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.

Selective expression of angiopoietin 1 and 2 in mesenchymal cells surrounding veins and arteries of the avian embryo.

The expression of angiopoietin1 and 2 (ang1 and 2) and their receptor tie-2 was studied in avian embryos using in situ hybridization. Ang1 was transcribed in the mesenchymal cells surrounding venous endothelium expressing tie-2. By contrast, ang2 was transcribed around arteries in which the endothelium down-regulated tie-2 mRNA. The aorta and large arteries of the heart outflow tract were never surrounded by ang2 positive cells and maintained tie-2 expression.

Ontogeny of the endothelial system in the avian model.

The avian model provides an experimental approach for dissecting the origin, migrations and differentiation of cell lineages in early embryos. In this model, the endothelial network was shown to take place through two processes depending on the origin of endothelial precursors: vasculogenesis when angioblasts emerge in situ, angiogenesis when angioblasts are extrinsic. Two different mesodermal territories produce angioblasts, the somite which only gives rise to endothelial cells and the splanchnopleural mesoderm which also produces hemopoietic stem cells. Potentialities of the mesoderm are determined by a positive influence from the endoderm and a negative control from the ectoderm. The presence of circulating endothelial precursors in the embryonic blood stream is also detected.

[Manipulation of angiopoietc/hematopoietic potentials in the avian embryo]. / Manipulation des potentialités angiopoïétique/hémangiopoïétique chez l'embryon d'oiseau.

The hypothesis that the endothelial and hemopoietic lineages have a common ontogenic origin is currently being revived. We have shown previously by means of quail/chick transplantations that two subsets of the mesoderm give rise to endothelial precursors: a dorsal one, the somite, produces pure angioblasts (angiopoietic potential), while a ventral one, the splanchnopleural mesoderm, gives rise to progenitors with a dual endothelial and hemopoietic potential (hemangiopoietic potential). To investigate the cellular and molecular controls of the angiopoietic/hemangiopoietic potential, we devised an in vivo assay based on the polarized homing of hemopoietic cell precursors to the floor of the aorta detectable in the quail/chick model. In the present work, quail mesoderm was grafted, after various pretreatments, onto the splanchnopleure of a chick host; the homing pattern and nature of graft-derived cells were analyzed thereafter using the QH1 monoclonal antibody which recognizes both quail endothelial and hemopoietic lineages. We report that transient contact with endoderm or ectoderm could change the behavior of cells derived from treated mesoderm, and that the effect of these germ layers could be mimicked by treatment with several growth factors VEGF, bFGF, TGF beta 1, EGF and TGF alpha, known to be involved in endothelial commitment and proliferation, and/or hemopoietic processes. The endoderm induced a hemangiopoietic potential in the associated mesoderm. Indeed, the association of paraxial or somatopleural mesoderm with endoderm promoted the "ventral homing" and the production of hemopoietic cells from mesoderm not normally endowed with this potential. The hemangiopoietic induction by endoderm could be mimicked by VEGF, bFGF and TGF beta 1. In contrast, a contact with ectoderm or EGF/TGF alpha treatments totally abrogated the hemangiopoietic capacity of the splanchnopleural mesoderm which produced pure angioblasts with no "ventral homing" behavior. We postulate that two gradients, one positive and one negative, modulate the angiopoietic/hemangiopoietic potential of the mesoderm.

[Emergence of the endothelial network during embryonic development]. / L'émergence du réseau endothélial pendant la vie embryonnaire.

The avian model provides an experimental approach for dissecting the origin, migrations, and differentiation of cell lineages in early embryos. In this model, the endothelial network was shown to stem from both the somites and the splanchnopleural mesoderm. The somite line age produces only endothelial cells, whereas the splanchnopleural line age also produces hematopoietic stem cells. Potentialities of the mesoderm are determined by a positive influence from the endoderm and a negative influence from the ectoderm. A novel mode of blood-borne angiogenesis is also described.

Distinct 5' SCL enhancers direct transcription to developing brain, spinal cord, and endothelium: neural expression is mediated by GATA factor binding sites.

