Primitive erythropoiesis and megakaryopoiesis in the yolk sac are independent of c-myb

Hematopoiesis initiates within the yolk sac of mammalian embryos in overlapping primitive and definitive waves, each containing erythroid and megakaryocyte progenitors. c-myb-null mouse fetuses lack definitive erythrocytes but contain primitive erythroblasts and hepatic megakaryocytes. However, it is unclear if c-myb-null embryos harbor definitive erythroid or any megakaryocyte progenitors. We determined that c-myb was not expressed in primitive erythroid precursors and that c-myb-null embryos had normal primitive erythroid and megakaryocyte progenitor numbers and kinetics between embryonic day (E) 7.0 and E9.0. While primitive hematopoiesis is c-myb-independent, no definitive erythroid potential was detected in c-myb-null embryos, confirming that definitive erythropoiesis, beginning at E8.25 in the yolk sac, is completely c-myb-dependent. In contrast, reduced numbers of megakaryocyte progenitors with restricted proliferative capacity persist in E10.5 yolk sac and E11.5 liver. Despite this impaired megakaryocyte potential, c-myb-null fetuses had normal platelet numbers at E12.5 but became thrombocytopenic by E15.5, suggesting that c-myb is required for sustained thrombopoiesis.     

The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis

In the adult, platelets are derived from unipotential megakaryocyte colony-forming cells (Meg-CFCs) that arise from bipotential megakaryocyte/erythroid progenitors (MEPs). To better define the developmental origin of the megakaryocyte lineage, several aspects of megakaryopoiesis, including progenitors, maturing megakaryocytes, and circulating platelets, were examined in the murine embryo. We found that a majority of hemangioblast precursors during early gastrulation contains megakaryocyte potential. Combining progenitor assays with immunohistochemical analysis, we identified 2 waves of MEPs in the yolk sac associated with the primitive and definitive erythroid lineages. Primitive MEPs emerge at E7.25 along with megakaryocyte and primitive erythroid progenitors, indicating that primitive hematopoiesis is bilineage in nature. Subsequently, definitive MEPs expand in the yolk sac with Meg-CFCs and definitive erythroid progenitors. The first GP1bbeta-positive cells in the conceptus were identified in the yolk sac at E9.5, while large, highly reticulated platelets were detected in the embryonic bloodstream beginning at E10.5. At this time, the number of megakaryocyte progenitors begins to decline in the yolk sac and expand in the fetal liver. We conclude that the megakaryocyte lineage initially originates from hemangioblast precursors during early gastrulation and is closely associated both with primitive and with definitive erythroid lineages in the yolk sac prior to the transition of hematopoiesis to intraembryonic sites.     

Yolk-sac hematopoiesis: the first blood cells of mouse and man

OBJECTIVE: To review the process of blood-cell formation in the murine and human yolk sac. DATA SOURCES: Most articles were selected from the PubMed database. DATA SYNTHESIS: The yolk sac is the first site of blood-cell production during murine and human ontogeny. Primitive erythroid cells originate in the yolk sac and complete their maturation, including enucleation, in the bloodstream. Though species differences exist, the pattern of hematopoietic progenitor cell emergence in the yolk sac is similar in mouse and man. In both species, there is a stage of development where both primitive red blood cells and definitive erythroid progenitors are produced in the yolk sac. An "embryonic" hematopoietic stem cell that engrafts in myeloablated newborn but not adult mice can be detected in the murine yolk sac and embryo. Stem-cell activity in the human yolk sac has not been reported. CONCLUSIONS: The yolk sac is the sole site of embryonic erythropoiesis. However, definitive erythroid, myeloid, and multipotential progenitors also originate in the yolk sac. The relationship between these progenitors and the "embryonic" hematopoietic stem cell has not been elucidated. Yolk sac-derived progenitor cells may seed the developing liver via the circulation and serve as the immediate source of the mature blood cells that are required to meet the metabolic needs of the rapidly growing fetus.     

