In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.

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. (Blood. 2012;120(2):314-322) IntroductionHematopoietic stem cells (HSCs) are pivotal to the continuous generation and maintenance of mature blood cells throughout adult life. Although they reside mainly in the bone marrow at the time of birth and thereafter, they are initially generated during embryonic life in large blood vessels located in the developing embryo. 1 Recent studies in mice 2-4 and zebrafish 5,6 have established that hematopoietic progenitors and stem cells are produced at these sites from endothelial cells by a process termed endothelial to hematopoietic transition (EHT).Besides these in vivo studies, the in vitro differentiation of embryonic stem (ES) cells represents a powerful system to study these early events of hematopoietic development. Using this system, it was shown that blood cell development is initiated by a clonal hemangioblast precursor, which gives rise to blast colonies with both endothelial and hematopoietic cells, 7 a finding subsequently confirmed in vivo. 8 More recently, hemangioblasts were shown to generate first a hemogenic endothelium intermediate cell population, which subsequently gives rise to hematopoietic progenitors. 9 During this process, hemogenic endothelial cells lose their endothelial identity by altering their flat, adherent appearance into the characteristic round shape of mobile hematopoietic precursor cells. 9,10 This transition critically relies on the presence of the transcription factor RUNX1. 3,9 Although these studies have together clearly established that the EHT process is the pivotal event in the generation of the first blood cells, 9,10 the molecular and cellular mechanisms orchestrating this critical transition remain essentially unknown.To identify downstream RUNX1 effectors that drive the development of hematopoietic precursor cells from the hemogenic endothelium, we compared the transcriptomes of Runx1 ϩ/Ϫ and Runx1 Ϫ/Ϫ hemogenic endothelium and identified the Gfi1 and Gfi1b genes as direc...

Key Points• Generated the first comprehensive RUNX1b-specific transcriptome and binding profile in HE.• RUNX1b induces a cell adhesion and migration program prior to the downregulation of endothelial genes and the emergence of blood cells.During ontogeny, the transcription factor RUNX1 governs the emergence of definitive hematopoietic cells from specialized endothelial cells called hemogenic endothelium (HE). The ultimate consequence of this endothelial-to-hematopoietic transition is the concomitant activation of the hematopoietic program and downregulation of the endothelial program. However, due to the rare and transient nature of the HE, little is known about the initial role of RUNX1 within this population. We, therefore, developed and implemented a highly sensitive DNA adenine methyltransferase identification-based methodology, including a novel data analysis pipeline, to map early RUNX1 transcriptional targets in HE cells. This novel transcription factor binding site identification protocol should be widely applicable to other low abundance cell types and factors. Integration of the RUNX1 binding profile with gene expression data revealed an unexpected early role for RUNX1 as a positive regulator of cell adhesion-and migration-associated genes within the HE. This suggests that RUNX1 orchestrates HE cell positioning and integration prior to the release of hematopoietic cells. Overall, our genome-wide analysis of the RUNX1 binding and transcriptional profile in the HE provides a novel comprehensive resource of target genes that will facilitate the precise dissection of the role of RUNX1 in early blood development. (Blood. 2014;124(11):e11-e20)