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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[prime] 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. Key Words: SCL/Tal-1; GATA; gene regulation; transgenic; midbrain; hindbrain; spinal cord; vasculogenesis; angiogenesis; haematopoiesis.

Long considered an accessory tubule of the male reproductive system, the epididymis is proving to be a key determinant of male fertility. In addition to its secretory role in ensuring functional maturation and survival of spermatozoa, the epididymis has a complex immune function. Indeed, it must manage both peripheral tolerance to sperm antigens foreign to the immune system and the protection of spermatozoa as well as the organ itself against pathogens ascending the epididymal tubule. Although our knowledge of the immunobiology of this organ is beginning to accumulate at the molecular and cellular levels, the organization of blood and lymphatic networks of this tissue, important players in the immune response, remains largely unknown. In the present report, we have taken advantage of a VEGFR3:YFP transgenic mouse model. Using high-resolution three-dimensional (3D) imaging and organ clearing coupled with multiplex immunodetections of lymphatic (LYVE1, PDPN, PROX1) and/or blood (PLVAP/Meca32) markers, we provide a simultaneous deep 3D view of the lymphatic and blood epididymal vasculature in the mature adult mouse as well as during postnatal development., Competing Interests: CD, AB, AC, CG, NP, LP, LP, CV, SB, RG, JT, FS, AK, MT, JD, JH No competing interests declared, (© 2023, Damon-Soubeyrand, Bongiovanni, Chorfa et al.)

This Françoise Dieterlen's homage gathers scientific and personal memories between 1984 and 2000, when I worked in her laboratory at Nogent-sur-Marne (France). I describe a clever woman who took care of her students and taught me all fundamental qualities to become a researcher: discipline, rigor and patience., (© Société de Biologie, 2023.)

Endothelial cells in veins differ in morphology, function and gene expression from those in arteries and lymphatics. Understanding how venous and arterial identities are induced during development is required to understand how arterio-venous malformations occur, and to improve the outcome of vein grafts in surgery by promoting arterialization of veins. To identify factors that promote venous endothelial cell fate in vivo, we isolated veins from quail embryos, at different developmental stages, that were grafted into the coelom of chick embryos. Endothelial cells migrated out from the grafted vein and their colonization of host veins and/or arteries was quantified. We show that venous fate is promoted by sympathetic vessel innervation at embryonic day 11. Removal of sympathetic innervation decreased vein colonization, while norepinephrine enhanced venous colonization. BMP treatment or inhibition of ERK enhanced venous fate, revealing environmental neurotransmitter and BMP signaling and intrinsic ERK inhibition as actors in venous fate acquisition. We also identify the BMP antagonist Noggin as a potent mediator of venous arterialization., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro , it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest., (Copyright: © 2020 Ferreira et al.)

Rationale: Arterial endothelial cells are morphologically, functionally, and molecularly distinct from those found in veins and lymphatic vessels. How arterial fate is acquired during development and maintained in adult vessels is incompletely understood., Objective: We set out to identify factors that promote arterial endothelial cell fate in vivo., Methods and Results: We developed a functional assay, allowing us to monitor and manipulate arterial fate in vivo, using arteries isolated from quails that are grafted into the coelom of chick embryos. Endothelial cells migrate out from the grafted artery, and their colonization of host arteries and veins is quantified. Here we show that sympathetic innervation promotes arterial endothelial cell fate in vivo. Removal of sympathetic nerves decreases arterial fate and leads to colonization of veins, whereas exposure to sympathetic nerves or norepinephrine imposes arterial fate. Mechanistically, sympathetic nerves increase endothelial ERK (extracellular signal-regulated kinase) activity via adrenergic α1 and α2 receptors., Conclusions: These findings show that sympathetic innervation promotes arterial endothelial fate and may lead to novel approaches to improve arterialization in human disease., (© 2016 American Heart Association, Inc.)

The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: The H2.0-like homeobox transcription factor (HLX) plays an essential role in visceral organogenesis in mice and has been shown to regulate angiogenic sprouting in vitro and in zebrafish embryos. We therefore examined the role of HLX in vascular development in mouse and avian embryos., Approach and Results: In situ hybridization showed that Hlx is expressed in a subset of sprouting blood vessels in postnatal mouse retinas and embryos. Hlx expression was conserved in quail embryos and upregulated in blood vessels at the onset of circulation. In vitro assays showed that Hlx is dynamically regulated by growth factors and shear stress alterations. Proangiogenic vascular endothelial growth factor induces Hlx expression in cultured endothelial cells, whereas signals that induce stalk cell identity lead to a reduction in Hlx expression. HLX was also downregulated in embryos in which flow was ablated, whereas injection of a starch solution, which increases blood viscosity and therefore shear stress, causes an upregulation in HLX. HLX knockdown in vitro resulted in a reduction in tip cell marker expression and in reduced angiogenic sprouting, but Hlx(-/-) embryos showed no defect in vascular sprouting at E8.5, E9.5, or E11.5 in vivo. Vascular remodeling of the capillary plexus was altered in Hlx(-/-) embryos, with a modestly enlarged venous plexus and reduction of the arterial plexus., Conclusions: Our findings indicate not only that Hlx regulates sprouting in vitro, but that its role in sprouting is nonessential in vivo. We find HLX is regulated by shear stress and a subtle defect in vascular remodeling is present in knockout embryos., (© 2014 American Heart Association, Inc.)

