Extraembryonic hematopoietic lineages—to macrophages and beyond

•Extraembryonic tissues give rise to the first hematopoietic cells in mice and humans.•Primitive hematopoiesis mainly gives rise to erythroblasts and megakaryocytes.•Erythro-myeloid progenitors differentiate into erythroid and myeloid cells in the yolk sac and the fetal liver.•Yolk sac contribution to blood and immune cells over time is cell type specific.•Lineage tracing shows the maintenance of yolk sac-derived macrophages into adulthood. The first blood and immune cells in vertebrates emerge in the extraembryonic yolk sac. Throughout the last century, it has become evident that this extraembryonic tissue gives rise to transient primitive and definitive hematopoiesis but not hematopoietic stem cells. More recently, studies have elucidated that yolk sac-derived blood and immune cells are present far longer than originally expected. These cells take over essential roles for the survival and proper organogenesis of the developing fetus up until birth. In this review, we discuss the most recent findings and views on extraembryonic hematopoiesis in mice and humans. The first blood and immune cells in vertebrates emerge in the extraembryonic yolk sac. Throughout the last century, it has become evident that this extraembryonic tissue gives rise to transient primitive and definitive hematopoiesis but not hematopoietic stem cells. More recently, studies have elucidated that yolk sac-derived blood and immune cells are present far longer than originally expected. These cells take over essential roles for the survival and proper organogenesis of the developing fetus up until birth. In this review, we discuss the most recent findings and views on extraembryonic hematopoiesis in mice and humans. Extraembryonic membranes, such as the amnion, chorion, yolk sac, and allantois, are essential for the survival of developing amniotic embryos [1Sheng G Foley AC. Diversification and conservation of the extraembryonic tissues in mediating nutrient uptake during amniote development.Ann N Y Acad Sci. 2012; 1271: 97-103Crossref PubMed Scopus (44) Google Scholar]. These membranes take over functions such as absorption of macro- and micronutrients, gas exchange, excretion of metabolic waste, and hematopoiesis, which is the production of blood cells [2Cindrova-Davies T Jauniaux E Elliot MG Gong S Burton GJ DS Charnock-Jones RNA-seq reveals conservation of function among the yolk sacs of human, mouse, and chicken.Proc Natl Acad Sci U S A. 2017; 114: E4753-E4761Crossref PubMed Scopus (69) Google Scholar]. In this review, we discuss the most important and recent findings on the contribution of extraembryonic hematopoiesis to blood and immune cell production in the mouse model, and we summarize the most recent advances in extraembryonic hematopoiesis in humans. Phylogenetically, the yolk sac is the oldest of the four extraembryonic membranes and evolved in fish over 500 million years ago [3Ross C Boroviak TE. Origin and function of the yolk sac in primate embryogenesis.Nat Commun. 2020 Jul 28; 11: 3760Crossref PubMed Scopus (87) Google Scholar]. It consists of an outer endodermal and an inner mesodermal lining (Figure 1). The endodermal layer of the yolk sac develops from the extraembryonic visceral endoderm (originating from the primitive endoderm). The mesodermal layer on the other hand emerges from the posterior epiblast after epithelial-mesenchymal migration (EMT) at the primitive streak during gastrulation (at embryonic day (E) 6.5) [4Arnold SJ Robertson EJ. Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo.Nat Rev Mol Cell Biol. 2009; 10: 91-103Crossref PubMed Scopus (0) Google Scholar]. Around 24 hours later, the emergence of the first blood cells from this mesoderm layer will mark the start of hematopoiesis. Initially, direct observation of avian embryo development showed that the first red blood cells of the embryo arise in the yolk sac, within vascular structures termed blood islands [5Wolff CF. Theorie von der Generation.1764https://www.deutschestextarchiv.de/book/view/wolff_theorie_1764/?p=189&hl=BlutDate accessed: July 29, 2024Google Scholar,6Pander C. Dissertatio inauguralis sistens historiam metamorphoseos quam ovum incubatum prioribus quinque diebus subit. 