Retinoic acid is a putative morphogen in limb formation in the chick and other vertebrates. In chick limb formation, it is thought that retinoic acid is released from the zone of polarizing activity (ZPA) and the concentration gradient of retinoic acid formed from the posterior to the anterior provides positional cues for digit formation. Implantation of a bead containing retinoic acid at the anterior margin of the limb bud induces a mirror-image symmetrical duplication of the digit pattern similar to that observed when the ZPA is grafted into the anterior margin of the host limb bud. Also, the level of endogenous retinoic acid (25 nM on average) is higher in the posterior one third of the limb bud. We found that when the bead containing either retinoic acid or an analogue but not the ZPA, was implanted in the anterior margin of the chick limb bud, expression of the retinoic acid receptor type-beta gene was induced around the bead within 4 h. These results indicate that exogenous retinoic acid is not identical with the ZPA morphogen. As the anterior tissue exposed to retinoic acid has polarizing activity, we conclude that the primary function of exogenous retinoic acid is to induce polarizing activity in the limb bud.

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The relationship between growth and pattern specification during development remains elusive. Some molecules known to function as growth factors are also potent agents of pattern formation. This raises the possibility that growth factors could act in pattern formation via an effect on the cell cycle. We have tested the significance of the length of the cell cycle for gene expression and pattern formation in developing chick limb buds by locally slowing the cell cycle. When anterior cell cycles are lengthened by reversible inhibition of DNA replication or by other means, some genes characteristic of the posterior polarizing region are expressed, and digit duplication is observed. Conversely, when posterior cell cycles are slowed, expression of some posterior-specific genes is inhibited, but the pattern is normal. These results indicate that control of the length of the cell cycle could play a primary role in pattern formation by influencing the complement of genes expressed in a particular region of the embryo.

We have previously shown that MRC-5 cells induce the duplication of the chick limb bud following the implantation into the anterior limb bud only during pre-limb-bud stages. We now report the process of duplicated pattern formation caused by MRC-5 cells. The duplicated patterns are also formed following the implantation into the center of the limb bud and an excess apical ectodermal ridge (AER) with Msx2 expression is induced prior to these duplicated pattern formulations. Only after the implantation into the anterior leg bud, the shh gene is expressed additionally in the anterior leg bud and the mirror-symmetric duplication along the anteroposterior (A-P) axis is formed. The map of the polarizing activity in stage 21 embryo suggests that the high polarizing activity of the normal flank region is responsible for the changes in the A-P polarity when MRC-5 cells are grafted into the anterior leg bud. These results indicate that MRC-5 cells induce the AER and that the excess AER produces the duplicated cartilage pattern of the limb bud.