Definitive Erythroblasts Research Articles

Primitive Erythropoiesis in the Mouse is Independent of DOT1L Methyltransferase Activity.

DOT1-like (DOT1L) histone methyltransferase is essential for mammalian erythropoiesis. Loss of DOT1L in knockout (Dot1l-KO) mouse embryos resulted in lethal anemia at midgestational age. The only recognized molecular function of DOT1L is its methylation of histone H3 lysine 79 (H3K79). We generated a Dot1l methyltransferase mutant (Dot1l-MM) mouse model to determine the role of DOT1L methyltransferase activity in early embryonic hematopoiesis. Dot1l-MM embryos failed to survive beyond embryonic day 13.5 (E13.5), similarly to Dot1l-KO mice. However, when examined at E10.5, Dot1l-MM embryos did not exhibit overt anemia like the Dot1l-KO. Vascularity and the presence of red blood cells in the Dot1l-MM yolk sacs as well as in the AGM region of Dot1l-MM embryos appeared to be similar to that of wildtype. In ex vivo cultures of yolk sac cells, Dot1l-MM primitive erythroblasts formed colonies comparable to those of the wildtype. Although ex vivo cultures of Dot1l-MM definitive erythroblasts formed relatively smaller colonies, inhibition of DOT1L methyltransferase activity in vivo by administration of EPZ-5676 minimally affected the erythropoiesis. Our results indicate that early embryonic erythropoiesis in mammals requires a DOT1L function that is independent of its intrinsic methyltransferase activity.

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.

Circulating primitive erythroblasts establish a functional, protein 4.1R-dependent cytoskeletal network prior to enucleating

Hematopoietic ontogeny is characterized by distinct primitive and definitive erythroid lineages. Definitive erythroblasts mature and enucleate extravascularly and form a unique membrane skeleton, composed of spectrin, 4.1R-complex, and ankyrinR-complex components, to survive the vicissitudes of the adult circulation. However, little is known about the formation and composition of the membrane skeleton in primitive erythroblasts, which progressively mature while circulating in the embryonic bloodstream. We found that primary primitive erythroblasts express the major membrane skeleton genes present in similarly staged definitive erythroblasts, suggesting that the composition and formation of this membrane network is conserved in maturing primitive and definitive erythroblasts despite their respective intravascular and extravascular locations. Membrane deformability and stability of primitive erythroblasts, assayed by microfluidic studies and fluorescence imaged microdeformation, respectively, significantly increase prior to enucleation. These functional changes coincide with protein 4.1 R isoform switching and protein 4.1R-null primitive erythroblasts fail to establish normal membrane stability and deformability. We conclude that maturing primitive erythroblasts initially navigate the embryonic vasculature prior to establishing a deformable cytoskeleton, which is ultimately formed prior to enucleation. Formation of an erythroid-specific, protein 4.1R-dependent membrane skeleton is an important feature not only of definitive, but also of primitive, erythropoiesis in mammals.

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.

Early Development of Definitive Erythroblasts from Human Pluripotent Stem Cells Defined by Expression of Glycophorin A/CD235a, CD34, and CD36

SummaryThe development of human erythroid cells has been mostly examined in models of adult hematopoiesis, while their early derivation during embryonic and fetal stages is largely unknown. We observed the development and maturation of erythroblasts derived from human pluripotent stem cells (hPSCs) by an efficient co-culture system. These hPSC-derived early erythroblasts initially showed definitive characteristics with a glycophorin A+ (GPA+) CD34lowCD36− phenotype and were distinct from adult CD34+ cell-derived ones. After losing CD34 expression, early GPA+CD36− erythroblasts matured into GPA+CD36low/+ stage as the latter expressed higher levels of β-globin along with a gradual loss of mesodermal and endothelial properties, and terminally suppressed CD36. We establish a unique in vitro model to trace the early development of hPSC-derived erythroblasts by serial expression of CD34, GPA, and CD36. Our findings may provide insight into the understanding of human early erythropoiesis and, ultimately, therapeutic potential.