All primitive and definitive hematopoietic progenitor cells emerging before E10 in the mouse embryo are products of the yolk sac

The relative contribution of yolk sac (YS)-derived cells to the circulating definitive hematopoietic progenitor cell (HPC) pool that seeds the fetal liver remains controversial due to the presence of systemic circulation and the onset of hematopoiesis within the embryo proper (EP) before liver seeding. Ncx1-/- embryos fail to initiate a heartbeat on embryonic day (E) 8.25, but continue to develop through E10. We detected normal numbers of primitive erythroid progenitors in Ncx1-/- versus wild type (WT) YS, but primitive erythroblasts did not circulate in the Ncx1-/- EP. While there was no significant difference in the number of definitive HPCs in Ncx1-/- versus WT YS through E9.5, the Ncx1-/- EP was nearly devoid of HPCs. Thus, primitive erythroblasts and essentially all definitive HPCs destined to initially seed the fetal liver after E9.5 are generated in the YS between E7.0-E9.5 and are redistributed into the EP via the systemic circulation.     

Properties of the earliest clonogenic hemopoietic precursors to appear in the developing murine yolk sac.

We have found that a variety of clonogenic hemopoietic cells can be obtained in a viable state from mouse conceptuses as early as day 7 of gestation when tissues are disaggregated in a crude collagenase solution containing fetal bovine serum. Examination of the time course of colony formation, and the ultimate size and lineages represented in colonies produced in semisolid medium containing methylcellulose, together with analysis of individual erythroid colonies stained with rabbit antisera specific for adult (HbA) and embryonic (HbE) mouse hemoglobins, revealed the presence on days 7 and 8 of gestation (but not later) of erythropoietic progenitors that give rise to mature erythroid colonies containing up to 100 HbE-containing erythroblasts after 4-6 days of growth in culture. These progenitors are highly sensitive to the disaggregation conditions used. Clonogenic progenitors of exclusively HbA-positive erythroblasts can also be detected in the day-7 conceptus. Assays of progenitors from separately disaggregated yolk sacs and embryos from day-8 conceptuses yielded colonies only from yolk sac suspensions, and again these contained either HbE- and HbA-positive erythroblasts or only HbA-positive erythroblasts. These findings demonstrate the very early appearance in the yolk sac of a population of erythroid progenitors with a number of unique properties. Although most of these yield HbE-positive erythroblasts in vitro, some produce erythroblasts containing HbA only. Such a developmental pattern is consistent with the hypothesis that definitive erythropoiesis in the mammalian fetal liver is derived from stem cells that originate in the yolk sac blood islands.     

Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development

We elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac-like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis.     

Ontogeny of erythropoiesis

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.     

alpha4-Integrin(+) endothelium derived from primate embryonic stem cells generates primitive and definitive hematopoietic cells

The mechanism of commencement of hematopoiesis in blood islands of the yolk sac and the aorta-gonad-mesonephros (AGM) region during primate embryogenesis remains elusive. In this study, we demonstrated that VE-cadherin(+)CD45(-) endothelial cells derived from nonhuman primate embryonic stem cells are able to generate primitive and definitive hematopoietic cells sequentially, as revealed by immunostaining of floating erythrocytes and colony-forming assay in cultures. Single bipotential progenitors for hematopoietic and endothelial lineages are included in this endothelial cell population. Furthermore, hemogenic activity of these endothelial cells is observed exclusively in the alpha4-integrin(+) subpopulation; bipotential progenitors are 4-fold enriched in this subpopulation. The kinetics of this hemogenic subpopulation is similar to that of hemogenic endothelial cells previously reported in the yolk sac and the AGM region in vivo in that they emerge for only a limited time. We suggest that VE-cadherin(+)CD45(-)alpha4-integrin(+) endothelial cells are involved in primitive and definitive hematopoiesis during primate embryogenesis, though VE-cadherin(-)CD45(-)alpha4-integrin(+) cells are the primary sources for primitive hematopoiesis.     

