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Notch activity acts as a sensor for extracellular calcium during vertebrate left–right determination

Nature volume 427, pages 121–128 (2004)Cite this article

Abstract

During vertebrate embryo development, the breaking of the initial bilateral symmetry is translated into asymmetric gene expression around the node and/or in the lateral plate mesoderm. The earliest conserved feature of this asymmetric gene expression cascade is the left-sided expression of Nodal, which depends on the activity of the Notch signalling pathway. Here we present a mathematical model describing the dynamics of the Notch signalling pathway during chick embryo gastrulation, which reveals a complex and highly robust genetic network that locally activates Notch on the left side of Hensen's node. We identify the source of the asymmetric activation of Notch as a transient accumulation of extracellular calcium, which in turn depends on left–right differences in H+/K+-ATPase activity. Our results uncover a mechanism by which the Notch signalling pathway translates asymmetry in epigenetic factors into asymmetric gene expression around the node.

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Figure 1: Asymmetries in the expression of Notch pathway components during chick gastrulation.
Figure 2: Mathematical model characterizing Notch activation dynamics.
Figure 3: Experimental test of the theoretical assumptions and predictions.
Figure 4: Differential H+/K+-ATPase activity regulates Notch signalling during LR determination.
Figure 5: Asymmetric domains of increased Ca2+ during LR asymmetry determination.
Figure 6: Extracellular Ca2+ concentrations directly regulate Notch activity and determine LR asymmetry.

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References

  1. Burdine, R. D. & Schier, A. F. Conserved and divergent mechanisms in left–right axis formation. Genes Dev. 14, 763–776 (2000)

    CAS  PubMed  Google Scholar 

  2. Capdevila, J., Vogan, K. J., Tabin, C. J. & Izpisúa Belmonte, J. C. Mechanisms of left–right determination in vertebrates. Cell 101, 9–21 (2000)

    Article  CAS  PubMed  Google Scholar 

  3. Mercola, M. & Levin, M. Left–right asymmetry determination in vertebrates. Annu. Rev. Cell Dev. Biol. 17, 779–805 (2001)

    Article  CAS  PubMed  Google Scholar 

  4. Hamada, H., Meno, C., Watanabe, D. & Saijoh, Y. Establishment of vertebrate left–right asymmetry. Nature Rev. Genet. 3, 103–113 (2002)

    Article  CAS  PubMed  Google Scholar 

  5. Bisgrove, B. W., Morelli, S. H. & Yost, H. J. Genetics of human laterality disorders: insights from vertebrate model systems. Annu. Rev. Genomics Hum. Genet. 4, 1–32 (2003)

    Article  CAS  PubMed  Google Scholar 

  6. Brennan, J., Norris, D. P. & Robertson, E. J. Nodal activity in the node governs left–right asymmetry. Genes Dev. 16, 2339–2344 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Saijoh, Y., Oki, S., Ohishi, S. & Hamada, H. Left–right patterning of the mouse lateral plate requires nodal produced in the node. Dev. Biol. 256, 160–172 (2003)

    Article  CAS  PubMed  Google Scholar 

  8. Krebs, L. T. et al. Notch signaling regulates left–right asymmetry determination by inducing Nodal expression. Genes Dev. 17, 1207–1212 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Raya, A. et al. Notch activity induces Nodal expression and mediates the establishment of left–right asymmetry in vertebrate embryos. Genes Dev. 17, 1213–1218 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Artavanis-Tsakonas, S., Rand, M. D. & Lake, R. J. Notch signaling: cell fate control and signal integration in development. Science 284, 770–776 (1999)

    Article  ADS  CAS  PubMed  Google Scholar 

  11. Hamburger, V. & Hamilton, H. L. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49–92 (1951)

    Article  CAS  PubMed  Google Scholar 

  12. Jouve, C., Iimura, T. & Pourquie, O. Onset of the segmentation clock in the chick embryo: evidence for oscillations in the somite precursors in the primitive streak. Development 129, 1107–1117 (2002)

    CAS  PubMed  Google Scholar 

  13. Irvine, K. D. Fringe, Notch, and making developmental boundaries. Curr. Opin. Genet. Dev. 9, 434–441 (1999)

    Article  CAS  PubMed  Google Scholar 

  14. Evrard, Y. A., Lun, Y., Aulehla, A., Gan, L. & Johnson, R. L. lunatic fringe is an essential mediator of somite segmentation and patterning. Nature 394, 377–381 (1998)

