Primitive Erythroid Lineage Research Articles

Multiple cell populations generate macrophage progenitors in the early yolk sac.

Yolk sac (YS) CSF1 receptor positive (CSF1R+) cells are thought to be the progenitors for tissue-resident macrophages present in various tissues. The YS progenitors for tissue-resident macrophages are referred to as erythroid-myeloid progenitors (EMPs). However, diverse types of hematopoietic progenitors are present in the early YS, thus it is not precisely known which type of hematopoietic cell gives rise to the CSF1R+ lineage. In this study, an analysis was conducted to determine when CSF1R+ progenitors appeared in the early YS. It showed that CSF1R+ cells appeared in the YS as early as embryonic day 9 (E9) and that the earliest hematopoietic progenitors that differentiate into CSF1R+ cells were found in E8. Since these progenitors possessed the capability to generate primitive erythroid cells, it was likely that primitive erythroid lineages shared progenitors with the CSF1R+ lineage. Mutual antagonism appears to work between PU.1 and GATA1 when CSF1R+ cells appear in the early YS. One day later (E9), multiple progenitors, including myeloid-restricted progenitors and multipotent progenitors, in the YS could immediately generate CSF1R+ cells. These results suggest that EMPs are not an exclusive source for the CSF1R+ lineage; rather, multiple hematopoietic cell populations give rise to CSF1R+ lineage in the early YS.

Stat3 Is Activated By ROS to Uniquely Regulate 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.

Ontogeny As a Critical Determinant of Natural Killer Cell Potential and Function

Hematopoietic development during mammalian embryogenesis is comprised of a restricted primitive program of primitive erythroid, megakaryocytic, and macrophage lineages, and a definitive program of definitive erythroid, myeloid and lymphoid potential emerging from hematopoietic stem cell (HSC)-independent and dependent processes. Interestingly, progenitors of natural killer (NK) cells, but not B- or T-cells, have been found in the early human yolk sac, suggesting that NK cells may arise from HSC-independent sources.NK cells recognize and kill virally infected cells and tumor cells, making them a highly desirable cell-type for adoptive immunotherapy. To bypass donor-related issues, human pluripotent stem cell (hPSC)-derived NK cells offer the possibility of uniform activity in a renewable “off-the-shelf” cell product. As the differentiation of hPSCs recapitulates early developmental processes, we sought to characterize the developmental origin of hPSC-derived NK cells. Studies in mice indicate that NK cells in the adult are derived from hematopoietic stem cells (HSCs) that commit to a lymphoid differentiation pathway. However, while NK cells, like HSCs, have been found in the fetal liver, the developmental origin of the fetal NK cell lineage remains poorly understood.We have developed a stage-specific hPSC differentiation method that separates WNT-independent (WNTi) hematopoietic progenitors that harbor “primitive” hematopoietic potential from WNT-dependent (WNTd) erythro-myeloid-(T-)lymphoid “definitive” hematopoietic progenitors. Using this system, we find that CD34+ cells from both populations harbor NK cell potential. NK cells from hPSC WNTi progenitors (WNTi-NK cells) mature rapidly, are significantly more granular, and express very high levels of CD16 in comparison to their hPSC WNTd counterparts (WNTd-NK cells) and cord blood-derived NK (cbNK) cells. Further, WNTi CD34+ progenitors always gave rise to a granulocyte population alongside NK cells, suggesting they may be derived from a myeloid progenitor. Both WNTi-NK and WNTd-NK cells robustly respond to tumor targets, antibody-dependent cell-mediated cytotoxicity (ADCC), and PMA/ionomycin stimulation in comparison to cbNK cells. In all cases, WNTi-NK cells exhibited a strong bias for cytolytic degranulation over cytokine production, while WNTd-NK cells were biased for IFNg secretion. Similarly, WNTi-NK cells exhibit superior ADCC-mediated cell killing of Raji cells.We then turned to the well-characterized murine embryo to determine whether HSC-independent NK cell progenitors are developmentally conserved. Assessing NK cell potential via explant culture, we found that as early as E7.5, yolk sac explants give rise to NK cells, as well as primitive and definitive erythroid progenitors. Further, we find that murine E9.5 yolk sac kit+CD41+CD16/32+ erythro-myeloid progenitors (EMP) give rise to NK cells ex vivo. Similar to hPSC WNTi-NK cells, EMP-derived NK cells were larger and more granular, and emerged alongside a granulocyte population in explant culture. Thus, the murine yolk sac harbors unique NK cell potential, from a committed myeloid progenitor, prior to HSC emergence.Collectively, these studies suggest that ontological origin is an unexpectedly important consideration in the design of hPSC-derived NK cell-based therapeutics, and raise new questions regarding the potential of early hematopoietic progenitors in the mammalian embryo. DisclosuresFehniger:Celgene: Research Funding; Cyto-Sen Therapeutics: Consultancy; Altor BioScience: Research Funding; Affimed: Research Funding; NIH/NCI: Other: R01 CA205239, P50CA171963. Palis:Rubies Therapeutics: Consultancy.

