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Functions of FGF signalling from the apical ectodermal ridge in limb development

Nature volume 418, pages 501–508 (2002)Cite this article

Abstract

To determine the role of fibroblast growth factor (FGF) signalling from the apical ectodermal ridge (AER), we inactivated Fgf4 and Fgf8 in AER cells or their precursors at different stages of mouse limb development. We show that FGF4 and FGF8 regulate cell number in the nascent limb bud and are required for survival of cells located far from the AER. On the basis of the skeletal phenotypes observed, we conclude that these functions are essential to ensure that sufficient progenitor cells are available to form the normal complement of skeletal elements, and perhaps other limb tissues. In the complete absence of both FGF4 and FGF8 activities, limb development fails. We present a model to explain how the mutant phenotypes arise from FGF-mediated effects on limb bud size and cell survival.

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Figure 1: Timing of Fgf4 and Fgf8 inactivation in hindlimb and forelimb buds.
Figure 2: Limb skeletal phenotypes.
Figure 3: AER morphology and gene expression in hindlimb buds.
Figure 4: Cell death and distribution of muscle cell precursors.
Figure 5: Cell proliferation and limb bud size.
Figure 6: A model for AER-FGF function in the limb.

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References

  1. Hinchliffe, J. R. & Johnson, D. R. The Development of the Vertebrate Limb: An Approach Through Experiment, Genetics, and Evolution (Clarendon, Oxford, 1980)

    Google Scholar 

  2. Johnson, R. L. & Tabin, C. J. Molecular models for vertebrate limb development. Cell 90, 979–990 (1997)

    Article  CAS  Google Scholar 

  3. Saunders, J. W. Jr The proximo-distal sequence of the origin of the parts of the chick wing and the role of the ectoderm. J. Exp. Zool. 108, 363–403 (1948)

    Article  Google Scholar 

  4. Summerbell, D. A quantitative analysis of the effect of excision of the AER from the chick limb bud. J. Embryol. Exp. Morphol. 32, 651–660 (1974)

    CAS  PubMed  Google Scholar 

  5. Rowe, D. A. & Fallon, J. F. The proximodistal determination of skeletal parts in the developing chick leg. J. Embryol. Exp. Morphol. 68, 1–7 (1982)

    CAS  PubMed  Google Scholar 

  6. Rubin, L. & Saunders, J. W. J. Ectodermal-mesodermal interactions in the growth of limb buds in the chick embryo: constancy and temporal limits of the ectodermal induction. Dev. Biol. 28, 94–112 (1972)

    Article  CAS  Google Scholar 

  7. Niswander, L., Tickle, C., Vogel, A., Booth, I. & Martin, G. R. FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 75, 579–587 (1993)

    Article  CAS  Google Scholar 

  8. Fallon, J. et al. FGF-2: Apical ectodermal ridge growth signal for chick limb development. Science 264, 104–107 (1994)

    Article  ADS  CAS  Google Scholar 

  9. Ornitz, D. M. & Itoh, N. Fibroblast growth factors. Genome Biol. 2, 3005.1–3005.12 (2001)

    Article  Google Scholar 

  10. Martin, G. R. The roles of FGFs in the early development of vertebrate limbs. Genes Dev. 12, 1571–1586 (1998)

    Article  CAS  Google Scholar 

  11. Sun, X. et al. Conditional inactivation of Fgf4 reveals complexity of signalling during limb bud development. Nature Genet. 25, 83–86 (2000)

    Article  CAS  Google Scholar 

  12. Feldman, B., Poueymirou, W., Papaioannou, V. E., DeChiara, T. M. & Goldfarb, M. Requirement of FGF-4 for postimplantation mouse development. Science 267, 246–249 (1995)

    Article  ADS  CAS  Google Scholar 

  13. Sun, X., Meyers, E. N., Lewandoski, M. & Martin, G. R. Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 13, 1834–1846 (1999)

    Article  CAS  Google Scholar 

  14. Colvin, J. S., Green, R. P., Schmahl, J., Capel, B. & Ornitz, D. M. Male-to-female sex reversal in mice lacking fibroblast growth factor 9. Cell 104, 875–889 (2001)

    Article  CAS  Google Scholar 

  15. Xu, J., Liu, Z. & Ornitz, D. M. Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and differentiation of midline cerebellar structures. Development 127, 1833–1843 (2000)