    Article  ADS  CAS  PubMed  Google Scholar 

  15. Zhang, N. & Gridley, T. Defects in somite formation in lunatic fringe-deficient mice. Nature 394, 374–377 (1998)

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Dale, J. K. et al. Periodic notch inhibition by lunatic fringe underlies the chick segmentation clock. Nature 421, 275–278 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  17. Geling, A., Steiner, H., Willem, M., Bally-Cuif, L. & Haass, C. A γ-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep. 3, 688–694 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Gibson, G. Developmental evolution: getting robust about robustness. Curr. Biol. 12, R347–R349 (2002)

    Article  CAS  PubMed  Google Scholar 

  19. Stern, C. D. et al. Activin and its receptors during gastrulation and the later phases of mesoderm development in the chick embryo. Dev. Biol. 172, 192–205 (1995)

    Article  CAS  PubMed  Google Scholar 

  20. Levin, M., Johnson, R. L., Stern, C. D., Kuehn, M. & Tabin, C. A molecular pathway determining left–right asymmetry in chick embryogenesis. Cell 82, 803–814 (1995)

    Article  CAS  PubMed  Google Scholar 

  21. Boettger, T., Wittler, L. & Kessel, M. FGF8 functions in the specification of the right body side of the chick. Curr. Biol. 9, 277–280 (1999)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  23. Wagner, M. K. & Yost, H. J. Left–right development: the roles of nodal cilia. Curr. Biol. 10, R149–R151 (2000)

    Article  CAS  PubMed  Google Scholar 

  24. Levin, M., Thorlin, T., Robinson, K., Nogi, T. & Mercola, M. Asymmetries in H+/K+-ATPase and cell membrane potentials comprise a very early step in left–right patterning. Cell 111, 77–89 (2002)

    Article  CAS  PubMed  Google Scholar 

  25. Stern, C. D. & Wolpert, L. Left–right asymmetry: all hands to the pump. Curr. Biol. 12, R802–R803 (2002)

    Article  CAS  PubMed  Google Scholar 

  26. Mercola, M. Left–right asymmetry: nodal points. J. Cell Sci. 116, 3251–3257 (2003)

    Article  CAS  PubMed  Google Scholar 

  27. Robinson, K. R. & Messerli, M. A. Left/right, up/down: The role of endogenous electrical fields as directional signals in development, repair and invasion. Bioessays 25, 759–766 (2003)

    Article  PubMed  Google Scholar 

  28. Rao, Z. et al. The structure of a Ca2+-binding epidermal growth factor-like domain: its role in protein-protein interactions. Cell 82, 131–141 (1995)

    Article  CAS  PubMed  Google Scholar 

  29. Rand, M. D., Lindblom, A., Carlson, J., Villoutreix, B. O. & Stenflo, J. Calcium binding to tandem repeats of EGF-like modules. Expression and characterization of the EGF-like modules of human Notch-1 implicated in receptor–ligand interactions. Protein Sci. 6, 2059–2071 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 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)

    Article  CAS  PubMed  Google Scholar 

  31. Hicks, C. et al. Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2. Nature Cell Biol. 2, 515–520 (2000)

    Article  CAS  PubMed  Google Scholar 

  32. Jarriault, S. et al. Signalling downstream of activated mammalian Notch. Nature 377, 355–358 (1995)

    Article  ADS  CAS  PubMed  Google Scholar 

  33. Rand, M. D. et al. Calcium depletion dissociates and activates heterodimeric notch receptors. Mol. Cell. Biol. 20, 1825–1835 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Jaffe, L. F. Organization of early development by calcium patterns. Bioessays 21, 657–667 (1999)

    Article  CAS  PubMed  Google Scholar 

  35. New, D. A. T. A new technique for the cultivation of the chick embryo in vitro. J. Embryol. Exp. Morphol. 3, 326–331 (1955)

    Google Scholar 

  36. Ryan, A. K. et al. Pitx2 determines left–right asymmetry of internal organs in vertebrates. Nature 394, 545–551 (1998)

    Article  ADS  CAS  PubMed  Google Scholar 

  37. Itasaki, N., Bel-Vialar, S. & Krumlauf, R. ‘Shocking’ developments in chick embryology: electroporation and in ovo gene expression. Nature Cell Biol. 1, E203–E207 (1999)

    Article  CAS  PubMed  Google Scholar 

  38. Izpisúa Belmonte, J. C., De Robertis, E. M., Storey, K. G. & Stern, C. D. The homeobox gene goosecoid and the origin of organizer cells in the early chick blastoderm. Cell 74, 645–659 (1993)