Autonomic sympathetic nerves innervate peripheral resistance arteries, thereby regulating vascular tone and controlling blood supply to organs. Despite the fundamental importance of blood flow control, how sympathetic arterial innervation develops remains largely unknown. Here, we identified the axon guidance cue netrin-1 as an essential factor required for development of arterial innervation in mice. Netrin-1 was produced by arterial smooth muscle cells (SMCs) at the onset of innervation, and arterial innervation required the interaction of netrin-1 with its receptor, deleted in colorectal cancer (DCC), on sympathetic growth cones. Function-blocking approaches, including cell type-specific deletion of the genes encoding Ntn1 in SMCs and Dcc in sympathetic neurons, led to severe and selective reduction of sympathetic innervation and to defective vasoconstriction in resistance arteries. These findings indicate that netrin-1 and DCC are critical for the control of arterial innervation and blood flow regulation in peripheral organs.

Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.

Vascular sprouting is a key process-driving development of the vascular system. In this study, we show that neuropilin-2 (Nrp2), a transmembrane receptor for the lymphangiogenic vascular endothelial growth factor C (VEGF-C), plays an important role in lymphatic vessel sprouting. Blocking VEGF-C binding to Nrp2 using antibodies specifically inhibits sprouting of developing lymphatic endothelial tip cells in vivo. In vitro analyses show that Nrp2 modulates lymphatic endothelial tip cell extension and prevents tip cell stalling and retraction during vascular sprout formation. Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment. To investigate whether this defect depends on Nrp2 interaction with VEGF receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous nrp2vegfr2 mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.

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.

Using quail-chick parabiosis and QH1 monoclonal antibody analysis, we have identified circulating endothelial cells and/or progenitors in the embryo. These cells were already present early in ontogeny, before the third embryonic day. Under normal conditions, they integrated into most tissues but remained scarce. When experimental angiogenic responses were induced by wounding or grafts onto the chorioallantoic membrane, circulating endothelial cells were rapidly mobilized and selectively integrated sites of neoangiogenesis. Their mobilization was not dependent on the presence of the bone marrow as it was effective before its differentiation. Surprisingly, mobilization was not effective during sprouting angiogenesis following VEGF treatment of chorioallantoic membrane. Thus, embryonic circulating endothelial cells were efficiently mobilized during the establishment of an initial vascular supply to ischemic tissues following wounding or grafting, but were not involved during classical sprouting angiogenesis.

Using quail-chick parabiosis and the QH1 monoclonal antibody, specific for the endothelial and hematopoietic cells of the quail species, as a marker, we identified circulating endothelial cells in the embryo. In normal conditions, these cells could integrate endothelia in many tissues but their number remained low. When artificial angiogenic responses were created, i.e., in grafting experiments on the chorioallantoic membrane or wound healing, the circulating endothelial cells were rapidly mobilized to reach the embryonic regions submitted to these processes and their number dramatically increased. Interestingly, 1) on one hand, these circulating endothelial cells were present early in ontogeny, before the third embryonic day in the quail embryo; 2) on the other hand, their mobilization was not dependent on the presence of the bone marrow since it was effective before the differentiation of this tissue.

Using quail/chick chimeras, we have previously shown that different embryonic territories are vascularized through two distinct mecanisms, angiogenesis and vasculogenesis. Angiogenesis occurs in tissues of somatopleural origin, vasculogenesis occurs in territories of splanchnopleural origin. The aim of this work was to establish if these modes of vascularization were conserved in the mammalian embryo. Since in vivo manipulations with mammalian embryos are difficult to perform, we used a quail/mouse chimera approach. Mouse limb buds of somatopleural origin, and visceral organ rudiments of splanchnopleural origin, were grafted into the coelomic cavity of 2.5 day-old quail embryos. After four to seven days, the hosts were killed and the origin of the endothelial cells in the mouse tissues was determined by double staining with the quail endothelial and hematopoietic cell-specific marker, QH1 and mouse-specific VEGFR2 and VEGFR3 probes. Our findings show that the great majority of vessels which developed in the mouse limbs was QH1+, indicating that these tissues were vascularized by angiogenesis. Conversely, visceral organs were vascularized through the vasculogenesis process by mouse endothelial cells which differentiated in situ. These results demonstrate for the first time that in the mouse embryo, as previously shown in avian species, the tissues from somatopleural origin are vascularized by angiogenesis, while rudiments of a splanchnopleural origin are vascularized by vasculogenesis, both at vascular and lymphatic levels.

