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Ontogeny of erythropoiesis James Palis University of Rochester Medical Center, Rochester, New York, USA Correspondence to James Palis, MD, University of Rochester Medical Center, Department of Pediatrics and Center for Pediatric Biomedical Research, Box 703, 601 Elmwood Ave., Rochester, NY 14642, USA Tel: +1 585 275 5098; fax: +1 585 276 0232; e-mail: [email protected] Current Opinion in Hematology 2008, 15:155– 161 Purpose of review The present study review examines the current understanding of the ontogeny of erythropoiesis with a focus on the emergence of the embryonic (primitive) erythroid lineage and on the similarities and differences between the primitive and the fetal/adult (definitive) forms of erythroid cell maturation. Recent findings Primitive erythroid precursors in the mouse embryo and cultured in vitro from human embryonic stem cells undergo ‘maturational’ globin switching as they differentiate terminally. The appearance of a transient population of primitive ‘pyrenocytes’ (extruded nuclei) in the fetal bloodstream indicates that primitive erythroblasts enucleate by nuclear extrusion. In-vitro differentiation of human embryonic stem cells recapitulates hematopoietic ontogeny reminiscent of the murine yolk sac, including overlapping waves of hemangioblast, primitive, erythroid, and definitive erythroid progenitors. Definitive erythroid potential in zebrafish embryos, like that in mice, initially arises prior to, and independent of, hematopoietic stem cell emergence in the region of the aorta. Maturation of definitive erythroid cells within macrophage islands promotes erythroblast–erythroblast and erythroblast–stromal interactions that regulate red cell output. Summary The study of embryonic development in several different model systems, as well as in cultured human embryonic stem cells, continues to provide important insights into the ontogeny of erythropoiesis. Contrasting the similarities and differences between primitive and definitive erythropoiesis will lead to an improved understanding of erythroblast maturation and the terminal steps of erythroid differentiation. Keywords definitive erythropoiesis, hemangioblast, primitive erythropoiesis, pyrenocyte Curr Opin Hematol 15:155–161 ß 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins 1065-6251 Introduction The red cells of mammals are unique in the animal kingdom as they circulate as enucleated cells. In contrast, the fully mature red cells of birds, amphibians, and fish remain nucleated [1]. A century ago, examination of mammalian embryos revealed the presence of distinct nucleated and enucleated red cells [2]. The continuous circulation of small, enucleated erythrocytes during fetal and postnatal life (‘definitive’ erythropoiesis) was distinguished from ‘primitive’ erythropoiesis, characterized by the transient circulation of large, nucleated red cells that originate in the yolk sac. Similarly, it was recognized that primitive erythropoiesis originates in the yolk sac and intermediate cell mass of chicken and zebrafish embryos, respectively. As mammalian primitive erythroblasts circulate as nucleated cells and are confined to the embryo, they have been thought of as a ‘primitive’ form of erythropoiesis that shares many characteristics with the nucleated red cells of nonmammalian vertebrates. Exam- ination of embryonic and fetal blood cell morphology, however, revealed that primitive red cells undergo a synchronous wave of maturation in the bloodstream. Four years ago, it was discovered that late-stage primitive erythroblasts in the mouse embryo complete their maturation by enucleating and continuing to circulate for several more days as erythrocytes [3]. The specification of hematopoiesis in mammalian embryos was discussed last in Current Opinion in Hematology 3 years ago [4]. Here, recent insights regarding the ontogeny of erythropoiesis and the maturation of the primitive and definitive erythroid lineages will be reviewed. Emergence of primitive erythropoiesis in the early embryo The initial generation of erythroid cells in the embryo of mammalian and nonmammalian organisms depends on the formation of mesoderm cells that migrate through the primitive streak and contribute to the formation of 1065-6251 ß 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 156 Erythroid system and its diseases intraembryonic as well as extraembryonic structures such as the yolk sac and the placenta in mammals. Immature primitive erythroid cells rapidly pool into so-called blood islands soon after the start of gastrulation in the yolk sac of mammalian and avian embryos [5,6]. These blood islands become enveloped by endothelial cells, which form the initial vascular plexus of the yolk sac (reviewed by Ferkowicz and Yoder, [7]). The appearance of primitive erythroid cells and endothelial cells at the same time and place within the early conceptus has long suggested that these lineages