3175 - Ontogeny as a Critical Determinant of Natural Killer Cell Potential and Function

Hematopoietic development during mammalian embryogenesis is comprised of a restricted primitive program of primitive erythroid, megakaryocytic, and macrophage lineages, and a definitive program of definitive erythroid, myeloid and lymphoid potential and hematopoietic stem cell (HSC)-independent and dependent processes. Interestingly, progenitors of natural killer (NK) cells, but not B- or T-cells, have been found in the early human yolk sac, suggesting that NK cells may arise from HSC-independent sources. NK cells are innate lymphoid cells and their derivation from human pluripotent stem cells (hPSCs) is an exciting alternative for adoptive immunotherapy strategies. We have developed an hPSC differentiation method that separates primitive-like hematopoietic progenitors from HSC-independent erythro-myeloid-(T)lymphoid definitive-like potential. Using this system, we find that CD34+ cells from both populations harbor NK cell potential. Further, we find that murine E9.5 yolk sac kit+CD41+CD16/32 + erythro-myeloid progenitors give rise to NK cells ex vivo. As early as E7.5, yolk sac explants give rise to NK cells, as well as primitive and definitive erythroid progenitors. Thus, the murine yolk sac harbors NK cell potential prior to HSC emergence. NK cells from hPSC primitive-like progenitors (pNK cells) mature rapidly, are significantly more granular, and express very high levels of CD16 in comparison to their hPSC definitive-like counterparts (dNK cells) and cord blood-derived NK (cbNK) cells. Both pNK and dNK cells robustly respond to tumor targets and antibody-dependent cell-mediated cytotoxicity (ADCC) in comparison to cbNK cells. In response to K562 cells, pNK cells exhibit a 3-fold preference for cytolytic degranulation, while dNK cells are 3-fold more biased for IFNg secretion. Similarly, in response to ADCC pNK cells display 3-fold more TNFa and degranulation than dNK or cbNK cells. Collectively, these studies suggest that ontological origin is an unexpectedly important consideration in the design of hPSC-derived NK cell-based therapeutics, and raise new questions regarding the potential of early hematopoietic progenitors in the mammalian embryo.

Interaction of the Macrophage and Primitive Erythroid Lineages in the Mammalian Embryo.

Two distinct forms of erythropoiesis, primitive and definitive, are found in mammals. Definitive erythroid precursors in the bone marrow mature in the physical context of macrophage cells in “erythroblastic islands.” In the murine embryo, overlapping waves of primitive hematopoietic progenitors and definitive erythro-myeloid progenitors, each containing macrophage potential, arise in the yolk sac prior to the emergence of hematopoietic stem cells. Primitive erythroblasts mature in the bloodstream as a semi-synchronous cohort while macrophage cells derived from the yolk sac seed the fetal liver. Late-stage primitive erythroblasts associate with macrophage cells in erythroblastic islands in the fetal liver, indicating that primitive erythroblasts can interact with macrophage cells extravascularly. Like definitive erythroblasts, primitive erythroblasts physically associate with macrophages through α4 integrin–vascular adhesion molecule 1-mediated interactions and α4 integrin is redistributed onto the plasma membrane of primitive pyrenocytes. Both in vitro and in vivo studies indicate that fetal liver macrophage cells engulf primitive pyrenocytes. Taken together, these studies indicate that several aspects of the interplay between macrophage cells and maturing erythroid precursor cells are conserved during the ontogeny of mammalian organisms.

