Scl and Notch Act Downstream of Hedgehog Signaling to Promote Embryonic Hematopoiesis From Endothelial Cells
Peter G. KimColleen E. AlbackerIl Ho JangGarrett C. HeffnerYoowon LimTeresa V. BowmanMichelle I. LinLeonard I. ZonSabine LoewerGeorge Q. Daley
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Hemangioblast
RUNX1
Hes3 signaling axis
Hemangioblast
RUNX1
Embryoid body
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•We describe the discovery of RUNX1 and its role in hemogenic endothelium (HE).•HE from different anatomic sites is molecularly distinct.•Novel bioinformatics approach predicts transcription factors in HE. The transcription factor RUNX1 is required in the embryo for formation of the adult hematopoietic system. Here, we describe the seminal findings that led to the discovery of RUNX1 and of its critical role in blood cell formation in the embryo from hemogenic endothelium (HE). We also present RNA-sequencing data demonstrating that HE cells in different anatomic sites, which produce hematopoietic progenitors with dissimilar differentiation potentials, are molecularly distinct. Hemogenic and non-HE cells in the yolk sac are more closely related to each other than either is to hemogenic or non-HE cells in the major arteries. Therefore, a major driver of the different lineage potentials of the committed erythro–myeloid progenitors that emerge in the yolk sac versus hematopoietic stem cells that originate in the major arteries is likely to be the distinct molecular properties of the HE cells from which they are derived. We used bioinformatics analyses to predict signaling pathways active in arterial HE, which include the functionally validated pathways Notch, Wnt, and Hedgehog. We also used a novel bioinformatics approach to assemble transcriptional regulatory networks and predict transcription factors that may be specifically involved in hematopoietic cell formation from arterial HE, which is the origin of the adult hematopoietic system. The transcription factor RUNX1 is required in the embryo for formation of the adult hematopoietic system. Here, we describe the seminal findings that led to the discovery of RUNX1 and of its critical role in blood cell formation in the embryo from hemogenic endothelium (HE). We also present RNA-sequencing data demonstrating that HE cells in different anatomic sites, which produce hematopoietic progenitors with dissimilar differentiation potentials, are molecularly distinct. Hemogenic and non-HE cells in the yolk sac are more closely related to each other than either is to hemogenic or non-HE cells in the major arteries. Therefore, a major driver of the different lineage potentials of the committed erythro–myeloid progenitors that emerge in the yolk sac versus hematopoietic stem cells that originate in the major arteries is likely to be the distinct molecular properties of the HE cells from which they are derived. We used bioinformatics analyses to predict signaling pathways active in arterial HE, which include the functionally validated pathways Notch, Wnt, and Hedgehog. We also used a novel bioinformatics approach to assemble transcriptional regulatory networks and predict transcription factors that may be specifically involved in hematopoietic cell formation from arterial HE, which is the origin of the adult hematopoietic system. An account of the developmental origin of blood cannot be told without the major protagonist of the story, RUNX1. RUNX was first discovered in the fly in a classic screen conducted by Nusslein-Volhard and Wieschaus to identify mutations that affect development [1Nüsslein-Volhard C Wieschaus E Mutations affecting segment number and polarity in Drosophila.Nature. 1980; 287: 795-801Crossref PubMed Scopus (3016) Google Scholar]. The mutation runt was named for the fact that it resulted in runted embryos, a defect caused by presegmentation patterning defects. The runt gene was cloned by Gergen et al. and the protein it encoded was shown to be nuclear, but its identity as a transcription factor was not discovered at that time [2Kania MA Bonner AS Duffy JB Gergen JP The Drosophila segmentation gene runt encodes a novel nuclear regulatory protein that is also expressed in the developing nervous system.Genes Dev. 1990; 4: 1701-1713Crossref PubMed Scopus (232) Google Scholar]. The human RUNX1 gene was the next to be cloned by Ohki et al. as one of a pair of genes disrupted by the (8;21)(q22;q22) translocation in acute myeloid leukemia (AML) [3Miyoshi H Shimizu K Kozu T Maseki N Kaneko Y Ohki M t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1.Proc Natl Acad Sci U S A. 1991; 88: 10431-10434Crossref PubMed Scopus (798) Google Scholar]. Homology to the Drosophila runt gene was noted [4Daga A Tighe JE Calabi F Leukaemia/Drosophila homology.Nature. 