BMP signalling differentially regulates distinct haematopoietic stem cell types.

Adult haematopoiesis is the outcome of distinct haematopoietic stem cell (HSC) subtypes with self-renewable repopulating ability, but with different haematopoietic cell lineage outputs. The molecular basis for this heterogeneity is largely unknown. BMP signalling regulates HSCs as they are first generated in the aorta-gonad-mesonephros region, but at later developmental stages, its role in HSCs is controversial. Here we show that HSCs in murine fetal liver and the bone marrow are of two types that can be prospectively isolated--BMP activated and non-BMP activated. Clonal transplantation demonstrates that they have distinct haematopoietic lineage outputs. Moreover, the two HSC types differ in intrinsic genetic programs, thus supporting a role for the BMP signalling axis in the regulation of HSC heterogeneity and lineage output. Our findings provide insight into the molecular control mechanisms that define HSC types and have important implications for reprogramming cells to HSC fate and treatments targeting distinct HSC types.

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.

Endothelio-mesenchymal interaction controls runx1 expression and modulates the notch pathway to initiate aortic hematopoiesis.

Hematopoietic stem cells (HSCs) are produced by a small cohort of hemogenic endothelial cells (ECs) during development through the formation of intra-aortic hematopoietic cell (HC) clusters. The Runx1 transcription factor plays a key role in the EC-to-HC and -HSC transition. We show that Runx1 expression in hemogenic ECs and the subsequent initiation of HC formation are tightly controlled by the subaortic mesenchyme, although the mesenchyme is not a source of HCs. Runx1 and Notch signaling are involved in this process, with Notch signaling decreasing with time in HCs. Inhibiting Notch signaling readily increases HC production in mouse and chicken embryos. In the mouse, however, this increase is transient. Collectively, we show complementary roles of hemogenic ECs and mesenchymal compartments in triggering aortic hematopoiesis. The subaortic mesenchyme induces Runx1 expression in hemogenic-primed ECs and collaborates with Notch dynamics to control aortic hematopoiesis.

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.

Human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development.

Hematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development.

Ventral embryonic tissues and Hedgehog proteins induce early AGM hematopoietic stem cell development.

Hematopoiesis is initiated in several distinct tissues in the mouse conceptus. The aorta-gonad-mesonephros (AGM) region is of particular interest, as it autonomously generates the first adult type hematopoietic stem cells (HSCs). The ventral position of hematopoietic clusters closely associated with the aorta of most vertebrate embryos suggests a polarity in the specification of AGM HSCs. Since positional information plays an important role in the embryonic development of several tissue systems, we tested whether AGM HSC induction is influenced by the surrounding dorsal and ventral tissues. Our explant culture results at early and late embryonic day 10 show that ventral tissues induce and increase AGM HSC activity, whereas dorsal tissues decrease it. Chimeric explant cultures with genetically distinguishable AGM and ventral tissues show that the increase in HSC activity is not from ventral tissue-derived HSCs, precursors or primordial germ cells (as was previously suggested). Rather, it is due to instructive signaling from ventral tissues. Furthermore, we identify Hedgehog protein(s) as an HSC inducing signal.

Embryonic stromal clones reveal developmental regulators of definitive hematopoietic stem cells.

Hematopoietic stem cell (HSC) self-renewal and differentiation is regulated by cellular and molecular interactions with the surrounding microenvironment. During ontogeny, the aorta-gonad-mesonephros (AGM) region autonomously generates the first HSCs and serves as the first HSC-supportive microenvironment. Because the molecular identity of the AGM microenvironment is as yet unclear, we examined two closely related AGM stromal clones that differentially support HSCs. Expression analyses identified three putative HSC regulatory factors, beta-NGF (a neurotrophic factor), MIP-1gamma (a C-C chemokine family member) and Bmp4 (a TGF-beta family member). We show here that these three factors, when added to AGM explant cultures, enhance the in vivo repopulating ability of AGM HSCs. The effects of Bmp4 on AGM HSCs were further studied because this factor acts at the mesodermal and primitive erythropoietic stages in the mouse embryo. In this report, we show that enriched E11 AGM HSCs express Bmp receptors and can be inhibited in their activity by gremlin, a Bmp antagonist. Moreover, our results reveal a focal point of Bmp4 expression in the mesenchyme underlying HSC containing aortic clusters at E11. We suggest that Bmp4 plays a relatively late role in the regulation of HSCs as they emerge in the midgestation AGM.

