As a result of interactions between the ectoderm and underlying mesoderm, formation occurs roughly around the fourth week of development.

[3] These signaling centers are crucial to the proper formation of a limb that is correctly oriented with its corresponding axial polarity in the developing organism.

[12] The FGF8 secreted by the AER acts to keep the cells of the limb mesenchyme in a mitotically active state and sustains their production of FGF10.

[14] Within the limb bud, expression of specific Hox genes varies as a function of the position along the anterior-posterior axis.

This statement is supported by the knowledge that Hox gene expression is initiated during gastrulation in primitive somitic mesoderm by FGF signaling which effects the primitive somitic mesoderm cells at different times depending on their axial location during organism development—and is even further specified with other anterior-posterior axis signals (such as retinoic acid).

[9] As previously stated, limb development is essentially autonomous after the signaling centers (AER) and ZPA) have been established.

[18] Though many of the finer details remain to be resolved, a number of significant connections between Hox gene expression and the impact on limb development have been discovered.

[19] The transition into the third phase is then marked by changes in how the limb bud mesenchymal cells responds to Shh signals.

[19] These three phases of regulation reveal a mechanism by which natural selection can independently modify each of the three limb segments – the stylopod, the zeugopod, and the autopod.

The ZPA first specifies anterior-posterior polarity (and dictates digit identity), and then, by sustaining AER activity, it ensures that the necessary cell proliferation occurs for normal formation of a five-digit limb.