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.

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Cellular Basis of Embryonic Hematopoiesis and Its Implications in Prenatal Erythropoiesis

International Journal of Molecular Sciences ◽

10.3390/ijms21249346 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9346

Author(s):

Toshiyuki Yamane

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Yolk Sac ◽

Hematopoietic Progenitors ◽

Adult Type ◽

Hematopoietic Stem ◽

Cellular Origin ◽

Developmental Switches ◽

Primitive Erythropoiesis

Primitive erythrocytes are the first hematopoietic cells observed during ontogeny and are produced specifically in the yolk sac. Primitive erythrocytes express distinct hemoglobins compared with adult erythrocytes and circulate in the blood in the nucleated form. Hematopoietic stem cells produce adult-type (so-called definitive) erythrocytes. However, hematopoietic stem cells do not appear until the late embryonic/early fetal stage. Recent studies have shown that diverse types of hematopoietic progenitors are present in the yolk sac as well as primitive erythroblasts. Multipotent hematopoietic progenitors that arose in the yolk sac before hematopoietic stem cells emerged likely fill the gap between primitive erythropoiesis and hematopoietic stem-cell-originated definitive erythropoiesis and hematopoiesis. In this review, we discuss the cellular origin of primitive erythropoiesis in the yolk sac and definitive hematopoiesis in the fetal liver. We also describe mechanisms for developmental switches that occur during embryonic and fetal erythropoiesis and hematopoiesis, particularly focusing on recent studies performed in mice.

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

Blood ◽

10.1182/blood.v104.11.566.566 ◽

2004 ◽

Vol 104 (11) ◽

pp. 566-566

Author(s):

James Palis ◽

Joanna Tober ◽

Radhika Vemishetti ◽

Anne Koniski ◽

Richard Waugh

Keyword(s):

Mouse Embryo ◽

Fetal Liver ◽

Primitive Streak ◽

Yolk Sac ◽

Erythroid Progenitors ◽

Mammalian Embryo ◽

Hematopoietic Organ ◽

Adult Mice ◽

Early Mammalian Embryo ◽

Megakaryocyte Progenitors

Abstract 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. Development126:5073, 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.

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Rac1 Regulates Migration of Yolk Sac Hematopoietic Progenitors into the Blood, a Process Essential for Subsequent Seeding of Fetal Liver Hematopoiesis.

Blood ◽

10.1182/blood.v106.11.518.518 ◽

2005 ◽

Vol 106 (11) ◽

pp. 518-518 ◽

Cited By ~ 1

Author(s):

Gabriel Ghiaur ◽

Michael J. Ferkowicz ◽

Jeff Bailey ◽

Mervin C. Yoder ◽

David A. Williams

Keyword(s):