The Essential Cell Cycle Regulator Setd8 Drives Chromatin Condensation during Terminal Erythroid Maturation

Chromatin condensation culminating in enucleation is a hallmark of erythropoiesis, however the mechanisms driving this process are incompletely understood. Setd8 is the sole enzyme that can mono-methylate histone H4, lysine 20 (H4K20me1) and is an important regulator of cell cycle progression, higher order chromatin structure, and genome stability. (Reviewed in Beck, Genes and Development, 2010) Setd8 and H4K20me1 are unique among epigenetic regulators in that their expression is dynamically regulated during the cell cycle. Setd8 expression peaks during G2/M, where it promotes mitotic chromatin condensation, and becomes undetectable during S-phase due to ubiquitin dependent destruction. (Oda, Mol Cell, 2010) The presence of H4K20me1 mirrors that of Setd8, peaking in G2/M, and reaching a nadir during S-phase due to removal by the histone demethylase PHF8. (Liu, Nature, 2010) Interestingly, Setd8 is expressed at levels 8- to 10- fold higher in CD71+ erythroblasts than in any other cell type, (Wu Genome Biology,2009) suggesting that it has an erythroid-specific function. We hypothesize that Setd8 drives chromatin condensation in maturing erythroblasts.In cell lines, forced accumulation of H4K20me1 during S-phase due to perturbation of either Setd8 or PHF8 results in pre-mitotic chromatin condensation (Centore Mol Cell 2010; Liu Nature 2010). We demonstrate that primary erythroblasts express Setd8 and accumulate H4K20me1 throughout the cell cycle, suggesting that Setd8 and H4K20me1 in promote chromatin condensation during terminal maturation. We further demonstrate that Setd8 is essential for erythropoiesis, with erythroid-specific Setd8 deletion resulting in profound anemia that is lethal by E12.5. The early onset of anemia indicates a defect in the primitive erythroid lineage, which emerges from the yolk sac at E8.5, and proliferates, matures, and enucleates in the circulation as a semi-synchronous cohort. (Kingsley, Blood, 2004) Detailed analyses of Setd8-null erythroblasts revealed severe defects in cell cycle progression, increased DNA content suggesting loss of genomic integrity, accumulation of DNA damage, and a modest increase in the rate of apoptosis. Global transcriptome analyses demonstrated that Setd8-null erythroblasts had activation of checkpoint genes such as CDKN1a and Gene Set Enrichment Analyses identified significant enrichment of cell cycle and p53 signaling pathways. Despite evidence of p53 activation, concomitant p53 deletion was not able to rescue the Set8-null phenotype, indicating that Setd8 has an essential role in promoting erythroid proliferation and survival that is independent of the p53 pathway.Consistent with our hypothesis that Setd8 drives chromatin condensation in maturing erythroblasts, the nuclear area of Setd8-null cells was nearly twice that of controls at E11.5 (119 and 69 um2, respectively p<0.0003). Transmission electron microscopy confirmed a profound defect in global chromatin condensation in the Setd8-null cells. Unexpectedly, heterochromatin was nearly absent from the Setd8-null cells, with the Setd8-null cells containing only a small amount of heterochromatin localized to the nuclear periphery. To determine the impact of Setd8 deletion on local chromatin structure, we performed ATAC-seq (Assay for Transposase Accessible Chromatin) on sorted populations of Setd8-null and control erythroblasts. Preliminary analyses of ATAC-seq data identified 364 ATAC peaks present in the Setd8-null cells but not in controls (p<0.001). Intriguingly, Gene Ontogeny analyses of the genes nearest to those regions was significant for multiple terms associated with higher order chromatin structure including "regulation of chromatin organization" and "positive regulation of histone deacetylation." Taken together, our results indicate that erythroblasts have adapted an essential cell cycle regulator to drive chromatin condensation during terminal maturation. DisclosuresNo relevant conflicts of interest to declare.

