Surgical removal of limb bud Sonic hedgehog results in posterior skeletal defects

Using Sonic Hedgehog (Shh) as a marker for polarizing region cells we have repeated the experiments of MacCabe et al. (1973) and Fallon and Crosby (1975) in an attempt to reexamine the question of a continuous role for the polarizing region during limb development. We report that the earlier experiments probably left Shh-expressing cells after surgery. Our results show that Shh-expressing cells do not regenerate and complete removal of the polarizing region results in truncations along the anteroposterior (A-P) axis; further, A-P patterning cannot be restored when a bead soaked in FGF is implanted in the limb bud mesenchyme to maintain outgrowth after extirpation of the polarizing region. However, in order to reproducibly remove all Shh-positive cells, it is possible that cells with posterior limb skeletal fate also must be removed. Therefore, microsurgical approaches do not permit an unequivocal answer to the question raised in this and the earlier papers and it remains a reasonable possibility that at least up to stage 20-21 the polarizing region plays a continuous role in patterning of the limb bud during its development.