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.

Temporally Distinct Developmental Waves of Erythropoiesis from Human Pluripotent Stem Cells

Mammalian erythropoiesis during embryogenesis occurs in several distinct stages or "waves" that vary according to timing, site of production, gene expression and physiology. The ontogeny of mammalian erythropoiesis is most thoroughly studied in mice where the earliest circulating erythroblasts released from the yolk sac are termed primitive. Later, the first definitive erythroid lineage is established by erythro-myeloid progenitors (EMPs) that originate in the yolk sac and migrate to the fetal liver for terminal differentiation. A second wave of definitive erythropoiesis is established from hematopoietic stem/progenitor cells that originate in the dorsal aorta and migrate to later stage fetal liver for terminal differentiation. Finally around birth, definitive erythropoiesis shifts to the bone marrow. The ontogeny of erythropoiesis overlaps in mice and humans, although less is known about the latter, as hematopoietic tissues from precisely staged early human embryos are difficult to obtain. We hypothesized that the initial steps of human erythroid ontogeny could be recapitulated by induced pluripotent stem cells (iPSCs) induced to undergo hematopoietic differentiation. We used a serum- and feeder-free protocol to differentiate iPSCs into embryoid bodies (EBs) that produced two sequential waves of distinctly different erythroid precursors. At day 8 of differentiation, EBs began to release hematopoietic precursors. Thereafter, erythroid precursors were released from the EBs in the presence of stem cell factor (SCF), erythropoietin (EPO) and insulin-like growth factor 1 (IGF-1). Erythroid precursors produced during wave 1 (days 12-23 of differentiation) were relatively large and expressed embryonic-type globins (zeta and epsilon), resembling those produced during primitive erythropoiesis. In contrast, wave 2 erythroblasts (day 27 and later) were smaller and expressed mainly gamma and alpha globins with some beta globin, suggestive of fetal-type definitive erythropoiesis. To investigate further the similarity of wave 1 and wave 2 erythroblasts to cells at the primitive and definitive stages of ontogeny, respectively, we used Affymetrix Genechips to analyze the global transcriptomes of stage-matched (CD235+ CD71high) cells. As primary human primitive erythroblasts were not available for comparison, we compared the transcriptomes from the iPSC-derived erythroblasts with those of primary murine definitive and primitive erythroblasts that were flow cytometry-purified from embryonic day 15.5 (E15.5) fetal liver and E10.5 bloodstream, respectively. The comparisons showed that wave 1 erythroblasts from human pluripotent cells resembled more closely the erythroid primitive lineage from mice, while wave 2 erythroblasts from the human cells resembled the erythroid definitive lineage of mice (P-value < 0.05 by a modified Kolmogorov-Smirnov test). For example, SOX6 and BCL11A, preferentially expressed during definitive erythropoiesis, were expressed at relatively high levels in wave 2 erythroblasts. In addition, gene set enrichment analysis (GSEA) demonstrated that wave 2 human iPSC-derived erythroblasts and primary murine definitive erythroblasts expressed numerous genes related to immune/inflammatory pathways that were shown previously to be important for the formation of definitive hematopoietic stem and progenitor cells in zebrafish and mouse embryos. Our findings demonstrate that human iPSC-derived embryoid bodies recapitulate early stages of erythroid ontogeny with respect to the timing of emerging lineages and their gene expression. Additionally, gene expression studies of human iPSC-derived primitive and definitive erythroblasts indicate inflammatory signaling as a potential regulator of the later stage of erythroid development. DisclosuresNo relevant conflicts of interest to declare.

Early exposure of murine embryonic stem cells to hematopoietic cytokines differentially directs definitive erythropoiesis and cardiomyogenesis in alginate hydrogel three-dimensional cultures.

