Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo

Remodeling of the primary vascular system of the embryo into arteries and veins has long been thought to depend largely on the influence of hemodynamic forces. This view was recently challenged by the discovery of several molecules specifically expressed by arterial or venous endothelial cells. We here analysed the expression of neuropilin-1 and TIE2, two transmembrane receptors known to play a role in vascular development. In birds, neuropilin-1 was expressed by arterial endothelium and wall cells, but absent from veins. TIE2 was strongly expressed in embryonic veins, but only weakly transcribed in most arteries. To examine whether endothelial cells are committed to an arterial or venous fate once they express these specific receptors, we constructed quail-chick chimeras. The dorsal aorta, carotid artery and the cardinal and jugular veins were isolated together with the vessel wall from quail embryos between embryonic day 2 to 15 and grafted into the coelom of chick hosts. Until embryonic day 7, all grafts yielded endothelial cells that colonized both host arteries and veins. After embryonic day 7, endothelial plasticity was progressively lost and from embryonic day 11 grafts of arteries yielded endothelial cells that colonized only chick arteries and rarely reached the host veins, while grafts of jugular veins colonized mainly host veins. When isolated from the vessel wall, quail aortic endothelial cells from embryonic day 11 embryos were able to colonize both host arteries and veins. Our results show that despite the expression of arterial or venous markers the endothelium remains plastic with regard to arterial-venous differentiation until late in embryonic development and point to a role for the vessel wall in endothelial plasticity and vessel identity.     

Differential expression of neuropilin-1 and neuropilin-2 in arteries and veins.

Neuropilin-1 (np1) and neuropilin-2 (np2) are receptors for class-3 semaphorins and for several isoforms of VEGF. We have cloned and characterized two chick isoforms of np2 cDNA. Expression patterns of np1, np2, and ephrin-B2 were compared in the developing vascular system of 24-72 h old chick embryos. We show for the first time that np2 is expressed in blood vessels in vivo from the earliest stages of their formation. In contrast to ephrin-B2, both np1 and np2 are expressed in blood islands of 24 h old chick embryos. At 48-72 h, np1 expression is localized preferentially in arteries with an expression pattern that resembles that of ephrin-B2. In contrast, np2 is expressed preferentially in veins. Thus, neuropilins may play a role in determining the arterial or venous identity of blood vessels.     

Ephrin-B2 selectively marks arterial vessels and neovascularization sites in the adult, with expression in both endothelial and smooth-muscle cells

The Eph receptor tyrosine kinases and their membrane-tethered ephrin ligands provide critical guidance cues at points of cell-to-cell contact. It has recently been reported that the ephrin-B2 ligand is a molecular marker for the arterial endothelium at the earliest stages of embryonic angiogenesis, while its receptor EphB4 reciprocally marks the venous endothelium. These findings suggested that ephrin-B2 and EphB4 are involved in establishing arterial versus venous identity and perhaps in anastamosing arterial and venous vessels at their junctions. By using a genetically engineered mouse in which the lacZ coding region substitutes and reports for the ephrin-B2 coding region, we demonstrate that ephrin-B2 expression continues to selectively mark arteries during later embryonic development as well as in the adult. However, as development proceeds, we find that ephrin-B2 expression progressively extends from the arterial endothelium to surrounding smooth muscle cells and to pericytes, suggesting that ephrin-B2 may play an important role during formation of the arterial muscle wall. Furthermore, although ephrin-B2 expression patterns vary in different vascular beds, it can extend into capillaries about midway between terminal arterioles and postcapillary venules, challenging the classical conception that capillaries have neither arterial nor venous identity. In adult settings of angiogenesis, as in tumors or in the female reproductive system, the endothelium of a subset of new vessels strongly expresses ephrin-B2, once again contrary to earlier views that such new vessels lack arterial/venous characteristics and derive from postcapillary venules. While earlier studies had focused on a role for ephrin-B2 during the earliest embryonic stages of arterial/venous determination, our current findings using ephrin-B2 as an arterial marker in the adult challenge prevailing views of the arterial/venous identity of quiescent as well as remodeling adult microvessels and also highlight a possible role for ephrin-B2 in the formation of the arterial muscle wall.     

