Experimental analysis of blood vessel development in the avian wing bud.

Shortly after its appearance, the avian limb bud becomes populated by a rich plexus of vascular channels. Formation of this plexus occurs by angiogenesis, specifically the ingrowth of branches from the dorsal aorta or cardinal veins, and by differentiation of endogenous angioblasts within limb mesoderm. However, mesenchyme located immediately beneath the surface ectoderm of the limb is devoid of patent blood vessels. The objective of this research is to ascertain whether peripheral limb mesoderm lacks angioblasts at all stages or becomes avascular secondarily during limb development. Grafts of core or peripheral wing mesoderm, identified by the presence or absence of patent channels following systemic infusion with ink, were grafted from quail embryos at stages 16-26 into the head region of chick embryos at stages 9-10. Hosts were fixed 3-5 days later and sections treated with antibodies that recognize quail endothelial cells and their precursors. Labeled endothelial cells were found intercalated into normal craniofacial blood vessels both nearby and distant from the site of implantation following grafting of limb core mesoderm from any stage. Identical results were obtained following grafting of limb peripheral mesoderm at stages 16-21. However, peripheral mesoderm from donors older than stage 22 did not contain endothelial precursors. Thus at the onset of appendicular development angioblasts are present throughout the mesoderm of the limb bud. During the fourth day of incubation, these cells are lost from peripheral mesoderm, either through emigration or degeneration.     

Experimental analysis of blood vessel development in the avian wing bud

Shortly after its appearance, the avian limb bud becomes populated by a rich plexus of vascular channels. Formation of this plexus occurs by angiogenesis, specifically the ingrowth of branches from the dorsal aorta or cardinal veins, and by differentiation of endogenous angioblasts within limb mesoderm. However, mesenchyme located immediately beneath the surface ectoderm of the limb is devoid of patent blood vessels. The objective of this research is to ascertain whether peripheral limb mesoderm lacks angioblasts at all stages or becomes avascular secondarily during limb development. Grafts of core or peripheral wing mesoderm, identified by the presence or absence of patent channels following systemic infusion with ink, were grafted from quail embryos at stages 16–26 into the head region of chick embryos at stage 9–10. Hosts were fixed 3–5 days later and sections treated with antibodies that recognize quail endothelial cells and their precursors. Labeled endothelial cells were found intercalated into normal craniofacial blood vessels both nearby and distant from the site of implantation following grafting of limb core mesoderm from any stage. Identical results were obtained following grafting of limb peripheral mesoderm at stages 16–21. However, peripheral mesoderm from donors older than stage 22 did not contain endothelial precursors. Thus at the onset of appendicular development angioblasts are present throughout the mesoderm of the limb bud. During the fourth day of incubation, these cells are lost from peripheral mesoderm, either through emigration or degeneration.     

The relationship between limb muscle and endothelial cells migrating from single somite

Somites contribute myogenic and endothelial precursor cells to the limb bud. Transplantations of single somites have shown the pattern of muscle cell distribution from individual somites to individual limb muscles. However, the pattern of the endothelial cell distribution from individual somites to the limb has not been characterized. We have mapped quail muscle and endothelial cell distribution in the distal part of the chick limb after single somite transplantation to determine if there is a spatial relationship between muscle and endothelial cells originating from the same somite. Single brachial somites from quail donor embryos were transplanted into chick embryos, and, following incubation, serial sections were stained with a quail-endothelial cell-specific monoclonal antibody (QH-1), an anti-quail antibody (QCPN) and an anti-desmin antibody to distinguish the quail endothelial and muscle cells from chick cells. Our results show that transplants of somite 16-21 each gave rise to quail endothelial cells in the wing. The anterioposterior position of the blood vessels formed by somitic endothelial cells corresponded to the craniocaudal position of the somite from which they have originated. Endothelial cells were located not only in the peri- and endomysium but also in the subcutaneous, intermuscular, perineural and periost tissues. There was no strict correlation between the distribution of muscle and endothelial cell from a single transplanted somite. Blood vessels formed by grafted quail endothelial cells could invade the muscle that did not contain any quail muscle cells, and conversely a muscle composed of numerous quail muscle cells was lacking any endothelial cells of quail origin. Furthermore, a chimeric limb with very little quail muscle cells was found to contain numerous quail endothelial cells and vice versa. These results suggest that muscle and endothelial cells derived from the same somite migrate on different routes in the developing limb bud.     