HepG2-conditioned medium (CM) facilitates early differentiation of murine embryonic stem cells (mESCs) into hematopoietic cells in two-dimensional cultures through formation of embryoid-like colonies (ELCs), bypassing embryoid body (EB) formation. We now demonstrate that three-dimensional (3D) cultures of alginate-encapsulated mESCs cultured in a rotating wall vessel bioreactor can be differentially driven toward definitive erythropoiesis and cardiomyogenesis in the absence of ELC formation. Three groups were evaluated: mESCs in maintenance medium with leukemia inhibitory factor (LIF, control) and mESCs cultured with HepG2 CM (CM1 and CM2). Control and CM1 groups were cultivated for 8 days in early differentiation medium with murine stem cell factor (mSCF) followed by 10 days in hematopoietic differentiation medium (HDM) containing human erythropoietin, m-interleukin (mIL)-3, and mSCF. CM2 cells were cultured for 18 days in HDM, bypassing early differentiation. In CM1, a fivefold expansion of hematopoietic colonies was observed at day 14, with enhancement of erythroid progenitors, hematopoietic genes (Gata-2 and SCL), erythroid genes (EKLF and β-major globin), and proteins (Gata-1 and β-globin), although ζ-globin was not expressed. In contrast, CM2 primarily produced beating colonies in standard hematopoietic colony assay and expressed early cardiomyogenic markers, anti-sarcomeric α-actinin and Gata-4. In conclusion, a scalable, automatable, integrated, 3D bioprocess for the differentiation of mESC toward definitive erythroblasts has been established. Interestingly, cardiomyogenesis was also directed in a specific protocol with HepG2 CM and hematopoietic cytokines making this platform a useful tool for the study of erythroid and cardiomyogenic development.

RhoA Gtpase Regulates Erythroblast Cytokinesis and Enucleation By Dynamic Coordination Of The Microtubule-Actomyosin Machineries For Successful Abscission

RhoA GTPase is known to regulate cell adhesion, actomyosin cytoskeleton, and cytokinesis. Specifically, it contributes to cytokinesis in sea urchin embryos, Xenopus and HeLa cells by associating with the microtubule ends at the abscission area marking the position for the daughter cell separation and by activating myosin through phosphorylation of the myosin regulatory light chain (MRLC) (Piekny et al, Trends in Cell Biology 2005). Erythroblast enucleation, the process through which orthochromatic erythroblasts expel their nucleus during the final stage of mammalian erythropoiesis, is a multi-step process resembling asymmetric cytokinesis (reviewed by Li, Developmental Cell 2013). It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring between reticulocyte and pyrenocyte (Konstantinidis et al, Blood 2012).To define the mechanistic contribution of RhoA signaling in terminal erythroid maturation, we generated mice with erythroid specific RhoA deletion (EpoR-GFPcre-driven). Erythroid-specific deletion of RhoA, confirmed in the CD71-positive cells by PCR and immunoblotting, caused severe anemia, fatal in utero by E15.5. RhoA-deficient peripheral primitive blood cells were large with significant poikilocytosis and anisocytosis, frequently binuclear or multinuclear with polyploidy of the genetic material as demonstrated by flow cytometry with propidium iodide, and with incomplete clearance of the Golgi network and mitochondria. RhoAΔ/Δ fetal livers had progressively decreased cellularity. The erythroblast populations as determined by CD71-Ter119-FSC flow cytometry as well as by multispectral high-speed cell imaging in flow, demonstrated progressive decline and significantly decreased reticulocyte production (n=3, p<0.05), compatible with a fatal intrauterine anemia. RhoAΔ/Δ definitive erythroblasts also exhibited increased frequency of polyploidy. These polyploid cells were Ki67-negative by immunohistochemistry, indicating that they were not actively mitotic. Apoptosis was not significantly increased in the RhoA-deficient erythroblasts as determined by Annexin-V positivity. However, the late RhoAΔ/Δ erythroblasts demonstrated a significant increase in necrosis as determined by 7AAD and Annexin-V double-positivity (n=3, p<0.05). Increased phosphorylation of p53 was evident by immunoblotting, likely functioning as a polyploidy checkpoint after failure of abscission due to RhoA deficiency. Further evidence that RhoA is significant for microtubule organization in erythroblasts was attained by immuno-localization of RhoA at the microtubule ends and by decreased nuclear polarization in EpoR-Cre RhoAΔ/Δ orthochromatic erythroblasts, as shown by decreased delta centroid Ter119-Draq5 (the distance between the centers of the erythroblast and the nucleus) in multispectral high-speed cell imaging in flow. Phosphorylated myosin regulatory light chain (pMRLC) was found significantly decreased by immunoblotting of RhoAΔ/Δ fetal liver erythroblasts, indicating that RhoA controls phosphorylation of MRLC in erythropoiesis. Increased PAK phosphorylation was noted pointing towards compensatory upregulation of Rac GTPases in the RhoA-deficient cells, which was confirmed by quantitative RT-PCR. We further examined the erythroblastic islands in RhoAΔ/Δfetal liver by transmission electron microscopy and found that RhoA-deficient erythroblasts, frequently dysplastic and binucleated, were loosely associated with the central macrophage and there was a paucity of intracellular vacuoles in comparison to WT erythroblasts, indicating possible defects in vesicular transport, which has also been shown to play a role in erythroblast enucleation (Keerthivasan et al, Blood 2010).Thus, RhoA GTPase regulates erythroblast differentiation and enucleation by dynamic coordination of the microtubule-actomyosin machineries to achieve successful abscission. In addition, RhoA may also regulate erythroblast adhesive interactions with the central macrophage in erythroblastic islands and intracellular vesicular transport. Our results reveal novel molecular components and cellular processes in erythropoiesis that may be important for improving the efficiency of red blood cell production in vitro. Disclosures:No relevant conflicts of interest to declare.