The SCL gene encodes a basic helix-loop-helix transcription factor with a pivotal role in the development of endothelium and of all hematopoietic lineages. SCL is also expressed in the central nervous system, although its expression pattern has not been examined in detail and its function in neural development is unknown. In this article we present the first analysis of SCL transcriptional regulation in vivo. We have identified three spatially distinct regulatory modules, each of which was both necessary and sufficient to direct reporter gene expression in vivo to three different regions within the normal SCL expression domain, namely, developing endothelium, midbrain, and hindbrain/spinal cord. In addition we have demonstrated that GATA factor binding sites are essential for neural expression of the SCL constructs. The midbrain element was particularly powerful and axonal lacZ expression revealed the details of axonal projections, thus implicating SCL in the development of occulomotor, pupillary, or retinotectal pathways. The neural expression pattern of the SCL gene was highly conserved in mouse, chicken, and zebrafish embryos and the 5' region of the chicken SCL locus exhibited a striking degree of functional conservation in transgenic mice. These data suggest that SCL performs critical functions in neural development. The regulatory elements identified here provide important tools for analyzing these functions.

Manipulation of the angiopoietic/hemangiopoietic commitment in the avian embryo.

The hypothesis that the endothelial and hemopoietic lineages have a common ontogenic origin is currently being revived. We have shown previously by means of quail/chick transplantations that two subsets of the mesoderm give rise to endothelial precursors: a dorsal one, the somite, produces pure angioblasts (angiopoietic potential), while a ventral one, the splanchnopleural mesoderm, gives rise to progenitors with a dual endothelial and hemopoietic potential (hemangiopoietic potential). To investigate the cellular and molecular controls of the angiopoietic/hemangiopoietic potential, we devised an in vivo assay based on the polarized homing of hemopoietic cell precursors to the floor of the aorta detectable in the quail/chick model. In the present work, quail mesoderm was grafted, after various pretreatments, onto the splanchnopleure of a chick host; the homing pattern and nature of graft-derived QH1(+) cells were analyzed thereafter. We report that transient contact with endoderm or ectoderm could change the behavior of cells derived from treated mesoderm, and that the effect of these germ layers could be mimicked by treatment with several growth factors VEGF, bFGF, TGFbeta1, EGF and TGF(&agr;), known to be involved in endothelial commitment and proliferation, and/or hemopoietic processes. The endoderm induced a hemangiopoietic potential in the associated mesoderm. Indeed, the association of somatopleural mesoderm with endoderm promoted the 'ventral homing' and the production of hemopoietic cells from mesoderm not normally endowed with this potential. The hemangiopoietic induction by endoderm could be mimicked by VEGF, bFGF and TGFbeta1. In contrast, contact with ectoderm or EGF/TGF(&agr;) treatments totally abrogated the hemangiopoietic capacity of the splanchnopleural mesoderm, which produced pure angioblasts with no 'ventral homing' behaviour. We postulate that two gradients, one positive and one negative, modulate the angiopoietic/hemangiopoietic potential of the mesoderm.

The c-ets1 protooncogene is expressed in human trophoblast during the first trimester of pregnancy.

Expression of the c-Ets1 protooncogene which codes for a transcription factor is associated with neovascularization and invasive processes. In order to determine c-Ets1 expression at the mRNA level, during the process of implantation during the first trimester of human pregnancy, samples of trophoblast were retrieved at the time of legal abortion and processed for in situ hybridization. We found that c-Ets1 mRNAs are transcribed in the endothelial cells of villous trophoblast and in the extravillous trophoblastic cells invading the uterine vessels. However, no transcript was found in maternal endothelial cells. We conclude that c-Ets1 plays a role in angiogenesis occurring in the development of the villous tree and is involved during the invasive process of the endometrium and maternal vessels by trophoblastic cells; this latter physiological event is crucial for a normal development of the fetus, its failure leading to pathological cases. We suggest that the role of the c-Ets1 protooncogene is related to the regulation of metalloproteinase genes transcription, a gene family which is known to be a target for Ets protein.

