GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment

Abstract

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...

A short history of hemogenic endothelium

“…However, they were unable to generate hematopoietic colonies in vitro, something CD41 + cells isolated from wild type ES cell cultures or embryos readily do. In complementary experiments, the loss of both Gfi1 and Gfi1b resulted in the generation of CD41 expressing adherent cells that maintained endothelial marker expression, which could only generate clonogenic progeny upon disaggregation [80]. Altogether these data indicate that the Runx1 target genes, Gfi1 and Gfi1b may be in part responsible for the repression of endothelial gene expression associated with the early steps of EHT.…”

“…The first insights of these studies showed that Runx1 represses the endothelial program whilst activating the hematopoietic program (reviewed in [51]). More recently, the Runx1 target genes Gfi1/Gfi1b, but not Pu.1 (Sfpi1) or c-myb, were shown to down-regulate expression of endothelial genes in Runx1 null hemogenic endothelium derived from ES cells [80]. In addition, the Gfi1/Gfi1b transduced hemogenic endothelial cells initiated morphological changes akin to the formation of hematopoietic progeny, forming round cells that express CD41.…”

The RUNX1–PU.1 axis in the control of hematopoiesis

“…TALE-effectormediated PU.1 repression in a murine ESC differentiation confirmed the presence of PU.1 at sites of mouse definitive hematopoiesis [35]. However, using the same experimental model, Lancrin et al [36] investigated the capability of the major downstream targets of RUNX1, i.e., GFI1, GFI1B and PU.1, to rescue the defective phenotype of Runx1…”

“…RUNX1 was shown to be essential for the formation of intra-aortic clusters, HSCs and hematopoietic progenitor formation [31], via facilitated expression of critical regulators of the EHT. These include GFI1 and GFIB, which trigger down-regulation of the endothelial markers Tek/ TIE2, VE-Cadherin/CDH5 and KIT, even in the absence of RUNX1 [36]. Genome-wide studies have shown that hematopoietic genes such as PU.1 are actually primed in the HE by occupancy of TAL1/SCL and FLI1 to their regulatory regions and that a RUNX1-mediated reorganization of these factors is critical during EHT [44].…”

Interplay between SOX7 and RUNX1 regulates hemogenic endothelial fate in the yolk sac

“…It is now widely accepted that the hematopoietic transcription factor RUNX1 is a key master regulator of this process (Chen et al, 2009;Lancrin et al, 2009;Lie-A-Ling et al, 2014). RUNX1 is expressed in HE and emerging HSPCs both in the yolk sac and in the embryo proper (Frame et al, 2015;Lancrin et al, 2009;North et al, 1999), and controls the downregulation and upregulation of endothelial and hematopoietic transcriptional programs, respectively (Lancrin et al, 2012;Tanaka et al, 2012;Thambyrajah et al, 2016). RUNX1 has been shown to act in a heptad of interacting transcription factors in hematopoietic progenitor cells (HPCs) comprising FLI1, ERG, TAL1, LYL1, LMO2 and GATA2 (Wilson et al, 2010).…”