Primitive Erythroid Lineage Research Articles

The fetal liver is a niche for maturation of primitive erythroid cells.

Primitive erythroid cells (EryP) are the earliest differentiated cell type of the mammalian embryo. They appear in the yolk sac by embryonic day 7.5, begin to enter the embryonic circulation 2 days later and continue to mature in a stepwise and synchronous fashion. Like their adult counterparts, EryP enucleate. However, EryP circulate throughout the embryo for several days before the first enucleated forms can be identified in the blood. We have used transgenic mouse lines in which GFP marks EryP to investigate this seemingly long lag and have identified a previously unrecognized developmental niche for EryP maturation. After exiting the yolk sac, EryP begin to express cell adhesion proteins, including alpha4, alpha5, and beta1 integrins, on their surface and migrate into the fetal liver (FL), where they interact with macrophages within erythroblastic islands. Binding of EryP to FL macrophages in vitro is stage-specific and partly depends on VCAM-1. The ability to tag and track EryP nuclei using a transgenic mouse line expressing an H2B-EGFP fusion allowed us to identify and characterize extruded EryP nuclei and to demonstrate that molecules such as alpha4, alpha5, and beta1 integrins are redistributed onto the plasma membrane surrounding the extruding nucleus. FL macrophages engulf extruded EryP nuclei in cocultures and in the native FL in vivo. We conclude that EryP home to, complete their maturation, and enucleate within the FL, a tissue that is just developing as EryP begin to circulate. Our observations suggest a simple solution for a puzzling aspect of the development of the primitive erythroid lineage.

Wnt, Activin, and BMP Signaling Regulate Distinct Stages in the Developmental Pathway from Embryonic Stem Cells to Blood

The embryonic stem cell differentiation system was used to define the roles of the Activin/Nodal, BMP, and canonical Wnt signaling pathways at three distinct developmental stages during hematopoietic ontogeny: induction of a primitive streak-like population, formation of Flk1(+) mesoderm, and induction of hematopoietic progenitors. Activin/Nodal and Wnt, but not BMP, signaling are required for the induction of the primitive streak. Although BMP is not required for primitive streak induction, it displays a strong posteriorizing effect on this population. All three signaling pathways regulate induction of Flk1(+) mesoderm. The specification of Flk1(+) mesoderm to the hematopoietic lineages requires VEGF and Wnt, but not BMP or Activin/Nodal signaling. Specifically, Wnt signaling is essential for commitment of the primitive erythroid, but not the definitive lineages. These findings highlight dynamic changes in signaling requirements during blood cell development and identify a role for Wnt signaling in the establishment of the primitive erythroid lineage.

Novel Insights into the Maturation and Enucleation of Primitive Erythroblasts.

Primitive erythroblasts (EryP), the first blood cell type to form during mammalian embryogenesis, arise in the yolk sac (YS) as a synchronous cohort before the establishment of the embryonic circulation. The large, nucleated EryP begin to circulate around midgestation, when connections between YS and embryo mature. Two to three days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and rapidly outnumber EryP within the circulation. Cells of the two erythroid lineages differ not only in size but also in their expression of globin genes. Recently it has been shown that EryP begin to enucleate around E12.5 in the mouse. We have begun to characterize EryP maturation during mouse embryogenesis, with a focus on circulating cells. Analysis of embryonic blood at different stages revealed a stepwise developmental progression within the primitive erythroid lineage: loss of nucleoli (E9.5–10.5); decrease in cell diameter and cross-sectional area (E10.5–11.5); progressive nuclear condensation (E10.5 onwards); and enucleation (E12.5 onwards). Thereafter, EryD are also present in the bloodstream. A major obstacle to the study of primitive erythroid development is the current inability to easily distinguish EryP from EryD and to cleanly separate these populations, particularly given that enucleation can no longer be considered an exclusively definitive erythroid characteristic. No EryP-specific surface antigens have been identified that would permit the detection and isolation of cells of this lineage. We have exploited the restricted expression of the human embryonic beta-like globin gene epsilon to generate EryP lineage-specific transgenic mouse and embryonic stem (ES) cell models in which green fluorescent protein (GFP) reporter expression is driven exclusively in EryP and have used this line to track quantitatively the differentiation and maturation of circulating EryP during embryogenesis. The maturation of primitive erythroblasts was reflected in their dynamic expression of Ter119 and CD71. Up-regulation of a number of nonerythroid-specific cell surface markers, including integrins and other adhesion molecules, was apparent beginning around E12.5, reflecting a previously unrecognized EryP maturation process. Interestingly, enucleation of EryP is accompanied by changes in surface antigen expression. Thus, the phenotype of primitive reticulocytes is distinct from that of their nucleated precursors. In contrast with the prevailing view that EryP are present only transiently in the embryo, we find that they comprise a stable cell population that persists through the end of gestation. The human epsilon-globin::GFP transgenic mouse model provides a novel system for defining, at the cellular and molecular level, the mechanisms involved in development of the primitive erythroid lineage.