Erythropoiesis in the Absence of Adult Hemoglobin

During erythropoiesis, hemoglobin (Hb) synthesis increases from early progenitors to mature enucleated erythrocytes. Although Hb is one of the most extensively studied proteins, the role of Hb in erythroid lineage commitment, differentiation, and maturation remains unclear. In this study, we generate mouse embryos and embryonic stem (ES) cells with all of the adult α and β globin genes deleted (Hb Null). While Hb Null embryos die in midgestation, adult globin genes are not required for primitive or definitive erythroid lineage commitment. In vitro differentiation of Hb Null ES cells generates viable definitive proerythroblasts that undergo apoptosis upon terminal differentiation. Surprisingly, all stages of Hb Null-derived definitive erythroblasts develop normally in vivo in chimeric mice, and Hb Null erythroid cells undergo enucleation to form reticulocytes. Free heme toxicity is not observed in Hb Null-derived erythroblasts. Transplantation of Hb Null-derived bone marrow cells provides short-term radioprotection of lethally irradiated recipients, whose progressive anemia results in an erythroid hyperplasia composed entirely of Hb Null-derived erythroblasts. This novel experimental model system enables the role played by Hb in erythroid cell enucleation, cytoskeleton maturation, and heme and iron regulation to be studied.

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.

Ontogeny of erythroid gene expression

AbstractErythroid ontogeny is characterized by overlapping waves of primitive and definitive erythroid lineages that share many morphologic features during terminal maturation but have marked differences in cell size and globin expression. In the present study, we compared global gene expression in primitive, fetal definitive, and adult definitive erythroid cells at morphologically equivalent stages of maturation purified from embryonic, fetal, and adult mice. Surprisingly, most transcriptional complexity in erythroid precursors is already present by the proerythroblast stage. Transcript levels are markedly modulated during terminal erythroid maturation, but housekeeping genes are not preferentially lost. Although primitive and definitive erythroid lineages share a large set of nonhousekeeping genes, annotation of lineage-restricted genes shows that alternate gene usage occurs within shared functional categories, as exemplified by the selective expression of aquaporins 3 and 8 in primitive erythroblasts and aquaporins 1 and 9 in adult definitive erythroblasts. Consistent with the known functions of Aqp3 and Aqp8 as H2O2 transporters, primitive, but not definitive, erythroblasts preferentially accumulate reactive oxygen species after exogenous H2O2 exposure. We have created a user-friendly Web site (http://www.cbil.upenn.edu/ErythronDB) to make these global expression data readily accessible and amenable to complex search strategies by the scientific community.