The lymphatic endothelium of the avian wing is of somitic origin.

It has recently been shown that there are lymphangioblasts in the early avian wing bud, but fate map studies on the origin of these cells have not yet been performed. The lymphatics in the wings of 10-day-old chick and quail embryos are characterized by both the position along with all major blood vascular routes and by the Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) expression. In the quail, the endothelium of both blood vessels and lymphatics can be marked with the QH1 antibody. We have grafted the dorsal halves of epithelial somites of 2-day-old quail embryos homotopically into chick embryos. The grafting was performed at the wing level and the host embryos were reincubated until day 10. The chimeric wings were studied with the QH1 antibody alone and with double staining consisting of VEGFR-3 in situ hybridization and QH1 immunofluorescence. Our results show that in the wing the endothelium of both the blood vessels and the lymphatics is derived from the somites. QH1-positive endothelial cells form the vasculature of the chimeric wings. Chimeric lymphatics of the wing can be identified because of their typical position and their VEGFR-3 and QH1 double-positivity. This shows that not only the blood vascular cells but also the lymphatic endothelial cells of the avian wing are born in the paraxial/somitic mesoderm.     

Differentiation of endothelial cells in avian embryos does not depend on gastrulation.

Unincubated quail eggs were treated with Cytochalasin B. By this means, gastrulation of the blastodiscs was inhibited. Fragments of these blastodiscs were grafted into wings buds of chick embryos, and the differentiation fate of graft-derived cells was studied. Results show that only endothelial cells differentiate from the grafts. They were even found outside the graft site in vessels made up of a chimeric endothelium. It can be concluded that determination, differentiation and migration of endothelial cells does not depend on gastrulation.     

Endogenous origin of the lymphatics in the avian chorioallantoic membrane.

The lymphatics of the intestinal organs have important functions in transporting chyle toward the jugulosubclavian junction, but the lymphangiogenic potential of the splanchnic mesoderm has not yet been tested. Therefore, we studied the allantoic bud of chick and quail embryos. It is made up of endoderm and splanchnic mesoderm and fuses with the chorion to form the chorioallantoic membrane (CAM) containing both blood vessels and lymphatics. In day 3 embryos (stage 18 of Hamburger and Hamilton [HH]), the allantoic mesoderm consists of mesenchymal cells that form blood islands during stage 19 (HH). The endothelial network of the allantoic bud, some intraluminal and some mesenchymal cells express the hemangiopoietic marker QH1. The QH1-positive endothelial cells also express the vascular endothelial growth factor receptor-3 (VEGFR-3), whereas the integrating angioblasts and the round hematopoietic cells are QH1-positive/VEGFR-3-negative. The ligand, VEGF-C, is expressed ubiquitously in the allantoic bud, and later predominantly in the allantoic epithelium and the wall of larger blood vessels. Allantoic buds of stage 17-18 (HH) quail embryos were grafted homotopically into chick embryos and reincubated until day 13. In the chimeric CAMs, quail endothelial cells are present in blood vessels and lymphatics, the latter being QH1 and VEGFR-3 double-positive. QH1-positive hematopoietic cells are found at many extra- and intraembryonic sites, whereas endothelial cells are confined to the grafting site. Our results show that the early allantoic bud contains hemangioblasts and lymphangioblasts. The latter can be identified with Prox1 antibodies and mRNA probes in the allantoic mesoderm of day 4 embryos (stage 21 HH). Prox1 is a specific marker of the lymphatic endothelium throughout CAM development.     

On the differentiation and origin of myoid cells in the avian thymus

Summary 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 quailchick 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.Supported by the Deutsche Forschungsgemeinschaft (Ch 44/8-1)     

Early mesoderm differentiation in the chick embryo

Summary Two groups of experiments were carried out. In the first group, grafts of quail mesoderm whose presumptive fate was to form somites or heart tissues, were taken from quail embryos (stage 4–5 of Hamburger and Hamilton 1951) and inserted beneath the ectoderm of chick embryos of stage 3–4 immediately lateral to the primitive streak. Whilst most grafts contributed to the somites and/or the heart, 22 out of a total of 46 were found to have contributed also to the pharyngeal endoderm. Although three of these grafts were known to have included some quail endoderm cells, the remainder were considered to consist of mesoderm alone. It is concluded that mesoderm at the primitive streak stages is still capable of forming endoderm.In the second group of experiments, grafts of quail somites (stage 10–14) were inserted beneath the ectoderm of chick embryos of stage 3–4. In 18 out of 23 cases the graft cells were found in somitic tissue, but they were also found in the endoderm (4 specimens), lateral plate (3 specimens) and endothelium (4 specimens). It is concluded that even at stages 10–14, the somite-derived cells are still not completely determined to form somite derivatives. In those cases where the grafted somites differentiated further, sclerotome cells which migrated from them did not necessarily move towards the host notochord.     