1992; 356: 484Crossref PubMed Scopus (106) Google Scholar], but because the biochemical function of the Drosophila runt protein was not known, the function of the human homolog remained a mystery. Not long afterward, however, three groups of investigators using two different approaches uncovered RUNX1's function. Our group (Speck) purified RUNX1 as a sequence-specific DNA-binding protein that regulated the disease specificity of a mouse retrovirus [5Wang S Speck NA Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.Mol Cell Biol. 1992; 12: 89-102Crossref PubMed Scopus (167) Google Scholar]. The team of Ito and Shigesada purified RUNX1's homolog RUNX2 based on its role in murine polyomavirus replication and transcription [6Kamachi Y Ogawa E Asano M et al.Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer.J Virol. 1990; 64: 4808-4819Crossref PubMed Google Scholar]. Both groups showed that the purified transcription factors consisted of two subunits, one that bound DNA directly (RUNX1 or RUNX2) and a common, non-DNA-binding subunit called core binding factor β (CBFβ) that increased the affinity of RUNX1 and RUNX2 for DNA [6Kamachi Y Ogawa E Asano M et al.Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer.J Virol. 1990; 64: 4808-4819Crossref PubMed Google Scholar, 7Ogawa E Inuzuka M Maruyama M et al.Molecular cloning and characterization of PEBP2b, the heterodimeric partner of a novel Drosophila runt-related DNA binding protein PEBP2a.Virology. 1993; 194: 314-331Crossref PubMed Scopus (439) Google Scholar, 8Wang S Wang Q Crute BE Melnikova IN Keller SR Speck NA Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor.Mol Cell Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (395) Google Scholar]. The name "core binding factor" (CBF) derives from the DNA motif in the polyomavirus and retrovirus enhancers to which the RUNX proteins bound, which had previously been named "Core" [9Weiher H Zonig M Gruss P Multiple point mutations affecting the simian virus 40 enhancer.Science. 1983; 219: 626-631Crossref PubMed Scopus (429) Google Scholar]. At around the same time that RUNX1 and CBFβ were purified and cloned, the Hiebert group used a "selection and amplification binding" technique to determine whether the human RUNX1 protein bound DNA and, if so, which DNA sequence it recognized [10Meyers S Downing JR Hiebert SW Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions.Mol Cell Biol. 1993; 13: 6336-6345Crossref PubMed Scopus (425) Google Scholar]. They identified the same DNA sequence that was used by us and the Ito/Shigesada groups to purify the proteins. Closing the circle, Liu et al. showed that the inv(16)(p13.1;q22) mutation associated with AML created a chimeric protein that fused the non-DNA-binding CBFβ subunit to the coiled-coil tail region of a smooth muscle myosin heavy chain [11Liu P Tarle SA Hajra A et al.Fusion between transcription factor CBFb/PEBP2b and a myosin heavy chain in acute myeloid leukemia.Science. 1993; 261: 1041-1044Crossref PubMed Scopus (650) Google Scholar]. Therefore, multiple lines of investigation converged linking RUNX1 to CBFβ, t(8;21) to inv(16), and human leukemia to mouse leukemia. These discoveries are a great example of the major contribution that the study of viruses and model organisms made to our understanding of human disease. Of all of the paths that led to the discovery of RUNX1 and CBFβ, only the chromosomal translocations hinted at an essential role at the earliest stages of blood cell formation. As background, hematopoiesis in the embryo unfolds in three waves and both RUNX1 and CBFβ are required in the last two waves. The first, primitive wave produces primitive erythrocytes, diploid megakaryocytes, and primitive macrophages, all of which differentiate from mesoderm in the yolk sac blood islands beginning at embryonic day 7.25 (E7.25) in the mouse [12Palis J Robertson S Kennedy M Wall C Keller G Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.Development. 