Tropomodulin3-Null Mice Are Embryonic Lethal with Anemia Due to Defects in Erythroblast Enucleation and Erythroblast-Macrophage Islands in the Fetal Liver

Abstract 84Tropomodulin1 (Tmod1) binds tropomyosin and caps the pointed ends of the short actin filaments in the spectrin-actin network of red blood cells (RBCs). Tmod1-null mice display a mild sphero-elliptocytic anemia due to mis-regulation of actin filament lengths and a disrupted membrane skeleton. The mild phenotype may be explained by the compensation of Tmod3, which is not found in wild-type RBCs but exists in Tmod1-null RBCs (one-fifth level of Tmod1). Our experiments with human erythroblasts show that the expression of Tmod1 increases during terminal differentiation while the level of Tmod3 is relatively constant, only decreasing at a very late stage. To investigate the role of Tmod3 in RBCs, we created a Tmod3 knockout mouse from ES cells (#RRF004, BayGenomics) with a gene-trap vector insertion into intron 1 of Tmod3. Both RT-PCR and western-blot results show that the expression of Tmod3 is abolished in Tmod3−/− mice. Tmod3+/− mice are viable and fertile, while Tmod3−/− animals are embryonic lethal, with most nulls dying between E16.5-E17.5. Tmod3−/− embryos are pale and anemic with a smaller fetal liver, suggesting that the lethality might be due to defective definitive erythropoiesis. This is supported by analysis of peripheral blood, which shows fewer definitive enucleated erythroblasts in Tmod3-null embryos. Flow-cytometry of fetal liver erythroblasts labeled with Ter119 and CD71 indicates that the late stage R3 population is reduced by about one-third in absence of Tmod3, while R1-R2 populations are somewhat increased. In addition, Annexin V staining shows a two-fold increase in apoptotic cells in the fetal liver, most of which are in the R1 population. Measurement of enucleation frequency in R populations shows a marked reduction of enucleated cells as the erythroblasts mature through the R3-R5 populations. These data indicate that definitive erythropoiesis is defective due to impaired erythroblast terminal differentiation in absence of Tmod3. To determine the underlying mechanisms, we used histology and confocal fluorescence microscopy to investigate the morphology and actin cytoskeleton of erythroblasts in process of enucleation. These experiments show abnormal nuclear morphology in orthochromatic Tmod3-null fetal liver erythroblasts, as well as defective F-actin contractile ring assembly in Tmod3−/− erythroblasts in process of nuclear expulsion, suggesting a role for Tmod3 in enucleation. Since macrophages are required for production of definitive erythroblasts and enucleation in vivo, we explored the role of macrophages in the Tmod3−/− phenotype. Immunofluorescence staining of fetal liver cryosections with F4/80, Ter119 and Hoechst reveals that macrophages display strikingly less dendritic morphologies in the Tmod3−/− mice, with macrophages sometimes containing Ter119-positive nucleated erythroblasts. Isolation of native erythroblast-macrophage islands from fetal liver demonstrates that islands isolated from Tmod3−/− fetal livers contain fewer erythroblasts compared with those from wild-type fetal liver. Further, reconstitution experiments indicate that erythroblasts from Tmod3−/− fetal liver are unable to form normal islands, indicating that Tmod3 function is required in erythroblasts. In conclusion, our study shows that knockout of Tmod3 leads to defective definitive erythropoiesis and embryonic lethality in mice, due to defects in island formation and abnormal enucleation. These data suggest that Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion as well as contractile ring assembly during erythroblast enucleation. Disclosures:No relevant conflicts of interest to declare.