share a common developmental origin. A unique blast colonyforming cell (Blast-CFC) containing both hematopoietic and endothelial cell potential has been identified in both cultured embryonic stem cells and mouse embryos [8,9]. Consistent with the hypothesis that the first blood cells arise from a hemangioblast precursor, clonal studies reveal that a small number of GATA1-expressing cells in blood islands of mouse yolk sacs have endothelial as well as primitive erythroid cell potential [10]. Furthermore, the transcription factors GATA2 and endoglin, both of which function in hematopoietic stem cells (HSC), have each been shown to regulate hemangioblast precursors [11,12]. The close association of the primitive erythroid and endothelial lineages is further supported by the finding that the primitive erythroid lineage in the mouse emerges from mesodermal cells that express markers found in adult endothelial cells, including flk-1, vascular endothelial cadherin, tie-2, and PECAM-1 [13]. There is an increasing evidence, however, that suggests that many, if not most, yolk sac vascular cells in the conceptus arise from unilineage angioblast precursors and not from hemangioblasts [14,15]. Thus, it is likely that all hematopoietic cells, but few endothelial cells, in the mammalian yolk sac arise from hemangioblast precursors. Terminal maturation of primitive erythroid cells Immature primitive erythroblasts in the blood islands of the mouse yolk sac begin to circulate at E8.25 coincident with, or soon after, the onset of cardiac contractions [16,17]. Over the next 8 days, primitive erythroid cells mature in a synchronous cohort as they undergo changes well recognized in maturing definitive erythroid precursors, including a limited number of cell divisions, accumulation of increasing amounts of hemoglobin, nuclear condensation, a progressive decrease in cell size, and ultimately enucleation [3,18]. Many of these findings have recently been confirmed using a transgenic mouse expressing the enhanced green fluorescent protein (eGFP) in primitive erythroid cells [19]. Interestingly, circulating primitive erythroblasts express several adhesion molecules that could mediate interactions with other cell types [19]. Coincident with primitive erythroblast enucleation, the appearance of a transient population of very small, nucleated cells with a rim of ey-globin-positive cytoplasm was noted in the circulation of mouse embryos [20]. These cells are reminiscent of extruded nuclei [21], which are the product of enucleation of late-stage erythroblasts (Fig. 1). The extruded nuclei from definitive erythroid cells undergo rapid loss of the phosphatidylserine asymmetry of the cell membrane and are engulfed by macrophage cells [22]. As the cell membrane plays an important role in the biology of these cells, extruded nucleus is an inadequate term and ‘pyrenocytes’, derived from the Greek word ‘pyren’ (the pit of a stone fruit), has been proposed as a more appropriate name for this very transient cell [20]. The discovery of primitive pyrenocytes in the fetal bloodstream suggests that late-stage primitive erythroblasts enucleate by nuclear extrusion. Unlike definitive erythroblasts, primitive erythroblasts do not enucleate spontaneously in vitro [20]. Nevertheless, they are capable, like definitive erythroblasts, of physically interacting with F4/80positive macrophage cells in vitro, in part through a4integrin-mediated interactions [20,23]. These findings, supported also by immunohistochemical studies [20], raise the intriguing likelihood that primitive erythroblasts enucleate while associated with macrophage cells in vivo. Figure 1 Enucleation of late-stage erythroblasts leads to the formation of two cells – a reticulocyte and a pyrenocyte n n Pyrenocyte n Reticulocyte Late-stage erythroblast Pyrenocytes lose phosphatidylserine asymmetry and are subsequently phagocytosed by macrophage cells. Reproduced with permission [22]. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Ontogeny of erythropoiesis Palis 157 Globin regulation in primitive erythroid cells Hemoglobin molecules contain globin chains derived both from the a-globin and b-globin gene loci. Although definitive erythroid cells in the mouse express a1-globin, a2globin, b1-globin, and b2-globin, primitive erythroid cells in addition express z-globin, bH1-globin, and ey-globin [24]. An extensive analysis of globin gene expression in primitive erythroid cells indicates that the initially expressed z- and bH1-globin genes are superseded by the a1-globin, a2-globin and ey-globin genes, respectively, as primitive proerythroblasts at E7.5 transition to reticulocytes at E15.5 [25]. This ‘maturational’ globin switching is associated with changes in RNA polymerase II density at the promoters of these various globin genes. Furthermore, the bH1-globin and ey-globin genes in primitive erythroid cells reside in a single large hyperacetylated domain, suggesting that this novel form of