Embryonic Development ◽

Fetal Liver ◽

Single Cells ◽

Flow Cytometric Analysis ◽

Yolk Sac ◽

Hematopoietic Progenitors ◽

Hematopoietic Progenitor ◽

Anatomic Site ◽

Littermate Control ◽

Hematopoietic Stem

Abstract Hematopoiesis originates in the mouse embryo at gastrulation with the formation of an initial wave of primitive erythrocytes in the yolk sac (YS) of the extra embryonic tissue. During embryonic development the anatomic site of hematopoiesis changes and hematopoietic stem cells (HSC) are postulated to migrate and seed successive embryonic sites. The origin of definitive hematopoiesis, which has been postulated to be either the YS or the PAS/AGM region, remains controversial, and both sites have previously been shown to contribute to the seeding of HSC/P to the fetal liver (FL). We utilized a genetic approach to study molecular pathways involved in the migration of definitive hematopoietic progenitor cells between the YS and the embryo (AGM and FL). We have recently shown that Rac1 regulates the engraftment phenotype of adult HSC/P (Cancelas et al. Nat Med 2005). In the present study, we induced deletion of Rac1 in a hematopoietic tissue-specific manner by crossing Rac1 flox/null mice with a Vav1-Cre transgenic mouse (Croker B et al. Immunity 2004). Using RosaLacZ reporter mice, we first documented the presence of Vav1Cre-induced recombination in mice in the extraembryonic tissue as early as E 7.5, and in an anatomical distribution concordant with emergence of primitive hematopoiesis. Flow cytometric analysis of single cells derived from the embryo body and from the YS of E 9.5 double transgenic (RosaGFP, Vav1Cre) embryos demonstrated complete recombination in definitive hematopoietic progenitors defined as CD41hiFlk1dim. These data demonstrate that Vav1Cre transgene can induce recombination of genomic flox sequences in both primitive as well as definitive hematopoietic cells early in embryonic development. In crosses of Vav1Cre, Rac1null/wt and Rac1flox/wt mice, no triple transgenic (Vav1Cre, Rac1flox/null) mice survived past E14.5 (0/29, p < 0.02). We next studied hematopoiesis in the developing embryos by enumerating hematopoietic progenitor content. At E9.5, CFU content of YS of Vav1Cre, Rac1flox/null embryos was equivalent to littermate control (Table). When compared with littermate controls at E10.5, even in the presence of normal YS hematopoiesis, we observed significantly decreased numbers of circulating CFU in the blood of Vav1Cre, Rac1flox/null embryos, suggesting defective migration of these cells out of the YS. At E10.5, the content of CFU in FL and embryo proper (after removal of FL and blood) was significantly reduced in Vav1Cre, Rac1flox/null embryos. The defect in FL hematopoiesis was more pronounced at E11.5 and E12.5. Consistent with these results, FL of E11.5 Vav1Cre, Rac1flox/null embryos were pale and had markedly reduced cellularity when compared with littermate controls (0.11x106±0.08x106 vs 1.2x106±0.3x106, n ≥ 5, p < 0.01), demonstrating a failure of fetal liver hematopoiesis. These data demonstrate that Rac1 regulates the migration of HSC/P from the YS into the blood and suggest that this migration is essential for subsequent seeding of both the embryo proper and the FL. Tissue Genotype E9.5 E10.5 E11.5 E12.5 Data are presented as mean±SD, * - p<0.01 YS control 1024±298 719±167 - - TgRac1 flox/null 1011±257 722±178 - - Blood control - 459±116 - - TgRac1 flox/null - 253±74 * - - FL control - 432±112 7496±2096 12889±2512 TgRac1 flox/null - 39±32 * 235±103 * 103±125 * Embryo proper control - 825±214 - - TgRac1 flox/null - 74±62 * - -

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Stat3 Is Activated By ROS to Uniquely Regulate Primitive Erythropoiesis

Blood ◽

10.1182/blood-2019-127229 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2216-2216

Author(s):

Zachary C. Murphy ◽

Kathleen E. McGrath ◽

James Palis

Keyword(s):

Hydrogen Peroxide ◽

Cell Cycle Progression ◽

Fetal Liver ◽

Yolk Sac ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Mammalian Embryo ◽

Stat3 Phosphorylation ◽

Murine Embryo ◽

Primitive Erythropoiesis

The mammalian embryo requires the sequential circulation of primitive and definitive erythroid cells for survival and growth. The central cytokine regulator of primitive and definitive erythropoiesis is erythropoietin (EPO). We have previously determined that primitive erythroid progenitors, unlike their definitive (CFU-E) counterparts, do not require EPO for survival. However, the mechanisms regulating the EPO-independent emergence of primitive erythropoiesis in the yolk sac remains poorly understood. Interestingly, maturing primitive erythroblasts in the murine embryo, unlike definitive erythroblasts in the fetal liver and adult bone marrow, express not only STAT5 but also STAT3, which is tyrosine phosphorylated at baseline and in response to EPO. These initial findings led is to hypothesize that STATs 5 and 3 differentially regulate terminal differentiation of primitive erythroid cells. To analyze the function of these two STATs in primary erythroid cells, we developed an imaging flow cytometry-based methodology to quantitate total and phosphorylated levels of STAT proteins in small numbers of cells isolated from staged murine embryos. We found that STAT5 plays conserved roles in primitive and definitive erythroblast survival and surface CD71 expression. In contrast, STAT3 regulates cell cycle progression and mitochondrial polarization specifically in primitive erythroblasts. In addition, STAT3, unlike STAT5, was phosphorylated in the absence of cytokine stimulation. We asked if reactive oxygen species (ROS) may be activating STAT3, since primary primitive erythroblasts, unlike definitive erythroblasts, specifically express Aquaporins 3 and 8, which can transport hydrogen peroxide, as well as water. Indeed, hydrogen peroxide exposure increased endogenous ROS, as well as phosphorylated STAT3, levels both in wild-type and EPOR-null primitive erythroblasts. Consistent with these findings, inhibition of aquaporin channel transport prevented STAT3 phosphorylation by exogenous hydrogen peroxide, but not by EPO. Taken together, our data support the concept that the primitive erythroid lineage, emerging in the hypoxic and EPO-low environment of the yolk sac in the pre-circulation murine embryo, uniquely integrates ROS-mediated STAT3 activation to regulate key aspects of terminal erythroid maturation. Disclosures Palis: Rubies Therapeutics: Consultancy.

