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An Fgf/Gremlin inhibitory feedback loop triggers termination of limb bud outgrowth

Abstract

During organ formation and regeneration a proper balance between promoting and restricting growth is critical to achieve stereotypical size. Limb bud outgrowth is driven by signals in a positive feedback loop involving fibroblast growth factor (Fgf) genes, sonic hedgehog (Shh) and Gremlin1 (Grem1)1. Precise termination of these signals is essential to restrict limb bud size2,3,4. The current model predicts a sequence of signal termination consistent with that in chick limb buds4. Our finding that the sequence in mouse limb buds is different led us to explore alternative mechanisms. Here we show, by analysing compound mouse mutants defective in genes comprising the positive loop, genetic evidence that FGF signalling can repress Grem1 expression, revealing a novel Fgf/Grem1 inhibitory loop. This repression occurs both in mouse and chick limb buds, and is dependent on high FGF activity. These data support a mechanism where the positive Fgf/Shh loop drives outgrowth and an increase in FGF signalling, which triggers the Fgf/Grem1 inhibitory loop. The inhibitory loop then operates to terminate outgrowth signals in the order observed in either mouse or chick limb buds. Our study unveils the concept of a self-promoting and self-terminating circuit that may be used to attain proper tissue size in a broad spectrum of developmental and regenerative settings.

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Figure 1: Fgf8 repression of Fgf4 expression is dependent on Grem1 but not Shh.
Figure 2: FGF signalling represses Grem1 expression.
Figure 3: AER-FGF repression of Grem1 expression is dose sensitive.
Figure 4: Model describing a self-promoting and self-terminating mechanism to control limb-bud outgrowth signals.

References

  1. Niswander, L. Interplay between the molecular signals that control vertebrate limb development. Int. J. Dev. Biol. 46, 877–881 (2002)

    CAS  PubMed  Google Scholar 

  2. Sanz-Ezquerro, J. J. & Tickle, C. Fgf signaling controls the number of phalanges and tip formation in developing digits. Curr. Biol. 13, 1830–1836 (2003)

    CAS  Article  Google Scholar 

  3. Pizette, S. & Niswander, L. BMPs negatively regulate structure and function of the limb apical ectodermal ridge. Development 126, 883–894 (1999)

    CAS  PubMed  Google Scholar 

  4. 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  Google Scholar 

  5. Aegerter-Wilmsen, T., Aegerter, C. M., Hafen, E. & Basler, K. Model for the regulation of size in the wing imaginal disc of Drosophila . Mech. Dev. 124, 318–326 (2007)

    CAS  Article  Google Scholar 

  6. Garcia-Bellido, A. C. & Garcia-Bellido, A. Cell proliferation in the attainment of constant sizes and shapes: the Entelechia model. Int. J. Dev. Biol. 42, 353–362 (1998)

    CAS  PubMed  Google Scholar 

  7. Hufnagel, L., Teleman, A. A., Rouault, H., Cohen, S. M. & Shraiman, B. I. On the mechanism of wing size determination in fly development. Proc. Natl Acad. Sci. USA 104, 3835–3840 (2007)

    ADS  CAS  Article  Google Scholar 

  8. Day, S. J. & Lawrence, P. A. Measuring dimensions: the regulation of size and shape. Development 127, 2977–2987 (2000)

    CAS  PubMed  Google Scholar 

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

    CAS  Article  Google Scholar 

  10. 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)

    ADS  CAS  Article  Google Scholar 

  11. Panman, L. et al. Differential regulation of gene expression in the digit forming area of the mouse limb bud by SHH and gremlin 1/FGF-mediated epithelial–mesenchymal signalling. Development 133, 3419–3428 (2006)

    CAS  Article  Google Scholar 

  12. Michos, O. et al. Gremlin-mediated BMP antagonism induces the epithelial–mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis. Development 131, 3401–3410 (2004)

    CAS  Article  Google Scholar 

  13. Khokha, M. K., Hsu, D., Brunet, L. J., Dionne, M. S. & Harland, R. M. Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning. Nature Genet. 34, 303–307 (2003)