The proepicardium delivers hemangioblasts but not lymphangioblasts to the developing heart

The mass of the myocardium and endocardium of the vertebrate heart derive from the heart-forming fields of the lateral plate mesoderm. Further components of the mature heart such as the epicardium, cardiac interstitium and coronary blood vessels originate from a primarily extracardiac progenitor cell population: the proepicardium (PE). The coronary blood vessels are accompanied by lymph vessels, suggesting a common origin of the two vessel types. However, the origin of cardiac lymphatics has not been studied yet. We have grafted PE of HH-stage 17 (day 3) quail embryos hetero- and homotopically into chick embryos, which were re-incubated until day 15. Double staining with the quail endothelial cell (EC) marker QH1 and the lymphendothelial marker Prox1 shows that the PE of avian embryos delivers hemangioblasts but not lymphangioblasts. We have never observed quail ECs in lymphatics of the chick host. However, one exception was a large lymphatic trunk at the base of the chick heart, indicating a lympho-venous anastomosis and a 'homing' mechanism of venous ECs into the lymphatic trunk. Cardiac lymphatics grow from the base toward the apex of the heart. In murine embryos, we observed a basal to apical gradient of scattered Lyve-1+/CD31+/CD45+ cells in the subepicardium at embryonic day 12.5, indicating a contribution of immigrating lymphangioblasts to the cardiac lymphatic system. Our studies show that coronary blood and lymph vessels are derived from different sources, but grow in close association with each other.     

Development of an arterial tree in C6 gliomas but not in A375 melanomas

The microcirculation of tumors is severely disturbed. Tumors are usually supplied by fragile capillaries and do not possess the natural hierarchy of blood vessels. The detection of specific markers for arterial and venous endothelial cells (ECs) now enables us to study the vascular tree in tumors. We have injected rat C6 glioma and human A375 melanoma cells into 3.5- to 4-day-old avian embryos. After 10-12 days of reincubation the tumor cells formed solid tumors vascularized by host ECs. In contrast to the melanomas, the gliomas induced an almost normal vascular tree with arterial and venous vessels. The arterial vessels express the arterial EC marker ephrin-B2, and possess a media of smooth muscle alpha-actin (alphaSMA)-positive cells. Venular vessels in the gliomas are ephrin-B2-negative/alphaSMA-positive. Although the gliomas may represent a rare case of vascular tree induction in tumors, the results underline the heterogeneity of tumor-induced angiogenesis. This has an impact on tumor blood flow and thereby also on the efficacy of chemotherapy and radiotherapy.     

Monoclonal antibodies specific to quail embryo tissues: their epitopes in the developing quail embryo and their application to identification of quail cells in quail-chick chimeras.

Quail-chick chimeras have been used extensively in the field of developmental biology. To detect quail cells more easily and to detect cellular processes of quail cells in quail-chick chimeras, we generated four monoclonal antibodies (MAb) specific to some quail tissues. MAb QCR1 recognizes blood vessels, blood cells, and cartilage cells, MAb QB1 recognizes quail blood vessels and blood cells, and MAb QB2 recognizes quail blood vessels, blood cells, and mesenchymal tissues. These antibodies bound to those tissues in 3-9-day quail embryos and did not bind to any tissues of 3-9-day chick embryos. MAb QSC1 is specific to the ventral half of spinal cord and thymus in 9-day quail embryo. No tissue in 9-day chick embryo reacted with this MAb. This antibody binds transiently to a small number of brain vesicle cells in developing chick embryo as well as in quail embryo. A preliminary application of two of these MAb, QCR1 and QSC1, on quail-chick chimeras of neural tube and somites is reported here.     