1999; 126: 5073-5084Crossref PubMed Google Scholar, 13Tober J Koniski A McGrath KE et al.The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis.Blood. 2007; 109: 1433-1441Crossref PubMed Scopus (222) Google Scholar, 14Bertrand JY Jalil A Klaine M Jung S Cumano A Godin I Three pathways to mature macrophages in the early mouse yolk sac.Blood. 2005; 106: 3004-3011Crossref PubMed Scopus (229) Google Scholar]. Wave 2 consists of the first "definitive" progenitors, which include erythro–myeloid progenitors (EMPs) that emerge in the yolk sac beginning at E8.25 [12Palis J Robertson S Kennedy M Wall C Keller G Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.Development. 1999; 126: 5073-5084Crossref PubMed Google Scholar, 15McGrath KE Frame JM Fegan KH et al.Distinct sources of hematopoietic progenitors emerge before HSCs and provide functional blood cells in the mammalian embryo.Cell Rep. 2015; 11: 1892-1904Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar] and lymphoid progenitors that appear at E9.5 in the yolk sac and in the caudal part of the embryo in the dorsal aorta, vitelline, and umbilical arteries [16Yoshimoto M Montecino-Rodriguez E Ferkowicz MJ et al.Embryonic day 9 yolk sac and intra-embryonic hemogenic endothelium independently generate a B-1 and marginal zone progenitor lacking B-2 potential.Proc Natl Acad Sci U S A. 2011; 108: 1468-1473Crossref PubMed Scopus (193) Google Scholar, 17Godin IE Garcia-Porrero JA Coutinho A Dieterlen-Lièvre F Marcos MAR Para-aortic splanchnopleura from early mouse embryos contain B1a cell progenitors.Nature. 1993; 364: 67-70Crossref PubMed Scopus (313) Google Scholar, 18Yoshimoto M Porayette P Glosson NL et al.Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence.Blood. 2012; 119: 5706-5714Crossref PubMed Scopus (114) Google Scholar, 19Boiers C Carrelha J Lutteropp M et al.Lymphomyeloid contribution of an immune-restricted progenitor emerging prior to definitive hematopoietic stem cells.Cell Stem Cell. 2013; 13: 535-548Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 20Lin Y Yoder MC Yoshimoto M Lymphoid progenitor emergence in the murine embryo and yolk sac precedes stem cell detection.Stem Cells Dev. 2014; 23: 1168-1177Crossref PubMed Scopus (47) Google Scholar, 21Yokota T Huang J Tavian M et al.Tracing the first waves of lymphopoiesis in mice.Development. 2006; 133: 2041-2051Crossref PubMed Scopus (74) Google Scholar]. Wave 3, the final wave of blood formation, includes pre-hematopoietic stem cells (pre-HSCs) that are unable to engraft adult mice directly, but colonize the fetal liver, where they mature into adult-repopulating HSCs [22Rybtsov S Sobiesiak M Taoudi S et al.Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region.J Exp Med. 2011; 208: 1305-1315Crossref PubMed Scopus (172) Google Scholar, 23Taoudi S Gonneau C Moore K et al.Extensive hematopoietic stem cell generation in the AGM region via maturation of VE-cadherin+CD45+ pre-definitive HSCs.Cell Stem Cell. 2008; 3: 99-108Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 24Medvinsky A Dzierzak E Definitive hematopoiesis is autonomously initiated by the AGM region.Cell. 1996; 86: 897-906Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar, 25Rybtsov S Batsivari A Bilotkach K et al.Tracing the origin of the HSC hierarchy reveals an SCF-dependent, IL-3-independent CD43(-) embryonic precursor.Stem Cell Rep. 2014; 3: 489-501Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 26Gordon-Keylock S Sobiesiak M Rybtsov S Moore K Medvinsky A Mouse extraembryonic arterial vessels harbor precursors capable of maturing into definitive HSCs.Blood. 2013; 122: 233-2345Crossref Scopus (71) Google Scholar, 27Kieusseian A Brunet de la Grange P Burlen-Defranoux O Godin I Cumano A Immature hematopoietic stem cells undergo maturation in the fetal liver.Development. 2012; 139: 3521-3530Crossref PubMed Scopus (66) Google Scholar]. Wave 3 also includes a small number of adult-repopulating HSCs in the dorsal aorta, vitelline, and umbilical arteries and in the placenta, which presumably have matured in situ from pre-HSCs [24Medvinsky A Dzierzak E Definitive hematopoiesis is autonomously initiated by the AGM region.Cell. 1996; 86: 897-906Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar, 26Gordon-Keylock S Sobiesiak M Rybtsov S Moore K Medvinsky A Mouse extraembryonic arterial vessels harbor precursors capable of maturing into definitive HSCs.Blood. 