How the blood system forms during embryonic development is a topic of intensive research, in part because of the potential importance of the process for regenerative medicine. Two main theories have emerged to explain the formation of the haematopoietic stem cells that eventually populate the adult born marrow. One idea is that the haematopoietic stem cell and the endothelial lineage arise independently from the mesoderm; the other is that some haematopoietic and endothelial lineages derive from a specialized progenitor called a haemangioblast. Three papers in this issue unify the two theories. Both are correct: the haemangioblast does generate haematopoietic cells, but via a haemogenic endothelium intermediate. There are two theories of how haematopoeitic cells arise during embryonic development. One is that haematopoietic cells arise from a mesodermal progenitor with both endothelial and haematopoietic potential called the haemangioblast. Another associates the first haematopoietic cells to an endothelial cell with haematopoietic potential, the haemogenic endothelium. This paper shows that both theories are correct. The haemangioblast generates haematopoietic cells through the formation of a haemogenic endothelium intermediate. It has been proposed that during embryonic development haematopoietic cells arise from a mesodermal progenitor with both endothelial and haematopoietic potential called the haemangioblast1,2. A conflicting theory instead associates the first haematopoietic cells with a phenotypically differentiated endothelial cell that has haematopoietic potential (that is, a haemogenic endothelium)3,4,5. Support for the haemangioblast concept was initially provided by the identification during mouse embryonic stem cell differentiation of a clonal precursor, the blast colony-forming cell (BL-CFC), which gives rise to blast colonies with both endothelial and haematopoietic components6,7. Although recent studies have now provided evidence for the presence of this bipotential precursor in vivo8,9, the precise mechanism for generation of haematopoietic cells from the haemangioblast still remains completely unknown. Here we demonstrate that the haemangioblast generates haematopoietic cells through the formation of a haemogenic endothelium intermediate, providing the first direct link between these two precursor populations. The cell population containing the haemogenic endothelium is transiently generated during BL-CFC development. This cell population is also present in gastrulating mouse embryos and generates haematopoietic cells on further culture. At the molecular level, we demonstrate that the transcription factor Tal1 (also known as Scl; ref. 10) is indispensable for the establishment of this haemogenic endothelium population whereas the core binding factor Runx1 (also known as AML1; ref. 11) is critical for generation of definitive haematopoietic cells from haemogenic endothelium. Together our results merge the two a priori conflicting theories on the origin of haematopoietic development into a single linear developmental process.

The hematopoietic and endothelial lineages derive from mesoderm and are thought to develop through the maturation of a common progenitor, the hemangioblast. To investigate the developmental processes that regulate mesoderm induction and specification to the hemangioblast, we generated an embryonic stem cell line with the green fluorescent protein (GFP) targeted to the mesodermal gene, brachyury. After the in vitro differentiation of these embryonic stem cells to embryoid bodies, developing mesodermal progenitors could be separated from those with neuroectoderm potential based on GFP expression. Co-expression of GFP with the receptor tyrosine kinase Flk1 revealed the emergence of three distinct cell populations, GFP^-Flk1^-, GFP⁺Flk1^- and GFP⁺Flk1⁺ cells, which represent a developmental progression ranging from pre-mesoderm to prehemangioblast mesoderm to the hemangioblast.

In this report we demonstrate a role for Runx1 (AML1) at the hemangioblast stage of hematopoietic and endothelial development in embryonic stem (ES) cell-derived embryoid bodies (EBs). Runx1 is expressed in EBs during the appearance of precursors with hemangioblast properties, the blast colony-forming cells (BL-CFCs). Cell sorting studies revealed that all BL-CFCs within EBs express Runx1. Runx1-deficient EBs consistently generate 10- to 20-fold fewer blast colonies than wild-type controls and display a complete block in definitive hematopoiesis. Despite this defect, Runx1-/- EBs and yolk sacs from mutant embryos generate normal numbers of primitive erythroid precursors. These observations clearly demonstrate that Runx1 functions early in hematopoietic development, and they support the interpretation that the primitive erythroid lineage is established early by a subset of BL-CFCs that develop in a Runx1-independent fashion.

Recent studies have established that during embryonic development, hematopoietic progenitors and stem cells are generated from hemogenic endothelium precursors through a process termed endothelial to hematopoietic transition (EHT). The transcription factor RUNX1 is essential for this process, but its main downstream effectors remain largely unknown. Here, we report the identification of Gfi1 and Gfi1b as direct targets of RUNX1 and critical regulators of EHT. GFI1 and GFI1B are able to trigger, in the absence of RUNX1, the down-regulation of endothelial markers and the formation of round cells, a morphologic change characteristic of EHT. Conversely, blood progenitors in Gfi1- and Gfi1b-deficient embryos maintain the expression of endothelial genes. Moreover, those cells are not released from the yolk sac and disseminated into embryonic tissues. Taken together, our findings demonstrate a critical and specific role of the GFI1 transcription factors in the first steps of the process leading to the generation of hematopoietic progenitors from hemogenic endothelium.

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