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E26 leukemia virus converts primitive erythroid cells into cycling multilineage progenitors

Blood ◽

10.1182/blood-2002-04-1050 ◽

2003 ◽

Vol 101 (3) ◽

pp. 1103-1110 ◽

Cited By ~ 6

Author(s):

Kelly M. McNagny ◽

Thomas Graf

Keyword(s):

Leukemia Virus ◽

Yolk Sac ◽

Erythroid Cells ◽

Multilineage Differentiation ◽

Cell Type ◽

Hematopoietic Progenitors ◽

Cell Type Specificity ◽

Erythroid Progenitors ◽

Avian Erythroblastosis Virus

Abstract Acute chicken leukemia retroviruses, because of their capacity to readily transform hematopoietic cells in vitro, are ideal models to study the mechanisms governing the cell-type specificity of oncoproteins. Here we analyzed the transformation specificity of 2 acute chicken leukemia retroviruses, the Myb-Ets– encoding E26 virus and the ErbA/ErbB-encoding avian erythroblastosis virus (AEV). While cells transformed by E26 are multipotent (designated “MEP” cells), those transformed by AEV resemble erythroblasts. Using antibodies to separate subpopulations of precirculation yolk sac cells, both viruses were found to induce the proliferation of primitive erythroid progenitors within 2 days of infection. However, while AEV induced a block in differentiation of the cells, E26 induced a gradual shift in their phenotype and the acquisition of the potential for multilineage differentiation. These results suggest that the Myb-Ets oncoprotein of the E26 leukemia virus converts primitive erythroid cells into proliferating definitive-type multipotent hematopoietic progenitors.

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A 2-kb c-mpl promoter fragment is sufficient to direct expression to the megakaryocytic lineage and sites of embryonic hematopoiesis in transgenic mice

Blood ◽

10.1182/blood-2002-01-0281 ◽

2002 ◽

Vol 100 (3) ◽

pp. 1072-1074 ◽

Cited By ~ 13

Author(s):

Sandra Ziegler ◽

Kurt Bürki ◽

Radek C. Skoda

Keyword(s):

Transgenic Mice ◽

Reporter Gene ◽

Fetal Liver ◽

Regulatory Elements ◽

Yolk Sac ◽

Placental Alkaline Phosphatase ◽

Hematopoietic Progenitors ◽

Thrombopoietin Receptor ◽

Human Placental Alkaline Phosphatase ◽

Dna Fragment

Abstract Thrombopoietin receptor c-mpl is expressed on hematopoietic progenitors and cells of the megakaryocytic lineage. The c-mpl promoter may, therefore, be useful for directing the expression of transgenes. We tested whether a 2-kb genomic DNA fragment comprising the putative c-mpl regulatory elements and most of the 5′-untranslated region of mouse c-mpl is able to direct the expression of a reporter gene to hematopoietic cells in transgenic mice. As a reporter gene we used the human placental alkaline phosphatase (PLAP). In adult transgenic mice, PLAP expression was specifically detected in megakaryocytes and platelets. Embryos showed PLAP reporter gene expression already in the yolk sac at embryonic day 6.5 (E6.5) and in blood islands at E7.5. At E9.5, expression was found in blood vessels of the yolk sac and the embryo proper, followed by high levels of expression in the fetal liver at E11.5. Expression in E6.5 yolk sac is compatible with a function of c-mpl and its ligand, thrombopoietin, in the earliest stages of embryonic hematopoiesis.