2013; 122: 233-2345Crossref Scopus (71) Google Scholar, 28de Bruijn MF Speck NA Peeters MC Dzierzak E Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo.EMBO J. 2000; 19: 2465-2474Crossref PubMed Scopus (448) Google Scholar, 29Ottersbach K Dzierzak E The murine placenta contains hematopoietic stem cells within the vascular labyrinth region.Dev Cell. 2005; 8: 377-387Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 30Gekas C Dieterlen-Lievre F Orkin SH Mikkola HK The placenta is a niche for hematopoietic stem cells.Dev Cell. 2005; 8: 365-375Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 31Li Z Lan Y He W et al.Mouse embryonic head as a site for hematopoietic stem cell development.Cell Stem Cell. 2012; 11: 663-675Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 32Müller AM Medvinsky A Strouboulis J Grosveld F Dzierzak E Development of hematopoietic stem cell activity in the mouse embryo.Immunity. 1994; 1: 291-301Abstract Full Text PDF PubMed Scopus (684) Google Scholar, 33Ivanovs A Rybtsov S Welch L Anderson RA Turner ML Medvinsky A Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region.J Exp Med. 2011; 208: 2417-2427Crossref PubMed Scopus (161) Google Scholar, 34Yoder MC Inducing definitive hematopoiesis in a dish.Nat Biotechnol. 2014; 32: 539-541Crossref PubMed Scopus (33) Google Scholar]. Knockouts of RUNX1 and CBFβ resulted in the absence of all wave 2 and 3 progenitors, including adult-repopulating HSCs [35Wang Q Stacy T Binder M Marín-Padilla M Sharpe AH Speck NA Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis.Proc Natl Acad Sci U S A. 1996; 93: 3444-3449Crossref PubMed Scopus (1027) Google Scholar, 36Wang Q Stacy T Miller JD et al.The CBFb subunit is essential for CBFa2 (AML1) function in vivo.Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 37Okuda T van Deursen J Hiebert SW Grosveld G Downing JR AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis.Cell. 1996; 84: 321-330Abstract Full Text Full Text PDF PubMed Scopus (1597) Google Scholar, 38Cai Z de Bruijn MFTR Ma X et al.Haploinsufficiency of AML1/CBFA2 affects the embryonic generation of mouse hematopoietic stem cells.Immunity. 2000; 13: 423-431Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 39Sasaki K Yagi H Bronson RT et al.Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core binding factor beta.Proc Natl Acad Sci U S A. 1996; 93: 12359-12363Crossref PubMed Scopus (327) Google Scholar, 40Niki M Okada H Takano H et al.Hematopoiesis in the fetal liver is impaired by targeted mutagenesis of a gene encoding a non-DNA binding subunit of the transcription factor, polyomavirus enhancer binding protein 2/core binding factor.Proc Natl Acad Sci U S A. 1997; 94: 5697-5702Crossref PubMed Scopus (164) Google Scholar]. To gain insight into the nature of this hematopoietic block, we introduced a lacZ reporter gene into the Runx1 locus to learn where RUNX1 was expressed in the blood lineage [41North TE Gu T-L Stacy T et al.Cbfa2 is required for the formation of intra-aortic hematopoietic clusters.Development. 1999; 126: 2563-2575Crossref PubMed Google Scholar]. We found RUNX1 only in the relatively rare wave 2 and 3 progenitors in the embryo, which are far outnumbered by primitive erythrocytes that are initially RUNX1+ but quickly lose RUNX1 expression. This restriction of RUNX1 expression to wave 2 and 3 progenitors (and also to wave 1 macrophages) was actually a key feature that enabled us to pinpoint their origin. Some RUNX1+ wave 2 and 3 progenitors could be found in the circulation and in the fetal liver, but we also found clusters of round RUNX1+ hematopoietic cells attached to RUNX1+ endothelial cells in arteries, specifically in anatomic sites where the first functional wave 2 and 3 progenitors and histological evidence of blood cell formation had been described previously [17Godin IE Garcia-Porrero JA Coutinho A Dieterlen-Lièvre F Marcos MAR Para-aortic splanchnopleura from early mouse embryos contain B1a cell progenitors.Nature. 