Trophoblasts Regulate the Placental Hematopoietic Niche through PDGF-B Signaling

The placenta is a hematopoietic organ that supports hematopoietic stem/progenitor cell (HSPC) generation and expansion without promoting differentiation. We identified PDGF-B signaling in trophoblasts as a key component of the unique placental hematopoietic microenvironment that protects HSPCs from premature differentiation. Loss of PDGF-B or its receptor, PDGFRβ, induced definitive erythropoiesis in placental labyrinth vasculature. This was evidenced by accumulation of CFU-Es and actively proliferating definitive erythroblasts that clustered around central macrophages, highly reminiscent of erythropoiesis in the fetal liver. Ectopic erythropoiesis was not due to a requirement of PDGF-B signaling in hematopoietic cells but rather in placental trophoblasts, which upregulated Epo in the absence of PDGF-B signaling. Furthermore, overexpression of hEPO specifically in the trophoblasts in vivo was sufficient to convert the placenta into an erythropoietic organ. These data provide genetic evidence of a signaling pathway that is required to restrict erythroid differentiation to specific anatomical niches during development.

STAT3 Is Differentially Activated Downstream of the Erythropoietin Receptor in Primitive Compared to Definitive Erythroblasts

Abstract 2383Erythropoietin (EPO) acting through its receptor (EPOR), is a critical regulator of definitive erythropoiesis. The EPOR signaling network has been extensively studied in cell lines overexpressing various forms of EPOR. Together with genetic models, these studies have defined an EPOR-JAK2-STAT5 pathway that is critical for the formation of red blood cells. However, the hematopoietic phenotype of mice with a truncated EPOR incapable of activating STAT5 (EPOR-HM) suggests that STAT5 activation is not the only pathway mediating the erythropoietic effects of EPO-EPOR signaling. Our recent studies indicate that EPO-EPOR signaling also regulates primitive erythroblast maturation in the mammalian embryo. To better understand EPOR signaling in the context of primary erythroid cells, we utilized primitive erythroblasts from wild-type and EPOR-HM murine embryos. Robust phosphorylation of STAT5 was detected by western blot in freshly isolated primitive erythroblasts from wild-type embryos. As expected, there is no STAT5 phosphorylation evident in primitive erythroblasts from EPOR-HM embryos. Surprisingly, EPOR-HM embryos produce nearly normal numbers of primitive erythroblasts and we detected equivalent induction of STAT target genes following EPO stimulation in wild-type and EPOR-HM primitive erythroblasts. To address the discrepancy between these data and the accepted model of EPOR-STAT5 signaling, we searched for compensatory signaling via other STAT family members. We found that STAT3, but not STAT1, was phosphorylated in primitive erythroblasts from EPOR-HM embryos, suggesting that phospho-STAT3 compensates for the loss of phospho-STAT5 in the EPOR-HM mice. We also detected a lower level of STAT3 phosphorylation in wild-type primitive erythroblasts that was increased upon in vitro EPO stimulation. These results indicate that EPOR-STAT3 signaling normally occurs in primitive erythroblasts. Furthermore, our preliminary data show that STAT3 transcript levels are higher in primitive erythroblasts compared to definitive erythroblasts from the fetal liver or bone marrow, suggesting that STAT3 activation during erythropoiesis may be lineage-dependent. To test this hypothesis, we utilized a small molecule inhibitor of STAT3 to examine the function of STAT3 signaling both in primitive and in definitive erythroblasts during in vitro maturation. The terminal maturation of definitive erythroblasts is unaffected by STAT3 inhibition. However, inhibition of STAT3 causes a 40% reduction in primitive erythroblast proliferation in a novel 2-step ex vivo culture system. We conclude that, in contrast to definitive erythroblasts, STAT3 is activated downstream of EPOR in primitive erythroid cells. EPOR-STAT3 signaling has been reported in non-erythroid cells as well as in several cancer cell types. Therefore the study of EPOR signaling in primary embryonic erythroblasts may provide novel insights regarding the function of EPO in these cells. Disclosures:No relevant conflicts of interest to declare.