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In situ hybridization reveals co-expression of embryonic and adult alpha globin genes in the earliest murine erythrocyte progenitors

Development ◽

10.1242/dev.116.4.1041 ◽

1992 ◽

Vol 116 (4) ◽

pp. 1041-1049 ◽

Cited By ~ 2

Author(s):

A. Leder ◽

A. Kuo ◽

M.M. Shen ◽

P. Leder

Keyword(s):

In Situ Hybridization ◽

Embryonic Development ◽

Fetal Liver ◽

Globin Gene ◽

Yolk Sac ◽

Globin Genes ◽

Globin Mrna ◽

Mature Adult ◽

Alpha Globin

Murine erythropoiesis begins with the formation of primitive red blood cells in the blood islands of the embryonic yolk sac on day 7.5 of gestation. By analogy to human erythropoiesis, it has been thought that there is a gradual switch from the exclusive expression of the embryonic alpha-like globin (zeta) to the mature adult form (alpha) in these early mouse cells. We have used in situ hybridization to assess expression of these two globin genes during embryonic development. In contrast to what might have been expected, we find that there is simultaneous expression of both zeta and alpha genes from the very onset of erythropoiesis in the yolk sac. At no time could we detect expression of embryonic zeta globin mRNA without concomitant expression of adult alpha globin mRNA. Indeed, adult alpha transcripts exceed those of embryonic zeta in the earliest red cell precursors. Moreover, the pattern of hybridization reveals co-expression of both genes within the same cells. Even in the fetal liver, which supersedes the yolk sac as the major site of murine fetal erythropoiesis, there is a brief co-expression of zeta and alpha genes followed by the exclusive expression of the adult alpha genes. These data indicate an important difference in hematopoietic ontogeny between mouse and that of human, where zeta expression precedes that of alpha. In addition to resolving the embryonic expression of these globin genes, our results suggest that the embryonic alpha-like globin gene zeta may be physiologically redundant, even during the earliest stages of embryonic development.

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Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo

Blood ◽

10.1182/blood-2002-06-1699 ◽

2003 ◽

Vol 101 (2) ◽

pp. 508-516 ◽

Cited By ~ 240

Author(s):

Hanna K. A. Mikkola ◽

Yuko Fujiwara ◽

Thorsten M. Schlaeger ◽

David Traver ◽

Stuart H. Orkin

Keyword(s):

Endothelial Cells ◽

Mouse Embryo ◽

Fetal Liver ◽

Stem Cell Differentiation ◽

Yolk Sac ◽

Embryoid Bodies ◽

Embryonic Stem Cell Differentiation ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Definitive Hematopoiesis

Murine hematopoietic stem cells (HSCs) originate from mesoderm in a process that requires the transcription factor SCL/Tal1. To define steps in the commitment to blood cell fate, we compared wild-type and SCL−/− embryonic stem cell differentiation in vitro and identified CD41 (GpIIb) as the earliest surface marker missing from SCL−/− embryoid bodies (EBs). Culture of fluorescence-activated cell sorter (FACS) purified cells from EBs showed that definitive hematopoietic progenitors were highly enriched in the CD41+ fraction, whereas endothelial cells developed from CD41− cells. In the mouse embryo, expression of CD41 was detected in yolk sac blood islands and in fetal liver. In yolk sac and EBs, the panhematopoietic marker CD45 appeared in a subpopulation of CD41+ cells. However, multilineage hematopoietic colonies developed not only from CD45+CD41+ cells but also from CD45−CD41+ cells, suggesting that CD41 rather than CD45 marks the definitive culture colony-forming unit (CFU-C) at the embryonic stage. In contrast, fetal liver CFU-C was CD45+, and only a subfraction expressed CD41, demonstrating down-regulation of CD41 by the fetal liver stage. In yolk sac and EBs, CD41 was coexpressed with embryonic HSC markers c-kit and CD34. Sorting for CD41 and c-kit expression resulted in enrichment of definitive hematopoietic progenitors. Furthermore, the CD41+c-kit+ population was missing from runx1/AML1−/− EBs that lack definitive hematopoiesis. These results suggest that the expression of CD41, a candidate target gene of SCL/Tal1, and c-kit define the divergence of definitive hematopoiesis from endothelial cells during development. Although CD41 is commonly referred to as megakaryocyte–platelet integrin in adult hematopoiesis, these results implicate a wider role for CD41 during murine ontogeny.