1817.https://www.summagallicana.it/Embriologia/Pander_Christian/Pander_Christian_latino/Pander_Christian_latino_inglese/Pander_latino_ingl_03.htm. Accessed July 29, 2024.Google Scholar], before the onset of circulation [7His, W. Untersuchungen über die erste Anlage des Wirbelthierleibes: Die erste Entwickelung des Hühnchens im Ei. 1868.Google Scholar]. Subsequent advances in histology at the beginning of the 20th century enabled pioneering scientists to observe the development of red blood cells, megakaryocytes, and macrophages in the mammalian yolk sac in cats, rabbits, mice, and rats [8Maximow AA. Untersuchungen uber blut und bindegewebe 1. Die fruhesten entwicklungsstadien der blut- und binde- gewebszellan bein saugetierembryo, bis zum anfang der blutbilding unden leber.Arch Mikroskop Anat. 1909; 73: 444-561Crossref Google Scholar] as well as in chickens [9Dantschakoff W. Untersuchungen über die Entwickelung des Blutes und Bindegewebes bei den Vögeln.Anatomische Hefte. I. Abteilung. 1908; : 37Google Scholar,10Sabin F. Studies on the origin of blood-vessels and of red blood-corpuscles as seen in the living blastoderm of chicks during the second day of incubation.Contributions to Embryology. 1920; : 36Google Scholar] and humans [11Bloom W Bartelmez GW. Hematopoiesis in young human embryos.American Journal of Anatomy. 1940; 67: 21-53Crossref Google Scholar]. We would like to highlight the illuminating work performed by embryologists Vera Dantschakoff and Florence Sabin that noticed the close relationship between hematopoietic cells and endothelial cells [9Dantschakoff W. Untersuchungen über die Entwickelung des Blutes und Bindegewebes bei den Vögeln.Anatomische Hefte. I. Abteilung. 1908; : 37Google Scholar,10Sabin F. Studies on the origin of blood-vessels and of red blood-corpuscles as seen in the living blastoderm of chicks during the second day of incubation.Contributions to Embryology. 1920; : 36Google Scholar]. It was hypothesized that hematopoietic stem cells might emerge during this developmental period. Work in the chicken embryo demonstrated that hematopoietic stem cells arise intraembryonically and not from the yolk sac, contrary to what was initially proposed [12Moore MAS Metcalf D. Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo.British Journal of Haematology. 1970; 18: 279-296Crossref PubMed Google Scholar,13Dieterlen-Lievre F. On the origin of haemopoietic stem cells in the avian embryo: An experimental approach.Development. 1975; 33: 607-619Crossref Google Scholar]. This was later confirmed in the mouse model by several research groups. They identified the para-aortic splanchnopleura (P-Sp), the precursor of the aorta-gonad mesonephros (AGM), in the embryo proper as the site of hematopoietic stem cell emergence [14Medvinsky AL Samoylina NL Müller AM Dzierzak EA. An early pre-liver intraembryonic source of CFU-S in the developing mouse.Nature. 1993; 364: 64-67Crossref PubMed Scopus (441) Google Scholar, 15Müller 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 Google Scholar, 16Medvinsky A Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region.Cell. 1996; 86: 897-906Abstract Full Text Full Text PDF PubMed Scopus (1229) Google Scholar, 17Cumano A Ferraz JC Klaine M Di Santo JP Godin I. Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution.Immunity. 2001; 15: 477-485Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar]. Within the AGM region, hematopoietic cells emerge from the endothelium of the dorsal aorta through a Runx1-dependent endothelial-to-hematopoietic transition (EHT) [18North T Gu T-L Stacy T et al.Cbfa2 is required for the formation on intra-aortic hematopoietic clusters.Development. 1999; 26: 2563-2575Crossref Google Scholar, 19de 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 (485) Google Scholar, 20Kissa K Herbomel P. Blood stem cells emerge from aortic endothelium by a novel type of cell transition.Nature. 2010; 464: 112-115Crossref PubMed Scopus (0) Google Scholar, 21Boisset 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 (727) Google Scholar, 22Bertrand 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 (814) Google Scholar], confirming the initial notion of a close relationship between hematopoietic and endothelial cells. The EHT process has been extensively reviewed [23Jaffredo T Nottingham W Liddiard K Bollerot K Pouget C de Bruijn M. From hemangioblast to hematopoietic stem cell: an endothelial connection?.Exp Hematol. 2005; 33: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar,24Ottersbach K. Endothelial-to-haematopoietic transition: an update on the process of making blood.Biochem Soc Trans. 