Basement membrane chondroitin sulfate proteoglycan and vascularization of the developing mammalian limb bud

We used immunocytochemistry to study the basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) distribution in mammalian limb bud and its relationship to and possible role in limb development. Anti-BM-CSPG immunostaining was examined in the developing limb buds of 24 Sprague-Dawley rats at embryonic days 12 to 14 and 19. BM-CSPG immunostaining was present in 3 regions. The first region was located peripherally in the limb bud ectodermal basement membrane (BM) that separates ectoderm from mesoderm and was present at all embryonic stages examined. The second region was in the mesenchymal extracellular matrix independent of the vascular system. This staining pattern was diffuse, granular, and often homogeneous, except for clustering adjacent to developing vessels, and was observed distally in the limb bud. In the mesenchymal extracellular matrix adjacent to the distal BM this staining pattern formed fibrils that were perpendicular and connected to the limb bud BM and extended into the underlying mesenchyme. The third region was localized to the BM of developing blood vessels of the limb bud. Blood vessel staining allowed analysis of limb bud vessel formation. The early developing blood vessels at the proximal limb bud were organized differently from those located distally. Large central vessels were present proximally, whereas a rich plexus of smaller vascular channels was present at the distal margin. A subectodermal avascular zone was observed at the margin of the limb bud, except beneath the apical ectodermal ridge where immunostained blood vessels extended from the distal vascular plexus toward the apical ectodermal ridge. The formation of central larger vessels occurs proximally, whereas formation of peripheral smaller vessels seems to take place locally and distally under the influence of the apical ectodermal ridge. BM-CSPG plays an important role in blood vessel formation and mammalian limb bud development. (J Hand Surg 2000; 25A:150-158.