The adult vascular system is composed of an arterial, a venous and a lymphatic compartment. These different compartments respectively provide oxygen and nutrients to peripheral organs, remove carbon dioxide and waste products and maintain an immune barrier to defend the host against foreign organisms. Malfunctions of the vascular system represent a major cause of mortality and disease in developed countries. Understanding of the molecular mechanisms regulating vascular system development and maintenance is thus crucial for the design of therapies to cure vascular diseases. The molecules implicated in the control of physiological and pathological angiogenesis in the adult already function during embryonic development. Indeed, the survival of the embryo also critically depends on the establishment of a functional circulatory loop. Here we review our current knowledge about the emergence of endothelial precursor cells in the embryo, of their assembly into the primary vascular plexus and of the remodeling of this plexus into arteries and veins. We also focus on the molecular mechanisms controlling the development of arteries, veins and lymphatic vessels.

Formation of the yolk sac vascular system and its connection to the embryonic circulation is crucial for embryo survival in both mammals and birds. Most mice with mutations in genes involved in vascular development die because of a failure to establish this circulatory loop. Surprisingly, formation of yolk sac arteries and veins has not been well described in the recent literature. Using time-lapse video-microscopy, we have studied arterial-venous differentiation in the yolk sac of chick embryos. Immediately after the onset of perfusion, the yolk sac exhibits a posterior arterial and an anterior venous pole, which are connected to each other by cis-cis endothelial interactions. To form the paired and interlaced arterial-venous pattern characteristic of mature yolk sac vessels, small caliber vessels of the arterial domain are selectively disconnected from the growing arterial tree and subsequently reconnected to the venous system, implying that endothelial plasticity is needed to fashion normal growth of veins. Arterial-venous differentiation and patterning are controlled by hemodynamic forces, as shown by flow manipulation and in situ hybridization with arterial markers ephrinB2 and neuropilin 1, which show that expression of both mRNAs is not genetically determined but plastic and regulated by flow. In vivo application of ephrinB2 or EphB4 in the developing yolk sac failed to produce any morphological effects. By contrast, ephrinB2 and EphB4 application in the allantois of older embryos resulted in the rapid formation of arterial-venous shunts. In conclusion, we show that flow shapes the global patterning of the arterial tree and regulates the activation of the arterial markers ephrinB2 and neuropilin 1.

This review focuses on the emergence of hemopoietic stem cells (HSC) in the embryonic aorta, which was analysed in the avian model. Intraaortic clusters, a characteristic vertebrate anatomical feature, were shown to derive from the splanchnopleural (ventral) mesoderm, which has the potential to give rise to both angioblasts and hemopoietic cells. In contrast, the somitic mesoderm was shown to give rise to angioblasts only. The derivation of hemopoietic progenitors from endothelial cells in the floor of the aorta was followed by means of in vivo labelling experiments. Finally, the expression of gene-encoding transcription factors involved in the emergence of HSC was restricted to the floor of the aorta immediately prior to and during the appearance of intraaortic clusters.

Neuropilin 2 is a receptor for class III semaphorins and for certain members of the vascular endothelial growth factor family. Targeted inactivation of the neuropilin 2 gene (Nrp2) has previously shown its role in neural development. We report that neuropilin 2 expression in the vascular system is restricted to veins and lymphatic vessels. Homozygous Nrp2 mutants show absence or severe reduction of small lymphatic vessels and capillaries during development. This correlated with a reduction of DNA synthesis in the lymphatic endothelial cells of the mutants. Arteries, veins and larger, collecting lymphatic vessels developed normally, suggesting that neuropilin 2 is selectively required for the formation of small lymphatic vessels and capillaries.

Embryonic endothelial cells (EC) are generated by two mechanisms, vasculogenesis and angiogenesis (1). The term vasculogenesis describes the de novo emergence of EC progenitors from the mesoderm, whereas angiogenesis corresponds to the generation of EC by sprouting from the pre-existing vascular network. Until recently, it was thought that vasculogenesis was restricted to the period of embryonic development, whereas in the adult, only angiogenesis contributed to EC proliferation. The discovery of circulating EC progenitors in adult bone marrow and peripheral blood has suggested that additional mechanisms besides angiogenesis can occur in the adult, and therefore have renewed interest in the embryonic origin and the development of these progenitor cells. Vasculogenesis in the chick embryo has been studied since the beginning of the 20th century. During early development, vasculogenesis is intimately linked to the emergence of hematopoietic cells (HC). The existence of a common precursor for both EC and HC, termed "hemangioblast," was postulated (2). The purpose of this review is to summarize the experimental evidence concerning the emergence of EC and HC during embryonic life.

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.

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.

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.

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.

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.

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.

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.

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.

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.