An Active Chromatin Hub Involving the Three Alternate Promoter/First Exons of the Ankyrin-1 (ANK-1) Gene

AbstractAbstract 3862The Ankyrin-1 (ANK-1) gene is an example of the many mammalian genes with alternate promoters in which transcripts containing distinct first exons are fused to a common exon 2 in different cell types. The ANK-1E promoter/first exon is active in only erythroid cells and is located between a promoter/first exon located 40 kb upstream that is expressed exclusively in brain and muscle cells (ANK-1B) and a promoter/first exon located 16 kb downstream that is expressed in many cell types (ANK-1A). We have previously shown that the ANK-1E promoter/first exon is flanked by two DNase I hypersensitive sites (HS). The ANK-1E 5′HS is located in the promoter region, while two adjacent HS (3′HS 1&2) are located ∼6 kb downstream. We hypothesize that the erythroid-specific activation of the ANK-1E promoter/first exon involves both changes in transcription factor occupancy and in chromatin structure. Using whole-genome Chromatin Immune Precipitation (ChIPSeq) and whole-genome mRNA analysis (RNASeq), we have made a detailed study of Erythroid Krupple-like Factor (EKLF) occupancy and mRNA levels in sorted mouse erythroid progenitor cells and definitive erythroblasts. In erythroid progenitor cells, EKLF occupies the Ank-1E 5′HS, and the level of Ank-1E mRNA is relatively low. In erythroblasts, EKLF is not detected at either Ank-1E 5′HS or the Ank-1E 3′HS and the Ank-1 mRNA level is increased more than 2-fold, consistent with EKLF acting as a transcriptional repressor. We have also shown that the ANK-1E 5′HS and the 3′HS region are both occupied by the transcription factors GATA-1 and NF-E2, although EMSA assays have shown that NF-E2 does not bind to the 5′HS region, and that GATA-1 does not bind to the 3′HS region in vitro. Addition of the NF-E2 site in 3′HS1 to the ANK-1E promoter increases expression 4-fold indicating a potential interaction of these two regions. We have recently demonstrated that both the ANK-1E 5′HS and 3′HS2 act as barrier elements that prevent gene silencing in cultured erythroid cells and transgenic mice. Consistent with the functional observations, in erythroid cells both ANK-1E 5′HS and the 3′HS region are occupied by the barrier-associated proteins USF-1 and -2, PRMT-1 and -4, CTCF and SRC1, while the ANK-1A region is occupied by the barrier-associated proteins USF-2, PRMT-1 and -4, but not USF-1 and CTCF. We hypothesized that the HS flanking the ANK-1E promoter/first exon form a chromatin loop that brings both the transcription factors and barrier proteins into proximity. Using the chromatin conformation capture assay we demonstrated an interaction between the Ank-1E 5′HS and the 3′HS region. Analysis of the three dimensional structure of the 100 kb region upstream of the mouse Ank-1 exon 2 region was performed using the chromatin conformation capture carbon copy (5C) assay. We designed a set of 12 forward primers on the three Ank-1 promoter/first exons and the HS that flank them, and a set of 264 reverse primers to analyze all interactions in this region. In mouse fetal liver erythroid cells, the strongest interaction is between Ank-1E 5′HS and a region 15 kb 3′ to the Ank-1B region. In addition, the Ank-1B promoter/first exon region interacts with both the Ank-1A region and a region 30 kb downstream of the Ank-1B region forming a ‘rosette’ that includes Ank-1E. We propose a model in which the formation of an active chromatin hub in erythroid cells which is facilitated by erythroid transcription factors and stabilized by the barrier-associated proteins. Disclosures:No relevant conflicts of interest to declare.

