In vivo generation of haematopoietic stem/progenitor cells from bone marrow-derived haemogenic endothelium.

It is well established that haematopoietic stem and progenitor cells (HSPCs) are generated from a transient subset of specialized endothelial cells termed haemogenic, present in the yolk sac, placenta and aorta, through an endothelial-to-haematopoietic transition (EHT). HSPC generation via EHT is thought to be restricted to the early stages of development. By using experimental embryology and genetic approaches in birds and mice, respectively, we document here the discovery of a bone marrow haemogenic endothelium in the late fetus/young adult. These cells are capable of de novo producing a cohort of HSPCs in situ that harbour a very specific molecular signature close to that of aortic endothelial cells undergoing EHT or their immediate progenies, i.e., recently emerged HSPCs. Taken together, our results reveal that HSPCs can be generated de novo past embryonic stages. Understanding the molecular events controlling this production will be critical for devising innovative therapies.

An in vitro model of hemogenic endothelium commitment and hematopoietic production.

Adult-type hematopoietic stem and progenitor cells are formed during ontogeny from a specialized subset of endothelium, termed the hemogenic endothelium, via an endothelial-to-hematopoietic transition (EHT) that occurs in the embryonic aorta and the associated arteries. Despite efforts to generate models, little is known about the mechanisms that drive endothelial cells to the hemogenic fate and about the subsequent molecular control of the EHT. Here, we have designed a stromal line-free controlled culture system utilizing the embryonic pre-somitic mesoderm to obtain large numbers of endothelial cells that subsequently commit into hemogenic endothelium before undergoing EHT. Monitoring the culture for up to 12 days using key molecular markers reveals stepwise commitment into the blood-forming system that is reminiscent of the cellular and molecular changes occurring during hematopoietic development at the level of the aorta. Long-term single-cell imaging allows tracking of the EHT of newly formed blood cells from the layer of hemogenic endothelial cells. By modifying the culture conditions, it is also possible to modulate the endothelial cell commitment or the EHT or to produce smooth muscle cells at the expense of endothelial cells, demonstrating the versatility of the cell culture system. This method will improve our understanding of the precise cellular changes associated with hemogenic endothelium commitment and EHT and, by unfolding these earliest steps of the hematopoietic program, will pave the way for future ex vivo production of blood cells.

Dorso-ventral contributions in the formation of the embryonic aorta and the control of aortic hematopoiesis.

The embryonic dorsal aorta plays a pivotal role in the production of the first hematopoietic stem cells (HSCs), the founders of the adult hematopoietic system. HSC production is polarized by being restricted to the aortic floor where a specialized subset of endothelial cells (ECs) endowed with hemogenic properties undergo an endothelial-to-hematopoietic production resulting in the formation of the intra-aortic hematopoietic clusters. This production is tightly time- and space-controlled with the transcription factor Runx1 playing a key role in this process and the surrounding tissues controlling the aortic shape and fate. In this paper, we shall review (a) how hemogenic ECs differentiate from the mesoderm, (b) how the different aortic components assemble coordinately to establish the dorso-ventral polarity, and (c) how this results in the initiation of Runx1 expression in hemogenic ECs and the initiation of the hematopoietic program. These observations should elucidate the first steps in HSC commitment and help in developing techniques to manipulate adult HSCs.

Endoglin expression level discriminates long-term hematopoietic from short-term clonogenic progenitor cells in the aorta.

CD105 is an auxiliary receptor for the transforming growth factor beta superfamily, highly expressed on proliferating endothelial cells and adult hematopoietic stem cells. Because CD105 mRNA expression was reported in the developing aortic region, we further characterized its expression profile in the aorta and examined the hematopoietic potential of CD105(+) cells. Aortic endothelial cells, intra-aortic hematopoietic cell clusters and the purified cell fraction enriched in progenitor/hematopoietic stem cell activity expressed CD105. Aortic hematopoietic short-term clonogenic progenitors were highly enriched in the CD105(intermediate) population whereas more immature long-term progenitors/hematopoietic stem cells are contained within the CD105(high) population. This places CD105 on the short list of molecules discriminating short-term versus long-term progenitors in the aorta. Furthermore, decreasing transforming growth factor beta signaling increases the number of clonogenic progenitors. This suggests that CD105 expression level defines a hierarchy among aortic hematopoietic cells allowing purification of clonogenic versus more immature hematopoietic progenitors, and that the transforming growth factor beta pathway plays a critical role in this process.