Essential role of Ufm1 conjugation in the hematopoietic system.

Protein modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins plays a pivotal role in a wide range of cellular functions and signaling pathways. The Ufm1 conjugation system is a novel ubiquitin-like system that consists of Ufm1, Uba5 (E1), Ufc1 (E2), and less defined E3 ligase(s) and targets. Despite its discovery more than a decade ago, its biological functions and working mechanism remains poorly understood. Recent genetic studies using knockout mouse models provide unambiguous evidence for the indispensable role of the Ufm1 system in animal development and hematopoiesis, especially erythroid development. In this short review, we summarize the recent progress on this important protein modification system and highlight potential challenges ahead. Further elucidation of the function and working mechanism of the ufmylation pathway would provide insight into disease pathogenesis and novel therapeutic targets for blood-related diseases such as anemia.

The modern primitives: applying new technological approaches to explore the biology of the earliest red blood cells.

One of the most critical stages in mammalian embryogenesis is the independent production of the embryo's own circulating, functional red blood cells. Correspondingly, erythrocytes are the first cell type to become functionally mature during embryogenesis. Failure to achieve this invariably leads to in utero lethality. The recent application of technologies such as transcriptome analysis, flow cytometry, mutant embryo analysis, and transgenic fluorescent gene expression reporter systems has shed new light on the distinct erythroid lineages that arise early in development. Here, I will describe the similarities and differences between the distinct erythroid populations that must form for the embryo to survive. While much of the focus of this review will be the poorly understood primitive erythroid lineage, a discussion of other erythroid and hematopoietic lineages, as well as the cell types making up the different niches that give rise to these lineages, is essential for presenting an appropriate developmental context of these cells.

Analysis of primitive erythroid cell proliferation and enucleation using a cyan fluorescent reporter in transgenic mice

Primitive erythropoiesis is a vital process for mammalian embryonic development. Here we report the generation and characterization of a new transgenic mouse line that expresses a histone H2B-CFP fusion protein in the nuclei of primitive erythroid cells. We demonstrate the potential of this ε-globin-histone H2B-CFP line for multicolor imaging and flow cytometry analysis. The ε-globin-H2B-CFP line was used to analyze the cell cycle distribution and proliferation of CFP-expressing primitive erythroblasts from E8.5-E13.5. We also evaluated phagocytosis of extruded CFP-positive nuclei by macrophages in fetal liver and placenta. The ε-globin-H2B-CFP transgenic mouse line adds to the available tools for studying the development of the primitive erythroid lineage.

Stat and interferon genes identified by network analysis differentially regulate primitive and definitive erythropoiesis.

BackgroundHematopoietic ontogeny is characterized by overlapping waves of primitive, fetal definitive, and adult definitive erythroid lineages. Our aim is to identify differences in the transcriptional control of these distinct erythroid cell maturation pathways by inferring and analyzing gene-interaction networks from lineage-specific expression datasets. Inferred networks are strongly connected and do not fit a scale-free model, making it difficult to identify essential regulators using the hub-essentiality standard.ResultsWe employed a semi-supervised machine learning approach to integrate measures of network topology with expression data to score gene essentiality. The algorithm was trained and tested on the adult and fetal definitive erythroid lineages. When applied to the primitive erythroid lineage, 144 high scoring transcription factors were found to be differentially expressed between the primitive and adult definitive erythroid lineages, including all expressed STAT-family members. Differential responses of primitive and definitive erythroblasts to a Stat3 inhibitor and IFNγ in vitro supported the results of the computational analysis. Further investigation of the original expression data revealed a striking signature of Stat1-related genes in the adult definitive erythroid network. Among the potential pathways known to utilize Stat1, interferon (IFN) signaling-related genes were expressed almost exclusively within the adult definitive erythroid network.ConclusionsIn vitro results support the computational prediction that differential regulation and downstream effectors of STAT signaling are key factors that distinguish the transcriptional control of primitive and definitive erythroid cell maturation.

