How studies on the avian embryo have opened new avenues in the understanding of development: a view about the neural and hematopoietic systems.

The chick embryo is as ancient a source of knowledge on animal development as the very beginning of embryology. Already, at the time of Caspar Friedrich Wolff, contemplating the strikingly beautiful scenario of the germ deploying on the yellow background of the yolk inspired and supported the tenants of epigenesis at the expense of the preformation theory. In this article, we shall mention some of the many problems of developmental biology that were successfully clarified by research on chick embryos.

The quest for hematopoietic stem cells in the embryo. An interview with Françoise Dieterlen-Lièvre. Interview by Thierry Jaffredo and Charles Durand.

Françoise Dieterlen-Lièvre is probably the scientist who has most contributed to our basic knowledge of developmental hematopoiesis. She has dedicated her career to answering cutting edge questions on the origin of hematopoietic stem cells in the embryo. Her seminal contributions, widely recognized by the scientific community, have paved the way for generations of developmental hematologists questioning the origins of hematopoietic stem cells.

Allantois and placenta as developmental sources of hematopoietic stem cells.

While the aortic region, the para-aortic splanchnopleura/aorta-gonads-mesonephros (P-Sp/AGM) is currently considered as the source of definitive hematopoietic stem cells during development, the mouse placenta has been found to generate large numbers of these cells and to remain functional in this respect for a longer period than the P-Sp/AGM. The fetal component, which derives from the fused allantois and chorion, is responsible for this activity. We and others have shown that the pre-fusion allantois (before the stage of 6 pairs of somites) is able to yield clonogenic progenitors, provided that it is pre-cultured in toto before it is dissociated into single cells and seeded in semi-solid medium.

Decoding the hemogenic endothelium in mammals.

A collection of recent Nature papers examines the relationship between endothelial precursors and hematopoietic cells. Two of these studies (Eilken et al., 2009; Lancrin et al.

Emergence of haematopoietic stem cells during development.

Self-renewable haematopoietic stem cells (HSCs) become segregated during development into a finite pool, from which they are mobilized upon physiological requirement. A central feature characterizing developmental haematopoiesis is that definitive organs become colonized by HSCs originating from a central source. The emission of HSCs occurs more or less continuously during a protracted period in parallel or successive sites.

Hematopoietic potential of the pre-fusion allantois.

We previously showed that the fetal component of the placenta has a vigorous hematopoietic activity. Whether this organ is an environmental niche where hematopoietic stem cells (HSC) proliferate and become committed to various lineages, or whether it is also a site for HSC emergence, was left open. This issue can be addressed only if the components that will give rise to the placenta are tested prior to vascularization.

Are intra-aortic hemopoietic cells derived from endothelial cells during ontogeny?

The aorta is recognized as an intraembryonic site that produces adult-type hemopoietic stem cells. A corpus of data indicates that hemopoietic cells arranged as clusters attached to the aortic floor derive from an endothelial intermediate. This review reports on experimental approaches carried out in the avian embryo to establish the developmental history of the aortic endothelium and trace the origin of associated hemopoietic cells.

Placenta as a site for hematopoietic stem cell development.

The discovery of a major hematopoietic stem cell (HSC) pool in mid-gestation mouse placenta has defined the placenta as yet another important anatomical site that participates in HSC development. Placental HSC activity starts in parallel with the AGM region, before HSCs are found in circulation or have colonized the fetal liver. Moreover, placental hematopoietic activity culminates in a rapid expansion of the definitive HSC pool, which occurs during the time when the fetal liver HSC reservoir begins to grow.

Commitment of hematopoietic stem cells in avian and mammalian embryos: an ongoing story.

During ontogeny, hematopoietic stem cells (HSC) become committed outside of hematopoietic organs. Once held to emerge from the yolk sac, they are currently thought to originate from the early aorta. However we now show that the allantois in the avian embryo and the placenta in the mouse embryo produce HSC in very large numbers.

The placenta is a niche for hematopoietic stem cells.

The hematopoietic system develops during embryogenesis at temporally and anatomically restricted sites. The anatomical origin of definitive HSCs is not fully resolved, and little is known about how the different fetal hematopoietic microenvironments direct HSC development. Here, we show that the mouse placenta functions as a hematopoietic organ that harbors a large pool of pluripotent HSCs during midgestation.

In vivo methods to analyze cell origins, migrations, homing, and interactions in the blood, vascular, and immune systems of the avian and mammalian embryo.