Aortic remodelling during hemogenesis: is the chicken paradigm unique?

Since the era of the ancient Egyptians and Greeks, the avian embryo has been a subject of intense interest to visualize the first steps of development. It has served as a pioneer model to scrutinize the question of hematopoietic development from the beginning of the 20th century. It's large size and easy accessibility have permitted the development of techniques dedicated to following the origins and fates of different cell populations. Here, we shall review how the avian model has brought major contributions to our understanding of the development of the hematopoietic system in the past four decades and how these discoveries have influenced our knowledge of mammalian hematopoietic development. The discovery of an intra-embryonic source of hematopoietic cells and the developmental link between endothelial cells and hematopoietic cells will be presented. We shall then point to the pivotal role of the somite in the construction of the aorta and hematopoietic production and demonstrate how two somitic compartments cooperate to construct the definitive aorta. We shall finish by showing how fate-mapping experiments have allowed the identification of the tissue which gives rise to the sub-aortic mesenchyme. Taken together, this review aims to give an overview of how and to what extent the avian embryo has contributed to our knowledge of developmental hematopoiesis.

Somite-derived cells replace ventral aortic hemangioblasts and provide aortic smooth muscle cells of the trunk.

We have previously shown that endothelial cells of the aortic floor give rise to hematopoietic cells, revealing the existence of an aortic hemangioblast. It has been proposed that the restriction of hematopoiesis to the aortic floor is based on the existence of two different and complementary endothelial lineages that form the vessel: one originating from the somite would contribute to the roof and sides, another from the splanchnopleura would contribute to the floor. Using quail/chick orthotopic transplantations of paraxial mesoderm, we have traced the distribution of somite-derived endothelial cells during aortic hematopoiesis. We show that the aortic endothelium undergoes two successive waves of remodeling by somitic cells: one when the aortae are still paired, during which the initial roof and sides of the vessels are renewed; and a second, associated to aortic hematopoiesis, in which the hemogenic floor is replaced by somite endothelial cells. This floor thus appears as a temporary structure, spent out and replaced. In addition, the somite contributes to smooth muscle cells of the aorta. In vivo lineage tracing experiments with non-replicative retroviral vectors showed that endothelial cells do not give rise to smooth muscle cells. However, in vitro, purified endothelial cells acquire smooth muscle cells characteristics. Taken together, these data point to the crucial role of the somite in shaping the aorta and also give an explanation for the short life of aortic hematopoiesis.

Widespread lipoplex-mediated gene transfer to vascular endothelial cells and hemangioblasts in the vertebrate embryo.

We report here a method that allows fast, efficient, and low-cost screening for gene function in the vascular system of the vertebrate embryo. Through intracardiac delivery of nucleic acids optimally compacted by a specific cationic lipid, we are able to induce in vivo endothelial cell-specific gain-of-function during development of the vascular network in the chick embryo. When the nucleic acids are delivered during the period of intraembryonic hematopoiesis, aortic hemangioblasts, the forerunners of the hematopoietic stem cells known to derive from the aortic endothelium, are also labeled. Similarly, we show that siRNA could be used to induce loss-of-function in vascular endothelial cells. This gene transfer technique was also applied to the mouse embryo with a high efficiency. The present method allows large-scale analysis and may represent a new and versatile tool for functional genomics.

Tracing the hemangioblast during embryogenesis: developmental relationships between endothelial and hematopoietic cells.

We review here the development of the hematopoietic system and its relationship to the endothelium, with a special focus on the characterisation of the hemangioblast, the putative ancestor for endothelial cells and hematopoietic cells. Using the avian model, we have traced in vivo the progeny of embryonic endothelial cells and shown that aortic-born hematopoietic cells (known to generate the definitive hematopoietic lineage) derive from endothelial cells in the floor of the aorta. During this process, endothelial cells undergo a switch from endothelial cells to hematopoietic cells characterised by a downgrading of endothelial cell-specific genes and the parallel upgrading of hematopoietic cell-specific genes. Using a similar approach, we have shown that generation of hematopoietic cells from endothelial cells also takes place during mouse embryonic development. We have thoroughly characterised the dynamics of key molecules (several of which we have cloned) specifically expressed by the yolk sac or aortic hemangioblast. The yolk sac hemangioblast is characterized by the specific expression of SCL/Tal-1 and Lmo2, whereas the aortic hemangioblast expresses Runx-1 (a runt domain transcription factor). Finally, we have demonstrated the existence of a new site for hematopoiesis, namely the allantois. Using quail/chick grafts, we show that this embryonic appendage autonomously produces endothelial cells and hematopoietic cells, these latter being endowed with the attributes of the definitive hematopoietic lineage.