Where do hematopoietic stem cells come from?

The experimental model constituted by a quail embryo grafted on a chick yolk sac has produced undisputable evidence according to which hematopoietic stem cells (HSC) colonizing the blood-forming rudiments are of intraembryonic origin in birds. Appropriate cell culture systems now make it feasible to demonstrate that ontogeny of the mouse hematopoietic system also involves at least two generations of HSC, one formed in the yolk sac and the other in the embryo, only the latter having lymphoid potential. Furthermore the developmental relationships between endothelial and hematopoietic cells are being analyzed in the avian model. Two distinct endothelial lineages are detected by means of interspecific transplantations, a dorsal one, somitic in origin, which is purely endothelial and a ventral one, splanchnopleural in origin, which is associated with the production of hematopoietic cells.

Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis.

We have shown previously by means of quail/chick transplantations that external and visceral organs, i.e., somatopleural and splanchnopleural derivatives, acquire their endothelial network through different mechanisms, namely immigration (termed angiogenesis) versus in situ emergence of precursors (or vasculogenesis). We have traced the distribution of QH1-positive cells in chick hosts after replacement of the last somites by quail somites (orthotopic grafts) or lateral plate mesoderm (heterotopic grafts). The results lead to the conclusion that the embryo becomes vascularized by endothelial precursors from two distinct regions, splanchnopleural mesoderm and paraxial mesoderm. The territories respectively vascularized are complementary, precursors from the paraxial mesoderm occupy the body wall and kidney, i.e., they settle along with the other paraxial mesoderm derivatives and colonize the somatopleure. The precursors from the two origins have distinct recognition and potentialities properties: endothelial precursors of paraxial origin are barred from vascularizing visceral organs and from integrating into the floor of the aorta, and are never associated with hemopoiesis; splanchnopleural mesoderm grafted in the place of somites, gives off endothelial cells to body wall and kidney but also visceral organs. It gives rise to hemopoietic precursors in addition to endothelial cells.

Does the paraxial mesoderm of the avian embryo have hemangioblastic capacity?

In a previous study of the hemangioblastic capacity of lateral plate mesoderm, we showed that the endoderm-associated splanchnopleural layer is capable of giving rise to both endothelial and hemopoietic cells while the ectoderm-associated somatopleural layer is not (Pardanaud and Dieterlen-Lièvre 1993a). In order to complete the inventory of territories able to produce these two cell lineages, we assayed the paraxial mesoderm, and report the results here. Quail somites or segmental plates were treated with mab QH1+complement in order to eliminate attached aortic endothelial cells, which cling to the ventral aspects of these structures. They were grafted in the limb bud or the coelom of chick host, since these sites promote the differentiation of endothelial and hemopoietic cells, respectively. Vascular development and hemopoietic cell emergence were analyzed using QH1 immunocytology. Segmental plate and somites both produced abundant endothelial cells. In addition, the segmental plate gave rise to small groups of hemopoietic cells when grafted in the coelom.

Expression of C-ETS1 in early chick embryo mesoderm: relationship to the hemangioblastic lineage.

In situ hybridization was used to detect the expression of the c-ets1 protooncogene during formation of the germ layers in the chick blastodisc. c-ets1 transcripts were present during the gastrulation process, i.e. when the mesodermal cells invaginated. The expression became down-regulated in lateral plate and the dorsal part of the somites while an intense signal was retained in the intermediate cell mass. When vasculogenesis started, c-ets1 transcripts labelled blood islands and endothelial cells. Before the mesoderm split, transcripts were present over the whole layer, more abundant however on its ventral side in contact with the endoderm. After the mesoderm split, silver grains became distributed asymmetrically: splanchnopleural mesoderm expressed c-ets1 messengers all over while expression in the somatopleural mesoderm was restricted to a few profiles corresponding to small endothelial cell groups. This asymmetrical distribution of c-ets1 transcripts is in agreement with our previous experimental findings establishing the different potentialities of the two mesodermal layers regarding hemopoiesis, vasculogenesis and angiogenesis processes.

