Institut dEmbryologie Cellulaire et Moléculaire CNRS FRE 2160, 49bis, Avenue de la Belle Gabrielle, Nogent-sur-Marne Cedex 94736, France
* These authors have contributed equally to this work
Author for correspondence (e-mail: eichmann{at}infobiogen.fr)
Accepted June 11, 2001
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SUMMARY |
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Key words: Neuropilin-1, TIE2, Endothelial cell, Artery, Vein, Vessel wall, Chick-quail chimera
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INTRODUCTION |
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Two growth factor families, VEGFs and angiopoietins, discovered over the last decade, are critical for vasculogenesis and angiogenesis (for reviews see Neufeld et al., 1999; Petrova et al., 1999; Yancopoulos et al., 2000). Moreover, several molecules have recently been shown to be expressed selectively by developing arterial or venous EC. Arterial-specific markers include the transcription factor gridlock (Zhong et al., 2000), and two membrane anchored molecules, the Notch ligand delta-like 4 (DLL4) (Shutter et al., 2000) and the transmembrane growth factor ephrinB2 (Wang et al., 1998; Adams et al., 1999). In contrast, venous EC specifically express the receptor for ephrin B2, EphB4 (Wang et al., 1998; Adams et al., 1999; Gerety et al., 1999). These observations raised, for the first time, the possibility of a genetic predetermination of blood vessel endothelium. Indeed, EC had previously been thought of as a homogeneous cell population, and arterial-venous differentiation was thought to occur as a consequence of hemodynamic forces (reviewed by Yancopoulos et al., 1998). However, targeted inactivation of the ephrinB2 gene in mice resulted in malformation of arteries as well as veins, suggesting that interaction of this growth-factor-receptor pair must be important for correct vascular patterning (Wang et al., 1998; Adams et al., 1998).
We present results of an expression analysis of two receptors known to be involved in vascular development, neuropilin-1 (NRP1) and TIE2. TIE2 is known as an EC-specific receptor for growth factors of the angiopoietin family, which play a role in angiogenesis and the assembly of the vascular wall (Gale and Yancopoulos, 1999; Yancopoulos et al., 2000, for reviews). NRP1 is a receptor for members of the semaphorin/collapsin family, which are negative mediators of neuronal guidance (He and Tessier-Lavigne, 1997; Kolodkin et al., 1997). In EC, NRP1 is a receptor for VEGF165 (Soker et al., 1998; Gluzman-Poltorak et al., 2000) and for several other members of the VEGF family, including PlGF, PlGF-2, VEGF-B and the VEGF-like protein from orf virus NZ2 (Migdal et al., 1998; Makinen et al., 1999; Wise et al., 1999). Expression of NRP1 in porcine aortic EC enhanced the ability of VEGF165 to bind to VEGFR2 and to stimulate chemotaxis via VEGFR2 (Soker et al., 1998). Overexpression of NRP1 in transgenic mice resulted in excess capillary and blood vessel formation and hemorrhaging in the embryo, contributing to embryonic lethality (Kitsukawa et al., 1995). Production of a NRP1 null mutant by homologous recombination of the NRP1 gene induced disorganization of nerve pathways (Kitsukawa et al., 1997) and also vascular defects, resulting in embryonic lethality by embryonic day (E)12.5 (Kawasaki et al., 1999).
We show that in the avian vascular system, both NRP1 and TIE2 are initially expressed in virtually all EC. Once arteries and veins differentiate, the NRP1 gene is transcribed exclusively in arterial EC and mesenchymal cells surrounding developing arteries. In contrast, the chick TIE2 gene (Jones et al., 1999) is a good marker for venous EC. Downregulation of NRP1 in veins and TIE2 in arteries has not been reported in rodents (Kawasaki et al., 1999; Schnurch and Risau, 1993; Sato et al., 1995) and may thus be specific for birds. To investigate whether EC are determined to an arterial or venous fate once they express specific molecules such as NRP1, TIE2 or the previously described ephrinB2 and EphB4 ligand and receptor, we have grafted embryonic arteries or veins from quail embryos at different stages of development into the coelom of chick hosts. This procedure has previously been shown to allow colonization of the host vasculature by donor EC, which can be visualized by staining with the QH1 monoclonal antibody (mAb) specific for quail EC and white blood cells (Pardanaud et al., 1987; Pardanaud and Dieterlen-Lièvre, 1999). We show that the embryonic aorta, taken from E2, E5 or E7 quail donors, still colonizes both arteries and veins of the host embryo. After E7, plasticity is progressively lost and EC from E11 to E15 quail aortae and carotid arteries are largely restricted to colonizing host arteries. EC from cardinal or jugular veins until E7 also reached both types of host vessels, while they were restricted to host veins after this stage. When E11 quail aortic EC were isolated from the vessel wall before grafting, they remained plastic and colonized both host arteries and veins. These results show that the endothelium remains plastic with regard to arterovenous differentiation long after it has acquired the expression of specific markers and that EC plasticity is restricted by the vessel wall.
