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Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds


Outgrowth and patterning of the vertebrate limb are controlled by reciprocal interactions between the posterior mesenchyme (polarizing region) and a specialized ectodermal structure, the apical ectodermal ridge (AER)1. Sonic hedgehog (SHH) signalling by the polarizing region modulates fibroblast growth factor (FGF)4 signalling by the posterior AER, which in turn maintains the polarizing region (SHH/FGF4 feedback loop)2,3. Here we report that the secreted bone-morphogenetic-protein (BMP) antagonist Gremlin4 relays the SHH signal from the polarizing region to the AER. Mesenchymal Gremlin expression is lost in limb buds of mouse embryos homozygous for the limb deformity (ld) mutation, which disrupts establishment of the SHH/FGF4 feedback loop5,6,7. Grafting Gremlin-expressing cells into ld mutant limb buds rescues Fgf4 expression and restores the SHH/FGF4 feedback loop. Analysis of Shh-null mutant embryos8,9 reveals that SHH signalling is required for maintenance of Gremlin and Formin (the gene disrupted by the ld mutations)10,11. In contrast, Formin, Gremlin and Fgf4 activation are independent of SHH signalling. This study uncovers the cascade by which the SHH signal is relayed from the posterior mesenchyme to the AER and establishes that Formin-dependent activation of the BMP antagonist Gremlin is sufficient to induce Fgf4 and establish the SHH/FGF4 feedback loop.

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Figure 1: Formin (Fmn) is a target of SHH signalling in the limb-bud mesenchyme and is essential to relay the SHH signal from the mesenchyme to the AER.
Figure 2: The BMP antagonist Gremlin (Gre) is a Fmn-dependent target of SHH signalling and is expressed by posterior-distal limb-bud mesenchymal cells.
Figure 3: Inhibition of BMP activity by Gremlin restores the SHH/FGF-4 feedback loop in ld mutant limb buds.
Figure 4: Analysis of Shh-deficient mouse limb buds9 shows that induction but not maintenance of Formin, Gremlin and Fgf4 are SHH independent.
Figure 5: Limb-bud mesenchyme to AER signalling and establishment of the SHH/FGF4 feedback loop through FMN- and GRE-mediated BMP antagonism.


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We thank G. Drossopoulu, M. Herlevsen and C. Tickle for advice in establishing the in vitro culturing system; T. Bouwmeester and S. Cohen for many stimulating discussions and support; C. McGuigan, B. Wagemakers and H. Goedemans for technical assistance; J. McMahon for breeding Shh-deficient mice; J. Jackson for mouse husbandry; D. Duprez, P. Brickell, H. Rohrer, R. Harland, T. Bouwmeester, S.-L. Ang and C. Niehrs for reagents; and G. Davidson, R. Dono, A. van Loon, P. Rørth and L. Panman for comments on the manuscript. This study was supported by EMBL and Utrecht University, grants from the European Community (to R.Z.), the NIH (to A.P.M.) and the Graduiertenkolleg “Experimentelle Nieren- und Kreislaufforschung” (to A.Z.).

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Correspondence to Rolf Zeller.

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Zúñiga, A., Haramis, AP., McMahon, A. et al. Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds. Nature 401, 598–602 (1999).

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