2019; 47: 591-601Crossref PubMed Scopus (49) Google Scholar]. Thanks to morphological and histological observations, transplantation and colony-forming assays, lineage tracing, bar-coding, and single-cell omics, it is now widely accepted that the blood and immune system develops in several consecutive waves. These progenitor waves are not only spatiotemporally distinct; analysis of mutant embryos for key transcription factors (such as Runx1 and Myb) also revealed that they are controlled by different molecular mechanisms. In mice, the extraembryonic yolk sac first gives rise to (1) a primitive wave of blood cells, followed by (2) a transient definitive wave consisting mainly of erythro-myeloid progenitors (EMPs) [8Maximow AA. Untersuchungen uber blut und bindegewebe 1. Die fruhesten entwicklungsstadien der blut- und binde- gewebszellan bein saugetierembryo, bis zum anfang der blutbilding unden leber.Arch Mikroskop Anat. 1909; 73: 444-561Crossref Google Scholar,12Moore MAS Metcalf D. Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo.British Journal of Haematology. 1970; 18: 279-296Crossref PubMed Google Scholar,25Palis 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, 26Gomez Perdiguero E Klapproth K et al.Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.Nature. 2015; 518: 547-551Crossref PubMed Scopus (1626) Google Scholar, 27Goh I Botting RA Rose A et al.Yolk sac atlas reveals multiorgan functions during early development.Science. 2023; 381: eadd7564Crossref PubMed Scopus (0) Google Scholar]. Hematopoiesis then switches to the embryo proper, wherein (3) embryonic multipotent progenitors and (4) hematopoietic stem cells emerge from the dorsal aorta in the AGM. Intra- but not extraembryonic hematopoiesis can sustain life-long hematopoiesis [14Medvinsky AL Samoylina NL Müller AM Dzierzak EA. An early pre-liver intraembryonic source of CFU-S in the developing mouse.Nature. 1993; 364: 64-67Crossref PubMed Scopus (441) Google Scholar,15Müller 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 Google Scholar,17Cumano A Ferraz JC Klaine M Di Santo JP Godin I. Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution.Immunity. 2001; 15: 477-485Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,28Patel SH Christodoulou C Weinreb C et al.Lifelong multilineage contribution by embryonic-born blood progenitors.Nature. 2022; 606: 747-753Crossref PubMed Scopus (52) Google Scholar,29Yokomizo T Ideue T Morino-Koga S et al.Independent origins of fetal liver haematopoietic stem and progenitor cells.Nature. 2022; 609: 779-784Crossref PubMed Scopus (48) Google Scholar] (Figure 2A). Although it was long thought that yolk sac-derived hematopoiesis is simply a transient wave important for the survival of the embryo until hematopoietic stem cells are mature enough to supersede blood and immune cell production, research of the last decades has provided ample evidence that yolk sac-derived cells remain in the organism long after the end of yolk sac hematopoiesis and even into adulthood [26Gomez Perdiguero E Klapproth K et al.Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.Nature. 2015; 518: 547-551Crossref PubMed Scopus (1626) Google Scholar,30Ginhoux F Greter M Leboeuf M et al.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (3667) Google Scholar, 31Schulz C Gomez Perdiguero E Chorro L et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (1942) Google Scholar, 32Gentek R Ghigo C Hoeffel G et al.Epidermal γδ T cells originate from yolk sac hematopoiesis and clonally self-renew in the adult.J Exp Med. 2018; 215: 2994-3005Crossref PubMed Scopus (72) Google Scholar, 33Soares-da-Silva F Freyer L Elsaid R et al.Yolk sac, but not hematopoietic stem cell-derived progenitors, sustain erythropoiesis throughout murine embryonic life.J Exp Med. 2021; 218e20201729Crossref PubMed Google Scholar] (Figure 2B, C). Here, we define primitive hematopoiesis as the production of mature blood cells from progenitors that arise directly from the mesoderm, without undergoing an endothelial-to-hematopoietic process. In the mouse, they emerge directly from the mesoderm in the blood islands between E7.5 and E8.5 [8Maximow AA. Untersuchungen uber blut und bindegewebe 1. Die fruhesten entwicklungsstadien der blut- und binde- gewebszellan bein saugetierembryo, bis zum anfang der blutbilding unden leber.Arch Mikroskop Anat. 1909; 73: 444-561Crossref Google Scholar,25Palis 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,34Ueno H Weissman IL. Clonal analysis of mouse development reveals a polyclonal origin for yolk sac blood islands.Dev Cell. 2006; 11: 519-533Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar] (Figure 1). Primitive cells are independent of c-Myb [31Schulz C Gomez Perdiguero E Chorro L et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (1942) Google Scholar,35Tober J McGrath KE Palis J. Primitive erythropoiesis and megakaryopoiesis in the yolk sac are independent of c-myb.Blood. 