“Maturational” globin switching in primary primitive erythroid cells

Mammals have 2 distinct erythroid lineages. The primitive erythroid lineage originates in the yolk sac and generates a cohort of large erythroblasts that terminally differentiate in the bloodstream. The definitive erythroid lineage generates smaller enucleated erythrocytes that become the predominant cell in fetal and postnatal circulation. These lineages also have distinct globin expression patterns. Our studies in primary murine primitive erythroid cells indicate that betaH1 is the predominant beta-globin transcript in the early yolk sac. Thus, unlike the human, murine beta-globin genes are not up-regulated in the order of their chromosomal arrangement. As primitive erythroblasts mature from proerythroblasts to reticulocytes, they undergo a betaH1- to epsilony-globin switch, up-regulate adult beta1- and beta2-globins, and down-regulate zeta-globin. These changes in transcript levels correlate with changes in RNA polymerase II density at their promoters and transcribed regions. Furthermore, the epsilony- and betaH1-globin genes in primitive erythroblasts reside within a single large hyperacetylated domain. These data suggest that this "maturational" betaH1- to epsilony-globin switch is dynamically regulated at the transcriptional level. Globin switching during ontogeny is due not only to the sequential appearance of primitive and definitive lineages but also to changes in globin expression as primitive erythroblasts mature in the bloodstream.

Characterization of Circulating Primitive Erythroid Cells during Embryogenesis Using a Human ε-Globin-GFP Transgenic Mouse Model.