Primitive and Definitive Erythropoiesis

Abstract SCI-37Studies in mammalian and nonmammalian vertebrate embryos indicate that erythropoiesis comes in two flavors: primitive and definitive. The primitive erythroid lineage in mammalian embryos is characterized by a transient wave of lineage-committed progenitors that emerge from the yolk sac and generate a wave of precursors that synchronously mature in the bloodstream. Primitive erythroid precursors dynamically regulate embryonic globin gene expression and ultimately enucleate to form erythrocytes. Primitive erythropoiesis is superseded by definitive erythroid cells that mature extravascularly in association with macrophage cells. Studies in the mouse embryo indicate that definitive erythropoiesis has two distinct developmental origins. The first is a transient wave of erythro-myeloid progenitors (EMP) that emerge from the yolk sac and seed the early fetal liver. The second is a long-term program of erythropoiesis derived from hematopoietic stem cells. Erythropoietin is the central regulator of definitive erythropoiesis, in part by regulating the survival of committed progenitors. In contrast, the role of erythropoietin in primitive erythropoiesis remains poorly understood. Recent studies indicate that erythropoietin does not regulate the primitive erythroid progenitor compartment, but rather plays a critical role in establishing an antiapoptotic state during the terminal maturation of primitive erythroblasts. EMP-derived proerythroblasts are capable of extensive self-renewal in vitro, while primitive erythroid progenitors are incapable of self-renewal under the same conditions. These studies, taken together, indicate that the primitive and definitive forms of erythropoiesis have fundamental differences in the regulation of red cell output. The overlapping emergence of primitive and definitive erythroid lineages in differentiating embryonic stem cells suggests that the transient yolk-sac-derived primitive and EMP-derived definitive erythroid programs are recapitulated in vitro. These studies offer the hope that human embryonic stem cells can serve as a source of functional definitive erythroid cells for transfusion therapy. Disclosures:No relevant conflicts of interest to declare.

Primitive Erythropoiesis Is Regulated by miR-126 via Nonhematopoietic Vcam-1+ Cells

Primitive erythropoiesis defines the onset of hematopoiesis in the yolk sac of the early embryo and is initiated by the emergence of progenitors assayed as colony-forming cells (EryP-CFCs). EryP-CFCs are detected for only a narrow window during embryonic development, suggesting that both their initiation and termination are tightly controlled. Using the embryonic stem differentiation system to model primitive erythropoiesis, we found that miR-126 regulates the termination of EryP-CFC development. Analyses of miR-126 null embryos revealed that thismiR also regulates EryP-CFCs invivo. We identified vascular cell adhesion molecule-1 (Vcam-1) expressed by a mesenchymal cell population asa relevant target of miR-126. Interaction of EryP-CFCs with Vcam-1 accelerated their maturation to ßh1-globin(+) and Ter119(+) cells through a Src family kinase. These findings uncover a cell nonautonomous regulatory pathway for primitive erythropoiesis that may provide insight into the mechanism(s) controlling the developmental switch from primitive to definitive hematopoiesis.

Deletion of the Scl +19 enhancer increases the blood stem cell compartment without affecting the formation of mature blood lineages

The stem cell leukemia (Scl)/Tal1 gene is essential for normal blood and endothelial development, and is expressed in hematopoietic stem cells (HSCs), progenitors, erythroid, megakaryocytic, and mast cells. The Scl +19 enhancer is active in HSCs and progenitor cells, megakaryocytes, and mast cells, but not mature erythroid cells. Here we demonstrate that in vivo deletion of the Scl +19 enhancer (SclΔ19/Δ19) results in viable mice with normal Scl expression in mature hematopoietic lineages. By contrast, Scl expression is reduced in the stem/progenitor compartment and flow cytometry analysis revealed that the HSC and megakaryocyte-erythroid progenitor populations are enlarged in SclΔ19/Δ19 mice. The increase in HSC numbers contributed to enhanced expansion in bone marrow transplantation assays, but did not affect multilineage repopulation or stress responses. These results affirm that the Scl +19 enhancer plays a key role in the development of hematopoietic stem/progenitor cells, but is not necessary for mature hematopoietic lineages. Moreover, active histone marks across the Scl locus were significantly reduced in SclΔ19/Δ19 fetal liver cells without major changes in steady-state messenger RNA levels, suggesting post-transcriptional compensation for loss of a regulatory element, a result that might be widely relevant given the frequent observation of mild phenotypes after deletion of regulatory elements.