1993; 364: 67-70Crossref PubMed Scopus (313) Google Scholar, 24Medvinsky A Dzierzak E Definitive hematopoiesis is autonomously initiated by the AGM region.Cell. 1996; 86: 897-906Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar, 32Müller AM Medvinsky A Strouboulis J Grosveld F Dzierzak E Development of hematopoietic stem cell activity in the mouse embryo.Immunity. 1994; 1: 291-301Abstract Full Text PDF PubMed Scopus (684) Google Scholar, 42Dantschakoff V Uber das erste aufreten der blut-elemente in hühnerembryo.Folia Haematol. 1907; 4: 159-166Google Scholar, 43Dieterlen-Lièvre F. On the origin of haematopoietic stem cells in avian embryos: an experimental approach.J Embryol Exp Morphol. 1975; 33: 609-619Google Scholar, 44Emmel VE The cell clusters in the dorsal aorta of mammalian embryos.Am J Anat. 1916; 19: 401-421Crossref Scopus (41) Google Scholar, 45Garcia-Porrero JA Godin IE Dieterlen-Lièvre F Potential intraembryonic hemogenic sites at pre-liver stages in the mouse.Anat Embryol. 1995; 192: 425-435Crossref PubMed Scopus (189) Google Scholar, 46Jordon HE Aortic cell clusters in vertebrate embryos.Proc Natl Acad Sci U S A. 1917; 3: 149-157Crossref Google Scholar, 47Jordon HE. A study of a 7mm human embryo: with special reference to its peculiar spirally twisted form, and its large aortic cell-clusters.Anat Rec. 1918; 14: 479-492Crossref Scopus (14) Google Scholar, 48Maximov AA Untersuchengen über blut und bindegewebe. I. Die frühesten entwicklingsstadien der blut und bindegewebzellen beim säugetier-embryo, bis zum anfang der blutbildung in der leber.Archiv für Mikroskopische Anatomie. 1909; 73: 444-450Crossref Scopus (63) Google Scholar, 49Smith RA Glomski CA "Hemogenic endothelium" of the embryonic aorta: does it exist?.Dev Comp Immunol. 1982; 6: 359-368Crossref PubMed Scopus (57) Google Scholar, 50Wood HB May G Healy L Enver T Morriss-Kay GM CD34 expression patterns during early mouse development are related to modes of blood vessel formation and reveal additional sites of hematopoiesis.Blood. 1997; 90: 2300-2311Crossref PubMed Google Scholar, 51Godin I Dieterlen-Lièvre F Cumano A Emergence of multipotent hematopoietic cells in the yolk sac and paraaortic splanchnopleura of 8.5 dpc mouse embryos.Proc Natl Acad Sci U S A. 1995; 92: 773-777Crossref PubMed Scopus (307) Google Scholar]. Most importantly, we showed that RUNX1 loss blocked the formation of wave 2 and 3 progenitors from this rare population of "hemogenic" endothelial cells. The concept that blood formed from hemogenic endothelium (HE) had been proposed in the early 20th century [44Emmel VE The cell clusters in the dorsal aorta of mammalian embryos.Am J Anat. 1916; 19: 401-421Crossref Scopus (41) Google Scholar, 52Jordon HE Evidence of hemogenic capacity of endothelium.Anat Rec. 1916; 10: 417-420Crossref Scopus (35) Google Scholar, 53Sabin FR Studies on the origin of blood vessels and of red corpuscles as seen in the living blastoderm of the chick during the second day of incubation.Contributions to Embryology. 1920; 9: 213-262Google Scholar] and, just prior to our work, several groups showed that blood could differentiate from endothelial cells [54Jaffredo T Gautier R Eichmann A Dieterlen-Lièvre F Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny.Development. 1998; 125: 4575-4583Crossref PubMed Google Scholar, 55Nishikawa SI Nishikawa S Kawamoto H et al.In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos.Immunity. 1998; 8: 761-769Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar]. Despite these compelling data, the notion that HE was the immediate precursor of blood was not widely accepted at the time of our discovery. Our identification of RUNX1 as the first specific marker of HE and our demonstration that RUNX1 was required for blood cell formation from HE [41North TE Gu T-L Stacy T et al.Cbfa2 is required for the formation of intra-aortic hematopoietic clusters.Development. 1999; 126: 2563-2575Crossref PubMed Google Scholar, 56Li Z Chen MJ Stacy T Speck NA Runx1 function in hematopoiesis is required in cells that express Tek.Blood. 2006; 107: 106-110Crossref PubMed Scopus (57) Google Scholar, 57Chen MJ Yokomizo T Zeigler BM Dzierzak E Speck NA Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter.Nature. 