globin switching is regulated by altered transcription factor presence instead of chromatin accessibility [25]. The GATA1 transcription factor plays an essential role in the regulation of erythroid-specific genes in both primitive and definitive erythroid cells and the loss of GATA1 leads to the arrest of both lineages at the proerythroblast stage of maturation [26,27]. Interestingly, different functional domains of GATA1 are required for activation of target genes in primitive versus definitive erythroid cells [28], suggesting that different transcriptional complexes may form in these lineages. Recent examination of murine erythroleukemia cells has led to the identification of novel transcriptional complexes involving a core complex (GATA1, Tal1, E2A, Lmo2, and Ldbd1) and newly identified Ldb1-binding partners Eto-2, Cdk9, and Lmo4 [29]. Forced downregulation of these latter transcription factors in zebrafish embryos reveals functional roles in definitive, but not primitive, hematopoiesis. These and other data are leading to increasing complex models of genetic regulatory networks in the emerging embryo and definitive erythroid cells [30]. Several transcription factors have been implicated in the regulation of globin gene expression in primitive erythroid cells, including several Kruppel-like transcription factors. Studies of klf2-null mouse embryos revealed significant decreases in bH1-globin and ey-globin transcripts in primitive erythroid precursors [31]. Interestingly, human e-globin transgenes were also reduced in mice lacking klf2, providing evidence that this transcription factor plays a similar role in humans. Morpholino knockdown of klf4 in zebrafish embryos leads to decreased embryonic globin gene expression [32]. Furthermore, klf4 preferentially binds the CACC sites in the promoters of the embryonic compared with the adult b-globin genes. The erythroid-specific Kruppellike factor (EKLF) was originally thought to primarily regulate the adult b-globin genes. Recent reexamination of primitive as well as definitive erythroid cells in EKLFnull mouse embryos, however, indicates that EKLF also regulates multiple erythroid-specific genes, including genes encoding AHSP, heme synthetic pathway proteins and cytoskeletal proteins such as ankyrin and band 3 [33,34]. Surprisingly, EKLF-null mouse embryos also have reduced accumulation of bH1-globin and ey-globin transcripts in primitive erythroblasts [35]. Furthermore, double EKLF/klf2-null mouse fetuses have a more severe reduction in embryonic globin gene expression than either single-null mutant, indicating that these two Kruppel-like factors play nonredundant roles in embryonic globin gene regulation. Erythropoietin is the central cytokine necessary for definitive erythroid cell maturation; however, its role in primitive erythropoiesis is less well understood. Targeted disruption of erythropoietin in mouse leads to a significant decrease in primitive erythroid cell numbers, but maturation does not seem to be disrupted for the remainder [36,37]. Interestingly, morpholino knockdown of erythropoietin in zebrafish embryos leads to a similar differential phenotype between primitive and definitive erythropoiesis [38]. Examination of erythropoietin signaling revealed several differences in mouse embryonic stem cell-derived primitive and definitive erythroid cells [39]. Primitive erythroblasts express higher levels of erythropoietin receptor and have a sustained and robust phosphorylation of the downstream STAT5 signaling molecule but barely detectable levels of the STAT5 inhibitory protein SHP-1. These differences predict a heightened sensitivity of primitive erythroblasts to erythropoietin signaling. Although these studies suggest that there may be differential roles for erythropoietin in primitive versus definitive erythropoiesis, it is likely that other cytokine signaling cascades differentially regulate primitive and definitive erythroid cell maturation. Hematopoiesis in the yolk sac is not confined to primitive erythropoiesis Examination of hematopoietic progenitors in carefully staged mouse embryos as well as cultured embryonic stem cells has revealed two overlapping waves of erythroid potential [40–42] (Fig. 2). The first wave consists of primitive erythroid progenitors (EryP-CFC) present in the yolk sac between E7.25 and E9.0 of gestation that is temporally associated with macrophage and megakaryocyte progenitors. Recent clonogenic studies in mouse embryos indicate that the primitive erythroid and megakaryocyte lineages are tied to a common bipotential primitive erythroid/megakaryocyte progenitor [43]. Thus, primitive hematopoiesis is at least bilineage in nature. The developmental origin of thrombocytes, however, has not yet been examined in other model organisms. The Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 158 Erythroid system and its diseases Figure 2 Simplified model of erythroid ontogeny in the mammalian embryo Current data