A. Signaling molecules (FGF-8?) are released from localized regions of the trunk, causing the expression of FGF-8 in the ectoderm over the future limb mesoderm.

B. Even before outgrowth occurs, the dorsoventral axis is established, initially in the mesoderm, but this information is soon taken over by the ectoderm. The dorsal ectoderm expresses Wnt-7a, which induces the expression of homeobox containing gene Lmx-1 in the dorsal mesoderm.

C. At essentially the same time, the ventral ectoderm is expressing En-1, which inhibits the expression of Wnt-7a on the ventral side.

D. The dorsal ectoderm is also expressing radical fringe, which is inhibited by the En-1 expressed in the ventral tissues. Just between the dorsal and ventral ectoderm, there is a thickening of ectoderm which forms the apical ectodermal ridge, or AER. The AER begins to express an array of genes.

A. The AER begins to secrete FGF-8. This combined with the Wnt-7a in the dorsal ectoderm causes the expression of sonic hedgehog in the zone of polarizing activity, or ZPA, the limb bud mesoderm just posterior to the AER. Sonic hedgehog in turn causes the production of FGF-4 and the two form a positive feedback loop in which each one stimulates the production of the other.

B. At the same time as the formation of this feedback loop between the AER and the ZPA, a slew of hox genes are expressed in the limb bud mesoderm. Hoxd genes appear to organize patterning along the anteroposterior axis, and Hoxa genes along the proximodistal axis.

The ZPA is primarily responsible for organization along the anteroposterior axis of the limb, through the stimulation of the production of BMP-2 in the posterior mesoderm.

The AER is primarily responsible for organization along the proximodistal axis of the limb through the control of the proliferation of the mesodermal cells. The cells in the progess zone just proximal to the AER are undefined and rapidly dividing. However, once they are beyond the influence of the AER, the morphogenetic paths of the cells are fixed such that the cells that leave earliest form the more proximal limb bones and those that remain under the influence of the AER longer form the more distal elements. The influence of the AER on the cells in the progress zone is seen in their expression of the gene Msx-1, which ceases upon their departure from the progress zone.

Stage 3: Formation of Skeletal Elements

In addition to maintaining the progress zone, the AER directly inhibits the differentiation of mesoderm cells into cartliage. As cells depart from the progress zone and condense to form precartilage, they express BMP-2 and BMP-4, which are later expressed only in the perichondrium and periosteum surrounding the forming bones. BMP-3 is expressed in cartilage, not precartilage, but it also later becomes only expressed in the perichondrium. This makes sense, as the BMP molecules are primarly responsible for the differentiation of skeletal tissues. Another BMP, BMP-6 is expressed in maturing cartilage, along with Indian hedgehog.