[From the primitive to the definitive aorta: angioblasts and hemangioblasts during aorta-associated haematopoiesis]. / De l'aorte primitive à l'aorte definitive: angioblastes et hémangioblastes au cours de l'hématopoïèse.

Intra-aortic haematopoiesis is a transient phenomenon, present in all the vertebrate species examined. Aorta-associated haematopoiesis produces Haematopoietic Stem Cells (HSC) that emerge from the ventral aortic endothelium through endothelial cells (EC) that switch to HSC. HSC emergence is followed by the colonization of definitive haematopoietic organs. Since intra-aortic haematopoiesis is born from EC of the aortic floor, we wondered how vascular integrity was maintained during haematopoietic production. Transplantation experiments have brought about evidence according to which two distinct endothelial lineages contribute to the embryonic vasculature. One comes from the splanchnic mesoderm and gives rise to EC and haematopoietic cells (HC). The other originates from the somite and is restricted to EC differentiation. We have used interspecific quail/chick grafts to study aortic organogenesis during the course of haematopoiesis. We demonstrate that: 1) before haematopoiesis, the aorta, originally entirely of splanchnic origin, is colonized by EC from the somite. This colonization contributes to create a new roof and sides, which are hence formed by somite-derived EC whereas the floor is contributed by splanchnopleural-derived EC; 2) as haematopoiesis proceeds, somite-derived EC begin to colonize the aortic floor and are found beneath HSC clusters; 3) after haematopoiesis, aortic hemangioblasts disappear from the endothelium and are replaced by somite-derived EC. At this stage, the whole aortic endothelium is derived from somitic cells; 4) we have identified a new cell population from the somite that contributes to the vascular smooth muscle cells (VSMC). This population appears distinct from the somite-derived EC. Using lineage tracing with non-replicative retroviral vectors, we show that EC do not give rise to VSMC as previously thought. Taken together, our results bring about new lights on aorta morphogenesis and the time-restricted production of haematopoiesis.

[Understanding the cellular and molecular aspects of haematopoietic stem cell emergence and their regulation by RUNX1/AML1]. / Aspects cellulaires et moléculaires de l'émergence des cellules souches hématopoïétiques: implication de RUNX1/AML1.

In the vertebrate embryo, the ventral wall of the aorta is the major site of Haematopoietic Stem Cell (HSC) production. HSC, which are at the basis of the adult blood cells hierarchy, are generated from Endothelial Cells (EC) through a complex cascade of molecular events. The transcription factor RUNX1/AML1 and its cofactor CBFbeta, disrupted in 20 % of acute myeloid leukaemia cases, are thought to control this process. A detailed gene expression analysis of RUNX1 and its associated factors in the chick embryo, prompted us to speculate on the molecular cascades involved in HSC production. The function of RUNX1 is however tightly regulated at several levels, rendering analysis through classical genetic approaches very difficult to manage. To offer new possibilities of investigation, we have designed a technique to target the blood forming system in vivo. Gene transfer was achieved by lipofection following delivery by intra-cardiac injection in the avian embryo. This method was optimised to allow a wide range of functional analysis, either by gain or loss of function, in a simple and efficient manner. In combination with experimental advantages of the avian embryo, this new system of genetic analysis allows us to perform a detailed study of RUNX1 function in HSC production from EC.

Avian HSC emergence, migration, and commitment toward the T cell lineage.

To date three sites of emergence of hemopoietin cells have been identified during early avian development: the yolk sac, the intraaortic clusters and recently the allantois. However, the contributions of the hematopoietic stem cell (HSC) populations generated by these different sites to definitive hematopoiesis and their migration routes are not fully unraveled. Experimental embryology as well as the establishment of the genetic cascades involved in HSC emergence help now to draw a better scheme of these processes.