2008; 111: 2636-2639Crossref PubMed Scopus (50) Google Scholar] and Runx1 [36Lacaud G Gore L Kennedy M Kouskoff V Kingsley P Hogan C Carlsson L Speck N Palis J Keller G. Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro.Blood. 2002; 100: 458-466Crossref PubMed Scopus (0) Google Scholar,37Potts KS Sargeant TJ Markham JF et al.A lineage of diploid platelet-forming cells precedes polyploid megakaryocyte formation in the mouse embryo.Blood. 2014; 124: 2725-2729Crossref PubMed Scopus (49) Google Scholar], which means that they can develop in the absence of these factors regardless of whether they express them (Figure 1). The term primitive can refer to different features and is further discussed in Box 1. Box 1Primitive versus definitive hematopoiesisTabled 1In the context of hematopoiesis, the terms "primitive" and "definitive" have been used inconsistently in the literature.Originally, the term "primitive" was introduced at the beginning of the 20th century to distinguish transient nucleated erythroblasts from the enucleated erythroblasts that replace the first [8Maximow AA. Untersuchungen uber blut und bindegewebe 1. Die fruhesten entwicklungsstadien der blut- und binde- gewebszellan bein saugetierembryo, bis zum anfang der blutbilding unden leber.Arch Mikroskop Anat. 1909; 73: 444-561Crossref Google Scholar,9Dantschakoff W. Untersuchungen über die Entwickelung des Blutes und Bindegewebes bei den Vögeln.Anatomische Hefte. I. Abteilung. 1908; : 37Google Scholar,38SaxerÜber die Entwickelung und den Bau normaler Lymphdrüsen.Anat Hefte. 1986; 6Google Scholar,39Jolly J. Sur l'évolution des globules rouges dans le sang des embryons des mammifères.Compt Rend Soc Biol. 1905; : T.57Google Scholar]. "Primitive" in this case refers to the resemblance to red blood cells of lower vertebrates in which only large, nucleated red blood cells are found [40Gulliver G. Observations on the sizes and shapes of red corpuscles of the blood of vertebrates, with drawings of them to a uniform scale, and extended and revised tables of measurement.Proc Zool Soc London. 1875; : 474-495Google Scholar]. The term also reflects the presence of embryonic hemoglobin. In this school of thought, blood cells emerging from the same ancestor as nucleated red blood cells will also be called primitive cells. Cells related to enucleated red blood cells without embryonic hemoglobin would be termed "definitive." Accordingly, EMPs and their progeny should be termed "definitive."However, the term "primitive" can also refer to the absence of full reconstitution potential in transplantation assays. By this definition, only bona fide stem cells and multipotent progenitors are "definitive" [30Ginhoux F Greter M Leboeuf M et al.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (3667) Google Scholar,41Orkin SH Zon LI. Hematopoiesis: an evolving paradigm for stem cell biology.Cell. 2008; 132: 631-644Abstract Full Text Full Text PDF PubMed Scopus (1860) Google Scholar].A third use of the term "primitive" is to describe cells not following the adult differentiation pathway. A well-known example of this is the differentiation of EMP-derived macrophages that develop without passing through a GMP or monocyte intermediate [26Gomez Perdiguero E Klapproth K et al.Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.Nature. 2015; 518: 547-551Crossref PubMed Scopus (1626) Google Scholar,27Goh I Botting RA Rose A et al.Yolk sac atlas reveals multiorgan functions during early development.Science. 2023; 381: eadd7564Crossref PubMed Scopus (0) Google Scholar,42Hoeffel G Chen J Lavin Y et al.C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.Immunity. 2015; 42: 665-678Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar].Lastly, "primitive" can also refer to immature morphology or distinct functions in comparison to adult cells, which would be the case for most embryonic mature immune cells. This is the case in regards to primitive macrophages as termed by Naito et al [43Naito M Yamamura F Nishikawa S Takahashi K. Development, differentiation, and maturation of fetal mouse yolk sac macrophages in cultures.J Leukoc Biol. 1989; 46: 1-10Crossref PubMed Google Scholar]. Yolk sac macrophages at E10.5 lacked the classical ultrastructural features of fetal liver and adult macrophages (hence "primitive" morphology) and they were observed before any monocytes were detected ("primitive" development that did not follow the classical adult differentiation cascade from monocytes) [43Naito M Yamamura F Nishikawa S Takahashi K. Development, differentiation, and