    CAS  PubMed  Google Scholar 

  16. Moon, A. M., Boulet, A. M. & Capecchi, M. R. Normal limb development in conditional mutants of Fgf4. Development 127, 989–996 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Lewandoski, M., Sun, X. & Martin, G. R. Fgf8 signalling from the AER is essential for normal limb development. Nature Genet. 26, 460–463 (2000)

    Article  CAS  Google Scholar 

  18. Moon, A. M. & Capecchi, M. R. Fgf8 is required for outgrowth and patterning of the limbs. Nature Genet. 26, 455–459 (2000)

    Article  CAS  Google Scholar 

  19. Meyers, E. N., Lewandoski, M. & Martin, G. R. An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nature Genet. 18, 136–141 (1998)

    Article  CAS  Google Scholar 

  20. Wright, E. et al. The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nature Genet. 9, 15–20 (1995)

    Article  CAS  Google Scholar 

  21. Min, H. et al. Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless. Genes Dev. 12, 3156–3161 (1998)

    Article  CAS  Google Scholar 

  22. Sekine, K. et al. Fgf10 is essential for limb and lung formation. Nature Genet. 21, 138–141 (1999)

    Article  CAS  Google Scholar 

  23. Revest, J. M. et al. Fibroblast growth factor receptor 2-IIIb acts upstream of Shh and Fgf4 and is required for limb bud maintenance but not for the induction of Fgf8, Fgf10, Msx1, or Bmp4. Dev. Biol. 231, 47–62 (2001)

    Article  CAS  Google Scholar 

  24. Prahlad, K. V., Skala, G., Jones, D. G. & Briles, W. E. Limbless: a new genetic mutant in the chick. J. Exp. Zool. 209, 427–434 (1979)

    Article  CAS  Google Scholar 

  25. Takahashi, M. et al. The role of Alx-4 in the establishment of anteroposterior polarity during vertebrate limb development. Development 125, 4417–4425 (1998)

    CAS  PubMed  Google Scholar 

  26. Rowe, D. A., Cairns, J. M. & Fallon, J. F. Spatial and temporal patterns of cell death in limb bud mesoderm after apical ectodermal ridge removal. Dev. Biol. 93, 83–91 (1982)

    Article  CAS  Google Scholar 

  27. Dudley, A. T., Ros, M. A. & Tabin, C. J. A re-examination of proximodistal patterning during vertebrate limb development. Nature 418, 539–544 (2002)

    Article  ADS  CAS  Google Scholar 

  28. Bober, E., Franz, T., Arnold, H. H., Gruss, P. & Tremblay, P. Pax-3 is required for the development of limb muscles: a possible role for the migration of dermomyotomal muscle progenitor cells. Development 120, 603–612 (1994)

    CAS  PubMed  Google Scholar 

  29. Baldwin, H. S. et al. Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): alternatively spliced, functionally distinct isoforms expressed during mammalian cardiovascular development. Development 120, 2539–2553 (1994)

    CAS  PubMed  Google Scholar 

  30. Gurley, L. R., D'Anna, J. A., Barham, S. S., Deaven, L. L. & Tobey, R. A. Histone phosphorylation and chromatin structure during mitosis in Chinese hamster cells. Eur. J. Biochem. 84, 1–15 (1978)

    Article  CAS  Google Scholar 

  31. Hornbruch, A. & Wolpert, L. Cell division in the early growth and morphogenesis of the chick limb. Nature 226, 764–766 (1970)

    Article  ADS  CAS  Google Scholar 

  32. Chiang, C. et al. Manifestation of the limb prepattern: limb development in the absence of Sonic hedgehog function. Dev. Biol. 236, 421–435 (2001)

    Article  CAS  Google Scholar 

  33. Kraus, P., Fraidenraich, D. & Loomis, C. A. Some distal limb structures develop in mice lacking Sonic hedgehog signalling. Mech. Dev. 100, 45–58 (2001)

    Article  CAS  Google Scholar 

  34. Alberch, P. & Gale, E. A. Size dependence during the development of the amphibian foot. Colchicine-induced digital loss and reduction. J. Embryol. Exp. Morphol. 76, 177–197 (1983)

    CAS  PubMed  Google Scholar 

  35. Wolpert, L., Tickle, C. & Sampford, M. The effect of cell killing by X-irradiation on pattern formation in the chick limb. J. Embryol. Exp. Morphol. 50, 175–193 (1979)