Cytokinetic studies on the aortic endothelium and limb bud vascularization in avian embryos

Summary 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.Supported by the Deutsche Forschungsgemeinschaft (Ch 44/9-1, Ch 44/9-2)This paper is dedicated to Prof. Dr. K.V. Hinrichsen on the occasion of his 65th birthday     

Morphological aspects of the vascularization in intraventricular neural transplants from embryo to embryo

Intraventricular transplants of neural tissues were performed in ovo from embryo to embryo. Fragments of the nervous wall of the optic lobe (tectum) from 14-day chick or 12-day quail embryos (donor) were inserted into the ventricle of the right optic lobe of 6-day chick or 5-day quail embryos (host). Chick-to-chick, chick-to-quail and quail-to-chick grafts were carried out. The vascularization changes occurring in the host tectum and in the grafted neural tissues were analysed under light, transmission, and scanning electron microscopes and by morphometric methods. In the host embryo tectum, the neural graft stimulates a statistically significant increment in vessel density and a vessel sprouting into the ventricle of the optic lobe. The vascular sprouts reach the transplanted tissue and establish connections with its native microvasculature. The chick-to-quail and quail-to chick grafts, submitted to immunoreaction with a quailspecific antibody which recognizes an antigen (MB1) present on endothelial cells, indicate that re-establishment of the circulation in the graft depends upon anastomoses between host and donor vasculatures and the rapid new growth of host-derived and donor-native vessels. The presence of macrophage-like cells escorting the new-growing vessels suggests that these cells are involved in the host and donor tissue angiogenesis.     

Vascularization of embryonic adrenal gland grafted onto chorioallantoic membrane

 Vascularization and endothelial phenotype expression were analysed in embryonic adrenal tissue grafted onto chorioallantoic membrane (CAM), by means of routine light microscopy and immunocytochemical staining, and of electron microscopy. Adrenal gland tissue from chick or quail embryos (donors) was grafted onto CAMs of chick or quail embryos (host). Vessels of chick origin were discriminated from those of quail origin by monoclonal antibodies, anti-MB1, specific for quail endothelial and haemopoietic cells, and QCPN, which labels quail cell nuclei. Vessels of adrenal type were distinguished from those of CAM-type by their ultrastructural endothelial phenotype – porous in the former and continuous in the latter. The observations carried out 6 days after implantation indicate that the adrenal gland develops and differentiates according to a virtually normal histological pattern. As regards the adrenal and CAM vascularization, the grafting procedure elicits angiogenic events consisting in the formation of peripheral anastomoses between the graft and the CAM original microvasculature and in new-growth of vessels from the CAM into the grafted tissue and vice versa. As to the endothelial phenotype, the ultrastructural results demonstrate that besides its own native vasculature, the adrenal tissue contains vessels with continuous endothelium and the CAM mesenchyme is supplied by adrenal-type, fenestrated vessels. Accepted: 27 April 1998     

Axial structures control laterality in the distribution pattern of endothelial cells

In the midline of the embryo an invisible barrier exists that keeps endothelial cells from migrating to the contralateral side. Interspecific grafting experiments between chick and quail were carried out in order to investigate the role of the axial structures in maintaining this barrier. The quail endothelial cells of the graft were therefore stained with QH1 antibody. In all experimental series quail paraxial mesoderm was used as a source of endothelial cells. First, a quail somite was transplanted either ipsilaterally or contralaterally. The results not only show the existence of laterality in the distribution pattern, but also demonstrate that the laterality does not depend on the origin of the graft but on the environment of the host embryo. Laterality in the distribution pattern of endothelial cells means that the endothelial cells of the two body halves migrate independently and do not change from one side to the other. Single cells do not know whether they are cells from the right or from the left half of the body. In the next series of experiments axial structures were removed in order to modify the barrier. In addition, paraxial mesoderm was exchanged with the corresponding quail tissue in order to determine the migration behaviour of the grafted endothelial cells. The removal of the neural tube does not influence the barrier. After notochordectomy, however, the endothelial cells exhibited a balanced distribution pattern over both halves of the embryo. We concluded that the notochord forms a barrier for endothelial cells that presumably operates on the basis of chemical substances. It is conceivable that our results can explain the lateralization of illnesses of the vascular system, as the Klippel-Trénaunay syndrome or the Sturge-Weber syndrome.     