2009; 457: 889-891Crossref Scopus (720) Google Scholar] lent considerable support to the notion that HE cells were the immediate precursor of the adult hematopoietic organ. Later, live-imaging studies performed by multiple groups showed blood cells forming from endothelial cells in real time, leading to the consensus that endothelial cells are the immediate precursor of blood cells [58Kissa K Herbomel P Blood stem cells emerge from aortic endothelium by a novel type of cell transition.Nature. 2010; 464: 112-115Crossref PubMed Scopus (674) Google Scholar, 59Eilken HM Nishikawa S Schroeder T Continuous single-cell imaging of blood generation from haemogenic endothelium.Nature. 2009; 457: 896-900Crossref PubMed Scopus (466) Google Scholar, 60Bertrand JY Chi NC Santoso B Teng S Stainier DY Traver D Haematopoietic stem cells derive directly from aortic endothelium during development.Nature. 2010; 464: 108-111Crossref PubMed Scopus (722) Google Scholar, 61Boisset JC van Cappellen W Andrieu-Soler C Galjart N Dzierzak E Robin C In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium.Nature. 2010; 464: 116-120Crossref PubMed Scopus (643) Google Scholar]. An interesting question is how different populations of HE cells produce embryonic blood progenitors with distinct properties. For example, HE cells in the yolk sac primarily give rise to EMPs, whereas HE cells in the major arteries give rise to pre-HSCs and HSCs. We hypothesize that differences in the lineage potential of hematopoietic stem and progenitor cells (HSPCs) produced in the yolk sac and major arteries likely originate, at least in part, from differences in the intrinsic properties of the HE cells from which they arise. Several lines of evidence support this hypothesis. For instance, HE cells that give rise to EMPs are both arterial and venous in origin, whereas pre-HSCs differentiate from arterial HE [62Frame JM Fegan KH Conway SJ McGrath KE Palis J Definitive hematopoiesis in the yolk sac emerges from Wnt-responsive hemogenic endothelium independently of circulation and arterial identity.Stem Cells. 2016; 34: 431-444Crossref PubMed Scopus (103) Google Scholar, 63Yzaguirre AD Speck NA Insights into blood cell formation from hemogenic endothelium in lesser-known anatomic sites.Dev Dyn. 2016; 245: 1011-1028Crossref PubMed Scopus (33) Google Scholar, 64Chen MJ Li Y De Obaldia ME et al.Erythroid/myeloid progenitors and hematopoietic stem cells originate from distinct populations of endothelial cells.Cell Stem Cell. 2011; 9: 541-552Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar]. Second, different signaling pathways are involved in EMP and pre-HSC formation. For example, EMP formation from HE in the yolk sac vascular plexus does not require Notch signaling, whereas HSPC production from arterial endothelium is strictly dependent on Notch [65Hadland BK Huppert SS Kanungo J et al.A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development.Blood. 2004; 104: 3097-3105Crossref PubMed Scopus (186) Google Scholar, 66Robert-Moreno A Espinosa L de la Pompa JL Bigas A RBPjkappa-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells.Development. 2005; 132: 1117-1126Crossref PubMed Scopus (215) Google Scholar, 67Kumano K Chiba S Kunisato A et al.Notch1 but not Notch2 is essential for generating hematopoietic stem cells from endothelial cells.Immunity. 2003; 18: 699-711Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 68Burns CE Traver D Mayhall E Shepard JL Zon LI Hematopoietic stem cell fate is established by the Notch-Runx pathway.Genes Dev. 2005; 19: 2331-2342Crossref PubMed Scopus (322) Google Scholar]. Similarly, inflammatory signaling regulates the numbers of HSPCs produced in the major arteries, but appears to have no influence on EMP numbers in the yolk sac [69Li Y Esain V Teng L et al.Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production.Genes Dev. 2014; 28: 2597-2612Crossref PubMed Scopus (168) Google Scholar, 70Espin-Palazon R Stachura DL Campbell CA et al.Proinflammatory signaling regulates hematopoietic stem cell emergence.Cell. 2014; 159: 1070-1085Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 71Sawamiphak S Kontarakis Z Stainier DY Interferon gamma signaling positively regulates hematopoietic stem cell emergence.Dev Cell. 2014; 31: 640-653Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 72He Q Zhang C Wang L et al.Inflammatory signaling regulates hematopoietic stem and progenitor cell emergence in vertebrates.Blood. 