support a model in which three waves of erythroid progenitors emerge in the mammalian embryo. The first wave consists of primitive erythroid progenitors (EryP-CFC) that originate in the yolk sac from hemangioblast precursors (HB) and generate a synchronous cohort of primitive erythroid precursors that mature in the bloodstream and enucleate to form reticulocytes and pyrenocytes. The second wave consists of a transient wave of definitive erythroid progenitors (BFU-E) that emerge from the yolk sac and seed the fetal liver. There they generate maturing definitive erythroid precursors that enucleate to become the first circulating definitive erythrocytes (RBC) of the fetus. The third wave consists of a continuous stream of definitive erythroid progenitors in the late gestation liver and postnatal marrow that originate from adult-repopulating hematopoietic stem cells (HSC). Unlike primitive erythroid cells, definitive erythroid precursors mature extravascularly within erythroblast islands. AGM, aorta–gonad–mesonephros region; mf, macrophage cell. second wave of definitive erythroid progenitors (BFU-E) emerges and expands in the yolk sac between E8.25 and E10.5 (see below), and is temporally linked to the expansion of multiple unilineage and multilineage myeloid progenitors [42]. Emergence of definitive erythroid potential in the embryo Primitiveerythroid cells fulfillthefunctionscritical for early postimplantation embryonic survival and growth, however the fetus requires even more red cells to meet the demands of growth at later stages of development. Prior to the formation of the bone marrow, the liver serves as the site of maturation of definitive erythroid cells in the mammalian fetus. The liver in the murine embryo is colonized by external hematopoietic elements at E9.5, soon after it begins to form as an organ. BFU-E and CFU-E subsequently expand exponentially in numbers for several days and generate definitive erythroid cells [44]. The developmental origin of the BFU-E that initially colonize the liver has been postulated to be the yolk sac, as BFU-E first emerge in the yolk sac at E8.25, before the onset of the circulation, and continue to preferentially expand in numberswithin theyolk sac forthenext48 h [40,42](Fig.2). Further evidence in support of the developmental origin of definitive erythroid potential comes from the mouse models lacking a functional circulation, as for example, vascular endothelial cadherin-null embryos [45]. A more recent model involves loss of NCX1, a sodium– calcium exchanger whose expression is confined to the heart in the mouse embryo. NCX1-null embryos lack a heartbeat and thus have no functional circulation, and yet have normal numbers of primitive and definitive erythroid progenitors emerging in the yolk sac [46]. Few definitive erythroid progenitors, however, are found within the embryo proper of these mutant mice, supporting the hypothesis that all definitive hematopoietic progenitors are initially generated in the yolk sac between E8.25 and E10 and are redistributed to the embryo proper at the onset of embryonic circulation. The NCX1-mutant embryo should continue to serve as a useful model to dissect the emergence of hematopoietic potential within the embryo proper and the placenta without the confounding factor of blood cell movement within the circulation. In aggregate, these studies indicate that a transient wave of definitive erythroid progenitors emerges from the yolk sac of mammalian embryos prior to the emergence of adult-repopulating hematopoietic stem cells and colonizes the fetal liver (Fig. 2). Interestingly, a wave of Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Ontogeny of erythropoiesis Palis 159 definitive ‘erythro-myeloid’ progenitors has recently been found in the posterior blood islands of zebrafish embryos after the appearance of primitive erythroid cells in the intermediate cell mass but before emergence of HSC in the region of the aorta [47]. These findings provide evidence that hematopoietic ontogeny is quite highly conserved between disparate species. Toward the end of gestation, definitive erythroid progenitors in the mammalian embryo transition from the liver to the newly formed bone marrow. Although fundamentally similar to their adult counterparts, fetal erythroid progenitors also have some distinctive features. First, fetal BFU-E have a greater and more rapid proliferative capacity. Second, fetal, but not steady-state adult, BFU-E proliferate in vitro in response to erythropoietin in theabsence of added colony-stimulating factors [48,49]. Finally, CFU-E in the murine fetus are more sensitive to erythropoietin [50]. Maturation of definitive erythroid cells In contrast to primitive erythroid cells that mature in the bloodstream, definitive erythroid precursors in the fetal liver and postnatal marrow mature while attached to macrophage cells of erythroblastic islands [51] (Fig. 2). Although macrophage cells engulf and digest