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Morphogenesis of the vertebrate limb bud depends upon reciprocal interactions between epithelial and mesenchymal tissues. A characteristic limb vascular pattern is essential for normal limb outgrowth. The vascular pattern in the distal portion of the wing bud was examined by ink injection and compared to the sites of cartilage differentiation, as indicated by [35S]-incorporation. During development, avascular areas arose in three distinct locations within the vascularized mesoderm. These areas corresponded to the distal skeletal elements, referred to as digits 2, 3, and 4. Incorporation of radioactive sulfate was high in the avascular areas and low in the adjacent vascular tissue. Examination of autoradiographs of ink-injected limbs suggested that the appearance of avascular regions preceded the accumulation of sulfated cartilage matrix. These results indicate that remodeling of the limb vasculature is related to the formation of the skeletal pattern.

The regression of blood vessels in the distal wing bud of chicken embryos from stages 19 to 31 was examined by light and electron microscopy. The vessels were double-labelled by an injection of Monastral blue B (MB) to label the regressing endothelial cells, followed 6-48 hours later with an injection of India ink which marked the lumens of patent vessels. Prior to stage 26 the vessels contained only India ink since the endothelial cells were not phagocytic at this stage. Vessels at stage 26 or later were often double-labelled, with MB sequestered in the endothelial cell cytoplasm and India ink in the vessel lumens. After stage 27 cells not associated with lumens, but labelled with MB, were observed in areas undergoing vascular regression. Ultrastructural changes in the endothelial cells as the vessels regressed included formation of luminal and abluminal processes, long complex junctions, and vacuoles containing MB. In many involuting vessels the endothelial cells appeared normal even though the lumens were collapsed. Occasionally, isolated pyknotic cells were observed in regions that had been previously vascularized. At stage 31 cells in the developing cartilage had vacuoles containing MB. Our study suggests that blood vessels may disappear from the prechondrogenic zone of the distal wing bud by several mechanisms. These could include a type of cell death that does not elicit a cellular infiltrate, migration of the endothelial cells away from vascularized regions, and/or transdifferentiation into cells that resembled chondrocytes.