and culture of whole postimplantation embryos and germ layer derivatives. Methods Enzymol. 225, 164–190 (1993)

    CAS  Article  PubMed  Google Scholar 

  32. Tanaka, Y., Kawahata, K., Nakata, T. & Hirokawa, N. Chronological expression of microtubule-associated proteins (MAPs) in EC cell P19 after neuronal induction by retinoic acid. Brain Res. 596, 269–278 (1992)

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank P. Tam and colleagues at CMRI for advice on performing embryo dissection and whole-embryo culture. We also thank S. Takeda, R. Takemura, J. Teng, Y. Noda, H. Sato, N. Onouchi, H. Fukuda, M. Sugaya, T. Akamatsu, T. Aizawa and others from the Hirokawa laboratory for providing materials, discussions and technical assistance.This study has been supported by a Center of Excellence Grant-in-Aid (to N.H.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.Author contributions Y.O. helped to produce Fig. 3.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Nobutaka Hirokawa.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Movie S1

Leftward nodal flow of fluorescent beads irrespective of FGF signalling. (MOV 1285 kb)

Supplementary Movie S2

Fluorescent images of mouse nodes in ventral views with unidirectional flow of NVPs towards the left. (MOV 822 kb)

Supplementary Movie S3

Ventral view of a mouse node, whose NVP flow is suppressed by 20 µM SU5402. (MOV 940 kb)

Supplementary Movie S4

Ball-throwing release of an NVP from the tip of a dynamically protruding microvillum of the right wall of a node. (MOV 641 kb)

Supplementary Movie S5

Crawling and smashed appearance of NVPs. (MOV 481 kb)

Supplementary Movie S6

Fragmentation of an NVP in the proximity of the left wall. (MOV 111 kb)

Supplementary Movie S7

Restored NVP flow by the addition of SHH-N peptide in the presence of SU5402. (MOV 2932 kb)

Supplementary Movie S8

NVP flow is restored by the addition of 10-7M RA in the presence of SU5402. (MOV 1643 kb)

Supplementary Movie S9

Ventral view of a node treated with SU5402 and IHH-N peptide. (MOV 1057 kb)

Supplementary Movie S10

Ventral view of an iv/iv mutant node. (MOV 2700 kb)

Supplementary Movie S11

Ventral view of a kif3a-/- mutant node lacking the nodal cilia. (MOV 2932 kb)

Supplementary Movie 12

Rotating view of a mouse ventral node fluorescently labelled by 5E1 antibody against SHH-N. (MOV 551 kb)

Supplementary Movie Legends (DOC 27 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tanaka, Y., Okada, Y. & Hirokawa, N. FGF-induced vesicular release of Sonic hedgehog and retinoic acid in leftward nodal flow is critical for left–right determination. Nature 435, 172–177 (2005). https://doi.org/10.1038/nature03494

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature03494

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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