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An in vitro bioassay for limb bud polarizing activity in the chick embryo has been verified by two procedures, demonstrating that the culture procedure mimics occurrences in vivo. First, no activity can be detected with the in vitro assay 24 hours after removal of the posterior region of the limb. In addition, after a positive assay for activity, the responding tissue develops into polarized limb structures when transplanted to a host embryo. After a negative assay, the transplanted responding tissue fails to develop into recognizable limb structures. Since polarizing activity is defined by its ability to induce polarized limb structures in vivo we conclude that the in vitro system provides a valid assay for limb polarizing activity.
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