    CAS  Article  Google Scholar 

  14. 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 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  18. Sun, X., Mariani, F. V. & Martin, G. R. Functions of FGF signalling from the apical ectodermal ridge in limb development. Nature 418, 501–508 (2002)

    ADS  CAS  Article  Google Scholar 

  19. Lu, P., Minowada, G. & Martin, G. R. Increasing Fgf4 expression in the mouse limb bud causes polysyndactyly and rescues the skeletal defects that result from loss of Fgf8 function. Development 133, 33–42 (2006)

    CAS  Article  Google Scholar 

  20. Xu, X., Qiao, W., Li, C. & Deng, C. X. Generation of Fgfr1 conditional knockout mice. Genesis 32, 85–86 (2002)

    Article  Google Scholar 

  21. Harfe, B. D. et al. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118, 517–528 (2004)

    CAS  Article  Google Scholar 

  22. Eswarakumar, V. P. et al. The IIIc alternative of Fgfr2 is a positive regulator of bone formation. Development 129, 3783–3793 (2002)

    CAS  PubMed  Google Scholar 

  23. Merino, R. et al. The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb. Development 126, 5515–5522 (1999)

    CAS  PubMed  Google Scholar 

  24. Nissim, S., Hasso, S. M., Fallon, J. F. & Tabin, C. J. Regulation of Gremlin expression in the posterior limb bud. Dev. Biol. 299, 12–21 (2006)

    CAS  Article  Google Scholar 

  25. 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)

    CAS  Article  Google Scholar 

  26. Selever, J., Liu, W., Lu, M. F., Behringer, R. R. & Martin, J. F. Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development. Dev. Biol. 276, 268–279 (2004)

    CAS  Article  Google Scholar 

  27. Ovchinnikov, D. A. et al. BMP receptor type IA in limb bud mesenchyme regulates distal outgrowth and patterning. Dev. Biol. 295, 103–115 (2006)

    CAS  Article  Google Scholar 

  28. Bandyopadhyay, A. et al. Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet 2, e216 (2006)

    Article  Google Scholar 

  29. Pajni-Underwood, S., Wilson, C. P., Elder, C., Mishina, Y. & Lewandoski, M. BMP signals control limb bud interdigital programmed cell death by regulating FGF signaling. Development 134, 2359–2368 (2007)

    CAS  Article  Google Scholar 

  30. Mishina, Y., Hanks, M. C., Miura, S., Tallquist, M. D. & Behringer, R. R. Generation of Bmpr/Alk3 conditional knockout mice. Genesis 32, 69–72 (2002)

    CAS  Article  Google Scholar 

  31. Verheyden, J. M., Lewandoski, M., Deng, C., Harfe, B. D. & Sun, X. Conditional inactivation of Fgfr1 in mouse defines its role in limb bud establishment, outgrowth and digit patterning. Development 132, 4235–4245 (2005)

    CAS  Article  Google Scholar 

  32. Neubuser, A., Peters, H., Balling, R. & Martin, G. R. Antagonistic interactions between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell 90, 247–255 (1997)

    CAS  Article  Google Scholar 

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Acknowledgements

We are grateful to J. Fallon, G. Martin, R. Bacon, G. Boekhoff-Falk, B. Harfe, M. Lewandoski, D. Wellik and members of the Sun laboratory, in particular L. Abler, for discussions and reading the manuscript. We thank R. Behringer, C. Chiang, C. Deng, B. Harfe, R. Harland, P. Lonai, Y. Mishina and C. Tabin for mouse strains. We are grateful to A. Lashua, M. Zhao and J. Heinritz for technical assistance. J.M.V. was supported by the predoctoral training program in genetics (5T32GM07133) funded by the National Institutes of Health. This work was supported by a March of Dimes Basil O’Connor award 5-FY03-13 (to X.S.) and a National Institutes of Health grant RO1 HD045522 (to X.S.).

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Verheyden, J., Sun, X. An Fgf/Gremlin inhibitory feedback loop triggers termination of limb bud outgrowth. Nature 454, 638–641 (2008). https://doi.org/10.1038/nature07085

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