Developing nervous tissue induces formation of blood-brain barrier characteristics in invading endothelial cells: A study using quail-chick transplantation chimeras

Brain capillaries have structural and functional characteristics that constitute a regulatory interface, or “barrier,” between the blood and the brain. We have investigated the role of the neural tissue environment in the differentiation of the endothelial barrier, by transplanting embryonic brain fragments to the coelomic cavity, where they were vascularized by nonneural vessels, and fragments of embryonic mesoderm to the brain, where they were vascularized by neural vessels. A major problem in this approach is that when embryonic tissues are transplanted to an ectopic site, their own blood vessels survive and form a part of the new vascular system. This has made the results of previous experiments difficult to interpret. We overcame this problem by transplanting fragments of tissue that had not yet been vascularized from very young quail embryos to host chick embryos. These grafts did not contain vascular channels that could form part of a new vascular system. Furthermore, the distinctive quail nuclear morphology allowed us to demonstrate that the grafted tissue was, in fact, vascularized by the host vessels. Abdominal vessels vascularizing grafted neural tissue formed structural, functional, and histochemical features of the blood-brain barrier. In contrast, brain vessels vascularizing grafted mesodermal tissue were devoid of barrier characteristics. These results indicate that endothelial blood-brain barrier characteristics develop in response to some aspect of the neural environment.     

Development of VIP in the peripheral nervous system of avian embryo

The distribution of the VIP containing structures was studied in the gut and in the paravertebral sympathetic ganglia of the quail and chick embryos by immunocytochemistry. In the gut, development of peptidergic nerves followed a craniocaudal gradient. Immunoreactive fibres were first visible in the oesophagus at day 9 in the quail and day 10 in the chick, at 12 days they extended over the whole length of the gut. Cell bodies were localized at day 9 in the foregut and observed in the mid- and hind-gut just before hatching. Transplantations on the chorioallantoic membrane of fragments of various parts of the digestive tract clearly demonstrated that VIP nerve cell bodies belonged to the intrinsic innervation of the gut. Besides the gut, sympathetic paravertebral ganglia contained cells with VIP immunoreactivity detected at day 9 and 10 in quail and chick respectively. In order to find out whether VIP containing neurons differentiated normally in chick embryos in which quail neural crest cells had been implanted at an early stage of development we looked for the appearance of peptidergic neurones in the following situations: when the quail neural primordium had been grafted orthotopically and isochronically into chick host (1) at the adrenomedullary (somites 18-24) and (2) at the vagal (somites 1-7) levels of the neural axis. In all conditions VIP immunoreactivity was observed in quail cells located either in the sympathetic paravertebral ganglia of the trunk at the level of the graft or in the enteric ganglia according to the graft was made at the adrenomedullary and vagal levels respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitogenic effect of muscle on the neuroepithelium of the developing spinal cord

A previous study revealed that segments of bowel grafted between the neural tube and somites of a younger chick host embryo would induce a unilateral increase in cellularity of the host's neural tube. The current experiments were done to test the hypotheses that muscle tissue in the wall of the gut is responsible for this growth-promoting effect and that the spinal cord enlargement is the result of a mitogenic action on the neuroepithelium. Fragments of skeletal (E8-15) or cardiac muscle (E4-14) were removed from quail embryos and grafted between the neural tube and somites of chick host embryos (E2). Both skeletal and cardiac muscle grafts mimicked the effect of bowel and induced an increase in cell number as well as a unilateral enlargement of the region of the host's neural tube immediately adjacent to the grafts. The growth-promoting effect of muscle-containing grafts was restricted to the neural tube itself and was not seen in proximate dorsal root or sympathetic ganglia. The action of the grafts of muscle was neither species- nor class-specific, since enlargement of the neural tube was observed following implantation of fetal mouse skeletal muscle into quail hosts. Grafts of skeletal muscle or gut increased the number of cells taking up [3H]thymidine in the host's neuroepithelium as early as 9 h following implantation of a graft. The increase in the number of cells entering the S phase of the cell cycle preceded the increase in cell number. These observations demonstrate that muscle-containing tissues can increase the rate of proliferation of neuroepithelial cells when these tissues are experimentally placed together.     