Primitive erythroid cells (EryP) are the first hematopoietic cell type to mature during embryonic development. EryP are characterized by expression of “embryonic” ε- and βh1-globin genes that are not expressed in fetal liver-derived “adult type” or definitive erythroid cells. Although EryP constitute the majority of the blood cells in mid-gestation embryos, EryP maturation remains poorly defined. Here, we utilize a transgenic mouse line in which green fluorescence protein (GFP) expression is driven by the human ε-globin minimal promoter to characterize the development of circulating EryP throughout embryogenesis. ε-globin(+)/GFP(+) EryP appear from 7.5 dpc within blood islands of the yolk sac. As expected, at 9.5 dpc, essentially all circulating blood cells express GFP. By 14.5 dpc, only 50% of the circulating cells express GFP. This sharp decrease in the numbers of circulating ε-globin(+)/GFP(+) cells continues until birth, by which stage fewer than 1% express the reporter gene. To further characterize the maturation of EryP, we analyzed the expression of surface antigens on ε-globin(+)/GFP(+) cells at various stages of embryonic development. From 9.5 dpc onwards, ε-globin(+)/GFP(+) cells express increasing levels of the erythroid marker Ter-119. The transferrin receptor CD71 is also expressed from early stages of development but is down-regulated as ε-globin(+)/GFP(+) cells mature. Increasing Ter-119 expression with concomitant loss of CD71 have previously been reported as hallmarks of maturing fetal liver erythroid cells (EryD) and we now report that ε-globin(+)/GFP(+) EryP demonstrate a similar developmental progression. Therefore, EryP contribute to the Ter119 and CD71 expression reported by others for total embryonic peripheral blood cells. The GPI-anchored surface marker CD24, present on EryD, is also found on ε-globin(+)/GFP(+) EryP. Interestingly, expression of the adhesion molecules CD44 and α4-integrin was upregulated on maturing ε-globin(+)/GFP(+) EryP, perhaps reflecting a requirement for interaction of EryP with other cells. ε-globin(+)/GFP(+) EryP lack expression of surface antigens typical of endothelial cells (Flk1, VCAM1), hematopoietic stem cells (c-kit, Sca1, CXCR4), myeloid (Gr1, Mac1) and lymphoid cells (CD19, CD3). Together, these data help to define the maturation pathway of the primitive erythroid lineage. It was recently shown by immunohistology that murine EryP enucleate, similar to their definitive counterparts. We used the cell permeable DNA-binding dye Draq5 to quantify enucleation in the circulating ε-globin(+)/GFP(+) population by FACS. We show that enucleated ε-globin(+)/GFP(+) cells are Draq5low/neg whereas those bearing nuclei are Draq5high. At 9.5 dpc, ε-globin(+)/GFP(+) cells are Draq5high. The frequency of nucleated EryPs decreases rapidly such that by 14.5 dpc, half of the circulating e-globin(+)/GFP(+) cells are Draq5low/neg. Shortly before birth, almost all EryP have enucleated. This system will allow us to separate nucleated from enucleated EryP by cell sorting and will help us assess changes in surface antigen expression during EryP maturation and enucleation. The human ε-globin-GFP transgenic mouse model is therefore a useful system for defining, at the cellular and molecular level, the developmental pathways of the primitive erythroid lineage.

Transcriptional regulation of the SCL locus: identification of an enhancer that targets the primitive erythroid lineage in vivo.

The stem cell leukemia (SCL) gene, also known as TAL-1, encodes a basic helix-loop-helix protein that is essential for the formation of all hematopoietic lineages, including primitive erythropoiesis. Appropriate transcriptional regulation is essential for the biological functions of SCL, and we have previously identified five distinct enhancers which target different subdomains of the normal SCL expression pattern. However, it is not known whether these SCL enhancers also regulate neighboring genes within the SCL locus, and the erythroid expression of SCL remains unexplained. Here, we have quantitated transcripts from SCL and neighboring genes in multiple hematopoietic cell types. Our results show striking coexpression of SCL and its immediate downstream neighbor, MAP17, suggesting that they share regulatory elements. A systematic survey of histone H3 and H4 acetylation throughout the SCL locus in different hematopoietic cell types identified several peaks of histone acetylation between SIL and MAP17, all of which corresponded to previously characterized SCL enhancers or to the MAP17 promoter. Downstream of MAP17 (and 40 kb downstream of SCL exon 1a), an additional peak of acetylation was identified in hematopoietic cells and was found to correlate with expression of SCL but not other neighboring genes. This +40 region is conserved in human-dog-mouse-rat sequence comparisons, functions as an erythroid cell-restricted enhancer in vitro, and directs beta-galactosidase expression to primitive, but not definitive, erythroblasts in transgenic mice. The SCL +40 enhancer provides a powerful tool for studying the molecular and cellular biology of the primitive erythroid lineage.

The Megakaryocyte Lineage Arises in the Yolk Sac and Generates an Initial Wave of Large Embryonic Platelets in the Early Mammalian Embryo.

