Ontogeny of Erythropoiesis James Palis

Ontogeny of erythropoiesis James Palis

University of Rochester Medical Center, Rochester, Purpose of review New York, USA The present study review examines the current understanding of the ontogeny of Correspondence to James Palis, MD, University of erythropoiesis with a focus on the emergence of the embryonic (primitive) erythroid Rochester Medical Center, Department of Pediatrics and Center for Pediatric Biomedical Research, Box lineage and on the similarities and differences between the primitive and the fetal/adult 703, 601 Elmwood Ave., Rochester, NY 14642, USA (definitive) forms of erythroid cell maturation. Tel: +1 585 275 5098; fax: +1 585 276 0232; e-mail: [email protected] 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 Current Opinion in Hematology 2008, 15:155– 161 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

ination of embryonic and fetal blood cell morphology, Introduction however, revealed that primitive red cells undergo a The red cells of mammals are unique in the animal synchronous wave of maturation in the bloodstream. Four kingdom as they circulate as enucleated cells. In contrast, years ago, it was discovered that late-stage primitive the fully mature red cells of birds, amphibians, and fish erythroblasts in the mouse embryo complete their matu- remain nucleated [1]. A century ago, examination of ration by enucleating and continuing to circulate for mammalian embryos revealed the presence of distinct several more days as erythrocytes [3]. The specification nucleated and enucleated red cells [2]. The continuous of hematopoiesis in mammalian embryos was discussed circulation of small, enucleated erythrocytes during fetal last in Current Opinion in Hematology 3 years ago [4]. Here, and postnatal life (‘definitive’ erythropoiesis) was distin- recent insights regarding the ontogeny of erythropoiesis guished from ‘primitive’ erythropoiesis, characterized by and the maturation of the primitive and definitive ery- the transient circulation of large, nucleated red cells that throid lineages will be reviewed. 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, Emergence of primitive erythropoiesis in the respectively. As mammalian primitive erythroblasts cir- early embryo culate as nucleated cells and are confined to the embryo, The initial generation of erythroid cells in the embryo they have been thought of as a ‘primitive’ form of of mammalian and nonmammalian organisms depends erythropoiesis that shares many characteristics with the on the formation of mesoderm cells that migrate through nucleated red cells of nonmammalian vertebrates. Exam- the primitive streak and contribute to the formation of

1065-6251 ß 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins

intraembryonic as well as extraembryonic structures such with, or soon after, the onset of cardiac contractions as the yolk sac and the placenta in mammals. Immature [16,17]. Over the next 8 days, primitive erythroid cells primitive erythroid cells rapidly pool into so-called blood mature in a synchronous cohort as they undergo changes islands soon after the start of gastrulation in the yolk sac of well recognized in maturing definitive erythroid precur- mammalian and avian embryos [5,6]. These blood islands sors, including a limited number of cell divisions, accu- become enveloped by endothelial cells, which form the mulation of increasing amounts of hemoglobin, nuclear initial vascular plexus of the yolk sac (reviewed by condensation, a progressive decrease in cell size, and Ferkowicz and Yoder, [7]). ultimately enucleation [3,18]. Many of these findings have recently been confirmed using a transgenic mouse The appearance of primitive erythroid cells and endo- expressing the enhanced green fluorescent protein thelial cells at the same time and place within the early (eGFP) in primitive erythroid cells [19]. Interestingly, conceptus has long suggested that these lineages share a circulating primitive erythroblasts express several adhe- common developmental origin. A unique blast colony- sion molecules that could mediate interactions with other forming cell (Blast-CFC) containing both hematopoietic cell types [19]. and endothelial cell potential has been identified in both cultured embryonic stem cells and mouse embryos [8,9]. Coincident with primitive erythroblast enucleation, the Consistent with the hypothesis that the first blood appearance of a transient population of very small, cells arise from a hemangioblast precursor, clonal studies nucleated cells with a rim of ey-globin-positive cyto- reveal that a small number of GATA1-expressing cells in plasm was noted in the circulation of mouse embryos blood islands of mouse yolk sacs have endothelial as well [20]. These cells are reminiscent of extruded nuclei as primitive erythroid cell potential [10]. Furthermore, [21], which are the product of enucleation of late-stage the transcription factors GATA2 and endoglin, both of erythroblasts (Fig. 1). The extruded nuclei from defini- which function in hematopoietic stem cells (HSC), have tive erythroid cells undergo rapid loss of the phosphati- each been shown to regulate hemangioblast precursors dylserine asymmetry of the cell membrane and are [11,12]. The close association of the primitive erythroid engulfed by macrophage cells [22]. As the cell membrane and endothelial lineages is further supported by the plays an important role in the biology of these cells, finding that the primitive erythroid lineage in the mouse extruded nucleus is an inadequate term and ‘pyreno- emerges from mesodermal cells that express markers cytes’, derived from the Greek word ‘pyren’ (the pit of a found in adult endothelial cells, including flk-1, vascular stone fruit), has been proposed as a more appropriate endothelial cadherin, tie-2, and PECAM-1 [13]. There is name for this very transient cell [20]. The discovery of an increasing evidence, however, that suggests that primitive pyrenocytes in the fetal bloodstream suggests many, if not most, yolk sac vascular cells in the conceptus that late-stage primitive erythroblasts enucleate by arise from unilineage angioblast precursors and not from nuclear extrusion. Unlike definitive erythroblasts, primi- hemangioblasts [14,15]. Thus, it is likely that all hema- tive erythroblasts do not enucleate spontaneously in vitro topoietic cells, but few endothelial cells, in the mamma- [20]. Nevertheless, they are capable, like definitive lian yolk sac arise from hemangioblast precursors. erythroblasts, of physically interacting with F4/80- positive macrophage cells in vitro,inpartthrougha4- integrin-mediated interactions [20,23]. These findings, Terminal maturation of primitive erythroid supported also by immunohistochemical studies [20], cells raise the intriguing likelihood that primitive erythro- Immature primitive erythroblasts in the blood islands of blasts enucleate while associated with macrophage cells the mouse yolk sac begin to circulate at E8.25 coincident in vivo.