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MATERIALS AND METHODS |
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Quail-chick grafting
Lateral splanchnopleural mesoderm and somites were retrieved as described (Pardanaud et al., 1996). E2 aorta and cardinal vein were isolated by pancreatin digestion and mechanical dissection. Aortic tubes were peeled off from the ventral part of the somites. Cardinal veins were isolated with the intermediate plate located laterally to the somites. Aortae, carotid arteries and jugular veins from E5 to E15 quail (Coturnix coturnix japonica) embryos were dissected together with their wall, and cut into rings of about 50-100 µm in length. For isolation of EC, aortae (16-25 per experiment) were longitudinally sectioned, pinned onto a black support and treated with collagenase (Sigma) for 30 minutes at 37°C. EC were then flushed out with a micropipette, placed in DMEM supplemented with 20% foetal calf serum (FCS), centrifuged and resuspended in 100 µl of the same medium. Two aliquots of 10 µl were cultured for 48 hours in a Petri dish to verify the presence of EC. Aliquots of 20 µl were placed overnight in hanging drop cultures for aggregate formation and grafting. For grafting, Indian ink, diluted 1:1 in PBS, was injected beneath the E2 chick (Gallus gallus, JA57) host blastodisc. Two types of grafts were performed. (1) Coelomic grafts, which were as described previously (Pardanaud and Dieterlen-Lièvre, 1999). (2) Dorsal grafts, in which 3 host somites at the wing level (somite 15-20) were substituted by quail tissues. A piece of ectoderm was removed above the somites, which were dissected after brief pancreatic digestion. The graft was inserted inside the cavity and the ectoderm was replaced. The host embryos were incubated for 2 days before autopsy, fixed in Bouin or Serra fluid and 5 µm paraffin sections were prepared.
Quantification of EC migration into host arteries and veins
The number of QH1+ EC reaching host arteries and veins was counted manually on every fifth section (x25 objective, final magnification x110). A positive cell was scored when it showed a nucleus, visible either by glychemalun or by QCPN labeling, and membrane QH1 staining. The arterial or venous identity of quail EC was obvious when they integrated great vessels (aorta, cardinal vein, omphalomesenteric artery and vein, subclavian and brachial arteries, umbilical vein, segmental arteries and veins). In the limb bud, we considered that all the EC present at the periphery belonged to the venous plexus while arterial EC were centrally located and connected to the brachial artery. QH1+ EC in the perineural vascular plexus were only quantified if we could trace the connection of these capillaries with a known artery or a vein. If we could not classify the arterial or venous identity of a donor EC, it was not counted. The mean number of these undetermined QH1+ EC reached 4.6% for 107 analyzed grafts and exceeded 10% in 8/107 grafts (not shown).
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RESULTS |
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Onset of NRP1 and TIE2 expression in the arterial and venous system
NRP1 transcripts were first detected before gastrulation in the hypoblast (not shown). During early somitic stages, NRP1 was expressed in the developing heart region and in yolk sac blood islands (Fig. 1A). With the onset of embryonic circulation at approximately the 12-somite stage (ss), NRP1 expression was detected in the dorsal aorta (Fig. 1B). NRP1 was progressively downregulated in the anterior part of the lateral mesoderm and the yolk sac vasculature (Fig. 1B). By the 18-20 ss, NRP1 expression was observed mainly in the posterior lateral mesoderm and yolk sac, corresponding to the prospective arterial compartment (Fig. 1C). In contrast, VEGFR2 expression was observed in both the posterior and anterior compartments of the lateral mesoderm and yolk sac (Fig. 1D). Following the development of the omphalomesenteric arteries and their connection to the embryo proper at the 21ss, NRP1 was strongly expressed in these vessels and their branches (Fig. 1E,F). Thus, NRP1 appeared to be restricted to arterial EC as soon as these vessels became morphologically distinguishable.