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Circulation is established in a stepwise pattern in the mammalian embryo

Blood ◽

10.1182/blood-2002-08-2531 ◽

2003 ◽

Vol 101 (5) ◽

pp. 1669-1675 ◽

Cited By ~ 166

Author(s):

Kathleen E. McGrath ◽

Anne D. Koniski ◽

Jeffrey Malik ◽

James Palis

Keyword(s):

Vascular System ◽

Fetal Liver ◽

Yolk Sac ◽

Red Cells ◽

Hematopoietic Progenitors ◽

Mammalian Embryo ◽

Hematopoietic Organ ◽

Significant Enrichment ◽

A Site ◽

The Relationship

To better understand the relationship between the embryonic hematopoietic and vascular systems, we investigated the establishment of circulation in mouse embryos by examining the redistribution of yolk sac–derived primitive erythroblasts and definitive hematopoietic progenitors. Our studies revealed that small numbers of erythroblasts first enter the embryo proper at 4 to 8 somite pairs (sp) (embryonic day 8.25 [E8.25]), concomitant with the proposed onset of cardiac function. Hours later (E8.5), most red cells remained in the yolk sac. Although the number of red cells expanded rapidly in the embryo proper, a steady state of approximately 40% red cells was not reached until 26 to 30 sp (E10). Additionally, erythroblasts were unevenly distributed within the embryo's vasculature before 35 sp. These data suggest that fully functional circulation is established after E10. This timing correlated with vascular remodeling, suggesting that vessel arborization, smooth muscle recruitment, or both are required. We also examined the distribution of committed hematopoietic progenitors during early embryogenesis. Before E8.0, all progenitors were found in the yolk sac. When normalized to circulating erythroblasts, there was a significant enrichment (20- to 5-fold) of progenitors in the yolk sac compared with the embryo proper from E9.5 to E10.5. These results indicated that the yolk sac vascular network remains a site of progenitor production and preferential adhesion even as the fetal liver becomes a hematopoietic organ. We conclude that a functional vascular system develops gradually and that specialized vascular–hematopoietic environments exist after circulation becomes fully established.

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Thrombopoietin Enhances Proliferation and Differentiation of Murine Yolk Sac Erythroid Progenitors

Blood ◽

10.1182/blood.v89.4.1207 ◽

1997 ◽

Vol 89 (4) ◽

pp. 1207-1213 ◽

Cited By ~ 21

Author(s):

Takumi Era ◽

Tomomi Takahashi ◽

Katsuya Sakai ◽

Kazuo Kawamura ◽

Toru Nakano

Keyword(s):

Colony Formation ◽

Es Cells ◽

Embryonic Stem ◽

Yolk Sac ◽

In Vitro Differentiation ◽

Hematopoietic Progenitors ◽

Erythroid Progenitors ◽

Proliferation And Differentiation ◽

Differentiation Status

Abstract Thrombopoietin (TPO), the ligand for the receptor proto-oncogene c-Mpl, has been cloned and shown to be the critical regulator of proliferation and differentiation of megakaryocytic lineage. Initially, TPO was not considered to have the activity on hematopoietic lineages other than megakaryocytes. Recently, however, TPO was reported to enhance the in vitro erythroid colony formation from human bone marrow (BM) CD34+ progenitors or from mouse BM cells in combination with other cytokines. We examined the effects of TPO on the colony formation of hematopoietic progenitors in mouse yolk sac. TPO remarkably enhanced proliferation and differentiation of erythroid-lineage cells in the presence of erythropoietin (Epo). This effect was observed even in the absence of Epo. Compared with adult BM, yolk sac turned out to have relatively abundant erythroid and erythro-megakaryocytic progenitors, which responded to TPO and Epo stimulation. TPO similarly stimulated erythroid colony formation from in vitro differentiation-induced mouse embryonic stem (ES) cells whose hematopoietic differentiation status was similar to that of yolk sac. These findings help to understand the biology of hematopoietic progenitors of the early phase of hematopoiesis. Yolk sac cells or in vitro differentiation-induced ES cells would be good sources to analyze the TPO function on erythropoiesis.

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