Figure 1 Enucleation of late-stage erythroblasts leads to the formation of two cells – a reticulocyte and a pyrenocyte

n Pyrenocyte

n n

Reticulocyte Late-stage erythroblast

Pyrenocytes lose phosphatidylserine asymmetry and are subsequently phagocytosed by macrophage cells. Reproduced with permission [22].

regulate the adult b-globin genes. Recent reexamination Globin regulation in primitive erythroid cells of primitive as well as definitive erythroid cells in EKLF- Hemoglobin molecules contain globin chains derived both null mouse embryos, however, indicates that EKLF also a b from the -globin and -globin gene loci. Although defini- regulates multiple erythroid-specific genes, including tive erythroid cells in the mouse express a1-globin, a2- genes encoding AHSP, heme synthetic pathway proteins globin, b1-globin, and b2-globin, primitive erythroid cells and cytoskeletal proteins such as ankyrin and band in addition express z-globin, bH1-globin, and ey-globin 3 [33,34]. Surprisingly, EKLF-null mouse embryos also [24]. An extensive analysis of globin gene expression in have reduced accumulation of bH1-globin and ey-globin primitive erythroid cells indicates that the initially transcripts in primitive erythroblasts [35]. Furthermore, expressed z- and bH1-globin genes are superseded by double EKLF/klf2-null mouse fetuses have a more the a1-globin, a2-globin and ey-globin genes, respect- severe reduction in embryonic globin gene expression ively, as primitive proerythroblasts at E7.5 transition to than either single-null mutant, indicating that these two reticulocytes at E15.5 [25]. This ‘maturational’ globin Kruppel-like factors play nonredundant roles in embryo- switching is associated with changes in RNA polymerase nic globin gene regulation. II density at the promoters of these various globin genes. Furthermore, the bH1-globin and ey-globin genes in Erythropoietin is the central cytokine necessary for primitive erythroid cells reside in a single large hyperace- definitive erythroid cell maturation; however, its role in tylated domain, suggesting that this novel form of globin primitive erythropoiesis is less well understood. Tar- switching is regulated by altered transcription factor pre- geted disruption of erythropoietin in mouse leads to a sence instead of chromatin accessibility [25]. significant decrease in primitive erythroid cell numbers, but maturation does not seem to be disrupted for the The GATA1 transcription factor plays an essential role in remainder[36,37].Interestingly,morpholinoknockdown the regulation of erythroid-specific genes in both primi- of erythropoietin in zebrafish embryos leads to a similar tive and definitive erythroid cells and the loss of GATA1 differential phenotype between primitive and definitive leads to the arrest of both lineages at the proerythroblast erythropoiesis [38]. Examination of erythropoietin sig- stage of maturation [26,27]. Interestingly, different func- naling revealed several differences in mouse embryonic tional domains of GATA1 are required for activation of stem cell-derived primitive and definitive erythroid cells target genes in primitive versus definitive erythroid cells [39]. Primitive erythroblasts express higher levels of [28], suggesting that different transcriptional complexes erythropoietin receptor and have a sustained and robust may form in these lineages. Recent examination of mur- phosphorylation of the downstream STAT5 signaling ine erythroleukemia cells has led to the identification of molecule but barely detectable levels of the STAT5 novel transcriptional complexes involving a core complex inhibitory protein SHP-1. These differences predict a (GATA1, Tal1, E2A, Lmo2, and Ldbd1) and newly heightened sensitivity of primitive erythroblasts to ery- identified Ldb1-binding partners Eto-2, Cdk9, and thropoietinsignaling.Althoughthesestudiessuggestthat Lmo4 [29]. Forced downregulation of these latter tran- there may be differential roles for erythropoietin in scription factors in zebrafish embryos reveals functional primitive versus definitive erythropoiesis, it is likely that roles in definitive, but not primitive, hematopoiesis. other cytokine signaling cascades differentially regulate These and other data are leading to increasing complex primitive and definitive erythroid cell maturation. models of genetic regulatory networks in the emerging embryo and definitive erythroid cells [30]. Hematopoiesis in the yolk sac is not confined Several transcription factors have been implicated in the to primitive erythropoiesis regulation of globin gene expression in primitive ery- Examination of hematopoietic progenitors in carefully throid cells, including several Kruppel-like transcription staged mouse embryos as well as cultured embryonic factors. Studies of klf2-null mouse embryos revealed stem cells has revealed two overlapping waves of ery- significant decreases in bH1-globin and ey-globin tran- throid potential [40–42] (Fig. 2). The first wave consists scripts in primitive erythroid precursors [31]. Interest- of primitive erythroid progenitors (EryP-CFC) present in ingly, human e-globin transgenes were also reduced in the yolk sac between E7.25 and E9.0 of gestation that is mice lacking klf2, providing evidence that this transcrip- temporally associated with macrophage and megakaryo- tion factor plays a similar role in humans. Morpholino cyte progenitors. Recent clonogenic studies in mouse knockdown of klf4 in zebrafish embryos leads to embryos indicate that the primitive erythroid and mega- decreased embryonic globin gene expression [32]. karyocyte lineages are tied to a common bipotential prim- Furthermore, klf4 preferentially binds the CACC sites itive erythroid/megakaryocyte progenitor [43]. Thus, in the promoters of the embryonic compared with the primitive hematopoiesis is at least bilineage in nature. adult b-globin genes. The erythroid-specific Kruppel- The developmental origin of thrombocytes, however, has like factor (EKLF) was originally thought to primarily not yet been examined in other model organisms. The