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Grafts of early mesodermal territories
In situ hybridization with NRP1 (see Fig. 1D) had shown high expression levels in the posterior, but not the anterior lateral mesoderm. We divided the lateral splanchnopleural mesoderm in posterior or anterior regions, corresponding respectively to the presumptive territory of the omphalomesenteric artery and vein (Fig. 5A). The capacity of these mesodermal compartments to give rise to arteries and veins was analyzed after grafting into the chick coelom. We also grafted somites of E2 quail embryos, which contain undifferentiated angioblasts (Pardanaud et al., 1996).
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To examine if the apparent loss of the capacity to colonize veins was observed with aortic EC only or was typical of all arterial EC, we performed grafts of carotid arteries from E4.5 to E14 quail donors. These grafts gave rise to EC, which behaved like aortic EC, i.e. they efficiently colonized both arteries and veins until E10 but were restricted to host arteries from E11 (Fig. 6E; Table 1B).
Grafts of veins
We next examined the behaviour of venous EC grafted in a novel environment. QH1+ EC from E2 cardinal vein or E5 to E7 jugular vein remained plastic and colonized host arteries and veins (Fig. 7A; Table 1C). Until E7, the distribution of vein- or artery-derived EC in the host vascular tree was very similar. No significant differences in the number of migrating EC or their migration distance was observed between arterial or venous grafts or between venous grafts at different ages. In situ hybridization with NRP1 (Fig. 7A) and ephrinB2 (not shown) showed that vein-derived QH1+ cells integrating arteries acquired expression of both markers, while those colonizing host veins remained NRP1 and ephrinB2 negative. After E7, the great majority of QH1+ EC derived from the jugular endothelium participated in the venous tree, and only a small number of QH1+ EC reached arteries (Fig. 7B; Table 1C). Colonisation of veins was mainly directed towards the umbilical vein and the cardinal vein and more rarely to the omphalomesenteric vein and the venous plexus of the limb.
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EC plasticity in the absence of a vascular wall
To examine the role of the vascular wall in EC plasticity, we isolated EC from E11 quail aortae by collagenase digestion. Isolated EC were cultured for 48 hours and the efficacy of removal of the vessel wall was tested by QH1 staining of the cultured cells (Fig. 8A,B). Cultures contained mainly QH1+ EC, as well as some other QH1- cell types. For grafting, isolated EC were cultured overnight in hanging drops to obtain small aggregates. These were introduced into the coelom of chick hosts (n=6), where they developed in contact with the body wall, the mesonephros and the dorsal mesentery, as observed for intact vessel rudiments. However, the distribution of grafted EC in the host vessels was strikingly different compared to grafts of intact E11 aortic rings: grafted EC colonized host arteries (59±17%) as well as host veins (41±17%). QH1+ EC reached the lateroventral endothelium of the aorta, the brachial artery, the cardinal vein and its branches, the umbilical and omphalomesenteric veins and the peripheral venous plexus of the wing bud (Fig. 8C,D).