Two distinct waves of hematopoietic progenitors originate in the yolk sac of the mammalian embryo. The first “primitive” wave contains primitive erythroid and macrophage progenitors that generate the embryo's first red cells (Palis, et al. Development 126:5073, 1999). The second “definitive” wave consists of definitive erythroid (BFU-E) and multiple myeloid progenitors that arise later in the yolk sac and are subsequently found in the fetal liver and postnatal marrow. While megakaryocyte progenitors have been detected in the yolk sac, the ontogeny of the megakaryocyte lineage is poorly understood. Furthermore, it is not been determined when platelets first enter the bloodstream of the mouse embryo. The presence and size of platelets in the embryonic bloodstream were examined by microscopy after staining with anti-GPV antibodies. Rare platelets were identified in 2 of 4 litters of mice at E10.5. These platelets were very large with a diameter of 4.2 ± 0.4 (mean ± SEM) microns. Platelet size remained large (4.0-3.8 microns) at E11.5–E12.5, but decreased to 3.3-3.2 microns in diameter between E13.5 and E15.5 of gestation. At birth, the mean platelet diameter (2.8 ± 0.04 microns) was similar to that of adult mice (2.7 microns). These results indicate that large, embryonic platelets begin to circulate in the mouse embryo beginning at E10.5 and raise the possibility that embryonic, fetal, and adult waves of platelets are produced during mammalian embryogenesis. Using a collagen-based culture system, megakaryocyte progenitors (Meg-CFC) were first identified in late primitive streak embryos (E7.25), concomitant with primitive erythroid progenitors. Meg-CFC numbers subsequently expand in the yolk sac along with BFU-E before the development of the fetal liver. To examine the relationship of the megakaryocyte and primitive erythroid lineages in the yolk sac, we stained hematopoietic colonies grown in collagen for 5–10 days with anti-GP1bβ (megakaryocyte) and anti-βH1-globin (primitive erythroid) antibodies. As expected, the majority of the stained colonies were primitive erythroid (containing only βH1-globin-positive cells) and approximately 15% of the colonies were megakaryocyte (containing only GP1bβ-positive cells). However, 15% of the colonies contained both GP1bβ- and βH1-globin-positive cells consistent with an origin from bipotential primitive erythroid/megakaryocyte progenitors. Furthermore, proplatelet formation was evident in both unipotential and bipotential megakaryocyte colonies cultured for 10 days. Our studies support the concept that the megakaryocyte and primitive erythroid lineages originate in the yolk sac from a bipotential precursor. This parallels lineage relationships in the bone marrow which contains bipotential definitive erythroid/megakaryocyte progenitors. Finally, we hypothesize that yolk sac-derived “primitive” Meg-CFC give rise to the first embryonic platelets that enter the bloodstream soon after the onset of circulation as the fetal liver becomes a hematopoietic organ.

Erythroblasts secrete the nonclassical HLA-G molecule from primitive to definitive hematopoiesis

AbstractThe initial steps of primitive hematopoiesis and endothelial vascular formation in the human embryo remain to be defined. Here, we report the identification of a novel marker, namely the nonclassical HLA-G class I molecule, which targets both primitive erythroid cells of the yolk sac and endothelial cells from developing vessels. Moreover, HLA-G was present in its soluble form in the erythropoietic lineage in all organs sustaining primitive to definitive erythropoiesis (ie, aorta-gonad-mesonephros, liver, spleen, and bone marrow). The alternatively spliced transcript coding the soluble HLA-G5 molecule was detected in erythroid cells. The corresponding intron 4–retaining 37-kDa HLA-G5 isoform was secreted from the erythroid progenitor stage to the reticulocyte but was lost in mature erythrocytes and in endothelial cells from differentiated vessels. This study constitutes the first description of an HLA class I antigen expression on the primitive erythroid lineage and provides a way of seeking both primitive and definitive erythropoiesis using HLA-G5. This new marker, previously known by its immunotolerogeneic properties, may be involved in erythroid differentiation, angiogenesis, or both.

Primitive erythropoiesis is regulated by Smad-dependent signaling in postgastrulation mesoderm