Positional control of mesoderm movement and fate during avian gastrulation and neurulation.

Segments of primitive streak from donor quail embryos at stages of gastrulation and neurulation were transplanted heterotopically and isochronically to primitive streaks of host chick embryos. The subsequent movement and fate of grafted cells was determined using the quail nucleolar marker to define grafted cells. The pattern of movement of grafted cells depended on their new position within the primitive streak, not on their original position. When cells of cranial regions were placed more caudally, they moved to mesodermal subdivisions that were located lateral to those they would have populated if left in their original position. When caudal segments were placed more cranially, they moved to more medial mesodermal subdivisions. Whether the fate of grafted cells corresponded to their original location or their new location depended on both their level of origin and their new position. Cells from heterotopically transplanted Hensen's nodes, which migrated to the somitic and more lateral mesoderm, self-differentiated notochords. Similarly, in some cases, heterotopically transplanted prospective somitic cells, which migrated to lateral plate mesoderm, formed ectopic somites. In other cases, however, grafted cells contributed to the host's somites, intermediate mesoderm, and lateral plate mesoderm. Moreover, prospective somitic cells, which migrated to the extraembryonic lateral plate mesoderm, changed their fate and formed extraembryonic lateral plate mesoderm; and prospective lateral plate mesoderm cells, which migrated to the somitic mesoderm, formed somites as well as intermediate mesoderm and lateral plate mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angioblast differentiation is influenced by the local environment: FGF-2 induces angioblasts and patterns vessel formation in the quail embryo.

The embryonic vasculature forms by the segregation, migration, and assembly of angioblasts from mesoderm, a process termed vasculogenesis. The initial role of fibroblast growth factor 2 (FGF-2) in vascular development appears to be in the induction of endothelial precursors, angioblasts. Quail somites transplanted into chick embryos will give rise to angioblasts of quail origin. The number of angioblasts present within the chimera is dependent on the host environment. Angioblast induction can be demonstrated in vitro by the addition of FGF-2 to cultures of dissociated somitic mesoderm, as assessed by QH-1 epitope expression. Manipulation of FGF-2 concentration in the quail/chick chimeras by FGF-2 peptide or neutralizing antibody injections increases or decreases angioblast induction in the predicted manner. To better control growth factor release in vivo we have implanted beads that release FGF-2 into the embryonic environment. FGF-2 beads implanted into the somite induce angioblast differentiation in the epithelial somite; whereas, beads lateral to the somitic mesoderm induce the formation of ectopic vessels. These studies suggest that FGF-2 is important for both the induction of angioblasts and the assembly of angioblasts into the initial vasculature pattern.     

Cardiac looping in the chick embryo: the role of the posterior precardiac mesoderm

Summary Grafts of mesoderm taken from the precardiac region of quail embryos of stages 5–7 were inserted into the precardiac mesoderm of chick embryos of stages 5–7. The experiments were of four types and were codenamed to indicate the origin and the destination of the graft. QACP: tissue from the anterior end of the quail precardiac area was inserted into the posterior end of the chick precardiac mesoderm; QPCA: tissue from the posterior end of the quail precardiac area was inserted into the anterior end of the chick precardiac mesoderm; QACA: tissue from the anterior end of the quail precardiac area was inserted into the anterior end of the chick precardiac mesoderm; QPCP: tissue from the posterior end of the quail precardiac area was inserted into the posterior end of the chick precardiac mesoderm. In no case was precardiac tissue removed from the host. Three main types of anomaly were obtained: inverted hearts, in which looping took place to the left rather than to the right; compact hearts, in which no looping occurred, and hearts in which extra tissues or regions were apparent. The incidence of compact hearts was significantly greater with QPCA than with any other category of experiment. When older donors were used (stages 8–9), the incidence of compact hearts fell. No variations in the origin of the graft, nor in its ultimate destination in the host, were found to affect the frequency of any of the anomalies. Sections showed that quail hearts tended to have thicker walls than chick hearts; although quail tissues were often incorporated into the host chick hearts, they retained the histological characteristics of the donors. The fact that no compact hearts resulted from the experiment QACA, or from the mock operations, leads us to conclude that failure to loop in the compact hearts was not due to mechanical trauma caused by the operation, but to some specific difference between grafts taken from the anterior and posterior precardiac mesoderm. The fact that compact hearts were obtained when chick donors were used instead of quails, shows that the effect is not species-specific. We propose that a morphogen is secreted by the posterior end of the precardiac mesoderm and this plays a role in controlling the cessation of looping.     

