Genetic evidence that FGFs have an instructive role in limb proximal–distal patterning

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Abstract
The developing limb bud possesses a small ridge, the apical ectodermal ridge (AER), that produces signals controlling development of the limb along the proximal–distal axis (from the upper arms to the finger tips). Fibroblast growth factors (FGFs) are known to be key AER signals, but as four FGFs are expressed in the mouse AER, it has been difficult to understand their roles. Mariani et al. used genetic techniques to delete different combinations of FGFs from the mouse limb, thereby revealing the contribution made by each FGF to the total AER-FGF signal. Only one of the four AER-FGFs, Fgf8, was found to be essential for normal limb development. This dispels a longstanding notion that there is a positive feedback loop between the three other FGF genes expressed in the posterior AER and the sonic hedgehog gene. They also provide the first genetic evidence that the AER-FGFs serve as distalizing factors for establishing limb patterning, suggesting a role of FGFs as patterning molecules. They present a model that synthesizes the new findings with several other controversial papers published in recent years on the validity of the 'progress zone' versus the 'early specification' model of limb development. Genetic techniques have been used to delete different combinations of fibroblast growth factors (FGFs) from the mouse limb, to study the contribution that each FGF makes to the total apical ectodermal ridge (AER)–FGF signal. Out of the four AER–FGFs, it is shown that only one of them, Fgf8 is sufficient for normal limb development. This dispels a longstanding notion that there is a positive feedback loop between the three other FGF genes expressed in the posterior AER, and the sonic hedgehog gene. Half a century ago, the apical ectodermal ridge (AER) at the distal tip of the tetrapod limb bud was shown to produce signals necessary for development along the proximal–distal (P–D) axis, but how these signals influence limb patterning is still much debated1,2. Fibroblast growth factor (FGF) gene family members are key AER-derived signals3,4, with Fgf4, Fgf8, Fgf9 and Fgf17 expressed specifically in the mouse AER5. Here we demonstrate that mouse limbs lacking Fgf4, Fgf9 and Fgf17 have normal skeletal pattern, indicating that Fgf8 is sufficient among AER-FGFs to sustain normal limb formation. Inactivation of Fgf8 alone causes a mild skeletal phenotype6,7; however, when we also removed different combinations of the other AER-FGF genes, we obtained unexpected skeletal phenotypes of increasing severity, reflecting the contribution that each FGF can make to the total AER-FGF signal. Analysis of the compound mutant limb buds revealed that, in addition to sustaining cell survival, AER-FGFs regulate P–D-patterning gene expression during early limb bud development, providing genetic evidence that AER-FGFs function to specify a distal domain and challenging the long-standing hypothesis that AER-FGF signalling is permissive rather than instructive for limb patterning. We discuss how a two-signal model for P–D patterning can be integrated with the concept of early specification to explain the genetic data presented here.