Vascularization in the murine allantois occurs by vasculogenesis without accompanying erythropoiesis

The aim of this study was to determine whether the blood vessels of the murine allantois are formed by vasculogenesis or angiogenesis. Morphological analysis revealed that differentiation of allantoic mesoderm into an outer layer of mesothelium and an inner vascular network begins in the distal region of the allantois, which is most remote from other tissues, as early as the late neural plate stage (approximately 7.75 days postcoitum). Nascent blood vessels were not found in the base of the allantois until 4-somite pairs had formed in the fetus (approximately 8.25 days postcoitum), and vascular continuity with the yolk sac and fetus was not present until the 6-somite-pair stage (approximately 8.5 days postcoitum). Immunohistochemical analysis demonstrated that flk-1, a molecular marker of early endothelial cells, is expressed in significantly more distal than basal core cells in the early allantois and never in mesothelium. Furthermore, synchronous grafting of donor yolk sac containing blood islands into blood islands of headfold-stage host conceptuses provided no evidence that the yolk sac contributes endothelial cells to the allantois. Finally, when removed from conceptuses and cultured in isolation, neural plate and headfold-stage allantoises formed a conspicuous vascular network that was positive for Flk-1. Hence, the vasculature of the allantois is formed intrinsically by vasculogenesis rather than extrinsically via angiogenesis from the adjacent yolk sac or fetus. Whether allantoic vasculogenesis is associated with erythropoiesis was also investigated. Benzidine-staining in situ revealed that primitive erythroid cells were not identified in the allantois until 6-somite pairs when continuity between its vasculature and that of the yolk sac was first evident. Nevertheless, a small number of allantoises removed from conceptuses at a considerably earlier stage were found to contain erythroid precursor cells following culture in isolation. To determine whether such erythroid cells could be of allantoic origin, host allantoises were made chimeric with lacZ-expressing donor allantoises that were additionally labeled with [3H]methyl thymidine. Following culture and autoradiography, many lacZ-expressing benzidine-stained cells were observed in donor allantoises, but none contained silver grains above background. Moreover, no cells of donor allantoic origin were found in the fetus or yolk sac. Hence, vasculogenesis seems to be independent of erythropoiesis in the allantois and to involve a distal-to-proximal gradient in differentiation of allantoic mesoderm into the endothelial cell lineage. Furthermore, this gradient is established earlier than reported previously, being present at the neural plate stage.