The ability to regenerate a lost body part during wound healing is largely restricted to amphibians and lower vertebrates. However, regenerative wound healing following human fetal surgery has exposed the potential for regeneration in higher vertebrates. Regeneration is dependent upon the activation of growth factors that drive cell proliferation and patterning factors that shape the growing tissue into the missing body part. Recently, an embryonic chick model has demonstrated that fibroblast growth factors (FGF) can drive regeneration of an amputated wing bud during development if applied to the posterior stump. Furthermore, the reactivation of sonic hedgehog (SHH), a potent patterning factor, surrounding the FGF-soaked bead was an early predictor of regeneration. In contrast, when FGF was introduced in the anterior stump no SHH induction or regeneration occurred. We hypothesized that the combined interaction between FGF and SHH was necessary for regeneration. To test this hypothesis we ectopically expressed both SHH (by electroporation) and FGF2-soaked beads to the anterior margin of stage 23 (HH) forelimb (wing) buds following amputation of the distal 500 μm. The chicks were grown to embryonic day 10; the wings were harvested and stained with alcian green for assessment of the skeleton. In limbs treated with ectopic SHH and FGF, 4 of 18 demonstrated regeneration with ulnarization of the radius, pattern reversal of the wrist, and a subsequent change in digit identity. The limbs treated with only SHH (10) or only FGF (11) failed to regenerate. These data demonstrate that both FGF and SHH are required for regeneration following wound healing and may indicate targets for reactivation in organisms that lack tissue regeneration.
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