2015; 125: 1098-1106Crossref PubMed Scopus (111) Google Scholar]. To determine whether HE cells in different anatomic sites are indeed molecularly distinct, we purified HE cells and non-HE cells from the yolk sac and major arteries at two different embryonic stages, E9.5 and E10.5, and determined their transcriptomes by RNA sequencing (RNA-Seq). We isolated major arteries (dorsal aorta, umbilical, vitelline) by dissecting the caudal region of the mouse embryo from which the head, heart, pulmonary regions, liver, gut tube, tail, and limb buds were removed. We used green fluorescent protein (GFP) expression from a Runx1:GFP knockin allele [73Lorsbach RB Moore J Ang SO Sun W Lenny N Downing JR Role of Runx1 in adult hematopoiesis: analysis of Runx1-IRES-GFP knock-in mice reveals differential lineage expression.Blood. 2004; 103: 2522-2529Crossref PubMed Scopus (108) Google Scholar] as a tool to separate HE from non-hemogenic endothelium (E). We purified HE from the arteries and yolk sac as Runx1:GFP+ CD31+ Kitlo/– CD45– CD41– cells and E as Runx1:GFP– CD31+ Kit– CD45– CD41– cells (Figures 1A and 1B). We confirmed the functional purity of the cells in ex vivo assays. Both HE and E cells could form endothelial tubes in culture (Figure 1C). Conversely, endothelial cells with the ability to form CD45+ blood cells in culture were highly enriched in the purified Runx1:GFP+ HE population (the frequency was 500 × higher in HE versus E from the arteries and 100 × higher in HE versus E from the yolk sac; Figure 1D). Purified HE and E cells produced very few hematopoietic colonies in methylcellulose cultures (0.5–1.5 colony-forming units per embryo equivalent) (Figure 1E), indicating that the HE cells were not significantly contaminated by HSPCs. We bulk sequenced approximately 20,000 cells, obtaining ∼74.7 million uniquely mapped reads per sample (accession number GSE103813). The RNA-seq data are highly reproducible, with an average transcriptome-wide correlation of biological replicates of 0.95 (not shown). Principal component analysis (PCA) of the HE and E RNA-seq data, along with data from E14.5 fetal liver (FL) HSCs (Lin–Sca1+Kit+CD48–CD150+) and adult bone marrow (BM) HSCs (Lin–Sca1+Kit+CD34–CD48–CD150+), clustered HE and E far away from FL and BM HSCs (Figure 2A). The tissue of origin was the strongest driver of clustering, with HE and E cells from yolk sacs clustered closely with each other and less closely with their equivalent populations in the arteries. Similarly, HE and E from the major arteries clustered more closely to each other than to the corresponding cells in the yolk sac. Therefore, HE and E from the major arteries are molecularly more closely related to each other than either is to HE or E from the yolk sac. We used EBSeq [74Leng N Dawson JA Thomson JA et al.EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments.Bioinformatics. 2013; 29: 1035-1
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The yolk sac is the first site of hematopoiesis during mammalian development. The yolk sac is also the first site of blood vessel development. Development of the blood islands in the yolk sac is an integrated process in which these two developmental events, hematopoiesis and vasculogenesis, proceed in concert. This review focuses on mouse yolk sac hematopoietic stem cells (YS-HSC), describing their differentiation in vitro and in vivo. YS-HSC go through a progressive series of changes prior to the initiation of lineage-specific differentiation. Experiments tracing their origins from postulated hemangioblasts, and the subsequent interaction between these stem cells and yolk sac endothelial cells are described. Differences between the extraembryonic YS-HSC and HSC found later within the embryo, perinatally or in adults, are described. YS-HSC have greater reproductive capability than HSC obtained from fetal liver, umbilical cord blood or adult bone marrow; they do not yet express major histocompatibility complex-associated antigens and they are able to reconstitute adult immunocompromised animals even when introduced in small numbers (< 100 cells/mouse). With recent results demonstrating the feasibility of expanding YS-HSC in vitro as well as of introducing new genes into these cells by transfection, the YS-HSC shows promise both as a means of achieving long-term restitution of hematopoiesis across histocompatibility barriers and as a self-renewing vehicle for gene transfer.