Regulation of Cell Number in Formation of the Dorsal Root Ganglion Revealed by Transplantation of Quail Neural Crest Cells into Chick Embryos

Neural crest cells appear transiently in early embryogenesis on the dorsal surface of the neural tube and subsequently migrate along specific pathways. Some migrate to between the neural tube and somites, aggregating to form the rudiments of dorsal root ganglia (DRG). The size of DRG at a given somite level is almost constant in all chick embryos. To determine the mechanisms controlling the size of DRG, we transplanted neural crest cells of 2.5-day-old quail embryos into 2.5-day-old chick embryos between the neural tube and the somites, and examined the size of DRG in these chimeric embryos with extra neural crest cells 2 days after the operation, when natural cell death in DRG had not yet occurred. The DRG on the operated side were composed of both chick and quail cells in various proportions. The cell numbers of these chimeric DRG were almost the same as those of the normal DRG on the opposite side. That is, there were significantly fewer chick cells in chimeric DRG than in DRG composed of only chick cells on the opposite unoperated side. This finding indicates that the size of DRG is not determined in migrating neural crest cells but is regulated by the circumstances.     

Netrin-1 inhibits sprouting angiogenesis in developing avian embryos

Netrin-1 is a bifunctional axonal guidance cue, capable of attracting or repelling developing axons via activation of receptors of the deleted in colorectal cancer (DCC) and uncoordinated 5 (UNC5) families, respectively. In addition to its role in axon guidance, Netrin-1 has been implicated in angiogenesis, where it may also act as a bifunctional cue. Attractive effects of Netrin-1 on endothelial cells appear to be mediated by an as yet unknown receptor, while repulsion of developing blood vessels in mouse embryos is mediated by the UNC5B receptor. To explore evolutionary conservation of vascular UNC5B expression and function, we have cloned the chick unc5b homologue. Chick and quail embryos showed unc5b expression in arterial EC and sprouting angiogenic capillaries. To test if Netrin-1 displayed pro- or anti-angiogenic activities in the avian embryo, we grafted cell lines expressing recombinant chick or human Netrin-1 at different stages of development. Netrin-1 expressing cells inhibited angiogenic sprouting of unc5b expressing blood vessels, but had no pro-angiogenic activity at any stage of development examined. Netrin-1 also had no effect on the recruitment of circulating endothelial precursor cells. Taken together, these data indicate that vascular unc5b expression and function is conserved between chick and mice.     

Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon.

Grafting a met-mesencephalic portion of neural tube from a 9.5-day mouse embryo into the prosencephalon of a 2-day chick embryo results in the induction of chick En-2 (ChickEn) expression in cells in contact with the graft (Martinez et al., 1991). In this paper we investigate the possibility of Wnt-1 being one of the factors involved in En-2 induction. Since Wnt-1 and En-2 expression patterns have been described as diverging during development of the met-mesencephalic region, we first compared Wnt-1 and En-2 expression in this domain by in situ hybridization in mouse embryos after embryonic day 8.5. A ring of Wnt-1-expressing cells is detected encircling the neural tube in the met-mesencephalic region at least until day 12.5. This ring consistently overlapped with the En-2 expression domain, and corresponds to the position of this latter gene's maximal expression. We subsequently studied ChickEn ectopic induction in chick embryos grafted with various portions of met-mesencephalon. When the graft originated from the level of the Wnt-1-positive ring, ChickEn induction was observed in 71% of embryos, and in these cases correlated with Wnt-1 expression in the grafted tissue. In contrast, this percentage dropped significantly when the graft was taken from more rostral or caudal parts of the mesencephalic vesicle. Taken together, these results are compatible with a prolonged role of Wnt-1 in the specification and/or development of the met-mesencephalic region, and show that Wnt-1 could be directly or indirectly involved in the regulation of En-2 expression around the Wnt-1-positive ring during this time. We also provide data on the position of the Wnt-1-positive ring relative to anatomical boundaries in the neural tube, which suggest a more general role for the Wnt-1 protein as a positional signal involved in organizing the met-mesencephalic domain.     

