part or another of the future limb). As a consequence, cells leave the PZ in a proximal to distal order of specification (i.e., the first to leave have the capacity to form the upper limb, the next to form the lower limb, and the last to form the hand or foot). When cells leave the PZ, they start to differentiate into cartilage, bone, and connective tissue of their specified limb level. Precursor cells of limb muscles migrate into the bud from the adjacent somitic myotomes, and nerves extend in from spinal ganglia and the spinal cord. Interactions between the AER and PZ are reciprocal (i.e., the AER maintains mitosis within the PZ by way of FGF, and the PZ maintains the thickened AER by way of a yet unknown signal).

The second axis of the limb, the anteroposterior axis, is also established through cell-cell interactions and secreted signals. The posterior portion of the limb bud contains a specialized region called the zone of polarizing activity (ZPA). This zone was originally recognized as a signaling center, because when it was transplanted to the anterior side of another limb bud, the bud develops a mirror-image duplicated limb. The ZPA secretes SHH, which diffuses across the anteroposterior dimension of the bud, establishing a gradient and setting off the formation of a second gradient of BMP2 and 4, two kinds of TGFβ signals. SHH is both sufficient and necessary to establish the anteroposterior pattern of the limb. Retinoic acid, acting via a nuclear receptor, might also have a role.

The third axis of the limb, the dorsoventral axis, is established through interactions between the nonridge dorsal ectoderm of the limb and the underlying bud mesoderm. That was initially shown by experiments in which the dorsal and ventral regions of the ectoderm were rotated with respect to the mesoderm. The dorsal ectoderm releases the WNT7A signal, which induces the expression of the Lmx-1 gene in the underlying mesoderm and which suppresses expression of the engrailed-1 gene, thereby restricting its expression to the ventral ectoderm. Mutants defective in WNT7A signaling develop double ventral limbs. Mutants defective in engrailed-1 expression develop double dorsal limbs with double sets of fingernails.

It remains to be learned how the signaling pathways of each axial dimension are coordinated with those of the other dimensions, and how the integration of these pathways leads to the formation of unique skeletal structures in precise locations within the limb. The limb exemplifies the advanced understanding of vertebrate organogenesis at a molecular genetic level (i.e., of the signal pathways and the genetic regulatory circuits involved in changing transcription and regulating cell proliferation). This kind of understanding is prerequisite to understanding the action of toxicants on embryogenesis. On the basis of new information, mechanisms of action of toxicants have been recently proposed, although these have yet to be tested. For example, thalidomide leads to a failure to form proximal parts of the limb (the upper and middle parts of the limb), but the hand or foot is usually formed. That developmental outcome is paradoxical, because other treatments, such as the removal or inactivation of the AER, lead to truncation of the limb in the reverse order; the upper and middle parts are present but the hand

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