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Hemangioblast
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SUMMARY Hematopoietic stem cells (HSCs) emerge from hemogenic endothelium (HE) localised in the embryonic dorsal aorta (DA). Here we show that Runx1, a transcription factor essential for HSC emergence, controls HE establishment in the absence of its non-DNA-binding partner, CBFβ, and that a CBFβ-binding-deficient Runx1 mutant form can activate the HE program in the DA. Nevertheless, CBFβ is also essential for HSC emergence by regulating the specification of definitive hemangioblasts (DHs), the precursors of the DA and HE, in the lateral plate mesoderm where it mediates VEGFA induction by BMP signalling. Surprisingly, no Runx gene is expressed in DHs and the pharmacological inhibition of CBFβ binding to Runx is not detrimental for DH, confirming that CBFβ functions independently of Runx. Thus, we have uncovered, for the first time, that CBFβ regulates gene expression without Runx, breaking the dogma in which CBFβ ‘s gene regulatory functions are strictly dependent on its binding to Runx. HIGHLIGHTS Runx1 and CBFβ play independent roles in the establishment of the HSC lineage Runx1 binding to CBFβ is not required for HE establishment CBFβ is downstream of BMP and regulates endogenous VEGFA expression in DH Binding to Runx is not obligatory for CBFβ function
RUNX1
Core binding factor
Hemangioblast
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Hemangioblast
RUNX1
Dorsal aorta
Conceptus
Hematopoietic stem cell
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Prior to the generation of hematopoietic stem cells (HSCs) from the hemogenic endothelial cells (HECs) mainly in the dorsal aorta in midgestational mouse embryos, multiple hematopoietic progenitors including erythro-myeloid progenitors and lymphoid progenitors are generated from yolk sac HECs. These HSC-independent hematopoietic progenitors have recently been identified as major contributors to functional blood cell production until birth. However, little is known about yolk sac HECs. Here, combining integrative analyses of multiple single-cell RNA-sequencing datasets and functional assays, we reveal that Neurl3-EGFP, in addition to marking the continuum throughout the ontogeny of HSCs from HECs, can also serve as a single enrichment marker for yolk sac HECs. Moreover, while yolk sac HECs have much weaker arterial characteristics than either arterial endothelial cells in the yolk sac or HECs within the embryo proper, the lymphoid potential of yolk sac HECs is largely confined to the arterial-biased subpopulation featured by the Unc5b expression. Interestingly, the B lymphoid potential of hematopoietic progenitors, but not for myeloid potentials, is exclusively detected in Neurl3-negative subpopulations in midgestational embryos. Taken together, these findings enhance our understanding of blood birth from yolk sac HECs and provide theoretical basis and candidate reporters for monitoring step-wise hematopoietic differentiation.
Hemangioblast
Dorsal aorta
Hematopoietic stem cell
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The yolk sac is the first observed site of hematopoiesis during mouse ontogeny. Primitive erythroid cells are the most well-recognized cell lineages produced from this tissue. In addition to primitive erythroid cells, several types of hematopoietic cells are present, including multipotent hematopoietic progenitors. Yolk sac-derived blood cells constitute a transient wave of embryonic and fetal hematopoiesis. However, recent studies have demonstrated that some macrophage and B cell lineages derived from the early yolk sac may persist to adulthood. This review discusses the cellular basis of mouse yolk sac hematopoiesis and its contributions to embryonic and adult hematopoietic systems.
Hemangioblast
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Hemangioblast
Multipotent Stem Cell
Compartment (ship)
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