The Higher Structure of Chromatin in the LCR of the β-Globin Locus Changes during Development

The β-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant β-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.

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.

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 E14.5–16.5 of mouse gestation (Kingsley, et al. Blood 104:19, 2004), however, it is unclear by what mechanism or even where primitive erythroblasts enucleate. Definitive erythroblasts in the fetal liver and adult marrow enucleate by nuclear extrusion, generating reticulocytes and small, nucleated cells with a thin rim of cytoplasm referred to as “extruded nuclei”, that rapidly lose cell membrane phosphatidylserine asymmetry and are engulfed by macrophages. Since the cell membrane plays an important role in the biology of these cells, “extruded nucleus” is a misleading term. We propose that these cells be termed “pyrenocytes”, derived from the Greek “pyren” (the pit of a stone fruit) and “cyte” (cell), reflecting their highly condensed nucleus and high nuclear to cytoplasmic ratio. Careful examination of murine fetal blood by immunohistochemistry and multispectral imaging flow cytometry (Amnis ImageStream) revealed a transient population of εy-globin-positive pyrenocytes temporally coincident with the enucleation of primitive erythroblasts (E14.5–15.5). A high percentage (40%) of these circulating primitive pyrenocytes were annexin V-positive, suggesting that they, like their definitive counterparts, generate a signal that can facilitate engulfment by macrophage cells. At E14.5–15.5, the highest frequency of macrophage cells within the conceptus is found in the liver. Immunohistochemical studies with anti-εy-globin and F4/80 antibodies revealed that many primitive erythroblasts in the liver, but not the spleen, are in close proximity to macrophages. Furthermore, the frequency of nucleated primitive erythroblasts was higher in the liver than in the bloodstream at E15.5. These results, taken together, suggest that late-stage primitive erythroblasts do not passively flow through the liver but may, rather, interact with macrophage cells there. Surprisingly, primitive erythroblasts, but not co-circulating fetal definitive erythrocytes, can reconstitute erythroblast islands by attaching to fetal liver-derived macrophages in vitro. α4 integrin blocking antibodies, but not isotype control antibodies, reduce erythroblast island reconstitution, indicating that these primitive erythroid-macrophage interactions are mediated in part by α4 integrin. Finally, we found that unlike definitive erythroblasts, late-stage primitive erythroblasts fail to autonomously enucleate in vitro unless co-cultured with macrophage cells. We conclude that primitive erythroblasts in the mammalian fetus enucleate by nuclear extrusion as a semi-synchronous cohort and generate a transient population of pyrenocytes. Furthermore, our studies suggest that this process occurs in the fetal liver in association with macrophage cells. Continued investigation of the differences and similarities between primitive and definitive erythropoiesis will lead to an improved understanding of the terminal steps of erythroid maturation.

CpG Hypomethylation in a Large Domain Encompassing the Embryonic β-Like Globin Genes in Primitive Erythrocytes

There is little evidence addressing the role of CpG methylation in transcriptional control of genes that do not contain CpG islands. This is reflected in the ongoing debate about whether CpG methylation merely suppresses retroelements or if it also plays a role in developmental and tissue-specific gene regulation. The genes of the beta-globin locus are an important model of mammalian developmental gene regulation and do not contain CpG islands. We have analyzed the methylation status of regions in the murine beta-like globin locus in uncultured primitive and definitive erythroblasts and other cultured primary and transformed cell types. A large ( approximately 20-kb) domain is hypomethylated only in primitive erythroid cells; it extends from the region just past the locus control region to before beta-major and encompasses the embryonic genes Ey, beta h1, and beta h0. Even retrotransposons in this region are hypomethylated in primitive erythroid cells. The existence of this large developmentally regulated domain of hypomethylation supports a mechanistic role for DNA methylation in developmental regulation of globin genes.