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Conditional inactivation of Fgf4 reveals complexity of signalling during limb bud development

Nature Genetics volume 25, pages 83–86 (2000)Cite this article

Abstract

Development of the vertebrate limb bud depends on reciprocal interactions between the zone of polarizing activity (ZPA) and the apical ectodermal ridge1 (AER). Sonic hedgehog (SHH) and fibroblast growth factors (FGFs) are key signalling molecules produced in the ZPA and AER, respectively1,2. Experiments in chicks suggested that SHH expression in the ZPA is maintained by FGF4 expression in the AER, and vice versa3,4, providing a molecular mechanism for coordinating the activities of these two signalling centres. This SHH/FGF4 feedback loop model is supported by genetic evidence showing that Fgf4 expression is not maintained in Shh−/− mouse limbs5. We report here that Shh expression is maintained and limb formation is normal when Fgf4 is inactivated in mouse limbs, thus contradicting the model. We also found that maintenance of Fgf9 and Fgf17 expression is dependent on Shh, whereas Fgf8 expression is not. We discuss a model in which no individual Fgf expressed in the AER (AER–Fgf) is solely necessary to maintain Shh expression, but, instead, the combined activities of two or more AER–Fgfs function in a positive feedback loop with Shh to control limb development.

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Figure 1: Strategy for production of Fgf4-mutant alleles and characterization of Cre activity in Msx2-Cre mice.
Figure 2: Analysis of Fgf4 expression in Msx2-Cre;Fgf4 mice.
Figure 3: Gene expression in Msx2-Cre;Fgf4 limb buds.
Figure 4: Fgf gene expression in Shh−/− limb buds.
Figure 5: A model for regulatory interactions between Shh and Fgf signalling in the mouse limb bud.

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References

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Niswander, L., Jeffrey, S., Martin, G.R. & Tickle, C. A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 371, 609–612 (1994).

    Article  CAS  PubMed  Google Scholar 

  4. Laufer, E., Nelson, C.E., Johnson, R.I., 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 

  5. Zuniga, A., Haramis, A.P., McMahon, A.P. & Zeller, R. Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds. Nature 401, 598–602 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. 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  CAS  PubMed  Google Scholar 

  7. Sauer, B. Inducible gene targeting in mice using the Cre/loxP system. Methods 14, 381–392 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. 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  PubMed  Google Scholar 

  9. Lewandoski, M., Meyers, E.N. & Martin, G.R. Analysis of Fgf8 gene function in vertebrate development. Cold Spring Harb. Symp. Quant. Biol. 62, 159–168 (1997).

    Article  CAS  PubMed  Google Scholar 

  10. Liu, Y.H. et al. Regulation of the Msx2 homeobox gene during mouse embryogenesis: a transgene with 439 bp of 5′ flanking sequence is expressed exclusively in the apical ectodermal ridge of the developing limb. Mech. Dev. 48, 187–197 (1994).

    Article  CAS  PubMed  Google Scholar 

  11. Lobe, C.G. et al. Z/AP, a double reporter for Cre-mediated recombination. Dev. Biol. 208, 281–292 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. Colvin, J.S., Feldman, B., Nadeau, J.H., Goldfarb, M. & Ornitz, D.M. Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene. Dev. Dyn. 216, 72–88 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Maruoka, Y. et al. Comparison of the expression of three highly related genes, Fgf8, Fgf17 and Fgf18, in the mouse embryo. Mech. Dev. 74, 175–177 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. Niswander, L. & Martin, G.R. Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114, 755–768 (1992).

    CAS  PubMed  Google Scholar 

  15. Ohuchi, H. et al. The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF8, an apical ectodermal factor. Development 124, 2235–2244 (1997).

    CAS  PubMed  Google Scholar 

  16. Minowada, G. et al. Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed. Development 126, 4465–4475 (1999).

    CAS  PubMed  Google Scholar 

  17. Lyons, K.M., Pelton, R.W. & Hogan, B.L. Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). Development 109, 833–844 (1990).

    CAS  PubMed  Google Scholar 

  18. Jones, C.M., Lyons, K.M. & Hogan, B.L. Involvement of bone morphogenetic protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development 111, 531–542 (1991).

    CAS  PubMed  Google Scholar 

  19. 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  PubMed  Google Scholar 

  20. Niswander, L. & Martin, G.R. FGF-4 and BMP-2 have opposite effects on limb growth. Nature 361, 68–71 (1993).

    Article  CAS  PubMed  Google Scholar 

  21. Chiang, C. et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407–413 (1996).

    Article  CAS  PubMed  Google Scholar 

  22. Xu, J., Liu, Z. & Ornitz, D.M. Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and differentiation of midline cerebellar structures. Development (in press).

  23. Mountford, P. et al. Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc. Natl Acad. Sci. USA 91, 4303–4307 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Dymecki, S.M. Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice. Proc. Natl Acad. Sci. USA 93, 6191–6196 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lewandoski, M., Wassarman, K.M. & Martin, G.R. Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line. Curr. Biol. 7, 148–151 (1997).

    Article  CAS  PubMed  Google Scholar 

  26. Bellusci, S., Grindley, J., Emoto, H., Itoh, N. & Hogan, B.L. Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung. Development 124, 4867–4878 (1997).

    CAS  PubMed  Google Scholar 

  27. Crossley, P.H. & Martin, G.R. The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121, 439–451 (1995).

    CAS  PubMed  Google Scholar 

  28. Echelard, Y. et al. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75, 1417–1430 (1993).

    Article  CAS  PubMed  Google Scholar 

  29. Hsu, D.R., Economides, A.N., Wang, X., Eimon, P.M. & Harland, R.M. The Xenopus dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities. Mol. Cell 1, 673–683 (1998).

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank W. Skarnes for providing CGR8 ES cells; A. Nagy for providing the Z/AP reporter line; C. Chiang for providing Shh+/− mice; R. Harland (gremlin), Genetics Institute (Bmp2, Bmp4), N. Itoh (Fgf10, Fgf17), A. McMahon (Shh) and D. Ornitz (Fgf9) for providing plasmids; M. Embry and C. Petersen for technical assistance; and our colleagues in the Martin laboratory for helpful discussion and critical readings of the manuscript. X.S. is the recipient of a postdoctoral fellowship from the American Cancer Society. E.N.M. is the recipient of Mentored Clinical Scientist Development Award HD01216 from the NIH. This work was supported by an HHMI Research Resources Program grant (76296-549901) to the UCSF School of Medicine, and NIH grants RO1 DE12450 (to R.E.M.) and RO1 HD34380 (to G.R.M.).

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Authors and Affiliations

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

    Xin Sun, Mark Lewandoski, Erik N. Meyers & Gail R. Martin

  2. Department of Biochemistry and Molecular Biology, USC/Norris Cancer Center, Los Angeles, California, USA

    Yi-Hsin Liu & Robert E. Maxson Jr

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  2. Mark Lewandoski
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Correspondence to Gail R. Martin.

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Sun, X., Lewandoski, M., Meyers, E. et al. Conditional inactivation of Fgf4 reveals complexity of signalling during limb bud development. Nat Genet 25, 83–86 (2000). https://doi.org/10.1038/75644

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