The vasculature of the avian limb bud takes its origin from the intersomitic vessels as can be shown by ink perfusion of the embryo. While the primitive vessels form a central network in the early limb bud, an area of about 100 p m in width from the ectoderm inward remains free from lumenized vessels. However, this subectodermal avascular zone contains isolated angioblasts, which can be demonstrated by confocal laser scanning microscopy in connection with QH-1-staining. QH-1-positive cells from the avascular zone are capable of giving rise to endothelial cells when grafted ectopically into a "permissive" environment such as the dorso-lateral paraxial mesoderm. Several grafting sites are compared regarding their permissiveness for capillary formation. In order to investigate the origin of the QH-1-positive angioblasts we carried out injections of DiI-Ac-LDL, which is specifically taken up by endothelial cells and macrophages, and found the lumenized vessels and a few isolated cells in the peripheral limb mesoderm stained. In double-labelling studies combining DiI-Ac-LDL and QH-1, it can be shown that there exists a pool of isolated angioblasts that are only QH-l-positive, but have not incorporated DiI-Ac-LDL. In contrast to the lumenized vessels in the core of the limb bud, we found that angioblasts in the avascular zone do not proliferate, as shown by proliferation studies applying the BrdU-method to semithin sections in connection with QH-l-labelled parallel sections. We conclude that the vascularization of the avian limb bud is achieved by a combination of angiotrophic growth (sprouting of vessels) and angioblastic growth (recruitment of angioblasts from the limb mesoderm).0 1995 Wiley-Liss, Inc.

Cytokinetic studies on the aortic endothelium using the BrdU/anti-BrdU-method were carried out on 2.5- to 6-day chick and quail embryos. The mitotic activity of the aortic endothelium is related temporally to the age of the avian embryo and spatially to the embryonic region where the aorta originates. The mitotic activity of the aortic endothelium decreases with increasing age of the embryos. In the limb buds, however, the mitotic rate of the aortic endothelial cells increases independently of the age of the embryo. This increase in the mitotic activity of the aortic endothelium at the appropriate levels coincides with the vascularization of the outgrowing limb buds. We concluded therefore that the aortic endothelium probably supplies endothelial cells for the formation of limb vessels at this stage. Thus our results suggest that angiogenesis (sprouting of capillaries from pre-existing vessels) takes place during limb vascularization in avian embryos. On the other hand, immunohistochemical studies with QH-1 or MB-1 antibody show, beside a capillary network in the central core of the wing bud, individual immunolabelled cells of mesenchymal character within the primarily avascular subectodermal region from the onset of vascularization onwards. We suggest that these cells have partly to be regarded as endothelial precursor cells, which have differentiated in situ from the local limb mesenchyme, and which will contribute to the developing vascular plexus. This means that not only angiogenesis, but also vasculogenesis (in situ from mesenchymal precursors differentiated endothelial cells) appears to be involved in limb vessel formation.

The avian thymus and its myoid cells were investigated paying special attention to the developmental and morphological differences between chick and quail. By means of light- and electron microscopy, and immunofluorescence technique using an anti-myosin antibody, the myoid cells were found to express characteristics corresponding to those of skeletal muscle cells. They change their appearance during embryonic development. In the chick the myoid cells become located singly and rounded, and their cross-striation disappears. In the quail they remain small, elongated, cross-striated, and become arranged in long cords. The origin of myoid cells was studied using the quail-chick marking technique: Cranial somites and the prechordal mesoderm were grafted from quail into chick embryos. After somite transplantation the host thymus does not contain graft-derived cells. The myoid cells are exclusively derived from the chick. After implantation of prechordal mesoderm, graft-derived quail cells are found in the central cores of all visceral arches and also within the early epithelial anlage of chimeric thymus. These findings indicate that the thymus myoid cells are derived from the axially located prechordal head mesoderm.

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