    Article  PubMed  Google Scholar 

  39. Rodríguez-Esteban, C. et al. The novel Cer-like protein Caronte mediates the establishment of embryonic left–right asymmetry. Nature 401, 243–251 (1999)

    Article  ADS  PubMed  Google Scholar 

  40. Laufer, E., Nelson, C. E., Johnson, R. L., Morgan, B. A. & Tabin, C. Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud. Cell 79, 993–1003 (1994)

    Article  CAS  PubMed  Google Scholar 

  41. Talora, C., Sgroi, D. C., Crum, C. P. & Dotto, G. P. Specific down-modulation of Notch1 signaling in cervical cancer cells is required for sustained HPV-E6/E7 expression and late steps of malignant transformation. Genes Dev. 16, 2252–2263 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lindsell, C. E., Shawber, C. J., Boulter, J. & Weinmaster, G. Jagged: a mammalian ligand that activates Notch1. Cell 80, 909–917 (1995)

    Article  CAS  PubMed  Google Scholar 

  43. Shi, S. & Stanley, P. Protein O-fucosyltransferase 1 is an essential component of Notch signaling pathways. Proc. Natl Acad. Sci. USA 100, 5234–5239 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  44. Panin, V. M., Papayannopoulos, V., Wilson, R. & Irvine, K. D. Fringe modulates Notch–ligand interactions. Nature 387, 908–912 (1997)

    Article  ADS  CAS  PubMed  Google Scholar 

  45. Bruckner, K., Perez, L., Clausen, H. & Cohen, S. Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406, 411–415 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  46. Moloney, D. J. et al. Fringe is a glycosyltransferase that modifies Notch. Nature 406, 369–375 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  47. Murray, J. D. Mathematical Biology (Springer, Berlin, 1993)

    MATH  Google Scholar 

  48. Bosenberg, M. W. & Massague, J. Juxtacrine cell signaling molecules. Curr. Opin. Cell Biol. 5, 832–838 (1993)

    Article  CAS  PubMed  Google Scholar 

  49. Collier, J. R., Monk, N. A., Maini, P. K. & Lewis, J. H. Pattern formation by lateral inhibition with feedback: a mathematical model of delta-notch intercellular signalling. J. Theor. Biol. 183, 429–446 (1996)

    Article  CAS  PubMed  Google Scholar 

  50. Owen, M. R., Sherratt, J. A. & Wearing, H. J. Lateral induction by juxtacrine signaling is a new mechanism for pattern formation. Dev. Biol. 217, 54–61 (2000)

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M-F. Schwarz and H. Pineda for their technical skills and dedication; A. Lehrman and H. Juguilon for help with the luciferase assays; G. Sternik for assistance in two-photon excitation microscopy analysis; all laboratory members for discussions; L. Hooks for help in preparing the manuscript; C. Fryers, K. Jones and C. Kintner for reagents and discussions; and T. Gridley and R. Johnson for sharing unpublished results. A.R. is partially supported by a postdoctoral fellowship from the Ministerio de Educación, Cultura y Deporte, Spain; J.R.L is supported by a fellowship from Fundação para a Ciencia e a Tecnologia, Portugal, and M.I. is partially supported by the Fulbright Program and Generalitat of Catalunya. This work was supported by grants from the Fundação Calouste Gulbenkian e Fundação para a Ciencia e a Tecnologia, the American Heart Association, the Human Frontier Science Program, the NIH and the G. Harold and Leila Y. Mathers Charitable Foundation.

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Author notes

  1. Ángel Raya, Yasuhiko Kawakami and Concepción Rodríguez-Esteban: These authors contributed equally to this work

Authors and Affiliations

  1. Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California, 92037, USA

    Ángel Raya, Yasuhiko Kawakami, Concepción Rodríguez-Esteban, Marta Ibañes, Diego Rasskin-Gutman, Dirk Büscher & Juan Carlos Izpisúa Belmonte

  2. Instituto Gulbenkian de Ciencia, Rua Da Quinta Grande n6, 2780-901, Oeiras, Portugal

    Joaquín Rodríguez-León & José A. Feijó

  3. Centro de Biotecnologia Vegetal, Faculdade de Ciencias, Universidade Lisboa, 1749-016, Lisboa, Portugal

    José A. Feijó

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Correspondence to Juan Carlos Izpisúa Belmonte.

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Raya, Á., Kawakami, Y., Rodríguez-Esteban, C. et al. Notch activity acts as a sensor for extracellular calcium during vertebrate left–right determination. Nature 427, 121–128 (2004). https://doi.org/10.1038/nature02190

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