Luc Pardanaud

Flow regulates arterial-venous differentiation in the chick embryo yolk sac

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.

Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3.

If neuropilin-2 and the growth factor VEGF-C don’t come together, lymphatic vessels don’t branch apart.

Emergence of endothelial and hemopoietic cells in the avian embryo

SummaryDuring 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.

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.

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.

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.

Early germ cell segregation and distribution in the quail blastodisc

The distribution and number of primordial germ cells has been analyzed in quail blastodiscs from the incubated state to the 13-somite stage, treated in toto with monoclonal antibody QH1. Some cells were already positive in unincubated blastulas some 18 h earlier than described with other markers in previous studies of avian development. The number of PGCs increased from 2-3 in the unincubated state to more than 100 at the early primitive streak stage. During following stages their numbers did not increase significantly. At first these cells were isolated, thereafter they often assembled in small groups and progressively gathered into Swifts crescent. It is concluded that PGCs begin segregating in birds at the blastula stage and that they multiply until the primitive streak stage.

Netrin-1 controls sympathetic arterial innervation

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.

Early haemopoietic stem cells in the avian embryo

Summary 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.

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.