The development of a characteristic vascular pattern is essential for the successful completion of limb morphogenesis. Congenital or experimental alteration in the vascular system results in abnormal development; nevertheless, we know very little about the way in which tissues interact during limb vasculogenesis. We have previously shown that embryonic ectoderm interacts with the underlying mesoderm to direct the pattern and distribution of blood vessels in the limb bud. The distal portion of the chick wing bud has been chosen as the experimental system because the dynamic changes that occur within the vasculature of this region coincide with the molecular changes that result in cytodifferentiation. Examination of India ink rejected, cleared embryos, at successive stages of development, will enable us to map the precise spatial and temporal pattern of the vasculature during the development of the distal skeletal elements of the limb. Areas destined to become cartilage appear to lose their vasculature before chondrogenesis begins. We will use 35S-autoradiography and a monoclonal antibody against keratan sulfate in combination with ink injection to determine whether changes in the distribution of the vasculature precede histodifferentiation. Intravascular injection of the colloidal dye, Monastral blue B, labels the vascular endothelial cells. Monastral blue and the histological marker exploited in chick-quail grafts will be used to trace the fate of endothelial cells in regions of the limb undergoing vascular regression. Light and electron microscopy along with morphometry will be used to examine the morphological changes that occur in the vascular endothelium during limb morphogenesis. The vasculature of the polydactylous talpid2 mutant will be examined and the relationship of blood vessels to the avascular chondrogenic regions will be compared with normal limbs. We will administer retinoic acid (a teratogen that causes limb abnormalities) to localized regions of the limbs by means of the slow-release polymer, ELVAX-40. The effects of retinoic acid on the distribution of blood vessels will be examined. A comprehensive examination of vasculogenesis will prove useful in understanding normal development, deformity and teratogenesis of the vertebrate limb.