Epo/Epor Signaling Critically Regulates Maturation of the Primitive Erythroid Lineage In the Mammalian Embryo

AbstractAbstract 813Erythropoiesis in both the embryo and the adult is characterized by the progressive maturation of lineage-specific progenitors through morphologically identifiable precursors that enucleate to form mature erythrocytes. In the adult, erythropoietin (Epo) binds its homodimeric receptor (EpoR) and activates the Jak2-Stat5 pathway to inhibit apoptosis and mediate the proliferation of definitive erythroid progenitors. Similar responses to Epo have also been reported in neurons, cardiomyocytes, and several cancer cell types. However, little is known regarding the function of Epo/EpoR in the first erythroid cells of the developing embryo, the yolk sac-derived, primitive erythroblasts that emerge from a transient wave of progenitors and mature as a synchronous cohort in the fetal vasculature. We have previously determined that EpoR transcripts are present in yolk-sac blood islands beginning at E7.5 (McGann et al., Exp Hem, 1997). Here, we examine the function of Epo/EpoR in embryonic erythropoiesis. Using a colony-forming assay, we found normal levels of primitive erythroid progenitor activity in E8.5 Epor-null embryos. However, these colonies were composed of markedly fewer cells, suggesting a defect in the erythroid precursor maturation. Consistent with these in vitro results, we found that E10.5 Epor-null embryos contain 60% fewer primitive erythroblasts compared to wild-type littermates. Cell cycle analysis revealed a block at the transition from G1- to S-phase with a greater than 2-fold increase in the proportion of erythroblasts in G1-phase in Epor-null versus wild-type embryos. Furthermore, utilizing multi-spectral imaging flow cytometry, we found that E10.5 and E11.5 Epor-null primitive erythroblasts exhibit decreased cell size, nuclear size, and RNA content. Transcriptional analysis revealed a higher εy:βH1 expression ratio in Epor-null erythroblasts consistent with a more rapid maturational globin switch. Most strikingly, we detected a 10-fold reduction in CD71 (transferrin receptor) cell surface expression, which led us to investigate its transcript levels along with other known Stat5-target genes. CD71, Myc, and Bcl-xl transcript levels were all reduced significantly in Epor-null compared to wild-type erythroblasts. Furthermore, we found robust phosphorylation of Stat5 in primitive erythroid precursors that increased upon exposure to Epo. Taken together, our findings indicate that Epo/EpoR signaling is not required for the specification or emergence of primitive erythroid progenitors. However, the subsequent proliferative maturation of primitive erythroid precursors is highly dependent on Epo/EpoR signaling and modulation of Stat5-target genes. The marked reduction of primitive erythroid precursors in Epor-null embryos is associated with cell cycle defects. Furthermore, Epor-null primitive erythroblasts exhibit a phenotype of premature maturation, characterized by decreased cell size, nuclear size, RNA content, and altered globin switching. Finally, the drastic reduction of CD71 expression supports the concept that Epo regulates iron metabolism through Stat5-mediated signaling. We conclude that erythropoietin is a critical regulator of primitive erythropoiesis in the mammalian embryo. However, unlike definitive erythropoiesis, Epo/EpoR primarily regulates the primitive lineage at the precursor rather than the progenitor stage of maturation. Disclosures:No relevant conflicts of interest to declare.