Developmental relationships between hemopoiesis and vasculogenesis.

Using avian chimeras, we have demonstrated earlier that the stem cells seeding the definitive hemopoietic organs form within the embryo rather than in the yolk sac. We now report experimental evidence indicating that hemopoietic progenitors appear in mouse embryos in the para-aortic splanchnopleura, a location similar to the one that produces stem cells in avian embryos. This structure obtained from E8.5 embryos was grafted under the kidney capsule of adult SCID mice. One compartment of the B lymphoid system became reconstituted with cells derived from the graft, that were identified with genetic and antigenic markers. In vitro data are also in favor of the production of progenitors from this structure. Finally a strategy designed to understand the developmental links between the endothelial network and hemopoietic cells is described. It is based on the expression patterns of two protooncogenes, c-ets1 and c-myb, activated during the amplification period of each of these lineages.

Emergence of endothelial and hemopoietic cells in the avian embryo.

During organogenesis, endothelial cells develop through two different mechanisms: differentiation of intrinsic precursors in organ rudiments constituted of mesoderm associated with endoderm, and colonization by extrinsic precursors in organs constituted of mesoderm associated with ectoderm (Pardanaud et al. 1989). On the other hand, both types of rudiment are colonized by extrinsic hemopoietic stem cells. In the present work we extend our former study by investigating the hemangioblastic (i.e. hemopoietic and angioblastic) potentialities of primordial germ layers in the area pellucida during the morphogenetic period. By means of interspecific grafts between quail and chick embryos, we show that splanchnopleural mesoderm gives rise to abundant endothelial cells, and to numerous hemopoietic cells in a permissive microenvironment, while somatopleural mesoderm produces very few cells belonging to these lineages, or none. Thus we confirm that the angioblastic capacities of the mesoderm differ radically, depending on its association with ectoderm or endoderm. Furthermore, at this embryonic period, both endothelial and hemopoietic potentialities are displayed by splanchnopleural mesoderm. However the site of emergence of intraembryonic hemopoietic stem cells appears spatially restricted by comparison to more widespread angioblastic capacities.

[Embryology of vessels]. / L'embryologie des vaisseaux.

Endothelial emergence is the best known aspect of vessel formation during embryogenesis. It has been analyzed in an avian model of chimeras at the time of organogenesis or morphogenesis. These chimeras involve two species, chick and quail, whose cells may be distinguished on the basis of distinct nuclear heterochromatin patterns or through the use of antibodies that are species and lineage specific. QH1, a monoclonal antibody obtained in our group, whose affinity is restricted to the quail hemangioblastic lineage (endothelial and hemopoietic cells), has been a sensitive probe to study the origin of these cells in various chimeric patterns. By transplanting organ rudiments or primordial germ layers, we have shown that endothelium emergence in organ rudiments occurs through two different mechanisms, vasculogenesis or in situ differentiation, and angiogenesis or colonization by extrinsic precursors. Vasculogenesis occurs in the mesoderm of internal organ rudiments while angiogenesis occurs in external rudiments. The conclusion is that associated endoderm exerts a positive influence on the emergence of endothelial progenitors from mesodermal precursors.

Complementary patterns of expression of c-ets 1, c-myb and c-myc in the blood-forming system of the chick embryo.

We have used in situ hybridization to study the spatial and temporal distribution of the transcription of three cellular oncogenes encoding DNA-binding proteins, c-ets 1, c-myb and c-myc during the development of the chick embryo. c-ets 1 mRNA expression appears linked to the mesodermal lineage and is strongly expressed in early endothelia; it subsequently becomes restricted to small vessel endothelia. Hemopoietic cells in extraembryonic blood islands at E2 express c-ets 1, while intraembryonic hemopoietic cells in aortic clusters (E3) and paraaortic foci (E6) express c-myb. c-myc transcripts are detected in cells undergoing hemopoiesis in both these extraembryonic and intraembryonic sites. Outside the blood-forming system, c-myc is transcribed in a large variety of cells; c-ets 1 displays tissue-specific expression in groups of mesodermal cells engaged in morphogenetic processes and appears excluded from all epithelia; finally the expression of c-myb is the most tightly linked to hemopoietic cells. In any case, it is clear that these three oncogenes display complementary expression in endothelial and hemopoietic cells where their patterns are modulated in relationship to multiplication and differentiation.

Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny.

Quail-chick intracoelomic grafts of organ rudiments were used to study the origin of endothelia and haemopoietic cells during avian organogenesis in conjunction with the monoclonal antibody QH1 which recognizes the quail haemangioblastic lineage. Results differed according to the germ-layer constitution of the grafted rudiments. In the case of the limb buds, endothelial cells from the host invaded the graft through an angiogenic process. Haemopoietic progenitors from the host also colonized the grafted bone marrow. In contrast, rudiments of internal organs provided their own contingent of endothelial precursors, a process termed vasculogenesis. Nevertheless, haemopoietic cells in these organs were all derived from the host. In the lung, this extrinsic cell population appeared regularly scattered around the parabronchi and had a macrophage-like phenotype. In the pancreas, the granulocytes which differentiate as dense aggregates located in the wall of the largest vessels were extrinsic. Similarly in the spleen, a mesodermal primordium that develops in close association with the pancreatic endoderm, endothelial cells were intrinsic and haemopoietic cells host-derived. This study demonstrates that, in ontogeny, vascularization obeys different rules depending on which germ layer the mesoderm is associated with: in mesodermal/ectodermal rudiments angiogenesis is the rule; in mesodermal/endodermal rudiments, vasculogenesis occurs. However, in these internal organs undergoing vasculogenesis, endothelial and haemopoietic cells have separate origins. We put forward the hypothesis that the endoderm induces the emergence of endothelial cells in the associated mesoderm. Formation of blood stem cells may also involve interactions between endoderm and mesoderm, but in this case the responding capacity of the mesoderm appears restricted to the paraaortic region.

Early haemopoietic stem cells in the avian embryo.

Using 'yolk sac chimaeras', we have previously demonstrated that stem cells, destined to colonize haemopoietic organs other than the yolk sac, arise in the embryo proper. We have now investigated the emergence and potentialities of these cells in vivo and in vitro. The in vivo approach consisted of interspecies grafting between quail and chick embryos. The cell progeny from the grafts was detected by means of QH1, a monoclonal antibody specific for the quail haemangioblastic lineage. When grafted into the dorsal mesentery of the chick embryo, which is a haemopoietic microenvironment, the region of the aorta from E3-E4 quail embryos generated large haemopoietic foci. When associated with a chick attractive thymic rudiment, cells left the quail aorta, entered this rudiment and underwent lymphopoiesis. Cell suspensions prepared from 40-50 chick aortae, seeded in appropriate semi-solid media, yielded macrophage, granulocyte or erythrocyte clones. These colony forming cells were two to eight times more frequent than in cell preparations from hatchling bone marrow. By contrast, cells prepared from the whole embryonic body deprived of the aorta were not clonogenic. By interspecies grafting of somatopleural (ectoderm + mesoderm, e.g. limb bud) or splanchnopleural rudiments (endoderm + mesoderm, e.g. lung, pancreas, intestine), the endothelial lining of blood vessels was shown to arise by two entirely different processes according to the rudiment considered: angiogenesis, i.e. invasion by extrinsic endothelial cells, in the limb bud, and vasculogenesis, i.e. in situ emergence of endothelial cells, in internal organs. The spleen, which first develops as a continuum to the pancreatic mesoderm, acquires its endothelial network by vasculogenesis, and is colonized by extrinsic haemopoietic stem cells. Granulopoietic cells in the pancreas and accessory cells in the lung are also extrinsic. Thus, in the case of endomesodermal rudiments, interspecies grafting reveals separate origins of endothelial and haemopoietic cells.