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

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Abstract
The question of how a tissue grows to a certain size and knows when to stop remains a major mystery of biology. It is a phenomenon seen not only in body formation, but also in organs such as the liver when regeneration is allowed to occur. Many ideas have been proposed to explain how a tissue senses the limit of size, and few have been supported by experimental evidence. Jamie Verheyden and Xin Sun have discovered a new principle for control of organ size. In a study of the termination of signalling factors controlling growth and expansion in the mouse limb bud, a combination of mutations revealed an inhibitory loop whereby fibroblast growth factor (Fgf) signalling represses Gremlin1 expression, these two factors being key positive signals in limb bud outgrowth. This inhibitory loop is triggered by a positive Fgf/Shh loop. This model differs from earlier published models, and may have implications far beyond limb development, for size control in other organs, in evolution, and in limb regeneration. A new principle for control of organ size is discovered by using the mouse limb bud to study how signals signalling growth and expansion are terminated. A combination of mutations were identified that revealed an inhibitory loop whereby FGF signalling represses Gremlin1, these two factors being key positive signals in limb bud outgrowth. This inhibitory loop is triggered by a positive FGF/Shh loop, and this model differs from earlier published models. 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.