Dose-dependent regulation of primitive erythroid maturation and identity by the transcription factor Eklf

AbstractThe primitive erythroid (EryP) lineage is the first to differentiate during mammalian embryogenesis. Eklf/Klf1 is a transcriptional regulator that is essential for definitive erythropoiesis in the fetal liver. Dissection of the role(s) of Eklf within the EryP compartment has been confounded by the simultaneous presence of EryP and fetal liver–derived definitive erythroid (EryD) cells in the blood. To address this problem, we have distinguished EryP from their definitive counterparts by crossing Eklf+/− mutant and ϵ-globin::histone H2B-GFP transgenic mice. Eklf-deficient EryP exhibit membrane ruffling and a failure to acquire the typical discoidal erythroid shape but they can enucleate. Flow cytometric analyses of H2B-GFP+ EryP revealed that Eklf heterozygosity results in the loss of Ter119 surface expression on EryP but not on EryD. Null mutation of Eklf resulted in abnormal expression of a range of surface proteins by EryP. In particular, several megakaryocyte markers were ectopically expressed by maturing Eklf-null EryP. Unexpectedly, the platelet tetraspanin CD9 was detected on nucleated wild-type EryP but not on mature EryD and thus provides a useful marker for purifying circulating EryP. We conclude that Eklf gene dosage is crucial for regulating the surface phenotype and molecular identity of maturing primitive erythroid cells.

Primitive erythropoiesis in the mammalian embryo

Erythropoiesis in adult mammals is characterized by the progressive maturation of hematopoietic stem cells to lineage-specific progenitors, to morphologically identifiable precursors which enucleate to form mature erythrocytes. In contrast, primitive erythropoiesis is characterized by the appearance within the yolk sac of a transient, lineage-restricted progenitor population which generates a wave of erythroid precursors. These precursors undergo progressive maturation in the bloodstream, characterized by nuclear condensation and embryonic hemoglobin accumulation. This process is dependent on erythropoietin signaling through its cognate receptor, as well as the function of several erythroid-specific transcription factors, including GATA1 and EKLF. Targeted disruption of genes in the mouse that result in failure of the emergence or maturation of the primitive erythroid lineage leads to early fetal death, indicating that the primitive erythroid lineage is necessary for survival of the mammalian embryo. While it was thought for over a century that primitive erythroid cells were uniquely nucleated mammalian red cells, it is now recognized that they, like their definitive erythroid counterparts, enucleate to form reticulocytes and pyrenocytes. This surprising finding indicates that the primitive erythroid lineage is indeed mammalian, rather than non-mammalian, in character.

Genome Expression Profiling of the First Hematopoietic Progenitors of the Mouse Embryo.

Abstract 2536Poster Board II-513The transcriptional networks that regulate lineage commitment and expansion of the earliest hematopoietic progenitors in the mammalian embryo have not been well studied, due to a lack of methods for isolating these cells. We have begun to address this problem by purifying the first hematopoietic-committed progenitors in the mouse embryo based on expression of a human ε-globin::H2B-EGFP transgene that is expressed exclusively within the primitive erythroid (EryP) lineage, as early as embryonic day (E) 7.5. EryP are the first lineage-specific cell type to form in the embryo. They arise in large numbers from yolk sac-derived progenitors at the end of gastrulation, enter the circulation as nucleated cells soon thereafter, and continue to mature in a stepwise, synchronous fashion until they enucleate. The early and lineage specific expression of the GFP reporter allowed us to isolate not only circulating EryP (E9.5-E11.5) but also a population from dispersed E7.5-8.5 embryos that is enriched in EryP progenitors. Genome expression profiling allowed us to define the transcriptome from each stage of development and revealed highly dynamic changes during the progression from progenitor to maturing erythroblast. Hierarchical clustering analysis was used to organize genes on the basis of overall similarity in expression patterns; six major cluster patterns were identified. Genes within these clusters comprised distinct functional classes. For example, particularly prominent increases in expression were detected for genes involved in ribosome biogenesis, translation, chromosome condensation, and autophagy. Genes that were downregulated included those involved in DNA replication, cell cycle, and nucleolar and organelle biogenesis. We have focused on the emergence of EryP in the yolk sac. Expression of Gata2 is high in the progenitor population at E7.5 and decreases dramatically by E8.5. In contrast, Gata1, Scl, and Eklf are all upregulated during maturation of EryP progenitors, suggesting that these transcription factors have distinct functions during primitive erythropoiesis. Consistent with expression of the GFP reporter as early as E7.5, we find that endogenous mouse embryonic globin genes are also expressed at this stage. Therefore, globin gene expression is an early feature of EryP development. Analysis of promoters of differentially expressed genes allowed us to identify candidate transcriptional regulators, some of which have not previously been implicated in erythroid development. This is the first lineage specific transcription profiling of a differentiating hematopoietic cell type in the early mouse embryo. While we have focused on the development of EryP, insights from this study should have broader relevance to the definitive erythroid lineage. Disclosures:No relevant conflicts of interest to declare.