AbstractThe bone morphogenetic proteins (BMPs) are required for the development of ventral mesoderm, which contributes to the ventral blood island and primitive (yolk sac stage) hematopoiesis. Primitive erythropoiesis is defective when BMP signaling is blocked during gastrulation of Xenopus embryos. This phenotype might be attributed to changes in mesoderm patterning leading indirectly to altered erythropoiesis. We developed an inducible system in order to block BMP signaling in a controlled fashion at later time points in development. For this purpose, an inhibitory Smad, xSmad6, was fused to the estrogen receptor ligand-binding domain. We show that ER-xSmad6 is inactive when expressed in developing embryos, but its activity is induced by estradiol. When induced early in development, ER-xSmad6 causes a dorsalized phenotype, equivalent to overexpression of native xSmad6. When ER-xSmad6 is induced after gastrulation, there is a specific defect in primitive erythropoiesis without any apparent effect on axial patterning. Our results identify an embryonic signal that is Smad-dependent, is required for maintaining expression of GATA-1, and functions within mesoderm and not the overlying ectoderm. Thus, BMP signaling is necessary both during mesoderm patterning and also following early specification events for proper regulation of the primitive erythroid lineage.

Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro

AbstractIn this report we demonstrate a role for Runx1 (AML1)at the hemangioblast stage of hematopoietic and endothelial development in embryonic stem (ES) cell–derived embryoid bodies (EBs).Runx1 is expressed in EBs during the appearance of precursors with hemangioblast properties, the blast colony-forming cells (BL-CFCs). Cell sorting studies revealed that all BL-CFCs within EBs express Runx1. Runx1-deficient EBs consistently generate 10- to 20-fold fewer blast colonies than wild-type controls and display a complete block in definitive hematopoiesis. Despite this defect, Runx1−/−EBs and yolk sacs from mutant embryos generate normal numbers of primitive erythroid precursors. These observations clearly demonstrate that Runx1 functions early in hematopoietic development, and they support the interpretation that the primitive erythroid lineage is established early by a subset of BL-CFCs that develop in aRunx1-independent fashion.

Autocrine and paracrine effects of an ES-cell derived, BCR/ABL-transformed hematopoietic cell line that induces leukemia in mice.

During differentiation in vitro, Embryonic Stem (ES) cells generate both primitive erythroid and definitive myeloid lineages in a process that mimics hematopoiesis in the mammalian yolk sac. To investigate leukemic transformation of these embryonic hematopoietic progenitors, we infected differentiating cultures of ES cells with the Chronic Myeloid Leukemia-specific BCR/ABL oncoprotein. Following a period of liquid culture, we isolated two transformed subclones, EB57 and EB67, that retained characteristics of embryonic hematopoietic progenitors and induced a fatal leukemia in mice characterized by massive splenomegaly and granulocytosis. Histopathology of the spleen revealed an abundance of undifferentiated blast-like cells. Investigation of the clonal origins of the granulocytes in the peripheral blood demonstrated that the injected donor cells contributed modestly to the granulocyte population while the majority were host-derived. EB57 secretes IL-3 and unidentified cytokines that can stimulate autocrine and paracrine cell proliferation, presumably accounting for the reactive granulocytosis in diseased mice. These BCR/ABL transformed hematopoietic derivatives of ES cells recapitulate the relationship of BCR/ABL expression to IL-3 production that has been described for primitive hematopoietic progenitors from human CML patients, and illustrates the potential for autocrine and paracrine effects of BCR/ABL-infected cells in murine models.

Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.

In this study, we have mapped the onset of hematopoietic development in the mouse embryo using colony-forming progenitor assays and PCR-based gene expression analysis. With this approach, we demonstrate that commitment of embryonic cells to hematopoietic fates begins in proximal regions of the egg cylinder at the mid-primitive streak stage (E7.0) with the simultaneous appearance of primitive erythroid and macrophage progenitors. Development of these progenitors was associated with the expression of SCL/tal-1 and GATA-1, genes known to be involved in the development and maturation of the hematopoietic system. Kinetic analysis revealed the transient nature of the primitive erythroid lineage, as progenitors increased in number in the developing yolk sac until early somite-pair stages of development (E8.25) and then declined sharply to undetectable levels by 20 somite pairs (E9.0). Primitive erythroid progenitors were not detected in any other tissue at any stage of embryonic development. The early wave of primitive erythropoiesis was followed by the appearance of definitive erythroid progenitors (BFU-E) that were first detectable at 1-7 somite pairs (E8.25) exclusively within the yolk sac. The appearance of BFU-E was followed by the development of later stage definitive erythroid (CFU-E), mast cell and bipotential granulocyte/macrophage progenitors in the yolk sac. C-myb, a gene essential for definitive hematopoiesis, was expressed at low levels in the yolk sac just prior to and during the early development of these definitive erythroid progenitors. All hematopoietic activity was localized to the yolk sac until circulation was established (E8.5) at which time progenitors from all lineages were detected in the bloodstream and subsequently in the fetal liver following its development. This pattern of development suggests that definitive hematopoietic progenitors arise in the yolk sac, migrate through the bloodstream and seed the fetal liver to rapidly initiate the first phase of intraembryonic hematopoiesis. Together, these findings demonstrate that commitment to hematopoietic fates begins in early gastrulation, that the yolk sac is the only site of primitive erythropoiesis and that the yolk sac serves as the first source of definitive hematopoietic progenitors during embryonic development.

