Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting in defective anterior pattern formation.

The ability of the anterior apical ectodermal ridge to promote outgrowth in the chick wing bud when disconnected from posterior apical ridge was examined by rotating the posterior portion of the stage-19/20 to stage-21 wing bud around its anteroposterior axis. This permitted contact between the anterior and posterior mesoderm, without removing wing bud tissue. In a small but significant number of cases (10/54), anterior structures (digit 2) formed spatially isolated from posterior structures (digits 3 and 4). Thus, continuity with posterior ridge is not a prerequisite for anterior-ridge function in the wing bud. Nevertheless, posterior-ridge removal does result in anterior limb truncation. To investigate events leading to anterior truncation, we examined cell death patterns in the wing bud following posterior-ridge removal. We observed an abnormal area of necrosis along the posterior border of the wing bud at 6-12 h following posterior-ridge removal. This was followed by necrosis in the distal, anterior mesoderm at 48 h postoperatively and subsequent anterior truncation. Clearly, healthy posterior limb bud mesoderm is needed for anterior limb bud survival and development. We propose that anterior truncation is the direct result of anterior mesodermal cell death and that this may not be related to positional specification of anterior cells. In our view, cell death of anterior mesoderm, after posterior mesoderm removal, should not be used as evidence for a role in position specification by the polarizing zone during the limb bud stages of development. We suggest that the posterior mesoderm that maintains the anterior mesoderm need not be restricted to the mapped polarizing zone, but is more extensively distributed in the limb bud.

Development of the apical ectodermal ridge in the chick leg bud and a comparison with the wing bud.

Histologic examination of the leg bud of stage-18 to stage-23 chick embryos was carried out with special reference to the development of the apical ectodermal ridge. The leg bud apical ectoderm, initially an irregular columnar epithelium with an overlying simple squamous periderm, began to thicken during stage 18 and was a pseudostratified epithelium by stage 19. A notch in the base of the thickened ectoderm was seen as early as stage 19. The notch represented the cross-sectional view of a groove, which developed in the base of the ridge. As development proceeded, the ridge and its associated groove lengthened. In addition, the groove became more prominent, and irregularities were seen in its width and depth along the apex. Ectodermal cell death was not consistently seen until stage 21 at which time most of the length of the thickened ectoderm had evidence of necrosis. Development of the leg bud ridge and wing bud ridge were compared. The temporal sequence of ectodermal thickening and ridge development was very similar in both the leg and wing buds with one exception; namely, that leg bud ridge development preceded wing bud ridge development by several hours. In addition, ectodermal cell death was not evident until stage 20 in the leg bud ridge, but could be seen at late stage 18 in the wing bud apical ectoderm. However, by stage 21, cell death was associated with most of the ridge in both the wing and leg buds. Finally, with respect to the axial line, the ridge with its associated groove extended further preaxially in the leg bud than in the wing bud, making the leg bud ridge more symmetrical.

Development of the apical ectodermal ridge in the chick wing bud.

Histological examination of the stage-18 to stage-23 chick wing bud apex revealed the following. Initially, the wing bud was covered by a cuboidal to columnar epithelium with an overlying periderm. Thickening of the apical ectoderm was not obvious until late stage 18 (36 pairs of somites), after the appearance of the wing bud. At late stage 18, cells of the inner layer of ectoderm had elongated slightly along an axis perpendicular to the epithelial-mesenchymal interface. Well-defined apical ectodermal ridge morphology, i.e., pseudostratified columnar epithelium with an overlying periderm, was not apparent until stage 20. Subsequently the ridge lengthened along the anteroposterior perimeter of the wing bud. We demonstrated histologically that the apical ectodermal ridge of the wing bud was asymmetric with respect to the anteroposterior axis, in that there was more ridge associated with posterior mesoderm. Other observations include the spatial and temporal location of a groove in the base of the thickest part of the ridge. The groove can be correlated with the specification of distal wing elements. The groove was first seen at stage 20 and became more prominent through stage 23. An anteroposterior progression of ectodermal cell death was also observed. This began at late stage 18 and continued through each of the stages examined.