Spatial and temporal emergence of high proliferative potential hematopoietic precursors during murine embryogenesis

During mouse embryogenesis, two waves of hematopoietic progenitors originate in the yolk sac. The first wave consists of primitive erythroid progenitors that arise at embryonic day 7.0 (E7.0), whereas the second wave consists of definitive erythroid progenitors that arise at E8.25. To determine whether these unilineage hematopoietic progenitors arise from multipotential precursors, we investigated the kinetics of high proliferative potential colony-forming cells (HPP-CFC), multipotent precursors that give rise to macroscopic colonies when cultured in vitro. No HPP-CFC were found at presomite stages (E6.5-E7.5). Rather, HPP-CFC were detected first at early somite stages (E8.25), exclusively in the yolk sac. HPP-CFC were found subsequently in the bloodstream at higher levels than the remainder of the embryo proper. However, the yolk sac remains the predominant site of HPP-CFC expansion (>100-fold) until the liver begins to serve as the major hematopoietic organ at E11.5. On secondary replating, embryonic HPP-CFC give rise to definitive erythroid and macrophage (but not primitive erythroid) progenitors. Our findings support the hypothesis that definitive but not primitive hematopoietic progenitors originate from yolk sac-derived HPP-CFC during late gastrulation.     

Yolk sac-derived primitive erythroblasts enucleate during mammalian embryogenesis

The enucleated definitive erythrocytes of mammals are unique in the animal kingdom. The observation that yolk sac-derived primitive erythroid cells in mammals circulate as nucleated cells has led to the conjecture that they are related to the red cells of fish, amphibians, and birds that remain nucleated throughout their life span. In mice, primitive red cells express both embryonic and adult hemoglobins, whereas definitive erythroblasts accumulate only adult hemoglobins. We investigated the terminal differentiation of murine primitive red cells with use of antibodies raised to embryonic beta H1-globin. Primitive erythroblasts progressively enucleate between embryonic days 12.5 and 16.5, generating mature primitive erythrocytes that are similar in size to their nucleated counterparts. These enucleated primitive erythrocytes circulate as late as 5 days after birth. The enucleation of primitive red cells in the mouse embryo has not previously been well recognized because it coincides with the emergence of exponentially expanding numbers of definitive erythrocytes from the fetal liver. Our studies establish a new paradigm in the understanding of primitive erythropoiesis and support the concept that primitive erythropoiesis in mice shares many similarities with definitive erythropoiesis of mammals.     

Immature erythroblasts with extensive ex vivo self-renewal capacity emerge from the early mammalian fetus

In the hematopoietic hierarchy, only stem cells are thought to be capable of long-term self-renewal. Erythroid progenitors derived from fetal or adult mammalian hematopoietic tissues are capable of short-term, or restricted (10(2)- to 10(5)-fold), ex vivo expansion in the presence of erythropoietin, stem cell factor, and dexamethasone. Here, we report that primary erythroid precursors derived from early mouse embryos are capable of extensive (10(6)- to 10(60)-fold) ex vivo proliferation. These cells morphologically, immunophenotypically, and functionally resemble proerythroblasts, maintaining both cytokine dependence and the potential, despite prolonged culture, to generate enucleated erythrocytes after 3-4 maturational cell divisions. This capacity for extensive erythroblast self-renewal is temporally associated with the emergence of definitive erythropoiesis in the yolk sac and its transition to the fetal liver. In contrast, hematopoietic stem cell-derived definitive erythropoiesis in the adult is associated almost exclusively with restricted ex vivo self-renewal. Primary primitive erythroid precursors, which lack significant expression of Kit and glucocorticoid receptors, lack ex vivo self-renewal capacity. Extensively self-renewing erythroblasts, despite their near complete maturity within the hematopoietic hierarchy, may ultimately serve as a renewable source of red cells for transfusion therapy.     