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DISCUSSION |
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During the phase of vasculogenesis, NRP1 and TIE2 were expressed throughout the yolk sac blood islands. During the subsequent remodeling of the primary vascular plexus into arteries and veins, NRP1 expression became restricted to arteries and TIE2 to venous EC. This chronology was reminiscent of the reported expression of ephrinB2 and its receptor EphB4 in the mouse embryo, both of which appear on EC only during the phase of remodeling (Wang et al., 1998; Adams et al., 1999; Gerety et al., 1999). In Zebrafish, two arterial-specific genes were recently cloned: gridlock (Zhong et al., 2000) and deltaC (Smithers et al., 2000), which encodes a ligand for members of the Notch family of receptors that control binary cell fate decisions (reviewed in Artavanis-Tsakonas et al., 1999). Interestingly, both gridlock and deltaC were expressed in presumptive arterial EC before the onset of blood flow. These observations raised the question of whether the mesoderm is pre-specified at the gastrulation stage to produce a posterior arterial and anterior venous domain, in order to achieve a functional vascular circuit with the onset of heartbeat? To address the question if EC are specified to an arterial or venous identity once they acquire expression of specific ligands or receptors, we performed quail-chick grafting experiments. Blood vessels at different stages of development were isolated from quail donors and grafted into the coelom of a chick host. The earliest vessel rudiments we isolated came from an E2 quail donor and corresponded to the first two major extraembryonic blood vessels, the omphalomesenteric artery and vein. At this stage, the posterior territory corresponding to the future omphalomesenteric artery strongly expressed NRP1, while the region of the future omphalomesenteric vein had already down-regulated NRP1 expression. EC from both territories could still colonize both arteries and veins of the host embryo. The same result was obtained with grafts from the aorta, the carotid artery or the jugular vein taken from quail embryos between E2 and E7. The expression of NRP1 and ephrinB2 was regulated according to the host vessel, which was colonized by the grafted EC, implying that the plasticity of expression of arterial- or venous-specific genes accompanies endothelial plasticity with regard to arterial-venous differentiation. The adaptation of EC to the novel vascular environment could explain the arterialization of vein grafts currently performed in therapeutic procedures (Henderson et al., 1986; Cahill et al., 1987; Wallner et al., 1999). An interesting question is whether constitutive expression of arterial or venous EC markers would lead to the loss of EC plasticity.
Vessel rudiments isolated from embryos older than E7 were still capable of colonizing the host vasculature. Thus, in spite of the presence of a fully differentiated vascular wall, EC were still able to exit the graft and to migrate into the host vessels. Comparison of the number of migrating quail EC and their migration distance failed to reveal any significant decrease between E7 or E11 aortae, carotid arteries or jugular veins. However, in striking contrast to the results obtained with early EC, plasticity was progressively lost after E7, and from E11 onward aortic EC mainly colonized the host aorta and other arteries. Thus, at late embryonic stages, aortic EC appeared restricted to an arterial fate. This behaviour was not specific to the aorta, since the same result was obtained using E11 and E14 carotid arteries. Venous EC showed a loss of ability to colonize host arteries between E7 and E11 of development, while they were still capable of colonizing host veins. To determine if the loss of plasticity was due to the site of engraftment, we perfomed dorsal grafts, in which 3 host somites were substituted by quail vessel rudiments. It had previously been shown that EC derived from somites selectively colonize only dorsal endothelium of the aorta in both coelomic and dorsal grafts, while splanchnopleural EC selectively colonize ventral aorta (Pardanaud et al., 1996; Pardanaud and Dieterlen-Lièvre, 1999). The loss of plasticity with respect to arterovenous differentiation was also observed in dorsal grafts. Moreover, these grafts showed that EC colonized the vessels closest to their site of engraftment: in the case of arteries, the dorsal aortic endothelium was colonized in dorsal grafts, while coelomic grafts led to colonization of the ventral aortic endothelium. Taken together, these results indicate that EC become restricted to an arterial or venous identity between E7 and E11 of development.
To understand the mechanisms regulating this loss of plasticity, we investigated the role of the vessel wall. EC from the E11 aorta were isolated from their wall and grafted into the coelom. Strikingly, this procedure fully restored their capacity to colonize host veins; about 60% of EC were found in arteries and 40% in veins of the hosts, as observed at earlier stages. A purely mechanical effect of the vessel wall cannot be definitively excluded, but seems unlikely, since migration distance and number of migrating cells were not significantly affected by the presence of the vessel wall, and longitudinal sectioning of vessels to directly expose the endothelium did not increase their migration. It therefore appears that a signal derived from the vessel wall determines EC identity.
The molecular mechanisms restricting arterial or venous EC identity remain to be explored. Our expression study with NRP1 and TIE2 shows that these arterial- and venous-specific genes are expressed long before restriction of plasticity occurs and thus make a direct role for these genes in the establishment of arterial and venous identity unlikely. It is however possible that these genes could be involved in the development and assembly of the arterial and venous vessel wall. In this respect, it is interesting to note that NRP1 and ephrinB2 are also expressed in cells of the arterial vessel wall in both chick and mouse embryos (Shin et al., 2001; Gale et al., 2001). The experiments reported here provide a framework for the investigation of the molecular mechanisms involved in restricting EC plasticity with respect to arterial-venous differentiation.
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ACKNOWLEDGMENTS |
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