    CAS  PubMed  Google Scholar 

  36. Li, S. & Muneoka, K. Cell migration and chick limb development: chemotactic action of FGF-4 and the AER. Dev. Biol. 211, 335–347 (1999)

    Article  CAS  Google Scholar 

  37. Saxton, T. M. et al. The SH2 tyrosine phosphatase Shp2 is required for mammalian limb development. Nature Genet. 24, 420–423 (2000)

    Article  CAS  Google Scholar 

  38. Stark, R. J. & Searls, R. L. The establishment of the cartilage pattern in the embryonic chick wing, and evidence for a role of the dorsal and ventral ectoderm in normal wing development. Dev. Biol. 38, 51–63 (1974)

    Article  CAS  Google Scholar 

  39. Summerbell, D., Lewis, J. H. & Wolpert, L. Positional information in chick limb morphogenesis. Nature 244, 492–496 (1973)

    Article  ADS  CAS  Google Scholar 

  40. Capdevila, J., Tsukui, T., Rodriquez Esteban, C., Zappavigna, V. & Izpisua Belmonte, J. C. Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin. Mol. Cell 4, 839–849 (1999)

    Article  CAS  Google Scholar 

  41. Mercader, N. et al. Conserved regulation of proximodistal limb axis development by Meis1/Hth. Nature 402, 425–429 (1999)

    Article  ADS  CAS  Google Scholar 

  42. Mercader, N. et al. Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes. Development 127, 3961–3970 (2000)

    CAS  PubMed  Google Scholar 

  43. Maini, P. K. & Solursh, M. Cellular mechanisms of pattern formation in the developing limb. Int. Rev. Cytol. 129, 91–133 (1991)

    Article  CAS  Google Scholar 

  44. Dahn, R. D. & Fallon, J. F. Interdigital regulation of digit identity and homeotic transformation by modulated BMP signalling. Science 289, 438–441 (2000)

    Article  ADS  CAS  Google Scholar 

  45. Kingsley, D. M. Genetic control of bone and joint formation. Novartis Found. Symp. 232, 213–222 (2001)

    CAS  PubMed  Google Scholar 

  46. Muneoka, K., Wanek, N. & Bryant, S. V. Mammalian limb bud development: in situ fate maps of early hindlimb buds. J. Exp. Zool. 249, 50–54 (1989)

    Article  CAS  Google Scholar 

  47. Carter, T. The genetics of luxate mice. IV. Embryology. J. Gent. 52, 1–35 (1954)

    Article  Google Scholar 

  48. Vargesson, N. et al. Cell fate in the chick limb bud and relationship to gene expression. Development 124, 1909–1918 (1997)

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M. Lewandoski, whose studies on the phenotype of Fgf8 single mutant embryos first suggested the model of AER-FGF function described here, for discussion at the outset of this work. We are grateful to J. Fallon, D. Kingsley and C. Tickle for discussion, and C. Tabin and A. Dudley for sharing unpublished information and helpful comments. We also thank D. Duboule, P. Gruss, R. Harland, B. Hogan, N. Itoh, J.-C. Izpisua-Belmonte, V. Lefebvre, A. McMahon, D. Ornitz, P. Sharpe, M. Scott and R. Wisdom for providing plasmids from which probes were prepared; D. Lakeland for help with statistical analysis; C. Larabell for technical advice on confocal microscopy; C. Petersen, Z. Serrano, J. Watanabe and R. Rantala for technical assistance; and H. Ingraham, J. Saunders, G. Schubiger and our colleagues in the Martin laboratory for comments on the manuscript. X.S. was the recipient of a postdoctoral fellowship from the American Cancer Society and was supported by an NIH Training grant. F.M. is the recipient of an individual National Research Service Award from the NIH. This work was supported by an HHMI Research Resources Program grant to the UCSF School of Medicine, and an NIH grant to G.R.M.

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Author notes

  1. Xin Sun

    Present address: Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin, 53706, USA

  2. Gail R. Martin: Correspondence and requests for materials should be addressed to G.R.M.

Authors and Affiliations

  1. Department of Anatomy and Program in Developmental Biology, School of Medicine, University of California at San Francisco, San Francisco, California, 94143-0452, USA

    Gail R. Martin

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Correspondence to Gail R. Martin.

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Sun, X., Mariani, F. & Martin, G. Functions of FGF signalling from the apical ectodermal ridge in limb development. Nature 418, 501–508 (2002). https://doi.org/10.1038/nature00902

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