Early hematopoiesis and developing lymphoid organs in the zebrafish

In zebrafish, the transparent and rapidly developing embryo and the potential for genetic screening offer a unique opportunity to investigate the early development of the vertebrate immune system. Here we describe the initial appearance of various blood lineages and the nature of accumulating hematopoietic tissue in the thymus and kidney, the main lymphoid organs of adult teleosts. The ultrastructure of the first site of hematopoiesis, the intermediate cell mass (ICM), is described from the 5-somite stage, about 11.5 hours post-fertilization (hpf) until 24 hpf. The ICM gives rise to the primitive erythroid lineage, which accounts for all circulating erythrocytes for the first 4 days pf. From 24 to 72 hpf, a few developing granulocytes are seen close to the yolk sac walls and in the caudal axial vein. The heart, previously proposed as an early blood-forming organ in zebrafish, did not contain hematopoietic cells. The thymic primordium, consisting of two layers of epithelial cells, appears at 60 hpf. By 65 hpf, it is colonized by immature lymphoblasts. The thymus gradually accumulates lymphocytes, and the lymphocytes and epithelial cells progressively differentiate for 3 weeks pf. At 96 hr, the pronephros contains hematopoietic cells, mainly developing erythrocytes and granulocytes. The amount of renal hematopoietic tissue increases rapidly; however, lymphocytes were not detected until 3 weeks pf. Dev Dyn 1999;214:323–336. © 1999 Wiley-Liss, Inc.

Early hematopoiesis and developing lymphoid organs in the zebrafish.

In zebrafish, the transparent and rapidly developing embryo and the potential for genetic screening offer a unique opportunity to investigate the early development of the vertebrate immune system. Here we describe the initial appearance of various blood lineages and the nature of accumulating hematopoietic tissue in the thymus and kidney, the main lymphoid organs of adult teleosts. The ultrastructure of the first site of hematopoiesis, the intermediate cell mass (ICM), is described from the 5-somite stage, about 11.5 hours post-fertilization (hpf) until 24 hpf. The ICM gives rise to the primitive erythroid lineage, which accounts for all circulating erythrocytes for the first 4 days pf. From 24 to 72 hpf, a few developing granulocytes are seen close to the yolk sac walls and in the caudal axial vein. The heart, previously proposed as an early blood-forming organ in zebrafish, did not contain hematopoietic cells. The thymic primordium, consisting of two layers of epithelial cells, appears at 60 hpf. By 65 hpf, it is colonized by immature lymphoblasts. The thymus gradually accumulates lymphocytes, and the lymphocytes and epithelial cells progressively differentiate for 3 weeks pf. At 96 hr, the pronephros contains hematopoietic cells, mainly developing erythrocytes and granulocytes. The amount of renal hematopoietic tissue increases rapidly; however, lymphocytes were not detected until 3 weeks pf.

Mechanisms of developmental control of transcription in the murine alpha- and beta-globin loci.