Quail melanoblast migration in two breeds of fowl and in their hybrids: evidence for a dominant genic control of the mesodermal pigment cell pattern through the tissue environment

In the Silkie fowl large numbers of melanocytes invade most internal tissues and organs. The factors involved in this internal pigment cell pattern were studied by grafting quail neural tube segments into White Leghorn, White Silkie, and F1 hybrids (White Silkie male X White Leghorn female). Sections of quail neural tube five somites long, excised at the level of the last formed somites, were grafted isotopically and ischoronically. Various tissues and organs (mesenteries, muscles, testis, ovary, mesonephros, metanephros, and adrenals) excised from the internal region corresponding to the peripheral transverse strip of quail melanocytes, were studied after staining by the Feulgen-Rossenbeck technique. Despite some variations in pigment cell density, Silkie and hybrid grafted embryos exhibited an extensive quail internal pigmentation similar to the melanocyte distribution in the Silkie breed. In white Leghorn host embryos, the internal pigmentation remained limited. These results show the part played by tissular factors in the expression of the Silkie pigment phenotype and that this genetic tissular character is dominant. On the contrary, White Leghorn embryos, grafted with Silkie neural tube segments, never exhibited any internal pigmentation; the melanocytes deriving from the grafted Silkie neural tube were only localized at the dermoepidermal level. Thus, the migrating and/or differentiating capabilities of the Silkie premelanoblasts are different from those of quail premelanoblasts. The sex-linked inhibitor of the White Leghorn tissue interferes at the level of the pigment cells of chickens but not of quails.     

Expression of ephrinB2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells, and marks subsets of microvessels at sites of adult neovascularization

The transmembrane ligand ephrinB2 and its receptor tyrosine kinase EphB4 are molecular markers of embryonic arterial and venous endothelial cells, respectively, and are essential for angiogenesis. Here we show that expression of ephrinB2 persists in adult arteries where it extends into some of the smallest diameter microvessels, challenging the classical view that capillaries have neither arterial nor venous identity. EphrinB2 also identifies arterial microvessels in several settings of adult neovascularization, including tumor angiogenesis, contravening the dogma that tumor vessels arise exclusively from postcapillary venules. Unexpectedly, expression of ephrinB2 also defines a stable genetic difference between arterial and venous vascular smooth muscle cells. These observations argue for revisions of classical concepts of capillary identity and the topography of neovascularization. They also imply that ephrinB2 may be functionally important in neovascularization and in arterial smooth muscle, as well as in embryonic angiogenesis.     

Histochemical identification and behavior of quail primordial germ cells injected into chick embryos by the intravascular route.

The behavior of quail primordial germ cells (PGC) after injection into chick embryos by the intravascular route was examined. The quail (donor) PGC, taken from the bloodstream of quail embryos (recipient) at stage 13-14, were injected into the vitelline vessels of chick embryos (recipient) at stage 15. In the recipient embryos, the PGC of the quail and the chick were histochemically distinguished by a double-staining technique involving a lectin, from Wistaria floribunda (WFA) and the PAS reaction. One day after injection, quail PGC appeared in the prospective gonadal region of recipient chick embryos, being localized among the recipient chick PGC. This result indicates that a staining technique specific for WFA lectin is useful for identification of quail PGC and that quail PGC can be transferred by a vascular route for the production of germline chimeras.     

Differentiation of avian neural crest cells in vitro: Absence of a developmental bias toward melanogenesis

Summary Neural crest cells from quail embryos grown in standard culture dishes differentiate almost entirely into melanocytes within 4 or 5 days when chick embryo extract (CEE) or occasional lots of fetal calf serum (FCS) are included in the medium. Gel fractionation showed that the pigment inducing factor(s) present in these media is of high molecular weight (> 400 K daltons). In the absence of CEE, the neural tube can also stimulate melanocyte differentiation. Culture medium supplemented by selected lots of FCS permits crest cell proliferation but little overt differentiation after up to 2 weeks in culture if the neural tube is removed within 18 h of explantation in vitro. Subsequent addition of CEE to such cultures promotes complete melanocyte differentiation. Crest cells from White leghorn chick embryos also differentiate into melanocytes in the presence of CEE, but do not survive well in its absence. Melanocyte differentiation of crest cells from both quail and chick embryos can by suppressed by culturing under a dialysis membrane, even in the presence of the neural tube and CEE, but neuronal differentiation appears greatly enhanced.     

Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neurectodermal mesenchymal derivatives, using a biological cell marking technique

By isotopic and isochronic transplantations of fragments of quail neural tube into chick, it has been previously shown that enteric ganglion cells arise from the “vagal” (somites 1–7) and the “lumbo-sacral” (behind somite 28) levels of the neural crest, while the trunk region (somites 8–28) gives rise to orthosympathetic ganglion chain and adrenomedullary cells. The latter originate precisely from the neural crest corresponding to somites 18–24 (i.e., “adrenomedullary” level of the crest). Heterotopic transplantations of fragments of quail neural tube into chick have been carried out in the present work. When the “adrenomedullary” level of the quail neural tube is grafted into the “vagal” region of a chick, the crest cells colonize the gut and differentiate into enteric ganglia of Auerbach's and Meissner's plexi. If quail cephalic neural crest is transplanted in the “adrenomedullary” level of a chick, quail cells migrate into the suprarenal glands and differentiate into adrenomedullary cells. Mesectodermal cells migrate laterally, and differentiate into cartilage, dermis and connective tissues. Thus it appears that preferential pathways located at precise levels of the embryo lead crest cells to their definitive sites. On the other hand the differentiation of the autonomic neuroblasts is controlled by the environment in which crest cells are localized at the end of their migration. On the contrary, mesenchymal derivatives of the cephalic neural crest appear to be early determined since they differentiate according to their presumptive fate when transplanted into the trunk.     

The vasculature of the rat embryo: an electron microscope study

The ultrastructure of portions of the arterial and venous systems of the 11.5 day old Wistar rat embryos has been studied by scanning and transmission electron microscopy. The vessels at this stage of development are in the form of capillaries, and the arterial and venous types can be distinguished by the morphology of the endothelial cells by SEM. The endothelial cells of the arterial vessels gave prominent nuclear bulges and numerous microvilli apart from their spindle shape, whilst those of the veins appear flattened, are polygonal in shape, and have few microvilli. Transmission electron microscopy shows that the endothelial cells of the arteries and veins are identical in structure. The ultrastructure of these cells resembles that of endothelial cells at later stages of development including the adult type in that mature forms of cytoplasmic organelles are obtained. In studies on the intercellular junctions and fenestrations with lanthanum nitrate, the impression is formed that the vessels at this stage are impermeable to small molecular size particles, compared with adult capillaries. This suggests that cytoplasmic vesicles must play a major role in the transport of macromolecules in the 11.5 day embryonic vessels.     

Relationships between terminal transferase expression, stem cell colonization, and thymic maturation in the avian embryo: studies in thymic chimeras resulting from homospecific and heterospecific grafts

Terminal deoxynucleotidyl transferase (TdT) can be detected in 11- to 12-day-old embryonic chick thymuses 5 to 6 days after the first influx of lymphoid stem cells into the thymic rudiment. To identify the main factors of TdT induction, grafting experiments were devised in such a way that the age of the grafted thymus and that of the host were different. Uncolonized embryonic chick thymuses were grafted into chick hosts of different ages. Under these conditions, lymphoid differentiation arose from host lymphoid stem cells (LSC) invading the thymic rudiment. TdT immunofluorescent detection in the first wave of thymocytes showed that the percentages of TdT+ cells were related to the total age of the explant and not to the age of the host (11 to 17 days). Similar results were obtained when the chick thymic rudiment was transplanted into quail embryos, showing that quail LSC have TdT inducibility similar to that of chick LSC while developing in a chick thymic environment. Colonized chick thymuses were also grafted into quail embryos to compare the TdT inducibility of the first lymphoid generation (of chick type) and of the second (of quail origin), taking advantage of the different chromatin structure of quail and chick cells. In these experiments, the majority of chick cells remained TdT negative for as long as 10 days, whereas most lymphocytes of the second generation became TdT+ soon after their arrival in the grafted thymus. Therefore, during embryonic life, most TdT+ cells were derived from the second wave of stem cells, but some early stem cells were also able to acquire the enzyme. In a final series of experiments, early thymic rudiments were cultured in vitro with 14- to 16-day-old bone marrow and then grafted into 3-day-old host embryos. Under these conditions, bone marrow LSC contributed to a variable proportion of the first generation of thymocytes. The percentage of TdT+ cells among the progeny of these bone marrow stem cells was found to be two times higher than that of thymocytes derived from host LSC. These results suggest that, in addition to intrathymic environmental factors, the origin of LSC influences the frequency of TdT expression in their progeny.