Dlx5 drives Runx2 expression and osteogenic differentiation in developing cranial suture mesenchyme.

Craniofacial bones derive from cephalic neural crest, by endochondral or intramembranous ossification. Here, we address the role of the homeobox transcription factor Dlx5 during the initial steps of calvaria membranous differentiation and we show that Dlx5 elicits Runx2 induction and full osteoblast differentiation in embryonic suture mesenchyme grown "in vitro". First, we compare Dlx5 expression to bone-related gene expression in the developing skull and mandibular bones. We classify genes into three groups related to consecutive steps of ossification. Secondly, we study Dlx5 activity in osteoblast precursors, by transfecting Dlx5 into skull mesenchyme dissected prior to the onset of either Dlx5 and Runx2 expression or osteogenesis. We find that Dlx5 does not modify the proliferation rate or the expression of suture markers in the immature calvaria cells. Rather, Dlx5 initiates a complete osteogenic differentiation in these early primary cells, by triggering Runx2, osteopontin, alkaline phosphatase, and other gene expression according to the sequential temporal sequence observed during skull osteogenesis "in vivo". Thirdly, we show that BMP signaling activates Dlx5, Runx2, and alkaline phosphatase in those primary cultures and that a dominant-negative Dlx factor interferes with the ability of the BMP pathway to activate Runx2 expression. Together, these data suggest a pivotal role of Dlx5 and related Dlx factors in the onset of differentiation of chick calvaria osteoblasts.

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.

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.

From hemangioblast to hematopoietic stem cell: an endothelial connection?

The developmental origin of hematopoietic stem cells has been the subject of much research. Now that the developmental link between the hematopoietic system and the vasculature has been well established, questions remain regarding the precise cellular origin of definitive hematopoietic cells and at what point they branch off from the endothelial lineage. Do they emerge directly from a hemangioblast-type cell, similar to what is proposed for primitive yolk sac hematopoiesis, or are they generated via an endothelial intermediate, the hemogenic endothelium? In this review, we will give an overview of the data obtained from the mouse and avian models on the cellular origins of the hematopoietic system.

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.

The embryonic origins of hematopoietic stem cells: a tale of hemangioblast and hemogenic endothelium.

The developmental origin of hematopoietic stem cells has been for decades the subject of great interest. Once thought to emerge from the yolk sac, hematopoietic stem cells have now been shown to originate from the embryonic aorta. Increasing evidence suggests that hematopoietic stem cells are produced from an endothelial intermediate designated by the authors as hemangioblast or hemogenic endothelium. Recently, the allantois in the avian embryo and the placenta in the mouse embryo were shown to be a site of hematopoietic cell production/expansion and thus appear to play a critical role in the formation of the hematopoietic system. In this review we shall give an overview of the data obtained from human, mouse and avian models on the cellular origins of the hematopoietic system and discuss some aspects of the molecular mechanisms controlling hematopoietic cell production.

[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.

Xenopus single-minded (xSim) is a nuclear factor allowing nuclear translocation of its cytoplasmic partner xArnt.

Transcription factors belonging to the basic helix-loop-helix Per-Arnt-Sim (bHLH/PAS) family control a wide variety of biological processes in mammalian and/or Drosophila. We have previously isolated bHLH/PAS Xenopus amphibian homologs of Single-minded (xSim) and aryl receptor nuclear translocator (xArnt) and characterized their expression pattern during embryogenesis. We show in this paper that xSim protein is a functional homolog of Drosophila or mammalian Sim(s). Biochemical analysis indicates that xSim forms a heterodimer with xArnt. Subcellular localization analysis of bHLH/PAS chimeric fluorescent versions in Xenopus or mammalian cell lines shows that xSim is constitutively localized in the nuclear compartment. On the opposite, xArnt appears to be predominantly expressed in the cytoplasm. In addition, we demonstrate that xArnt nuclear localization depends on the presence of xSim. Thus xSim appears to be an essential factor in the nuclear translocation of the xSim/xArnt complex. In perfect agreement, we show that the C-terminal half of xSim contains the information for this nuclear localization.

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. 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.