A test of positional properties of avian wing-bud mesoderm

Supernumerary wing structures are readily produced by grafting pieces of wing-bud mesoderm into different locations of host wing buds, but the mechanism underlying their formation remains obscure. The major aim of this study was to examine the ability of posterior quail wing-bud mesoderm, cultured in vitro long enough to lose ZPA (zone of polarizing activity) activity, to stimulate or participate in the formation of supernumerary structures when grafted into anterior slits of host chick wing buds. Small pieces of anterior and posterior quail wing-bud mesoderm (HH stages 21-23) were placed in in vitro culture for up to 3 days. After 2 days, ZPA activity of cultured mesoderm was lost. After the grafting of 2- to 3-day cultured anterior quail wing-bud mesoderm into posterior slits of host chick wing-buds, a consistently high percentage (70%-90%) of grafts result in formation of supernumerary cartilage; in this experiment, however, only a low percentage of grafts resulted in supernumerary cartilage when 2- to 3-day cultured posterior mesoderm was grafted into anterior slits. Taken with controls, these results show that positional differences exist between cultured anterior and posterior wing-bud mesoderm. Serial-section analysis of numerous operated wings has shown several patterns of contribution to supernumerary structures by cells of graft and host. Single supernumerary digits induced by grafts of ZPA mesoderm into anterior slits were normally composed entirely of host cells, but graft cells regularly contributed to skeletal elements of more complex supernumerary structures. Cartilage rods produced by anterior-to-posterior grafts were composed mostly of graft cells, but cartilage nodules and the bases of some rods were often mosaics of chick and quail cells. The results support the proposition that mesodermal cells of the quail wing-bud possess a form of anteroposterior positional memory, but its nature and the means by which the memory of grafted cells interacts with host mesoderm are still not clear.     

Is chemotaxis a factor in the migration of precardiac mesoderm in the chick?

Summary The chick heart is formed from bilateral patches of presumptive cardiac mesoderm cells which migrate over the endoderm and fuse in the midline. We have tested the possibility that this migration is controlled, at least in part, by a chemotactic substance exuded by the anterior end of the endoderm. We have used chick/quail combinations to follow naturally marked cells during the course of their migration. Chimaeric embryos were formed by fusing together parts of chick and quail embryos of stage 5–6. Each embryo possessed two pairs of precardiac regions, the quail pair lying immediately anterior to that of the chick. These chimaeras were then explanted in embryo culture. In the event of chemotaxis, cells from the posterior end of the quail precardiac mesoderm might be expected to invade the chick area. Samples of explants and chimaeras were examined at intervals from 2 to 24 h, but in no case were cells found to have changed their direction of migration as a result of the proximity of anterior endoderm. It is concluded that this work does not provide evidence for a chemotactic attraction by the anterior end of the endoderm. Supported by the following grants: NIH HD 21048, HD 06819, and AHA 880696 (JWL); the British Heart Foundation, and Action Research (R.B.); and an SERC postgraduate studentship (HSE).     

Vascular endothelial cells migrate centripetally within embryonic arteries

Summary Migration of vascular endothelial cells was traced in quail-chick chimeras. After heterospecific transplantations of quail limb bud pieces, or other tissues containing blood vessels, into the limbs or the coelomic cavity, the immunohistochemically stained endothelial cells of the quail were found to invade the chick host vessels, favouring the arteries. Within these vessels the endothelial cells regularly reach the host aorta, where they contribute to the endothelium on the ipsilateral side. It is concluded that the endothelial cells activity migrate, because microinjections of a synthetic peptide which contains the RGD-sequence and mimics fibronectin, stop the invasion of endothelial cells.Supported by the Dutch Heart Foundation, The Netherlands Organization for Pure Research N.W.O., the Jo Keurfonds, the Drie Lichten and the Deutsche Forschungsgemeinschaft (Ch 44/9-1)