We have characterized mRNA expression and transcription of the mouse alpha- and beta-globin loci during development. S1 nuclease and primary transcript in situ hybridization analyses demonstrate that all seven murine globin genes (zeta, alpha1, alpha2, epsilony, betaH1, betamaj, and betamin) are transcribed during primitive erythropoiesis, however transcription of the zeta, epsilony, and betaH1 genes is restricted to the primitive erythroid lineage. Transcription of the betamaj and betamin genes in primitive cells is EKLF-dependent demonstrating EKLF activity in embryonic red cells. Novel kinetic analyses suggest that multigene expression in the beta locus occurs via alternating single-gene transcription whereas coinitiation cannot be ruled out in the alpha locus. Transcriptional activation of the individual murine beta genes in primitive cells correlates inversely with their distance from the locus control region, in contrast with the human beta locus in which the adult genes are only activated in definitive erythroid cells. The results suggest that the multigene expression mechanism of alternating transcription is evolutionarily conserved between mouse and human beta globin loci but that the timing of activation of the adult genes is altered, indicating important fundamental differences in globin gene switching.

Overexpression of HOX11 Leads to the Immortalization of Embryonic Precursors With Both Primitive and Definitive Hematopoietic Potential

Primitive and definitive erythropoiesis represent distinct hematopoietic programs that differ with respect to stage of development, transcriptional control, and growth regulation. Although these differences have been recognized for some time, the relationship of the two erythroid lineages to each other is not well established. We have used a model system based on the hematopoietic development of embryonic stem (ES) cells in culture to investigate the origins of the earliest hematopoietic populations. Using ES cells transduced with a retrovirus that overexpresses the HOX11 gene, we have established factor-dependent hematopoietic cell lines that represent novel stages of embryonic hematopoiesis. Analysis of three of these cell lines indicates that they differ with respect to cytokine responsiveness, cell surface markers, and developmental potential. Two of the cell lines, EBHX1 and EBHX11, display the unique capacity to generate both primitive and definitive erythroid progeny as defined by morphology and expression of βH1 and βmajor globin. The third line, EBHX14, has definitive erythroid and myeloid potential, but is unable to generate cells of the primitive erythroid lineage. Analysis of the cytokine responsiveness of the two lines with primitive erythroid potential has indicated that exposure to leukemia inhibitory factor (LIF) results in the upregulation of βH1 and a change in cellular morphology to that of primitive erythrocytes. These findings are the first demonstration of a clonal cell line with primitive and definitive hematopoietic potential and support the interpretation that these lineages may arise from a common precursor in embryonic life. In addition, they suggest that LIF could play a role in the regulation of primitive erythropoiesis.© 1998 by The American Society of Hematology.

Overexpression of HOX11 Leads to the Immortalization of Embryonic Precursors With Both Primitive and Definitive Hematopoietic Potential

AbstractPrimitive and definitive erythropoiesis represent distinct hematopoietic programs that differ with respect to stage of development, transcriptional control, and growth regulation. Although these differences have been recognized for some time, the relationship of the two erythroid lineages to each other is not well established. We have used a model system based on the hematopoietic development of embryonic stem (ES) cells in culture to investigate the origins of the earliest hematopoietic populations. Using ES cells transduced with a retrovirus that overexpresses the HOX11 gene, we have established factor-dependent hematopoietic cell lines that represent novel stages of embryonic hematopoiesis. Analysis of three of these cell lines indicates that they differ with respect to cytokine responsiveness, cell surface markers, and developmental potential. Two of the cell lines, EBHX1 and EBHX11, display the unique capacity to generate both primitive and definitive erythroid progeny as defined by morphology and expression of βH1 and βmajor globin. The third line, EBHX14, has definitive erythroid and myeloid potential, but is unable to generate cells of the primitive erythroid lineage. Analysis of the cytokine responsiveness of the two lines with primitive erythroid potential has indicated that exposure to leukemia inhibitory factor (LIF) results in the upregulation of βH1 and a change in cellular morphology to that of primitive erythrocytes. These findings are the first demonstration of a clonal cell line with primitive and definitive hematopoietic potential and support the interpretation that these lineages may arise from a common precursor in embryonic life. In addition, they suggest that LIF could play a role in the regulation of primitive erythropoiesis.© 1998 by The American Society of Hematology.