Anaemia and its association with month and blood phenotype in blood donors in Fako division,Cameroon

Background

Mammalian erythropoiesis can be divided into two distinct types, primitive and definitive, in which new cells are derived from the yolk sac and hematopoietic stem cells, respectively. Primitive erythropoiesis occurs within a restricted period during embryogenesis. Primitive erythrocytes remain nucleated, and their hemoglobins are different from those in definitive erythrocytes. Embryonic type hemoglobin is expressed in adult animals under genetically abnormal condition, but its later expression has not been reported in genetically normal adult animals, even under anemic conditions. We previously reported that injecting animals with nitrogen-containing bisphosphonate (NBP) decreased erythropoiesis in bone marrow (BM). Here, we induced severe anemia in a mouse model by injecting NBP injection in combination with phenylhydrazine (PHZ), and then we analyzed erythropoiesis and the levels of different types of hemoglobin.

Methods

Splenectomized mice were treated with NBP to inhibit erythropoiesis in BM, and with PHZ to induce hemolytic anemia. We analyzed hematopoietic sites and peripheral blood using morphological and molecular biological methods.

Results

Combined treatment of splenectomized mice with NBP and PHZ induced critical anemia compared to treatment with PHZ alone, and numerous nucleated erythrocytes appeared in the peripheral blood. In the BM, immature CD71-positive erythroblasts were increased, and extramedullary erythropoiesis occurred in the liver. Furthermore, embryonic type globin mRNA was detected in both the BM and the liver. In peripheral blood, spots that did not correspond to control hemoglobin were observed in 2D electrophoresis. ChIP analyses showed that KLF1 and KLF2 bind to the promoter regions of β-like globin. Wine-colored capsuled structures were unexpectedly observed in the abdominal cavity, and active erythropoiesis was also observed in these structures.

Conclusion

These results indicate that primitive erythropoiesis occurs in adult mice to rescue critical anemia because primitive erythropoiesis does not require macrophages as stroma whereas macrophages play a pivotal role in definitive erythropoiesis even outside the medulla. The cells expressing embryonic hemoglobin in this study were similar to primitive erythrocytes, indicating the possibility that yolk sac-derived primitive erythroid cells may persist into adulthood in mice.

     

Disruption of palladin leads to defects in definitive erythropoiesis by interfering with erythroblastic island formation in mouse fetal liver

Palladin was originally found up-regulated with NB4 cell differentiation induced by all-trans retinoic acid. Disruption of palladin results in neural tube closure defects, liver herniation, and embryonic lethality. Here we further report that Palld(-/-) embryos exhibit a significant defect in erythropoiesis characterized by a dramatic reduction in definitive erythrocytes derived from fetal liver but not primitive erythrocytes from yolk sac. The reduction of erythrocytes is accompanied by increased apoptosis of erythroblasts and partial blockage of erythroid differentiation. However, colony-forming assay shows no differences between wild-type (wt) and mutant fetal liver or yolk sac in the number and size of colonies tested. In addition, Palld(-/-) fetal liver cells can reconstitute hematopoiesis in lethally irradiated mice. These data strongly suggest that deficient erythropoiesis in Palld(-/-) fetal liver is mainly due to a compromised erythropoietic microenvironment. As expected, erythroblastic island in Palld(-/-) fetal liver was found disorganized. Palld(-/-) fetal liver cells fail to form erythroblastic island in vitro. Interestingly, wt macrophages can form such units with either wt or mutant erythroblasts, while mutant macrophages lose their ability to bind wt or mutant erythroblasts. These data demonstrate that palladin is crucial for definitive erythropoiesis and erythroblastic island formation and, especially, required for normal function of macrophages in fetal liver.     