A common bipotent progenitor generates the erythroid and megakaryocyte lineages in embryonic stem cell–derived primitive hematopoiesis

AbstractThe megakaryocytic (MK) and erythroid lineages are tightly associated during differentiation and are generated from a bipotent megakaryocyte-erythroid progenitor (MEP). In the mouse, a primitive MEP has been demonstrated in the yolk sac. In human, it is not known whether the primitive MK and erythroid lineages are generated from a common progenitor or independently. Using hematopoietic differentiation of human embryonic stem cells on the OP9 cell line, we identified a primitive MEP in a subset of cells coexpressing glycophorin A (GPA) and CD41 from day 9 to day 12 of coculturing. This MEP differentiates into primitive erythroid (GPA+CD41−) and MK (GPA−CD41+) lineages. In contrast to erythropoietin (EPO)–dependent definitive hematopoiesis, KIT was not detected during erythroid differentiation. A molecular signature for the commitment and differentiation toward both the erythroid and MK lineages was detected by assessing expression of transcription factors, thrombopoietin receptor (MPL) and erythropoietin receptor (EPOR). We showed an inverse correlation between FLI1 and both KLF1 and EPOR during primitive erythroid and MK differentiation, similar to definitive hematopoiesis. This novel MEP differentiation system may allow an in-depth exploration of the molecular bases of erythroid and MK commitment and differentiation.

Numb mediates the interaction between Wnt and Notch to modulate primitive erythropoietic specification from the hemangioblast

During embryonic development, the establishment of the primitive erythroid lineage in the yolk sac is a temporally and spatially restricted program that defines the onset of hematopoiesis. In this report, we have used the embryonic stem cell differentiation system to investigate the regulation of primitive erythroid development at the level of the hemangioblast. We show that the combination of Wnt signaling with inhibition of the Notch pathway is required for the development of this lineage. Inhibition of Notch signaling at this stage appears to be mediated by the transient expression of Numb in the hemangioblast-derived blast cell colonies. Activation of the Notch pathway was found to inhibit primitive erythropoiesis efficiently through the upregulation of inhibitors of the Wnt pathway. Together, these findings demonstrate that specification of the primitive erythroid lineage is controlled, in part, by the coordinated interaction of the Wnt and Notch pathways, and position Numb as a key mediator of this process.

Eena Promotes Myeloid Proliferation through Stimulating ERK1/2 Phosphorylation in Zebrafish

The EEN (extra eleven nineteen) gene is one of the fusion partners of mixed-lineage leukemia, located on chromosome 19p13. Here we cloned two een genes (designated as eena and eenb) in zebrafish, which are assigned to linkage groups 8 and 2, respectively. Whole-mount in situ hybridization assay showed that eena and eenb have overlapping but distinct expression patterns during embryogenesis. Ubiquitous or targeted overexpression of eena, but not eenb, into wild-type or transgenic embryos (green fluorescent protein-labeled myeloid progenitors) induced a significant proliferation and ectopic distribution of myeloid progenitors in the yolk sac. Using a morpholino antisense gene knockdown approach, we showed that the number of myeloid progenitors and their downstream mature myelomonocytic cells was significantly decreased in the eena- deficient embryos. Mechanistically, overexpression of eena selectively stimulated ERK phosphorylation and increased the level of transcription factor c-Fos in vitro and in vivo, whereas eena lacking the Src homology 3 domain completely abolished these effects. Furthermore, a MAPK/ERK kinase (MEK) inhibitor, PD98059, blocked the eena-induced cell proliferation and activation of ERK signaling. The results suggest that eena plays an important role in the development of the myeloid cell through activation of the ERK pathway and may provide a valuable reference for future studies of the role of EEN in leukemogenesis.