In vivo experimental approaches that have been designed to study the ontogeny of the hematopoietic system in higher vertebrates are described in the present chapter. The avian embryo is directly available to manipulations in ovo during gastrulation and organogenesis. This permissiveness has led to the design of various approaches that provided crucial insights into the ontogeny of the hematopoietic system, particularly regarding traffic of progenitors between different compartments.

From the hemangioblast to self-tolerance: a series of innovations gained from studies on the avian embryo.

During the last decades of the 20th century, studies on the vertebrate hematopoietic and immune systems have largely been performed, on mammalian models. The mouse has been the preferred material for several cogent reasons: (i) numerous well defined genetic strains are available; (ii) this species has been and still is instrumental in the study of gene activity through transgenesis; and (iii) in vitro culture techniques and in vivo assays for blood cells together with a wide array of antibodies and nucleic acid probes have been developed to investigate the cellular interactions occurring during hematopoiesis and immune reactivity. However, important and fundamental notions have emerged from using another higher vertebrate model, the avian embryo.

Mouse placenta is a major hematopoietic organ.

Placenta and yolk sac from 8- to 17-day-old (E8-E17) mouse embryos/fetuses were investigated for the presence of in vitro clonogenic progenitors. At E8-E9, the embryonic body from the umbilicus caudalwards was also analysed. Fetal liver was analysed beginning on E10.

Hemangioblasts and hemopoietic stem cells during ontogeny.

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.

Hemangioblast commitment in the avian allantois: cellular and molecular aspects.

We recently identified the allantois as a site producing hemopoietic and endothelial cells capable of colonizing the bone marrow of an engrafted host. Here, we report a detailed investigation of some early cytological and molecular processes occurring in the allantoic bud, which are probably involved in the production of angioblasts and hemopoietic cells. We show that the allantois undergoes a program characterized by the prominent expression of several "hemangioblastic" genes in the mesoderm accompanied by other gene patterns in the associated endoderm.

[Lineage-switching by pluripotent cells derived from adults].

When proceeding normally, embryonic morphogenesis begins with germ layer formation through the process of gastrulation. Each primordial germ layer gives rise to a particular set of lineages. Until recently, it was considered that fate switches between germ layers were impossible.

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.

Tracing the progeny of the aortic hemangioblast in the avian embryo.

A population of hematopoietic progenitors becomes committed within the embryo proper in the floor of the aorta (P-Sp/AGM in the mouse). In birds, this first aspect of intraembryonic hematopoiesis is prominent during embryonic day 3 (E3) as endothelium-associated "intra-aortic clusters." Between E6 and E8, diffuse hematopoiesis then occurs as "para-aortic foci" located in the dorsal mesentery ventral to the aorta.

The splanchnopleura/AGM region is the prime site for the generation of multipotent hemopoietic precursors, in the mouse embryo.

The first hemopoietic cells were found in the yolk sac of mammalian embryos. Based on this observation it was postulated that hemopoietic stem cells are generated in this location. In the last few years, however, increasing evidence indicates that there is an independent site of hemopoietic cell generation, in the embryo proper, designated the paraaortic splanchnopleura/aorta, gonad, mesonephros region.

Lmo2 and GATA-3 associated expression in intraembryonic hemogenic sites.

It is now widely accepted that hemopoietic cells born intraembryonically are the best candidates for the seeding of definitive hemopoietic organs. To further understand the mechanisms involved in the generation of definitive hemopoietic stem cells, we analysed the expression of the hemopoietic-related transcription factors Lmo2 and GATA-3 during the early steps of mouse development (7-12 dpc), with a particular emphasis on intraembryonic hemogenic sites. We show here that both Lmo2 and GATA-3 are present in the intraembryonic regions known to give rise to hemopoietic precursors in vitro and in vivo, suggesting that they act together at key points of hemopoietic development.

[Manipulation of angiopoietc/hematopoietic potentials 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.

[Affiliation between endothelial and intra-embryonic hematopoietic stem cells].

We have investigated the developmental relationship of the hemopoietic and endothelial lineages in the floor of the chicken aorta, a site of hemopoietic progenitor emergence in the embryo proper. We show that, prior to the onset of hemopoiesis, the aortic endothelium uniformly expresses the endothelium-specific membrane receptor VEGF-R2. The onset of hemopoiesis can be precisely determined by detecting the common leukocyte antigen CD45.