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) Further evidence in support of the developmental origin emerges and expands in the yolk sac between E8.25 and of definitive erythroid potential comes from the mouse E10.5 (see below), and is temporally linked to the expan- models lacking a functional circulation, as for exam- sion of multiple unilineage and multilineage myeloid ple, vascular endothelial cadherin-null embryos [45]. A progenitors [42]. 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 Emergence of definitive erythroid potential in a heartbeat and thus have no functional circulation, and the embryo yet have normal numbers of primitive and definitive Primitiveerythroidcellsfulfillthefunctionscriticalforearly erythroid progenitors emerging in the yolk sac [46]. postimplantation embryonic survival and growth, however Few definitive erythroid progenitors, however, are found the fetus requires even more red cells to meet the demands within the embryo proper of these mutant mice, support- of growth at later stages of development. Prior to the ing the hypothesis that all definitive hematopoietic pro- formation of the bone marrow, the liver serves as the site genitors are initially generated in the yolk sac between ofmaturationofdefinitiveerythroidcellsinthemammalian E8.25 and E10 and are redistributed to the embryo proper fetus. The liver in the murine embryo is colonized by at the onset of embryonic circulation. The NCX1-mutant external hematopoietic elements at E9.5, soon after it embryo should continue to serve as a useful model to begins to form as an organ. BFU-E and CFU-E sub- dissect the emergence of hematopoietic potential within sequently expand exponentially in numbers for several the embryo proper and the placenta without the con- days and generate definitive erythroid cells [44]. The founding factor of blood cell movement within the circu- developmental origin of the BFU-E that initially colonize lation. In aggregate, these studies indicate that a transient the liver has been postulated to be the yolk sac, as BFU-E wave of definitive erythroid progenitors emerges from the first emerge in the yolk sac at E8.25, before the onset of yolk sac of mammalian embryos prior to the emergence of the circulation, and continue to preferentially expand in adult-repopulating hematopoietic stem cells and colo- numberswithintheyolksacforthenext48 h[40,42](Fig.2). nizes the fetal liver (Fig. 2). Interestingly, a wave of