Generation of a Primitive Erythroid Cell Line and Promotion of Its Growth by Basic Fibroblast Growth Factor

An immortalized cell line representing the primitive erythroid (EryP) lineage was established from in vitro–differentiated progeny (embryoid bodies [EBs]) of embryonic stem (ES) cells using a retroviral insertional mutation, and has been termed EB-PE for embryoid body–derived primitive erythroid. Even though EB-PE cells are immortalized, they show characteristics of normal EryP cells, such as gene expression and growth factor dependency. In addition, EB-PE cells can differentiate further in culture. Investigation of growth factor requirements of EB-PE cells showed that basic fibroblast growth factor (bFGF) and erythropoietin (Epo) play unique roles in EB-PE proliferation and differentiation. While bFGF was a strong mitogen, Epo was required for both proliferation and differentiation. The unique proliferative response to bFGF coincided with upregulation of its receptor, fibroblast growth factor receptor (fgfr-1), and downregulation of erythropoietin receptor (EpoR) gene expression. Studies of primary EryP cells derived from early EBs, when tested in a colony-formation assay, also provided evidence for the mitogenic role of bFGF in concert with Epo.

Generation of a Primitive Erythroid Cell Line and Promotion of Its Growth by Basic Fibroblast Growth Factor

AbstractAn immortalized cell line representing the primitive erythroid (EryP) lineage was established from in vitro–differentiated progeny (embryoid bodies [EBs]) of embryonic stem (ES) cells using a retroviral insertional mutation, and has been termed EB-PE for embryoid body–derived primitive erythroid. Even though EB-PE cells are immortalized, they show characteristics of normal EryP cells, such as gene expression and growth factor dependency. In addition, EB-PE cells can differentiate further in culture. Investigation of growth factor requirements of EB-PE cells showed that basic fibroblast growth factor (bFGF) and erythropoietin (Epo) play unique roles in EB-PE proliferation and differentiation. While bFGF was a strong mitogen, Epo was required for both proliferation and differentiation. The unique proliferative response to bFGF coincided with upregulation of its receptor, fibroblast growth factor receptor (fgfr-1), and downregulation of erythropoietin receptor (EpoR) gene expression. Studies of primary EryP cells derived from early EBs, when tested in a colony-formation assay, also provided evidence for the mitogenic role of bFGF in concert with Epo.

Commitment to Hematopoietic Fates Begins During Early Gastrulation in the Mammalian Embryo • 292

During mammalian embryogenesis, erythropoiesis occurs in two distinct waves. The first wave, primitive erythropoiesis, characterized by nucleated megaloblasts expressing embryonic globins, occurs in yolk sac blood islands. The second wave, definitive erythropoiesis, is characterized by adult globincontaining anucleate cells, that differentiate later in the fetal liver and bone marrow. To determine where and when embryonic cells become committed to hematopoietic fates, we previously examined the spatial and temporal expression of transcription factors critical to yolk sac hematopoiesis. SCL/tal-1 and LMO2/rbtn-2 were found exclusively in mesoderm cells as early as the mid-primitive streak stage, just 12 hours after the start of gastrulation. These findings suggest that commitment of mesoderm cells to blood cell fates occurs during early gastrulation. We tested this hypothesis by culturing cells from carefully staged gastrulating mouse embryos in methylcellulose with cytokines that support the growth of both primitive and definitive erythroid colonies. No hematopoietic progenitors were evident prior to, or at the onset of, primitive streak formation (E6.5). Blood cell progenitors, consisting of primitive erythroid and macrophage lineages, were first present at the mid-primitive streak stage (E7.0). Definitive erythroid progenitors first appeared at early somite pair stages (E8.5) exclusively within the yolk sac. We conclude that commitment of embryonic cells to hematopoietic fates occurs soon after the onset of mesoderm formation and that definitive erythroid potential is evident in the yolk sac prior to the development of the fetal liver. These findings suggest that hematopoietic stem cells with primitive and definitive hematopoietic potential arise during gastrulation in the yolk sac.