maturation of fetal mouse yolk sac macrophages in cultures.J Leukoc Biol. 1989; 46: 1-10Crossref PubMed Google Scholar].We propose that progenitors arising from an endothelial-to hematopoietic transistion (EHT) should be termed as "definitive", irrespectively of the "primitive" or immature features of the mature cells they give rise to. In addition, the potential of primitive progenitors is restricted to a single lineage (either erythroid or myeloid). Recently, some papers have distinguished between primitive EMP and definitive EMP. EMP are defined as having erythroid and myeloid potential at the single-cell level (and they arise from an EHT), therefore they should not be referred to as primitive. To distinguish EMPs from bona fide HSPCs the terms "pro-definitive" [44Canu G Ruhrberg C First blood: the endothelial origins of hematopoietic progenitors.Angiogenesis. 2021 May; 24: 199-211Crossref PubMed Scopus (37) Google Scholar] or "transient-definitive" [45Calvanese V Mikkola HKA. The genesis of human hematopoietic stem cells.Blood. 2023; 142: 519-532Crossref PubMed Scopus (14) Google Scholar] have been introduced. Open table in a new tab Tabled 1In the context of hematopoiesis, the terms "primitive" and "definitive" have been used inconsistently in the literature.Originally, the term "primitive" was introduced at the beginning of the 20th century to distinguish transient nucleated erythroblasts from the enucleated erythroblasts that replace the first [8Maximow AA. Untersuchungen uber blut und bindegewebe 1. Die fruhesten entwicklungsstadien der blut- und binde- gewebszellan bein saugetierembryo, bis zum anfang der blutbilding unden leber.Arch Mikroskop Anat. 1909; 73: 444-561Crossref Google Scholar,9Dantschakoff W. Untersuchungen über die Entwickelung des Blutes und Bindegewebes bei den Vögeln.Anatomische Hefte. I. Abteilung. 1908; : 37Google Scholar,38SaxerÜber die Entwickelung und den Bau normaler Lymphdrüsen.Anat Hefte. 1986; 6Google Scholar,39Jolly J. Sur l'évolution des globules rouges dans le sang des embryons des mammifères.Compt Rend Soc Biol. 1905; : T.57Google Scholar]. "Primitive" in this case refers to the resemblance to red blood cells of lower vertebrates in which only large, nucleated red blood cells are found [40Gulliver G. Observations on the sizes and shapes of red corpuscles of the blood of vertebrates, with drawings of them to a uniform scale, and extended and revised tables of measurement.Proc Zool Soc London. 1875; : 474-495Google Scholar]. The term also reflects the presence of embryonic hemoglobin. In this school of thought, blood cells emerging from the same ancestor as nucleated red blood cells will also be called primitive cells. Cells related to enucleated red blood cells without embryonic hemoglobin would be termed "definitive." Accordingly, EMPs and their progeny should be termed "definitive."However, the term "primitive" can also refer to the absence of full reconstitution potential in transplantation assays. By this definition, only bona fide stem cells and multipotent progenitors are "definitive" [30Ginhoux F Greter M Leboeuf M et al.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (3667) Google Scholar,41Orkin SH Zon LI. Hematopoiesis: an evolving paradigm for stem cell biology.Cell. 2008; 132: 631-644Abstract Full Text Full Text PDF PubMed Scopus (1860) Google Scholar].A third use of the term "primitive" is to describe cells not following the adult differentiation pathway. A well-known example of this is the differentiation of EMP-derived macrophages that develop without passing through a GMP or monocyte intermediate [26Gomez Perdiguero E Klapproth K et al.Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.Nature. 2015; 518: 547-551Crossref PubMed Scopus (1626) Google Scholar,27Goh I Botting RA Rose A et al.Yolk sac atlas reveals multiorgan functions during early development.Science. 2023; 381: eadd7564Crossref PubMed Scopus (0) Google Scholar,42Hoeffel G Chen J Lavin Y et al.C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.Immunity. 