definitive ‘erythro-myeloid’ progenitors has recently podocalyxin, and thus regulating reticulocyte egress from been found in the posterior blood islands of zebrafish the marrow [56]. These studies highlight an emerging role embryos after the appearance of primitive erythroid cells for adhesion factors in the terminal differentiation of in the intermediate cell mass but before emergence of definitive erythroid cells. HSC in the region of the aorta [47]. These findings provide evidence that hematopoietic ontogeny is quite MicroRNAs are endogenous molecules that target highly conserved between disparate species. mRNAs in a sequence-specific manner and regulate various cellular functions. Recent screens have identified Toward the end of gestation, definitive erythroid progen- numerous microRNAs in definitive erythroid cells that itors in the mammalian embryo transition from the liver to are either upregulated or downregulated with maturation the newly formed bone marrow. Although fundamentally [57–59]. Initial functional studies in murine erythroleu- similar to their adult counterparts, fetal erythroid progeni- kemia cells indicate that the abundantly expressed miR- tors also have some distinctive features. First, fetal BFU-E 451 facilitates erythroid maturation [57]. The continued have a greater and more rapid proliferative capacity. Sec- study of microRNAs and their targets will lead to the ond, fetal, but not steady-state adult, BFU-E proliferate identification of genes critical for hematopoietic differ- invitroinresponsetoerythropoietinintheabsenceofadded entiation and erythroid lineage maturation throughout colony-stimulating factors [48,49]. Finally, CFU-E in ontogeny. the murine fetus are more sensitive to erythropoietin [50].

Human embryonic stem cells serve as a new Maturation of definitive erythroid cells model of embryonic erythropoiesis In contrast to primitive erythroid cells that mature in the The culture of human embryonic stem cells has emerged bloodstream, definitive erythroid precursors in the fetal as an increasingly important model of early embryonic liver and postnatal marrow mature while attached to events. Recent studies indicate that the in-vitro differ- macrophage cells of erythroblastic islands [51] (Fig. 2). entiation of human embryonic stem cells recapitulates Although macrophage cells engulf and digest pyrenocytes hematopoietic ontogeny reminiscent of the in-vivo yolk following enucleation [22], it is unclear what other func- sac, including overlapping waves of hemangioblast, tions macrophage cells perform to enhance erythroid primitive erythroid, and definitive erythroid potential maturation. It has recently been shown that coculture of [60–62]. Interestingly, culture of differentiating human maturing Friend leukemia virus infected erythroid cells embryonic stem cells in liquid culture has recently pro- with macrophage cells in vitro enhanced their proliferation vided evidence that human primitive erythroid cells, like but did not alter their intrinsic ability to enucleate [52]. their murine counterparts, undergo ‘maturational’ z-glo- Potential ‘nurse’ functions of macrophage cells, such as the bin to a-globin switching [63]. Thus, human embryonic provision of iron and cytokines, remain to be proven. stem cells can provide access to hematopoietic cells that are not otherwise available for study. Erythroblast islands bring maturing definitive erythroid precursors in contact not only with macrophage cells but also with other erythroblasts. Fas and FasL signaling Conclusion between maturing erythroblasts has been invoked as a Primitive erythropoiesis serves as a useful model of mam- mechanism controlling erythroid cell output. Both nega- malian erythroid differentiation, as primitive erythroblasts tive feedback of more mature erythroblasts on immature in mammals mature in a synchronous cohort in the blood- erythroblasts as well as autoregulatory loops at the proer- stream and ultimately enucleate. In contrast, definitive ythroblast stage of maturation have been proposed erythroid precursors mature attached to macrophage cells [53,54]. As definitive erythroblasts mature extravascu- in erythroblast islands within the complex cellular milieu larly, they are also in contact with stromal elements. of the fetal liver and postnatal bone marrow (Fig. 2). Recent evidence suggests that definitive erythroblasts Erythroblastic islands can thus facilitate erythroblast– transition from an erthropoietin-dependent phase to a erythroblast and erythroblast–stromal interactions that fibronectin-dependent phase, and that both erythropoietin play important roles in the regulation of definitive ery- and fibronectin deliver antiapoptotic signals to maturing thropoiesis. The continued study of the similarities erythroblasts [55]. Alpha 4 integrin was shown to mediate and differences between primitive and definitive erythro- this erythroblast–fibronectin interaction, raising the intri- poiesis will lead to an improved understanding of eryth- guing question of whether similar signals are mediated to roblast maturation and the terminal steps of erythroid erythroblasts by VCAM1 on macrophage cells. Surpris- differentiation. ingly, erythropoietin was recently shown to play a role at very late stages of erythroid maturation by specifically The comparative investigation of embryonic development altering the expression of adhesion molecules, including in multiple model organisms, including mouse, chicken,

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