Distribution of type IV collagen during avian limb bud development

Normal limb development is dependent on an epithelial-mesenchymal interaction between the overlying apical ectodermal ridge (AER) and the underlying mesenchyme. The basement membrane between the epithelium and the mesenchyme has been proposed to play an important role in regulating epithelial-mesenchymal interactions during development. To explore the role basement membrane type IV collagen may play during limb development we investigated the distribution of type IV collagen by immunolocalization. Developing avian leg buds were examined at 2 developmental stages: stage 23, when the AER is inductively active, and stage 28, when the AER is regressing. The proximal basement membrane in stage 23 limb buds stained much more intensely than the distal basement membrane. This proximal-distal immunostaining difference was less in stage 28 limb buds. We used the monoclonal antibody IIB12, which recognizes an epitope adjacent to the initial collagenase cleavage site on the type IV collagen molecule, to explore whether this proximal-distal difference in basement membrane staining could result from the loss of type IV collagen. The distal basement membrane of stage 23 limb buds demonstrated little immunostaining with the IIB12 antibody, suggesting enhanced collagenase-associated degradation. The immunostaining was increased in stage 28 limb buds. Consistent with a loss of type IV collagen, we also found that unfixed stage 23 leg bud cryostat sections stored at 4 degrees C lost their immunostaining for type IV collagen, in contrast to stored stage 28 limb bud cryostat sections. These results demonstrate that type IV collagen is distributed in a proximal-distal pattern in the basement membrane of the developing chick limb bud and suggest that this pattern may be the result of a selective degradation of type IV collagen in the basement membrane underlying the active AER. These results are consistent with the hypothesis that the basement membrane plays a role in regulating the epithelial-mesenchymal interaction responsible for induction of limb outgrowth.