Distribution of type IV collagen during avian limb bud development

Normal limb development is dependent on an epithelial-mesenchymal interacti on between the overlying apical ectodermal ridge (AER) and the underlying m esenchyme. The basement membrane between the epithelium and the mesenchyme has been proposed to play an important role in regulating epithelial-mesenc hymal interactions during development. To explore the role basement membran e type IV collagen may play during limb development we investigated the dis tribution of type IV collagen by immunolocalization. Developing avian leg b uds were examined at 2 developmental stages: stage 23, when the AER is indu ctively active, and stage 28, when the AER is regressing. The proximal base ment membrane in stage 23 limb buds stained much more intensely than the di stal basement membrane. This proximal-distal immunostaining difference was less in stage 28 limb buds. We used the monoclonal antibody IIB12, which re cognizes an epitope adjacent to the initial collagenase cleavage site on th e type IV collagen molecule, to explore whether this proximal-distal differ ence in basement membrane staining could result from the loss of type IV co llagen. The distal basement membrane of stage 23 limb buds demonstrated lit tle 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 unfixe d stage 23 leg bud cryostat sections stored at 4 degrees C lost their immun ostaining for type IV collagen, in contrast to stored stage 28 limb bud cry ostat sections. These results demonstrate that type IV collagen is distribu ted 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 select ive degradation of type IV collagen in the basement membrane underlying the active AER. These results are consistent with the hypothesis that the base ment membrane plays a role in regulating the epithelial-mesenchymal interac tion responsible for induction of limb outgrowth. Copyright (C) 1999 by the American Society for Surgery of the Hand.