The zone of polarizing activity (ZPA) is an area of mesenchyme that contains signals which instruct the developing limb bud to form along the anterior/posterior axis.

[1] Twenty years later, the same group did transplantation studies in chick limb bud and identified the ZPA.

These results showed that the cells of the apical ectoderm have a precise fate to form specific regions of the wing.

The first hypothesis came from Tickle et al. who showed that when retinoic acid is placed in the anterior margin of the limb bud, mirror image duplications result.

[10] The idea that Shh is required for proper ZPA signaling and anterior/posterior limb formation needed to be tested.

Riddle et al. took Saunders and Gasselings findings to the next step and proved that Shh is the morphogen within the ZPA that is required for anterior posterior patterning.

[9] By isolating the Shh gene and implanting it into the anterior limb bud, mirror image digit duplications formed.

Isolation was conducted by designing PCR primers that correspond to sequences of Shh that are conserved in Drosophila and mouse and involved in limb bud formation.

The group ectopically expressed the gene by taking advantage of a retroviral vector to insert the cDNA into chick cells.

Therefore, this group used a retroviral vector termed RCAS-E, which lacks a type E envelope protein, and is able to infect certain chick embryo fibroblasts with Shh.

[8] Hoxb-8 signaling is active in the early embryo, beginning at the posterior end of the lateral plate mesoderm and extending to the anterior region.

Experiments done by Heikinheimo et al. show that when the AER is removed, beads that express FGF are sufficient to induce Shh signaling in the ZPA.

Genes that are targets of Shh signaling encode factors that lead to the formation of the stylopod, zeugopod and autopod.

Overall, the molecular ZPA requires input for several signaling centers, but acts as an organizer itself, inducing anterior-posterior pattering of the chick limb bud.