pyrenocytes following enucleation [22], it is unclear what other functions macrophage cells perform to enhance erythroid maturation. It has recently been shown that coculture of maturing Friend leukemia virus infected erythroid cells with macrophage cells in vitro enhanced their proliferation but did not alter their intrinsic ability to enucleate [52]. Potential ‘nurse’ functions of macrophage cells, such as the provision of iron and cytokines, remain to be proven. Erythroblast islands bring maturing definitive erythroid precursors in contact not only with macrophage cells but also with other erythroblasts. Fas and FasL signaling between maturing erythroblasts has been invoked as a mechanism controlling erythroid cell output. Both negative feedback of more mature erythroblasts on immature erythroblasts as well as autoregulatory loops at the proerythroblast stage of maturation have been proposed [53,54]. As definitive erythroblasts mature extravascularly, they are also in contact with stromal elements. Recent evidence suggests that definitive erythroblasts transition from an erthropoietin-dependent phase to a fibronectin-dependent phase, and that both erythropoietin and fibronectin deliver antiapoptotic signals to maturing erythroblasts [55]. Alpha 4 integrin was shown to mediate this erythroblast–fibronectin interaction, raising the intriguing question of whether similar signals are mediated to erythroblasts by VCAM1 on macrophage cells. Surprisingly, erythropoietin was recently shown to play a role at very late stages of erythroid maturation by specifically altering the expression of adhesion molecules, including podocalyxin, and thus regulating reticulocyte egress from the marrow [56]. These studies highlight an emerging role for adhesion factors in the terminal differentiation of definitive erythroid cells. MicroRNAs are endogenous molecules that target mRNAs in a sequence-specific manner and regulate various cellular functions. Recent screens have identified numerous microRNAs in definitive erythroid cells that are either upregulated or downregulated with maturation [57–59]. Initial functional studies in murine erythroleukemia cells indicate that the abundantly expressed miR451 facilitates erythroid maturation [57]. The continued study of microRNAs and their targets will lead to the identification of genes critical for hematopoietic differentiation and erythroid lineage maturation throughout ontogeny. Human embryonic stem cells serve as a new model of embryonic erythropoiesis The culture of human embryonic stem cells has emerged as an increasingly important model of early embryonic events. Recent studies indicate that the in-vitro differentiation of human embryonic stem cells recapitulates hematopoietic ontogeny reminiscent of the in-vivo yolk sac, including overlapping waves of hemangioblast, primitive erythroid, and definitive erythroid potential [60–62]. Interestingly, culture of differentiating human embryonic stem cells in liquid culture has recently provided evidence that human primitive erythroid cells, like their murine counterparts, undergo ‘maturational’ z-globin to a-globin switching [63]. Thus, human embryonic stem cells can provide access to hematopoietic cells that are not otherwise available for study. Conclusion Primitive erythropoiesis serves as a useful model of mammalian erythroid differentiation, as primitive erythroblasts in mammals mature in a synchronous cohort in the bloodstream and ultimately enucleate. In contrast, definitive erythroid precursors mature attached to macrophage cells in erythroblast islands within the complex cellular milieu of the fetal liver and postnatal bone marrow (Fig. 2). Erythroblastic islands can thus facilitate erythroblast– erythroblast and erythroblast–stromal interactions that play important roles in the regulation of definitive erythropoiesis. The continued study of the similarities and differences between primitive and definitive erythropoiesis will lead to an improved understanding of erythroblast maturation and the terminal steps of erythroid differentiation. The comparative investigation of embryonic development in multiple model organisms, including mouse, chicken, Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 160 Erythroid system and its diseases Xenopus and zebrafish, will continue to provide important insights into the ontogeny of erythropoiesis. The in-vitro culture of human embryonic system cells is now established as an important model system to investigate the early ontogeny of human hematopoiesis and offers the potential for cell-based therapies for the treatment of hematopoietic disorders. Acknowledgements I thank coworkers and colleagues for many stimulating discussions. This study was supported in part by the National Institutes of Health grants DK09361 and DK071116. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 256–257). 18 Fantoni A, de la Chapelle A, Marks PA. 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