Figure 1. (a) Origin of tissue types within the limb. The cartilage, tendon and connective tissue of the limb arise from mesenchyme of the lateral plate somatopleure (shown by a bracket in the diagram of a stage 14 chick embryo on left side). Muscle cell progenitors arise from the somite (purple) where they delaminate from the lateral aspect of the dermamyotome and migrate into the limb (see diagram of stage 20 chick embryo on right side). The nerves (green) arise from the spinal cord and migrate into the limb. The ectoderm will give rise to the skin. The apical ectodermal ridge (AER; see stage 20 diagram) forms at the boundary between the dorsal and ventral limb ectoderm.

(b, c) Experimental determination of the dorsoventral (D-V) signalling centres.

(b) Prelimb bud embryo (stage 14). Rotation of the ectoderm 180° relative to the lateral plate somatopleure results in a limb bud with D-V polarity that corresponds to the mesenchyme. This indicates that D-V patterning information resides in the mesenchyme at this early stage.

(c) Limb bud embryo (stage 20). Rotation of the ectoderm 180° relative to the limb bud mesenchyme results in formation of distal skeletal elements with D-V polarity that corresponds to the ectoderm. This indicates that D-V patterning information resides in the ectoderm by this stage. Similar experiments indicate that D-V information is transferred from the mesenchyme to the ectoderm around stage 15.

(d) Molecular determinants of D-V signalling. Before stage 15 the molecular basis of the early mesenchymal D-V signal is not known. After stage 15, the dorsal ectoderm expresses Wnt7a (purple). WNT7a induces Lmx1 (green) in the mesenchyme. WNT7a and LMX1 are required for specification of dorsal cell fates. Engrailed-1 (En-1; blue) is expressed in the ventral ectoderm and it directs development of ventral limb structures.

AER, apical ectodermal ridge; D, dorsal; DE, dorsal ectoderm; DM, dorsal mesenchyme; V, ventral; VE, ventral ectoderm; VM, ventral mesenchyme.

Figure 2. (a) The proximodistal signalling centre: the apical ectodermal ridge (AER). On the left is a diagram of a stage 20 chick limb bud and on the right is the normal pattern of skeletal elements. The AER signals (arrows) to the underlying progress zone mesenchyme (red stipple) to maintain it in an undifferentiated and proliferative state. The pattern of the skeletal elements from proximal to distalare humerus (H), radius (R) and ulna (U), and digits (2, 3, 4 from anterior to posterior).

(b, c) Experimental determination of proximodistal signalling centre. Surgical removal of the AER results in truncation of the limb along the proximodistal axis. (c) Approximate level of truncation following removal of the AER at the indicated stages.

(d) Molecular determinants of the proximodistal signalling centre. Left: Proposed medial to lateral cascade of fibroblast growth factor (FGF) signalling to initiate limb development. Fgf8 (red) and Fgf10 (purple) from the mesonephros (M) signal to induce Fgf10 in the lateral plate mesenchyme (LP) which then induces Fgf8 in the surface ectoderm (SE). Right: FGF signalling from the AER signalling centre. Fgfs are expressed in the AER and can substitute for the AER to allow proximodistal outgrowth of the limb. Fgf4 is expressed in the posterior half of the AER (green) whereas Fgf8 and Fgf9 are expressed throughout the AER (red). Fgf2 is present in the AER and mesenchyme. Fgf10 is expressed in the progress zone mesenchyme.

A, anterior; Fgf, fibroblast growth factor; H, humerus; LP, lateral plate mesenchyme; M, mesonephros; NT, neural tube; P, posterior; R, radius; S, somite; SE, surface ectoderm; U, ulna; 2, 3, 4, digits 2, 3 and 4 respectively.

Figure 3. (a) Experimental determination of the anteroposterior signalling centre: the zone of polarizing activity ZPA). Transplantation of mesenchyme from the posterior portion of a stage 20 donor limb bud to the anterior region of a stage 20 host limb results in repolarization of the host limb such that additional digits are formed in a mirror image of the normal digits.

(b) Molecular determinant of the ZPA signalling centre. Sonic Hedgehog (SHH) is expressed in the posterior region of the limb bud and, when introduced anteriorly into the limb, causes repolarization of the limb bud, resulting in mirror-image skeletal duplications.

A, anterior; H, humerus; NT, neural tube; P, posterior; S, somite; SHH, Sonic Hedgehog; U, ulna; 2, 3, 4, digits 2, 3 and 4 respectively.