Podocalyxin is a CD34-related marker of murine hematopoietic stem cells and embryonic erythroid cells

Podocalyxin/podocalyxin-like protein 1 [PCLP1]/thrombomucin/MEP21 is a CD34-related sialomucin. We have performed a detailed analysis of its expression during murine development and assessed its utility as a marker of hematopoietic stem cells (HSCs) and their more differentiated progeny. We find that podocalyxin is highly expressed by the first primitive hematopoietic progenitors and nucleated red blood cells to form in the embryonic yolk sac. Likewise, podocalyxin is expressed by definitive multilineage hematopoietic progenitors and erythroid precursors in fetal liver. The level of podocalyxin expression gradually declines with further embryo maturation and reaches near-background levels at birth. This is followed by a postnatal burst of expression that correlates with the seeding of new hematopoietic progenitors to the spleen and bone marrow. Shortly thereafter, podocalyxin expression gradually declines, and by 4 weeks postpartum it is restricted to a rare population of Sca-1(+), c-kit(+), lineage marker(-) (Lin(-)) cells in the bone marrow. These rare podocalyxin-expressing cells are capable of serially reconstituting myeloid and lymphoid lineages in lethally irradiated recipients, suggesting they have HSC activity. In summary, we find that podocalyxin is a marker of embryonic HSCs and erythroid cells and of adult HSCs and that it may be a valuable marker for the purification of these cells for transplantation.     

Enucleation of primitive erythroid cells generates a transient population of "pyrenocytes" in the mammalian fetus

Enucleation is the hallmark of erythropoiesis in mammals. Previously, we determined that yolk sac-derived primitive erythroblasts mature in the bloodstream and enucleate between embryonic day (E)14.5 and E16.5 of mouse gestation. While definitive erythroblasts enucleate by nuclear extrusion, generating reticulocytes and small, nucleated cells with a thin rim of cytoplasm ("pyrenocytes"), it is unclear by what mechanism primitive erythroblasts enucleate. Immunohistochemical examination of fetal blood revealed primitive pyrenocytes that were confirmed by multispectral imaging flow cytometry to constitute a distinct, transient cell population. The frequency of primitive erythroblasts was higher in the liver than the bloodstream, suggesting that they enucleate in the liver, a possibility supported by their proximity to liver macrophages and the isolation of erythroblast islands containing primitive erythroblasts. Furthermore, primitive erythroblasts can reconstitute erythroblast islands in vitro by attaching to fetal liver-derived macrophages, an association mediated in part by alpha4 integrin. Late-stage primitive erythroblasts fail to enucleate in vitro unless cocultured with macrophage cells. Our studies indicate that primitive erythroblasts enucleate by nuclear extrusion to generate erythrocytes and pyrenocytes and suggest this occurs in the fetal liver in association with macrophages. Continued studies comparing primitive and definitive erythropoiesis will lead to an improved understanding of terminal erythroid maturation.     

Maturation and enucleation of primitive erythroblasts during mouse embryogenesis is accompanied by changes in cell-surface antigen expression

Primitive erythroblasts (EryPs) are the first hematopoietic cell type to form during mammalian embryogenesis and emerge within the blood islands of the yolk sac. Large, nucleated EryPs begin to circulate around midgestation, when connections between yolk sac and embryonic vasculature mature. Two to 3 days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and rapidly outnumber EryPs in the circulation. The development and maturation of EryPs remain poorly defined. Our analysis of embryonic blood at different stages reveals a stepwise developmental progression within the EryP lineage from E9.5 to E12.5. Thereafter, EryDs are also present in the bloodstream, and the 2 lineages are not easily distinguished. We have generated a transgenic mouse line in which the human epsilon-globin gene promoter drives expression of green fluorescent protein exclusively within the EryP lineage. Here, we have used this line to characterize changes in cell morphology and surface-marker expression as EryPs mature and to track EryP numbers and enucleation throughout gestation. This study identifies previously unrecognized synchronous developmental stages leading to the maturation of EryPs in the mouse embryo. Unexpectedly, we find that EryPs are a stable cell population that persists through the end of gestation.     

Circulation is established in a stepwise pattern in the mammalian embryo

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.