2015; 42: 665-678Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar].Lastly, "primitive" can also refer to immature morphology or distinct functions in comparison to adult cells, which would be the case for most embryonic mature immune cells. This is the case in regards to primitive macrophages as termed by Naito et al [43Naito M Yamamura F Nishikawa S Takahashi K. Development, differentiation, and maturation of fetal mouse yolk sac macrophages in cultures.J Leukoc Biol. 1989; 46: 1-10Crossref PubMed Google Scholar]. Yolk sac macrophages at E10.5 lacked the classical ultrastructural features of fetal liver and adult macrophages (hence "primitive" morphology) and they were observed before any monocytes were detected ("primitive" development that did not follow the classical adult differentiation cascade from monocytes) [43Naito M Yamamura F Nishikawa S Takahashi K. Development, differentiation, and maturation of fetal mouse yolk sac macrophages in cultures.J Leukoc Biol. 1989; 46: 1-10Crossref PubMed Google Scholar].We propose that progenitors arising from an endothelial-to hematopoietic transistion (EHT) should be termed as "definitive", irrespectively of the "primitive" or immature features of the mature cells they give rise to. In addition, the potential of primitive progenitors is restricted to a single lineage (either erythroid or myeloid). Recently, some papers have distinguished between primitive EMP and definitive EMP. EMP are defined as having erythroid and myeloid potential at the single-cell level (and they arise from an EHT), therefore they should not be referred to as primitive. To distinguish EMPs from bona fide HSPCs the terms "pro-definitive" [44Canu G Ruhrberg C First blood: the endothelial origins of hematopoietic progenitors.Angiogenesis. 2021 May; 24: 199-211Crossref PubMed Scopus (37) Google Scholar] or "transient-definitive" [45Calvanese V Mikkola HKA. The genesis of human hematopoietic stem cells.Blood. 2023; 142: 519-532Crossref PubMed Scopus (14) Google Scholar] have been introduced. Open table in a new tab Primitive erythroblasts are the first hematopoietic cells that develop in the vertebrate yolk sac and are essential for embryonic survival in mammals [46Fujiwara Y Browne CP Cunniff K Goff SC Orkin SH. Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1.Proc Natl Acad Sci USA. 1996; 93: 12355-12358Crossref PubMed Scopus (0) Google Scholar]. The development of primitive erythroblasts within the yolk sac blood islands is the best studied. Blood islands are mesodermal cell masses proliferating between the visceral endoderm and mesothelial cell layer. Although the outer cells differentiate into endothelial cells, mesodermal cells in the center of the cluster differentiate into primitive erythroblasts [47Haar JL Ackerman GA. A phase and electron microscopic study of vasculogenesis and erythropoiesis in the yolk sac of the mouse.Anat Rec. 1971; 170: 199-223Crossref PubMed Google Scholar,48Ferkowicz MJ Yoder MC. Blood island formation: longstanding observations and modern interpretations.Exp Hematol. 2005; 33: 1041-1047Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar]. These are larger than their definitive counterpart and contain both embryonic ζ-hemoglobin (εy- and βH1-globin) and adult α- and β-hemoglobin (α1-, α2-, β1- and β2-globin) [49Barker JE. Development of the mouse hematopoietic system. I. Types of hemoglobin produced in embryonic yolk sac and liver.Dev Biol. 1968; 18: 14-29Crossref PubMed Google Scholar] and mature in circulation while retaining their nucleus until E12.5 [50Kingsley PD Malik J Fantauzzo KA Palis J. Yolk sac-derived erythroblasts enucleate during mammalian embryogenesis.Blood. 2004; 104: 19-25Crossref PubMed Scopus (0) Google Scholar]. The primitive wave is the only source of red blood cells until E12.5 and can sustain the embryo until E15.5 [50Kingsley PD Malik J Fantauzzo KA Palis J. Yolk sac-derived erythroblasts enucleate during mammalian embryogenesis.Blood. 2004; 104: 19-25Crossref PubMed Scopus (0) Google Scholar,51Mucenski ML McLain K Kier AB et al.A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis.Cell. 1991; 65: 677-689Abstract Full Text PDF PubMed Scopus (944) Google Scholar]. By E17.5, most primitive erythroblasts are gone, but few are still detectable as late as 5 days after birth (Figure 2B) [50Kingsley PD Malik J Fantauzzo KA Palis J. Yolk sac-derived erythroblasts enucleate during mammalian embryogenesis.Blood. 2004; 104: 19-25Crossref PubMed Scopus (0) Google Scholar]. Concomitantly with primitive erythroblasts, megakaryocyte potential emerges in the yolk sac around E7.5, peaks around E8.5, and is no longer detectable after E13.5 [8Maximow AA. Untersuchungen uber blut und bindegewebe 1. Die fruhesten entwicklungsstadien der blut- und binde- gewebszellan bein saugetierembryo, bis zum anfang der blutbilding unden leber.Arch Mikroskop Anat. 1909; 73: 444-561Crossref Google Scholar,52Xu M J Matsuoka S Yang FC et al.Evidence for the presence of murine primitive megakaryocytopoiesis in the early yolk sac.Blood. 2001; 97: 2016-2022Crossref PubMed Scopus (94) Google Scholar,53Tober J Koniski A McGrath KE et al.The